Cellular communication network factor 1 promotes retinal leakage in diabetic retinopathy via inducing neutrophil stasis and neutrophil extracellular traps extrusion

Cellular communication network factor 1 promotes retinal leakage in diabetic retinopathy via inducing neutrophil stasis and neutrophil extracellular traps extrusion | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Cellular communication network factor 1 promotes retinal leakage in diabetic retinopathy via inducing neutrophil stasis and neutrophil extracellular traps extrusion Ting Li, Yixia Qian, Haicheng Li, Tongtong Wang, Qi Jiang, Yuchan Wang, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3845429/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Diabetic retinopathy (DR) is a major cause of blindness and is characterized by dysfunction of the retinal microvasculature. Neutrophil stasis, resulting in retinal inflammation and the occlusion of retinal microvessels, is a key mechanism driving DR. These plugging neutrophils subsequently release neutrophils extracellular traps (NETs), which further disrupts the retinal vasculature. Nevertheless, the primary catalyst for NETs extrusion in the retinal microenvironment under diabetic conditions remains unidentified. In recent studies, cellular communication network factor 1 (CCN1) has emerged as a central molecule modulating inflammation in pathological settings. Additionally, our previous research has shed light on the pathogenic role of CCN1 in maintaining endothelial integrity. However, the precise role of CCN1 in microvascular occlusion and its potential interaction with neutrophils in diabetic retinopathy have not yet been investigated. Methods We first examined the circulating level of CCN1 and NETs in our study cohort and analyzed related clinical parameters. To further evaluate the effects of CCN1 in vivo , we used recombinant CCN1 protein and CCN1 overexpression for gain-of-function, and CCN1 knockdown for loss-of-function by intravitreal injection in diabetic mice. The underlying mechanisms were further validated on human and mouse primary neutrophils and dHL60 cells. Results We detected increases in CCN1 and neutrophil elastase in the plasma of DR patients and the retinas of diabetic mice. CCN1 gain-of-function in the retina resulted in neutrophil stasis, NETs extrusion, capillary degeneration, and retinal leakage. Pre-treatment with DNase I to reduce NETs effectively eliminated CCN1-induced retinal leakage. Notably, both CCN1 knockdown and DNase I treatment rescued the retinal leakage in the context of diabetes. In vitro , CCN1 promoted adherence, migration, and NETs extrusion of neutrophils. Conclusion In this study, we uncover that CCN1 contributed to retinal inflammation, vessel occlusion and leakage by recruiting neutrophils and triggering NETs extrusion under diabetic conditions. Notably, manipulating CCN1 was able to hold therapeutic promise for the treatment of diabetic retinopathy. Diabetic retinopathy retinal inflammation CCN1 Retinal leakage Blood-retinal barrier Neutrophils Neutrophil extracellular traps Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Diabetic retinopathy (DR) stands as one of the most prevalent complications of diabetes mellitus (DM) and poses a significant threat to vision[ 1 ]. In 2020, DR was reported as the fifth most prevalent cause of blindness among individuals aged 50 years and older[ 2 ]. Pathologically, the increased infiltration of neutrophils and heightened adhesion between neutrophils and the endothelium result in retinal inflammation and vessel leukostasis, contributing to the progression of DR[ 3 ]. To date, studies have confirmed a positive correlation between diabetic retinopathy and both the neutrophil-to-lymphocyte ratio (NLR) and the neutrophil percentage-to-albumin ratio (NAR) in peripheral blood[ 4 ]. NETosis is a specific cell death process of neutrophils[ 5 ] and is characterized by the release of neutrophil extracellular traps (NETs), which are extracellular structures composed of chromatin and proteins such as myeloperoxidase (MPO), citrullinated histone H3 (Cit-H3), and neutrophil elastase (NE)[ 6 ]. NETs hallmarks including NE and DNA-histone complexes were identified as independent risk factors for DR[ 7 ]. Beyond peripheral blood, NETs have been detected in both vitreous bodies[ 8 ] and retina[ 9 ] of DR patients. The components of NETs cause vascular dysfunction by promoting retinal cells’ oxidative stress, senescence[ 10 ], apoptosis[ 11 ], thrombosis[ 12 ], inflammation pathway activation[ 13 , 14 ], and wrecked cell-to-cell integrity[ 15 ]. As a result, the increased trafficking of leukocytes in capillaries and the presence of NETs ultimately lead to the disruption of the blood-retinal barrier (BRB)[ 13 ]. These findings underscore the importance of identifying the key molecule that precisely modulates NETs extrusion for the treatment of DR at different disease stages. Cellular communication network factor 1 (CCN1), also known as cysteine-rich protein 61, is a matricellular protein secreted by various cell types including endothelial cells[ 16 ]. CCN1 is recognized for its involvement in a multitude of cellular processes, encompassing proliferation, differentiation, angiogenesis, apoptosis, and the formation of the extracellular matrix[ 17 ]. A previous study has unveiled elevated CCN1 levels in the vitreous humor of DR patients when compared to individuals without diabetes-related ocular conditions[ 18 – 20 ]. The upregulation of CCN1 in the retinas was also reported in diabetic mice[ 20 – 22 ] and rats[ 18 ]. Notably, intravitreal injection of anti-CCN1 antibody has been confirmed to reduce retinal neovascularization[ 23 ]. Beyond its role in promoting angiogenesis, CCN1 is now emerging as a pivotal molecular player in the modulation of inflammation under pathological conditions[ 24 – 27 ]. A growing body of research considers CCN1 as a regulator in immune cell trafficking, which attracts and locally immobilizes immune cells including monocytes[ 28 – 30 ], macrophages[ 24 , 31 ], leukocytes[ 27 ] and lymphocytes[ 32 ]. Moreover, CCN1 has been reported to stimulate the production of reactive oxygen species (ROS) through the activation of Rac1 and NADPH oxidase (NOX) 2[ 27 ]. In line with this, our prior research has demonstrated that CCN1 stimulates ROS production by activating NOX4 in diabetic retinopathy[ 21 ]. Considering the well-established critical role of NOX/ROS activation in NETs extrusion[ 33 – 35 ], the aforementioned evidence suggests a potential connection between CCN1 and neutrophils. Yet, the mechanism underlying the interaction between CCN1 and neutrophils remains largely unclear and necessitates further investigation. In this study, we delved into the intricate interplay between CCN1 and neutrophils and examined their roles in exacerbating retinal leakage in the context of diabetes. Our study cohort unveiled a marked elevation of circulating CCN1 in DR patients. Notably, circulating CCN1 was positively correlated with several key clinical parameters, including the absolute neutrophil count, NLR, NE, and the duration of diabetes. Mechanistically, CCN1 promoted neutrophil stasis within microvasculature by enhancing the adhesion, migration, and NETs extrusion of neutrophils. Significantly, CCN1 knockdown effectively counteracted the retinal leakage of diabetic mice. In summary, our study uncovers the substantial role played by CCN1 in retinal inflammation and retinal leakage by promoting neutrophil stasis and NETs extrusion during the progression of DR. Material and methods Study participants and blood sample collection This study was approved by the Third Affiliated Hospital of Sun Yat-sen University Network Ethics Committee following the principles of the Helsinki Declaration. Individuals with diabetes or healthy adults were respectively recruited from the Department of Endocrinology and Metabolism and Physical Examination Center of the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China, in 2023. In addition, healthy adults were enrolled with written informed consent. The study population comprised 77 consecutive patients with diabetes and 12 volunteers without chronic disease history between 18 and 70 years old. Of 77 patients with diabetes, 17 patients with diabetic retinopathy, and 50 patients without retinopathy were enrolled in the study. The diagnosis of diabetes is based on the 1999 World Health Organization (WHO) criteria[ 36 ]. The 7-field color fundus photographs were taken by a trained ophthalmic technician using VISUCAM Lite Digital Fundus Camera (Carl Zeiss Medite, AG, Jena, Germany) to diagnose diabetic retinopathy. Patients were excluded from this study if they had a history of renal failure with estimated glomerular filtration rate (eGFR) < 30 ml/min; acute infectious disease at the time of evaluation; a history of malignancy, mental disorders, autoimmune diseases, or severe heart or liver dysfunction; and history of solid or hematological neoplasia or active neoplasia. Those who had a history of eye diseases were also excluded. For each patient, a personal interview was conducted to collect basic demographic data regarding age, sex, height, and body weight. Information about medical history, duration of diabetes (years), diabetes treatment, and chemobiological examination results were also collected. Peripheral blood was collected in sodium citrate tubes (Becton Dickinson, San Jose, CA, USA). Whole blood samples were centrifuged for 15 min at 1550 g to isolate plasma for further analysis. Human peripheral blood and mouse neutrophil isolation Isolation of neutrophils from human whole blood and mouse bone marrow was performed as MojoSort Human Neutrophils Isolation Kit (Biolegend, California, United States) and MojoSort Mouse Neutrophils Isolation Kit (Biolegend, California, United States) according to the manufacturer’s instructions. Mice and diabetes modeling Mice Conventional Specific Pathogen Free (SPF) NOD/ShiLtJ mice and wild-type C57BL/6J mice were purchased from Guangdong Province Medical Experimental Animal Center. All mice were bred and maintained at the animal facility of Sun Yat-sen University under specific pathogen-free conditions. Mice were group housed in a controlled environment under the 12-h cycles of light-darkness, with free access to water and a standard chow diet. Mice were randomly assigned to each experimental group. The animal experiment was approved by the Animal Care Committee assigned by Sun Yat-sen University. For NOD/ShiLtJ mice, 25-week-old female mice with random blood glucose levels above 16.7 mmol/L were assigned to the experiment group, and the age-matched female mice (with random blood glucose levels below 16.7 mmol/L) were assigned to the control group. As for C57BL/6J mice, 6-week-old male mice were used in animal experiments. For the modeling of diabetes, multiple low doses of STZ (55mg/kg, Sigma-Aldrich, Steinheim, Germany), dissolved in citrate buffer (0.1M, pH = 4.7), were intraperitoneally injected into animals (STZ-group) for continues 5 days. Meanwhile, the control group received an equal 0.1M citrate buffer volume. Random blood glucose levels above 16.7 mmol/L were considered as diabetes one week after a 5-day injection. Intravitreal injection The intravitreal injection was operated as reported[ 37 ]. In our study, recombinant CCN1 protein (MedchemExpress, NJ, USA) was expressed in HEK 293 cells system. In intravitreal injection, 2ul volume of rCCN1 (10ug/ml) or vehicle control (PBS) was applied. Injection of rCCN1 was operated on day 1 and day 4, and the retina was dissected on day 7 and day 14 respectively. Lentivirus overexpressed CCN1 (LV-CCN1, pSLenti-CMV-CCN1-3xFLAG-PGK-Puro-WPRE) was synthesized and purified by OBIO Technology (Shanghai, China) Corp.Ltd. and the titer was 4.57E + 08/ml. In intravitreal injection, 2ul of virus was applied to overexpress CCN1 in the mouse eye. The overexpression efficiency was confirmed on day 14. Further experiments were performed on day 30 and day 60. To knock down CCN1 in the mouse eyes, lentivirus-silenced CCN1 (LV-siCCN1, target sequence: CTTCTACAGGCTGTTCAAT) and vehicle (LV-Con, target sequence: TTCTCCGAACGTGTCACGT) were synthesized by GENECHEM Technology (Shanghai, China) Corp.Ltd. The titer was 2E + 8/ml and the volume used in intravitreal injection was 2ul per eye. Intravitreal injection was performed at 12 weeks after diabetes modeling. The left eye of the mice accepted LV-Con injection, and the right eye accepted LV-siCCN1 injection. Mice were sacrificed one month after the virus interfered. For the DNase I treatment experiment, 2ul of DNase I (Thermo, Massachusetts, USA) or vehicle control (PBS) was intravenously administered to mice 30 minutes before rCCN1 treatment on day 1 and day 7. Mice were sacrificed on day 14 and the retina was collected. In STZ-DM mice, 2ul of DNase I was administered to the left eye, and an equal volume was administered to the right eye 8 weeks after diabetes modeling. The intravitreal injection was performed once per week for continuously 4 weeks, and the mice were sacrificed 1 week after the 4th injection. Evans Blue Permeability Assay Evans Blue (EB, 45 mg/kg, Sigma-Aldrich, Missouri, USA) was injected intravenously and then mice were placed on thermal blankets for 2h. For visualization of EB leakage, eyes were then enucleated, fixed for 1 h in 4% PFA, and dissected to collect the retina for flat-mount. To quantify EB leakage, the mice were perfused with 20 ml of PBS to clear EB in vessels after 2h-circulation on thermal blankets. Retinas were dissected and weighed. Then retinal tissues were homogenized and sonicated in formamide, followed by a 65°C-metal bath for 18h. The lysates were centrifuged, and the supernatant was used for EB quantification with a BioTek Synergy H1(excitation at 620 nm, emission at 680 nm). Immunofluorescence staining For the retina paraffin sections, samples were dewaxed and antigen-retrieved before staining. For the retina frozen sections, samples were restored to room temperature before staining. For retina flat-mount, retinas were treated as reported[ 38 ]. Briefly, eyeballs were enucleated from mice and then fixed in 4% PFA made up in 2x PBS at room temperature for 20 min. Retinas were dissected, flattened, and fixed again by cold methanol for 20 min at -20°C. Samples were blocked and permeabilized in 10% goat serum and 0.1% triton-X for 1 hour at room temperature and then incubated with primary antibodies at 4◦C overnight. Secondary antibodies conjugated fluorescence were used to incubate samples for 1 hour at room temperature. At last, samples were applied with mounting medium with DAPI (Abcam, MA, USA). Between each step, samples were washed with PBS 3 times. Slides were photographed under confocal microscopy (Leica TCS SP5, Wetzlar, Germany). Quantification was determined from 3 or 4 non-overlapping fields per sample and the average was used for statistics in every experiment. Both fluorescence intensity and related areas were analyzed with ImageJ software. The antibodies used for immunofluorescence staining were listed as follows: Isolectin GS-IB4 (1:200, Invitrogen#I21411, California, USA), CCN1 (Abcam#24448, 1:200), Ly6G (Biolegend#108401, 1:400), IL-1b (Cell Signaling Technology#12242S, 1:500), MPO (Thermo Fisher Scientific#MA1-34067, 1:400), MPO (Cell Signaling Technology#79623, 1:400), NE (Cell Signaling Technology#89241; 1:200), Collagen IV (Sigma Aldrich#SAB4500375, 1:500), CD31 (Cell Signaling Technology#3528S, 1:800), GFAP (Cell Signaling Technology#3670, 1:1000), Histone H3 (citrulline R2 + R8 + R17) (Abcam#5103, 1:400), PAD4 (Thermo Fisher Scientific# PA5-18318,1:200), Iba1 (Abcam#5067, 1:800), goat anti-mouse conjugated with Alexa Fluor®555 (Cell Signaling Technology#4409, 1:1000), goat anti-mouse conjugated with Alexa Fluor®647 (Abcam#150115, 1:1000), goat anti-rabbit conjugated with Alexa Fluor®555 (Cell Signaling Technology#4413, 1:1000), goat anti-mouse conjugated with Alexa Fluor®488 (Abcam#150077, 1:1000). ELISA The concentrations of CCN1 (#EK10933) and NE (#EK1447) in the plasma of human peripheral blood were assayed with ELISA kits (Signalway Antibody, Maryland, USA) according to the manufacturer’s instructions. Western blot Western Blot was performed according to our previous study[ 39 ]. Briefly, retinal tissues were homogenized and sonicated and lysates were analyzed by SDS-PAGE (10% acrylamide). Specific primary antibodies were used to incubate the membranes overnight at 4°C. Secondary antibodies were used to incubate the membranes for 1 hour at room temperature. Band density was then photographed by the ChemiDOC XRS + system (Biorad) and quantified using the Image Lab software. The detailed antibodies information for Western Blot was listed as follows: CCN1 (Abcam#24448, 1:1000), IL-1b (Cell Signaling Technology#12242S, 1:2000), MPO (Thermo Fisher Scientific#MA1-34067, 1:1000), HSP90 (Cell Signaling Technology#14793, 1:2000), HRP conjugated goat anti-mouse antibody (Biorad#1706516, 1:5000), HRP conjugated goat anti-rabbit secondary (Biorad#1706515, 1:5000). Cell culture Human retinal vascular endothelial cell (HRVEC), 293T, and HL60 cell line were obtained from Otwo Biotech (Shanghai, China) and cultured at 37◦C with 5% CO2. Both HRVEC and 293T were maintained in Dulbecco’s modified Eagle’s medium (Corning#10-014-CVR) containing 10% fetal bovine serum (Hyclone#SH30406.05) and 1% penicillin/streptomycin (Hyclone#SV30010-5). HL60 were cultured in RPMI 1640 (Gibco#C11875500CP-10) with L-glutamine (Gibco #25030081) with 25 mM HEPES (HyClone#SH30237.01), 1% penicillin/streptomycin and 10% fetal bovine serum. To differentiate HL60 cells into granulocyte-like cells, the cells were incubated for 5 days with DMSO (1.3%). Cells in passages 3–6 were used to perform experiments. Adherence assay HRVEC were plated in 48-well plates and cultured for 48h to 80% confluence. To evaluate the adherence of neutrophils to HRVEC, neutrophils were isolated as previously described and then co-cultured with HRVEC for 4h. Medium and non-adherent neutrophils were then removed, and adherent cells were washed 3 times with PBS. The number of adherent neutrophils to HRVEC was counted under the microscope after fixation and 0.5% crystal violet staining. Direct adherence of neutrophils to wells was evaluated using 48-well plates. Conditioned medium to suspend neutrophils was collected from HRVEC Veh and HRVEC CCN1 OE . Neutrophils in different mediums were then seeded into each well and incubated for 4h. After the removal of non-adherent cells, the wells were fixed and stained with crystal violet, and the adherence cell number was counted under the microscope. Transwell chemotaxis assay HRVEC were seeded on the bottom chamber of a transwell with 3.0 µm Pore Polycarbonate Membrane (Corning, NY, USA) for 48 h. Neutrophils were placed in the top chamber and incubated for 2h. Neutrophils migrated into the bottom chambers were harvested with PBS containing 5 mM EDTA and their absolute numbers were determined by counter. In the conditioned medium-treated experiment, the conditioned medium was collected as described previously and added into the bottom chamber instead of HRVEC. Concentration of supernatant The supernatant derived from HRVEC Veh and HRVEC CCN1 OE was concentrated via Amicon® Ultra-2 centrifugal filter devices (MilliporeSigma#1145U09, Darmstadt, Germany) according to the manufacturer’s instructions. The supernatant pre- and post-concentrated, as well as the depleted part, were used in our experiment with 1:50 dilution in RPMI-1640 medium for further treatment. NETs assay Neutrophils were plated at 20,000 cells per well in 48-well plates. After incubation in the RPMI 1640 for 1 h, cells were pre-treated with SYTOX Green (Invitrogen#S7020, 1 µM) and then stimulated with rCCN1 (4 ug/ml), ionomycin (4 µM), or an equal volume of PBS for 2.5 h. For experiments that involve CM treatment, CM was added to the wells with 1:50 dilution for 2.5 h. Extracellular DNA release was examined by measuring the green fluorescence with a microplate fluorescence reader (excitation: 485 nm, emission: 527 nm). Neutrophils were then fixed in 4% PFA, followed by Hoechst 33342 (Invitrogen#H3570,1:10,000) staining for 20 min. The quantification of NETs was performed as reported[ 40 ]. Briefly, the NETs area was quantified by subtracting the Hoechst 33342 signal from the SYTOX Green signal to remove the nucleus in the quantification. In immunocytochemistry experiments, the medium was removed, and cells were washed with PBS 3 times after 2.5 h treatment. Cells were then fixed with 4% PFA instantly for 20 min. After fixation, cells were blocked and permeabilized in 10% goat serum and 0.1% triton-X for 1 hour at room temperature and then incubated with primary antibodies at 4◦C overnight. Secondary antibodies conjugated fluorescence were used to incubate samples for 1 hour at room temperature. At last, samples were applied with mounting medium with DAPI (Abcam#104139). Slides were photographed under confocal microscopy (Leica TCS SP5, Wetzlar, Germany). Quantification was determined from 3 or 4 non-overlapping fields per sample and the average was used for statistics in every experiment. Both fluorescence intensity and related areas were analyzed with ImageJ software. Statistical analysis Clinical data analyses were performed using R ( www.Rproject.org ) or Prism (Version 9.5.0, GraphPad Software, LLC.) software, and we considered P value < 0.05 to be statistically significant. The Chi-square test or Fisher's exact test was used for the analysis of categorical variables. Student's t-test was used for comparing continuous variables. Logistic regression analysis was used to identify significant risk factors for diabetic retinopathy. Results CCN1 is positively related to the progression of DR. To gain insight into the role of CCN1 in DR, we first enrolled participants including control participants without diabetes (Non-DM), diabetes participants without diabetic retinopathy (DM), and diabetes participants with retinopathy complication (DR) to examine the CCN1 protein level change in peripheral blood. 88 participants participated in the study including 12 Non-DM, 49 DM, and 27 DR (Fig. 1 A). The mean age of Non-DM, DM, and DR was 54.0, 53.0, and 54.0 years respectively (Table 1 ). According to the previous report[ 41 ], insulin treatment, elevated FBG levels, and higher HbA1c concentration were considered risk factors for a higher prevalence of DR in people with DM. In our study cohort, the percentage of HbA1c and fasting plasma glucose (FBG) rose in diabetic participants as expected (both P < 0.001, Table 1 ), although no significant differences were found between the DR and DM groups. Among these groups, the level of uric acid (UA), estimated glomerular filtration rate (eGFR), Creatinine (Cr) and Serum cystatin C (CysC) was higher in DR than DM (Table S1), indicating poor kidney function in diabetic patients with retinopathy. Medication in DM and DR groups has no statistical difference in our study cohort (Table S2). Table 1 Characteristics of participants by the presence of DM or DR Variables Non-DM( N = 12 ) DM( N = 49 ) DR( N = 27 ) P overall P a Sex (Female) 6 (50.0%) 18 (36.7%) 15 (55.6%) 0.262 0.045* Age (years) 54.0 [49.5;59.2] 53.0 [47.0;59.0] 54.0 [50.5;60.5] 0.750 0.457 BMI (kg/m 2 ) 25.6 [23.5;26.6] 25.1 [22.9;26.7] 24.8 [23.1;26.0] 0.530 0.931 Diabetes duration (years) 0.00 [0.00;0.00] 5.00 [0.50;9.00] 11.0 [6.75;17.1] < 0.001* < 0.001* HbA1c (%) 5.80 [5.60;6.40] 8.30 [7.00;10.3] 8.30 [7.20;9.30] < 0.001* 0.987 FBG (mmol/L) 5.81 [5.14;5.87] 7.86 [6.77;9.17] 8.55 [5.89;10.1] < 0.001* 0.353 CCN1 (pg/ml) 124 [116;133] 134 [116;169] 160 [128;247] 0.007* 0.036* P values < 0.05 indicates the statistical significance and are shown with an asterisk. Results were presented as mean ± SD for continuous normally distributed variables and or medians (quartile 1, quartile 3) for not normally distributed variables, or n (%) for categorical variables. Abbreviations: BMI: body mass index; FBG: fasting plasma glucose; HbA1c: glycated hemoglobin A1c; CCN1: cellular communication network factor 1; NE: neutrophil elastase. P a : statistical significance between DM and DR group. We then performed an ELISA assay to detect whether the circulating level of CCN1 differed from each group (Fig. 1 A). The circulating level of CCN1 in the plasma of DR was higher than that of DM or Non-DM (Fig. 1 B). This was also confirmed by the Western blot (Fig. 1 D-E). To delve further into the relationship between CCN1 and the progression of diabetic retinopathy, we performed linear correlation analysis, revealing a positive correlation between CCN1 and the duration of diabetes (Fig. 1 C). This suggests that increased CCN1 levels are associated with the prevalence of DR. We further investigate whether CCN1 was involved in the progress of DR. No elevated circulating CCN1 level was observed in DR patients in proliferation stage or with macular edema or microaneurysms (Fig. 1 F-H). However, the level of circulating CCN1 in DR patients with hard exudates was higher than those without hard exudates (Fig. 1 I), suggesting that CCN1 levels begin to rise in the early stages of DR, and that there is a positive association between CCN1 and active DR. Building on this, we turned our attention to the retinas, the primary site of DR lesions, to investigate whether CCN1 is similarly upregulated in this critical context (Fig. 1 J). Non-obese diabetic (NOD) mouse is an experimental model for type 1 diabetes mellitus that spontaneously develops diabetes in a manner akin to humans[ 42 ]. Our immunofluorescence analysis revealed that CCN1 expression was notably upregulated in the retinas of NOD mice that had spontaneously progressed to diabetes (random blood glucose levels exceeding 16.7 mmol/L) when compared to their diabetes-free littermates (Fig. 1 K-L, Fig S1A-B). This trend was also observed in the retinas of streptozocin (STZ)-induced diabetic mice (Fig. 1 M-N). Notably, the CCN1 fluorescence is highly co-localized with isolectin B4 (IB4)-positive and CD31-positive endothelial cells (Fig. 1 K&M, Fig S1A). Furthermore, we validated the expression of CCN1 in publicly available single-cell sequencing (scRNA-Seq) data of retinas from various diabetes models. In the retinas of STZ-induced diabetic mice, CCN1 was predominantly expressed in Müller cells and endothelial cells (Fig S1E). In STZ and high-fat diet (HFD)-treated rats, CCN1 was primarily expressed in endothelial cells (Fig S1F). It is noteworthy that in both diabetic conditions, there was a discernible trend of increased CCN1 expression, particularly within the endothelial cell population (Fig S1E-F). Additionally, in scRNA-Seq data from an oxygen-induced retinopathy (OIR) model, which is commonly used to mimic ischemic retinopathies such as retinopathy of prematurity and proliferative diabetic retinopathy[ 43 ], we observed CCN1 highly expressed in Müller cells, pericytes, endothelial cells, neural stem cells, and astrocytes (Fig S1G). Under normoxia conditions, the retinal vasculature of neonatal mice matures within 14 days. As an angiogenic factor, CCN1 was highly expressed at postnatal day 14 (P14) and was rarely detected at P17 in the endothelial cells of retinas in normoxia-exposed mice (NORM) (Fig S1C). In the OIR model, pathological neovasculature proliferates until P17 and then begins to regress[ 44 ]. Intriguingly, the pattern of CCN1 mRNA expression was entirely reversed in OIR retinas; it exhibited a robust upregulation at P17 compared to P14 (Fig S1G). This result indicated that under physiological conditions CCN1 primarily participates in retinal vasculature development, while under OIR conditions, it may play a role in the regression of pathological vasculature. In summary, our findings indicate that CCN1 is expressed in various cell types during different stages of retinal development but is primarily found in Müller cells and endothelial cells in the mature retinas of mice. Collectively, these results suggest that CCN1 is upregulated in both retinas and peripheral blood under diabetic conditions, and the elevated levels of CCN1 may contribute to the progression of diabetic retinopathy. CCN1 promotes retinal leakage. To evaluate the contribution of CCN1 to DR pathogenesis, we then administered rCCN1 directly into the eyes of mice via intravitreal injection (Fig. 2 A). Elevated CCN1 within the vitreous cavity led to a trend of increased vascular permeability on day 7 following the first rCCN1 injection but was not statistically significant (Fig. 2 B-C). However, by day 14, there was a significant increase in vascular permeability in the retinas of mice treated with rCCN1 (Fig. 2 D). To further explore the impact of CCN1 on retinal vasculature, we examined various vessel parameters. Interestingly, rCCN1 injection did not result in changes to the total vessel length or the number of junctions or endpoints of the retinal vasculature (Fig S2A, B-E). The average vessel length appeared to be longer in the retinas treated with rCCN1 (Fig S2C). Astrocytes play a crucial role in BRB, and their support is essential for maintaining retinal vascular integrity. Our data revealed that the number of astrocytes per field and the extent of astrocyte coverage were not significantly altered by the rCCN1 treatment (Fig. 2 E-G). Of particular interest, we investigated non-functional empty basement membrane sleeves, which are acellular and express collagen IV but not IB4 and are associated with limited flow[ 45 ]. An increased number of these acellular empty sleeves suggests heightened capillary degeneration, which is a significant contributor to the progression of DR[ 46 ]. Strikingly, we observed an elevated number of acellular empty sleeves in the deep vessel plexus (Fig. 2 H-I), but not in the intermediate vessel plexus (Fig S2F-G), of the retinas that had been injected with rCCN1 on day 14, suggesting the role of CCN1 in promoting capillary degeneration. Considering the limited duration of recombinant protein administration, we established a model of continuous CCN1 overexpression within the eye by intravitreal injection of lentivirus (Fig. 2 J). The successful overexpression of CCN1 was confirmed through immunofluorescent staining (Fig. 2 K-L, S2H-I) and western blot analysis (Fig. 3 F, H) on day 14. Our findings revealed a significant increase in vascular permeability in the retinas with CCN1 overexpression on day 30, and this effect was sustained through day 60 (Fig. 2 M-O). Importantly, the total vessel length, average vessel length, and the number of junctions or endpoints of the retinal vasculature remained unaltered following lentivirus-mediated CCN1 overexpression (Fig S2I-M). However, we observed a reduction in astrocyte coverage after CCN1 overexpression (Fig. 2 P-Q), even though the number of astrocytes remained unchanged (Fig. 2 R). These results collectively support the role of CCN1 in promoting retinal leakage through the induction of capillary degeneration. CCN1 boosts neutrophil stasis and NETs extrusion. Our previous findings have shown that elevated levels of CCN1 in the retina resulted in capillary degeneration and increased retinal leakage. To further elucidate the role of CCN1 in DR, we sought to determine whether CCN1 overexpression also leads to retinal neurodegeneration. However, CCN1 overexpression did not alter the number of neuron cells or the thickness of retinal layers (Fig S3F-H). This suggests that the increase in CCN1 primarily contributes to the augmentation of retinal leakage rather than neuronal degeneration in DR. Microvessels, especially capillaries, are the sites most susceptible to neutrophil stasis and represent the severest sites of leakage in the progression of DR[ 47 ]. Therefore, we proceeded to investigate whether the overexpression of CCN1 in retinas leads to an upregulation of neutrophil stasis within capillaries. Upon examination of the entire eye section, we observed a significant accumulation of cells, including neutrophils, in the vitreous cavity (Fig. 3 A-B). Furthermore, aggregated neutrophils were observed at the branch points of vessels, and NETs appeared around vessels on day 30 following LV-CCN1 injection, as indicated by the increased MPO and Ly6G positive area on retina flat-mount (Fig. 3 C-E). Consistently, MPO levels were markedly increased in parallel with the elevation of CCN1 throughout the whole retina (Fig. 3 F-H). To gain further insight into the location of NETs within the retina, we performed immunofluorescent staining of retinal frozen sections on day 60 after injection. Our observations revealed an accumulation of immune cells on the surface of the inner retina, and we noted that NETs were present on retinas overexpressed CCN1, as indicated by the colocalization of MPO and Ly6G (Fig. 3 I-K) or MPO and Cit-H3 (Fig. 3 L-N). This suggests that CCN1 modulates the adhesion, migration, and NETs extrusion by neutrophils. In accordance with CCN1 overexpression by lentivirus, the supplementation of rCCN1 also led to neutrophil stasis and NETs formation on day 7, characterized by the colocalization of MPO and Ly6G (Fig. 3 O, Q, S) or MPO and NE (Fig. 3 P, R, S). However, these effects diminished on day 14 (Fig S3D-E). Remarkably, despite the reduction in NETs, the vascular permeability on day 14 was significantly increased (Fig. 2 D). As previously reported, NETs disrupt endothelial integrity and cause vascular leakage[ 15 , 48 ]. Hence, the abnormal neutrophil stasis and NETs in the retina appear to be resolved over time, while the disruption of retinal vasculature can persist for a longer duration. In the retinas of STZ-DM mice, more Ly6G + fluorescence was evident and colocalized with CCN1 and IB4 (Fig. 3 T-U), suggesting increasing neutrophil adherence to retinal vessels under diabetic conditions. In summary, these findings collectively support the role of CCN1 in modulating neutrophil stasis and the NETs extrusion in the context of DR. Neutrophil stasis and subsequent inflammation are significant contributors to the pathogenesis of DR, and this complex process involves various cell types, including microglia, Müller cells, and leukocytes[ 49 ]. In our study, we observed a substantial increase in the disorganized IB4 + area in retinas treated with rCCN1 supplementation (Fig S3A). This area extended from the inner retina to the outer retina, in contrast to the control retinas where the IB4 + area was primarily confined to the inner retina (Fig S3A). It’s worth noting that while IB4 is often used as a vascular indicator, it can also be utilized to label microglia and macrophages[ 50 ]. Furthermore, microglial cells predominantly reside in the inner plexiform layer (IPL) and outer plexiform layer (OPL) of the retina and remain in a quiescent or resting state under normal conditions. However, they become activated and migrate toward the outer retina in response to diabetic conditions[ 51 , 52 ]. Hence, rCCN1 supplementation may trigger an inflammatory cascade and gliosis in the retina, as confirmed by the co-staining of IB4 and CD31 (Fig S3B-C). This observation was further corroborated by co-staining with IBA1, a marker for microglia cells in the retinas (Fig S3I). Consistent with the activation of microglia cells in rCCN1-injected retinas, we also observed an increase in both IB4 + and IBA1 + areas in the retinas injected with LV-CCN1 (Fig S3I-K). On day 60 after LV-CCN1 injection, the IBA1 + area was notably expanded in the IPL and OPL (Fig S3I), indicating that the elevated CCN1 in the retina induced both NETs extrusion and microglial cell activation. In summary, our findings suggest that CCN1 is also a catalyst for microglial cell activation in retinas. CCN1 enhances the adhesion and migration of neutrophils. To further explore the potential involvement of CCN1 in modulating neutrophil behavior, we delved into the underlying mechanisms of how CCN1 interacts with neutrophils in vitro . We first established an HRVECs cell line overexpressing CCN1 (HRVEC CCN1 OE ) and isolated human primary neutrophils from whole peripheral blood for adhesion assay (Fig. 4 A). Our cell-cell adhesion assay revealed that a greater number of neutrophils adhered to HRVEC CCN1 OE when compared to HRVEC Veh (Fig. 4 B-C), indicating that CCN1 mediated neutrophils-to-endothelial cell adhesion. Beyond its role in modulating cell-cell adhesion, we found that pre-treatment with a conditioned medium enriched with CCN1 (CCN1-CM) increased the number of adherent neutrophils (Fig. 4 D-F) and dHL60s (Fig S4A-B), which indicated that CCN1 also directly supported cell-matrix adhesion. To assess the impact of CCN1 on neutrophil migration, we conducted migration assays (Fig. 4 G). In both co-culture with HRVECs CCN1 OE and treatment with CCN1-CM, the number of migrated neutrophils exhibited a significant increase compared to the control conditions (Fig. 4 H-I). Therefore, the above results collectively suggest that CCN1 promotes the adhesion and migration of neutrophils and potentially captures neutrophils within retinas. To further confirm that CCN1 contributes to NETs extrusion, we utilized rCCN1 and concentrated conditioned medium to treat human primary neutrophils and mouse bone marrow neutrophils (Fig. 4 J) in vitro . Under rCCN1 treatment, NETs was detected in both human (Fig S4C-E) and mouse neutrophils (Fig S4F) indicated by increased SYTOX Green fluorescence or area. Moreover, co-staining of DNA and Cit-H3 further confirmed that CCN1 contributed to NETs extrusion (Fig. 4 K-L, S4H-J). Additionally, ROS production was induced by rCCN1 treatment in both human (Fig S4G) and mouse neutrophils (Fig S4K). This further supports the idea that CCN1 activates neutrophils and leads to NETs extrusion. Furthermore, concentrated CCN1-CM also strongly induced NETs (Fig. 4 N-Q). In conclusion, our experimental evidence underscores the role of CCN1 in modulating neutrophil adherence, migration, and NETs extrusion. Suppressing CCN1 attenuates retinal leakage in diabetic mice. Based on the accumulating evidence, CCN1 might increase retinal leakage by promoting neutrophil stasis and triggering NETs extrusion. Therefore, targeting CCN1 offers a potential strategy to alleviate retinal leakage under diabetic conditions. To further evaluate whether reducing CCN1 could diminish neutrophil stasis and consequently ameliorate retinal leakage, we conducted intravitreal injections to knock down CCN1 expression in STZ-DM mice (Fig. 5 A). It’s worth noting that the intravitreal injection did not impact blood glucose levels (Fig S5A). The LV-siCCN1 treatment led to a significant decrease in CCN1 expression in the retinas of STZ-DM mice (Fig S5B-C). Consistent with CCN1 reduction, LV-siCCN1 effectively mitigated retinal leakage in STZ-DM mice (Fig. 5 B-C). However, LV-siCCN1 did not elicit alterations in the vasculature parameters (Fig S5D-H) and astrocyte coverage (Fig S5I-J). We also observed a significant increase in pathological empty sleeves in the retinas of STZ-DM mice, which declined after CCN1 knockdown (Fig. 5 D-E). As expected, CCN1 knockdown also resulted in a reduction of neutrophil stasis (Fig. 5 F-G), strongly suggesting that CCN1 is responsible for neutrophil stasis, capillary degeneration, and retinal leakage in DR. This finding further underscores the pivotal role of CCN1 in facilitating the progression of DR. DNase I alleviates CCN1-dependent retinal leakage. To further investigate the contribution of NETs in CCN1-dependent retinal leakage, we injected DNase I into the vitreous humor 30 minutes before rCCN1 injection to determine if DNase I pretreatment would abrogate the negative effects caused by rCCN1 (Fig. 6 A). As a result, DNase I pretreatment significantly improved retinal leakage (Fig. 6 B-C) and reduced the number of empty sleeves (Fig. 6 D-E), although it did not affect astrocyte coverage (Fig. 6 F-G). We further explored the effects of DNase I treatment in diabetic retinas to observe whether DNase I could improve retinal leakage (Fig. 6 H). Consequently, the inhibition of NETs through DNase I significantly ameliorated retinal leakage (Fig. 6 I-J) and decreased the number of empty sleeves (Fig. 6 K-L). Interestingly, DNase I treatment also significantly increased astrocyte coverage in the retinas of STZ-DM mice (Fig. 6 M-N). Notably, DNase I treatment promoted the degradation of NETs and ameliorated retinal inflammation (Fig. 6 O-Q). In summary, these comprehensive findings suggest that CCN1 induces retinal leakage by promoting neutrophil stasis within the retinal microvasculature and modulating NETs extrusion. By suppressing CCN1 or abolishing NETs, retinal leakage can be significantly reduced, providing a novel insight into the treatment of diabetic retinopathy. Circulating CCN1 and NE are potentially valuable markers for the assessment of DR. Finally, we investigated the circulating level of NE in our study cohort. Our findings indicated that the circulating level of NE was increased in both DM and DR patients when compared to Non-DM (Fig. 7 A). However, there were no significant differences between DR and DM patients. Nevertheless, NE was found to be positively correlated with CCN1 (Fig. 7 B). Additionally, the NLR is a marker of systemic inflammation and has previously been reported to positively correlate with DR[ 4 ], as is NAR[ 53 ]. We also observed a positive correlation between circulating CCN1 and NLR (Fig. 7 C), but no statistically significant correlation was identified between circulating CCN1 and NAR in our study population (Fig S6A). CCN1 also exhibited a positive correlation with the absolute number of neutrophils in the peripheral blood (Fig. 7 D). Circulating NE was positively correlated with HbA1c, FBG, DM duration, and TG (Fig. 7 E-H), thereby supporting the potential role of NE in the pathology of diabetes. Consequently, we constructed receiver operating characteristic (ROC) curves to determine whether circulating CCN1 or NE could better predict the presence of DR and DM. As illustrated in Fig. 7 I, the area under the curve (AUC) of a combination of CCN1 and NE was higher than that of HbA1c, suggesting that combining CCN1 and NE can effectively identify the presence of DM. To predict DR, CCN1 outperformed NE and HbA1c (Fig. 7 J). In conclusion, these findings lead us to conclude that CCN1 is intricately involved in the pathogenesis of DR, and it may play a role in modulating the physiological behavior of circulating neutrophils, making it a potentially valuable marker for the assessment of DR (Fig. 8 ). Discussion Initially, CCN1 was primarily characterized as an angiogenesis factor[ 17 ] and was found to exert influence over pathological angiogenesis in patients with PDR[ 18 , 20 ] and within the retinas of diabetic animal models[ 22 , 54 , 55 ]. Furthermore, previous research had established a positive correlation between CCN1 levels and the extent of neutrophil infiltration within lesion tissues[ 56 ]. This observation prompted us to embark on an exploration of the potential link between CCN1 and neutrophils. In our previous study, we unveiled the capacity of CCN1 to induce injury in endothelial cells[ 21 ]. In this present investigation, we not only observed an elevation in CCN1 levels in the peripheral blood of patients with DR and in the retinas of diabetic mice, but provided conclusive evidence demonstrating that CCN1 instigated the adhesion, migration, and NETs extrusion of neutrophils. Additionally, treatment with LV-siCCN1 or DNase I yields significant amelioration of neutrophil adhesion to the endothelial lining, consequently reversing the diabetes-induced retinal permeability. Collectively, these findings unequivocally validate the pivotal role of CCN1 in shaping the biological behavior of neutrophils, expanding its functional repertoire beyond its previously reported functions. More importantly, it stands to reason that the targeting of CCN1 may hold considerable promise in curtailing neutrophil stasis and, by extension, halting the progression of DR. The influence of CCN1 on BRB disruption is discernible across all the constituent cell types comprising the BRB. Firstly, CCN1 exhibits the capacity to incite sterile inflammation, even in the absence of infection[ 24 ], therefore causing endothelial cell injury. Secondly, CCN1 triggers pericyte apoptosis, exacerbating the compromise of BRB integrity[ 57 ]. Thirdly, our investigation reveals that CCN1 overexpression corresponds to a reduction in astrocyte coverage. Lastly, we observe that CCN1 overexpression induces gliosis and activates microglial cells. Contrary to our findings, Lulu Yan et al. have reported that the loss of CCN1 led to microglial cell activation in the retinas of the OIR model[ 58 ]. Given that microglial cells function as the resident macrophages of the retina and are responsive to inflammatory cues[ 59 ], we posit that the activation of microglial cells detected in our study is more likely attributed to inflammation rather than a direct consequence of CCN1. Furthermore, the dynamic nature of CCN1 expression in various cell types within the retina, as previously described, may account for the divergent conclusions stemming from its differential roles at distinct life stages. The elevation of CCN1 in the postnatal retina appears to play a pivotal role in maintaining retinal homeostasis during vasculature development. However, within the context of the adult retina under diabetic conditions, CCN1 assumes a contradictory role. Last but not least, despite the anticipated damaging effects of a pronounced inflammatory cascade upon neurons[ 60 ], our study does not reveal any significant alterations in neuron numbers following CCN1 treatment. This intriguing phenomenon may be attributable to CCN1’s multifunctionality, as it has been reported to exhibit neuroprotective properties within retinal tissue[ 61 ]. Consequently, the role of CCN1 in DR is indisputably multifaceted, rather than singular. CCN1 can be likened to a double-edged sword, capable of inflicting catastrophe upon the BRB while simultaneously conferring neuroprotective benefits. Our data demonstrated a noteworthy correlation between CCN1 and the duration of diabetes, in keeping with findings from the study by Bin Feng et al.[ 62 ] and Zhao-Yu Xiang et al.[ 63 ]. Notably, we have provided new and compelling evidence, showcasing elevated levels of circulating CCN1 in DR patients presenting with hard exudates. This newfound insight further substantiates the role of CCN1 in promoting retinal leakage, shedding light on its multifaceted impact on DR progression. Additionally, a multitude of studies have consistently demonstrated the elevation of circulating NETs markers in DR patients when compared to diabetic patients without DR[ 8 , 64 , 65 ]. Among these markers, circulating NE has been identified as an independent risk factor of DR[ 7 ]. However, no significant variance was detected between the DM and DR groups in our study. This observation may be attributed, in part, to the relatively small number of participants in our study. Additionally, it is worth noting that elevated circulating NE is not exclusive to retinopathy complications; it is also observed in other microvascular and macrovascular complications, such as atherosclerosis, nephropathy, and neuropathy[ 66 ]. Importantly, we did not exclude patients with these complications in the DM group, potentially contributing to the higher levels of circulating NE. Moreover, our findings reveal a positive correlation between circulating NE and diabetes duration, FBG, and HbA1c, in line with previous research[ 64 ]. Given the associations of circulating NE with multiple clinical indicators and its link to various diabetic complications, the absence of a difference in NE levels between the DR and DM groups may, at least in part, be attributed to these confounding factors. Consequently, circulating CCN1, rather than NE, emerges as a potential risk factor and biomarker for DR. However, it is noteworthy that our results highlight that the combination of circulating CCN1 and NE yields higher diagnostic accuracy for DM than HbA1c. This suggests that the combined assessment of circulating CCN1 and NE may also serve as a risk factor and biomarker for DM. However, the precise mechanism by which CCN1 activates neutrophils and triggers NETs extrusion remained unelucidated in our present study. The mechanisms of NETosis (the process of NETs extrusion) are still controversial and warrant further investigation. To date, three distinct mechanisms have been identified in NETosis, categorized as suicidal NETosis, vital NETosis, and mitochondrial NETosis[ 67 ]. Suicidal NETosis is dependent on NOX and culminates in neutrophil death[ 68 ]. In contrast, vital NETosis and mitochondrial NETosis are believed to result in NETs release without causing neutrophil death[ 69 , 70 ]. In DR, NETs triggered by hyperglycemia are dependent on NOX and ROS production, signifying that neutrophils confined within the retinal microvasculature undergo suicidal NETosis[ 8 ]. Additionally, activated endothelial cells can enhance NETs formation by releasing cytokines like IL-1β and ROS, while NETs can induce endothelial cell activation through protease components like histones, creating a positive feedback loop[ 71 , 72 ]. Notably, our prior research has revealed that CCN1 promotes NOX4 activation and increases ROS production in human retinal vascular endothelial cells[ 21 ]. In this study, heightened ROS production was also observed in neutrophils after rCCN1 treatment. Therefore, CCN1 may induce suicidal NETosis by activating the NOX/ROS signaling pathway. As a secreted protein, CCN1 initiates intracellular signaling pathways through interactions with cell membrane receptors, such as integrins[ 24 , 67 ]. Furthermore, it has been reported that CCN1 directly binds to Toll-like receptors to activate neutrophil mobilization[ 24 ]. Hence, CCN1 may potentially stimulate NETs extrusion in a NOX-dependent manner via interaction with surface receptors on neutrophils, although this hypothesis necessitates further validation. Moreover, prior research has recognized CCN1 as a mediator of leukocyte migration and vascular inflammation[ 28 ]. Our data reinforces this concept by confirming that CCN1 can attract and locally immobilize neutrophils, thereby expanding the conventional notion of CCN1 as a cell-matrix adhesion molecule. Conclusion Here, we present evidence establishing a pivotal link between CCN1 and NETs in the progression of DR. By orchestrating the adhesion, migration, and NETs extrusion by neutrophils, CCN1 promotes vascular occlusion, ultimately culminating in capillary degeneration and subsequent retinal leakage (Fig. 8 ). These findings not only shed light on the etiology of leukostasis in the early stages of DR but also underscore the potential therapeutic benefits of CCN1 knockdown or the prevention of NETs extrusion in ameliorating retinal leakage in the context of diabetes. Abbreviations CCN1: Cellular Communication Network Factor 1; DR: Diabetic Retinopathy; DM: Diabetes Mellitus; NPDR: Non-Proliferative Diabetic Retinopathy; PDR: Proliferative Diabetic Retinopathy; NETs: Neutrophil Extracellular Traps; MPO: Myeloperoxidase; Cit-H3: Citrullinated Histone H3; NE: Neutrophil Elastase; BRB: Blood-Retinal Barrier; ELISA: Enzyme-Linked Immunosorbent Assay; IB4: Isolectin B4; STZ: Streptozocin; scRNA-Seq: Single-cell RNA Sequencing; OIR: Oxygen-Induced Retinopathy; rCCN1: Recombinant CCN1 protein; LV: Lentivirus; HRVECs: Human Retinal Vascular Endothelial Cells; LV-siCCN1: Lentivirus carrying small interfering RNA targeting CCN1; DNase I: Deoxyribonuclease I; AUC: Area Under the Curve; NOX: NADPH Oxidase; ROS: Reactive Oxygen Species; EB: Evans Blue; IL-1b: Interleukin-1 beta; SPF: Specific Pathogen Free; PFA: Paraformaldehyde; DMSO: Dimethyl Sulfoxide; OIR: Oxygen-Induced Retinopathy; NORM: Normoxia Control; GCL: ganglion cell layer; IPL: Inner Plexiform Layer; INL: inner nuclear layer; OPL: Outer Plexiform Layer; ONL: outer nuclear layer; RPE: retinal pigment epithelium; WBC: white blood cell; RBC: red blood cell; PLT: platelet; HGB: hemoglobin; NEUT: neutrophils; LYMPH: lymphocytes; EO: eosinophils; BASO: basophils; MONO: monocytes; NLR: neutrophil to lymphocyte ratio; NAR: neutrophil to albumin ratio; TC: total cholesterol; TG: triglycerides; LDL: low-density lipoprotein; HDL: high-density lipoprotein; AST: aspartate aminotransferase; ALT: alanine aminotransferase; ALB: albumin; BUN: blood urea nitrogen; Cr: creatinine; CysC: serum cystatin C; UA: uric acid; eGFR: estimated glomerular filtration rate. Declarations Ethics approval and consent to participate This study was approved by the Third Affiliated Hospital of Sun Yat-sen University Network Ethics Committee following the principles of the Helsinki Declaration. The animal experiment was approved by the Animal Care Committee assigned by Sun Yat-sen University. Consent for publication All participants consented to submit the manuscript to the journal. Availability of data and materials Data of this study could be accessed freely under reasonable request. Competing interests All authors declare no potential conflicts of interest. Funding This work was supported by grants from the National Natural Science Foundation of China (No. 82270886, China), Clinical Research 5010 Program (No. 2023006, China), Sci-Tech Research Development Program of Guangzhou City (No. 202201020589, China), and Fundamental Research Funds for the Central Universities, Sun Yat-sen University to Y.C. This work was also supported by grants from the National Natural Science Foundation of China (No. 32000621, China), the Guangdong Basic and Applied Basic Research Foundation (No.s 2022A1515220129, 2023A1515010526, China) and the Science and Technology Projects in Guangzhou (No. 202102010338, China) to Y.L. Authors’ contributions Yan Lu and Yanming Chen conceived the study and acquitted funding. Yanming Chen, Yan Lu, Wenru Su, Guojun Shi, Xuemin He, Yanhua Zhu, and Shasha Li supervised this study. Ting Li designed the experiments and wrote the manuscript. Ting Li, Yixia Qian, and Tongtong Wang carried out the animal and cellular experiments. Haicheng Li, Wenru Su, Yuchan Wang, and Qi Jiang recruited the patients and collected clinical samples. Yixia Qian and Haicheng Li, blinded to the group information, assisted in analyzing the experiment data. All authors have reviewed and approved the final version of the manuscript. 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Feng B, Xu G, Sun K, Duan K, Shi B, Zhang N. Association of serum Cyr61 levels with peripheral arterial disease in subjects with type 2 diabetes. Cardiovasc Diabetol. 2020;19:194. Xiang Z-Y, Chen S-L, Qin X-R, Lin S-L, Xu Y, Lu L-N, et al. Changes and related factors of blood CCN1 levels in diabetic patients. Front Endocrinol (Lausanne). 2023;14:1131993. Magaña-Guerrero FS, Aguayo-Flores JE, Buentello-Volante B, Zarco-Ávila K, Sánchez-Cisneros P, Castro-Salas I, et al. Spontaneous Neutrophil Extracellular Traps Release Are Inflammatory Markers Associated with Hyperglycemia and Renal Failure on Diabetic Retinopathy. Biomedicines. 2023;11:1791. Evaluation of Circulating Markers of Neutrophil Extracellular Trap (NET) Formation as Risk Factors for Diabetic Retinopathy in a Case-Control Association Study - PubMed. [cited 2023 May 14]; Available from: https://pubmed.ncbi.nlm.nih.gov/27420129/ Shafqat A, Abdul Rab S, Ammar O, Al Salameh S, Alkhudairi A, Kashir J, et al. Emerging role of neutrophil extracellular traps in the complications of diabetes mellitus. Front Med. 2022;9:995993. Zhao Z, Pan Z, Zhang S, Ma G, Zhang W, Song J, et al. Neutrophil extracellular traps: A novel target for the treatment of stroke. Pharmacology & Therapeutics. 2023;241:108328. Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS, et al. Neutrophil Extracellular Traps Kill Bacteria. Science. 2004;303:1532–5. Takishita Y, Yasuda H, Shimizu M, Matsuo A, Morita A, Tsutsumi T, et al. Formation of neutrophil extracellular traps in mitochondrial DNA-deficient cells. J Clin Biochem Nutr. 2020;66:15–23. Yipp BG, Kubes P. NETosis: how vital is it? Blood. 2013;122:2784–94. Gupta AK, Joshi MB, Philippova M, Erne P, Hasler P, Hahn S, et al. Activated endothelial cells induce neutrophil extracellular traps and are susceptible to NETosis‐mediated cell death. FEBS Letters. 2010;584:3193–7. Saffarzadeh M, Juenemann C, Queisser MA, Lochnit G, Barreto G, Galuska SP, et al. Neutrophil Extracellular Traps Directly Induce Epithelial and Endothelial Cell Death: A Predominant Role of Histones. PLOS ONE. 2012;7:e32366. Additional Declarations No competing interests reported. Supplementary Files FigS1.jpg Supplemental Figure 1. The expression pattern of CCN1 in the retina. (A-B) Representative images (A) and MFI quantification (B) of CCN1 and IB4 on retinas frozen section for NOD mice (n=4). (C-D) Representative immunoblots (C) and quantification of CCN1 (D) in retina tissue of Non-DM and STZ-DM mice (n = 3). (E) Violin plots illustrating CCN1 gene expression across various cell types in retina single-cell RNA sequencing data from STZ-treated diabetic mice and control mice (GSE178121). (F) Violin plots displaying CCN1 gene expression in different cell types based on retina single-cell RNA sequencing data from rats fed with high-fat diets and STZ-treated diabetic rats, as compared to control rats (GSE209872). CD: Chow Diets; HFD: High-Fat Diets. (G) Violin plots representing CCN1 gene expression in various cell types as observed in retina single-cell RNA sequencing data from mice with oxygen-induced retinopathy (OIR) and normoxia control (NORM) mice at postnatal day 14 (p14) and p17 (GSE150703). *: P <0.05, **: P <0.01. Data are shown as mean ± SEM. Statistical differences were assessed using unpaired, 2-tailed Student’s t-test. FigS2.jpg Supplemental Figure 2. Exogenous CCN1 does not impact retinal vasculature. (A-E) Vasculature representative images (A) of deep (red), intermedium (green), and superficial (blue) layers day 14 post rCCN1 injection (n=3 per condition), the total vessel length (B), the average vessel length (C), the number of vessel endpoints (D), and the number of junctions (E). (F-G) Representative images (F) and the number of empty sleeves (G) in retinal intermedium layers day 14 post rCCN1 injection (n=3 per condition). (H) Representative confocal imaging and 3D reconstruction of retina flat mounts showing a view of the inner retina surface and a z section of the retina stained with CCN1 and CD31 on day 7 post lentivirus injection. (I-M) Representative images (I) and quantification of the total vessel length (J), the average vessel length (K), the number of vessel endpoints (L), and the number of junctions (M). *: P <0.05. Data are shown as mean ± SEM. Statistical differences were examined by unpaired, 2-tailed Student’s t-test in data. FigS2.jpg Supplemental Figure 2. Exogenous CCN1 does not impact retinal vasculature. (A-E) Vasculature representative images (A) of deep (red), intermedium (green), and superficial (blue) layers day 14 post rCCN1 injection (n=3 per condition), the total vessel length (B), the average vessel length (C), the number of vessel endpoints (D), and the number of junctions (E). (F-G) Representative images (F) and the number of empty sleeves (G) in retinal intermedium layers day 14 post rCCN1 injection (n=3 per condition). (H) Representative confocal imaging and 3D reconstruction of retina flat mounts showing a view of the inner retina surface and a z section of the retina stained with CCN1 and CD31 on day 7 post lentivirus injection. (I-M) Representative images (I) and quantification of the total vessel length (J), the average vessel length (K), the number of vessel endpoints (L), and the number of junctions (M). *: P <0.05. Data are shown as mean ± SEM. Statistical differences were examined by unpaired, 2-tailed Student’s t-test in data. FigS4.jpg Supplemental Figure 4. CCN1 mediates the adherence, migration, and NETs extrusion. (A-B) Visual assessment of dHL60s adherence to the plate after 4-hour co-culture with CM (A) and the number of adherent neutrophils per field (B) was assessed by crystal violet staining (n=5). (C-E) Representative images of Hoechst33342 and SYTOX Green (C), and SYTOX Green fluorescence measurement (D) and quantification of SYTOX Green+ area (E) for human primary neutrophils treated with PBS, rCCN1 or Ionomycin (Iono) for 2.5 h (n=5), PBS served as a negative control, and Iono served as a positive control. (F) The SYTOX Green fluorescence measurement for mouse bone marrow neutrophils treated with PBS, rCCN1, or ionomycin for 2.5 h (n=6). (G) ROS production of human primary neutrophils treated with PBS, rCCN1, or Ionomycin (Iono) for 2.5 h (n=4). (H-K) Representative images of mouse bone marrow primary neutrophils stained with Cit-H3 and DNA (SYTOX Green) (H), and quantification of Cit-H3 + area (I, n=3). (J) The SYTOX Green fluorescence measurement for mouse bone marrow neutrophils treated with PBS, rCCN1, or phorbol myristate acetate (PMA) for 2.5 h (n=5). (K) ROS production of mouse bone marrow neutrophils treated with PBS, rCCN1, or PMA for 2.5 h (n=4). **: P <0.01, ***: P <0.001, ****: P <0.0001. Data are shown as mean ± SEM. Statistical differences were assessed using unpaired, 2-tailed Student’s t-test. FigS5.jpg Supplemental Figure 5. CCN1 knockdown in diabetic retina does not cause vessel change in the short term. (A) The random blood glucose of STZ-modeling mice. (B-C) Representative retinal flat mount images stained with CCN1 (B) and MFI of CCN1 1 month after lentivirus injection (C, n=3 per condition). (D-H) Retinal vasculature representative images (D) of deep (red), intermedium (green), and superficial (blue) layers 1 month after lentivirus injection (n=3 per condition), the total vessel length (E), the average vessel length (F), the number of vessel endpoints (G), and the number of junctions (H), n=3 per condition. (I-J) Representative images of CD31 and GFAP stained retina flat mount (I) and the percentage of astrocytes’ endfeet coverage around retinal blood vessels (J), n=4 per condition. *: P <0.05, **: P <0.01, ***: P <0.001, ****: P <0.0001. Data are shown as mean ± SEM. Statistical differences were assessed using unpaired, 2-tailed Student’s t-test. FigS6.jpg Supplemental Figure 6. Correlation of each index. (A) Heatmap of Spearman’s correlation between each test outcome. Red represents the correlation coefficient closer to 1 and blue represents the correlation coefficient closer to -1. *: P <0.05, **: P <0.01, ***: P <0.001. The Spearman correlation test was conducted. SupplementalTables.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3845429","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":266039027,"identity":"9c0e73bd-1466-4f7a-a22b-fd650b8b4a05","order_by":0,"name":"Ting Li","email":"","orcid":"","institution":"Third Affiliated Hospital of Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Ting","middleName":"","lastName":"Li","suffix":""},{"id":266039028,"identity":"6f139035-ac57-4237-8ec4-8faf66bc78e3","order_by":1,"name":"Yixia Qian","email":"","orcid":"","institution":"Third Affiliated Hospital of Sun Yat-sen 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12:51:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3845429/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3845429/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49440546,"identity":"96436e53-cabb-4140-8e6c-615e9a154585","added_by":"auto","created_at":"2024-01-10 21:54:40","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3439832,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCCN1 is upregulated during the progeression of DR.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Overview of study groups and experimental design. (B) Measurement of circulating CCN1 protein levels in peripheral blood across study groups: Non-DM (\u003cem\u003eN\u003c/em\u003e=12), DM without DR (\u003cem\u003eN\u003c/em\u003e=49), and DR (\u003cem\u003eN\u003c/em\u003e=27). (C) Linear correlation between circulating CCN1 level and DM duration, correlation was measured using Spearman correlation coefficient. (D-E) Representative immunoblots (D) and densitometry quantification (E) of CCN1 protein level in plasma among three study populations. (F-I) Circulating CCN1 protein level in DR groups with or without proliferation (F), diabetic macular edema (G), microaneurysms (H), hard exudates (I). (J) Schematic presentation of the diabetic mouse model. (K-L) Representative images (K) and mean fluorescence intensity (MFI) quantification (L) of CCN1 and CD31 on retinas flat mount of NOD mice with high blood glucose (\u0026gt;16.7mm) or normal blood glucose (\u0026lt;16.7mm) (n=5). (M-N) Representative images and MFI quantification of CCN1 and IB4 on retinas of Non-DM and STZ-DM mice (n=3). *: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01. Data are shown as mean ± SEM. Statistical differences were assessed using unpaired, 2-tailed Student’s t-test.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3845429/v1/47cffbdb9ea0f42412f530e0.jpg"},{"id":49440934,"identity":"a5647161-3325-440e-873e-30a88ff0f050","added_by":"auto","created_at":"2024-01-10 22:02:40","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4753147,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCCN1 induced capillary degeneration and retinal leakage.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Experimental flow chart for rCCN1 injection. (B-D) Representative images (B) and quantification of Evans blue extravasation on day 7 (C, n=3) and day 14 (D, n=4) after rCCN1 or PBS injection. (E) Representative images showing Col IV, CD31, GFAP staining of the rCCN1- or PBS-injected retinas (n=3) on day 14. (F-G) The percentage of astrocytes’ endfeet coverage around retinal blood vessels (F) and the number of astrocytes per field (G) on day 14 after rCCN1 or PBS injection. (H-I) Representative images (H) and the number of empty sleeves in retinal deep layer vessels plexus (I, n=3), the white arrows indicate empty sleeves on the image. (J) Experimental workflow chart for lentivirus (LV-Con or LV-CCN1) injection. (K-L) Representative images of CD31, CCN1 stained retina frozen sections (K), and MFI of CCN1 (L) on day 14 post lentivirus injection. (M-O) Representative images (M) and quantification of Evans blue extravasation on day 30 (N, n=4) and day 60 (O, n=8). (P-R) Representative images of Col IV, CD31, GFAP staining on day 60 post lentivirus injection (n=7), the percentage of astrocytes’ endfeet coverage around retinal blood vessels (Q), and the number of astrocytes per field (R). GCL: ganglion cell layer; INL: inner nuclear layer; ONL: outer nuclear layer; RPE: retinal pigment epithelium. *: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ***: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, ****: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001. Data are shown as mean ± SEM. Statistical differences were examined by unpaired, 2-tailed Student’s t-test.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3845429/v1/c1dbbdec33dfdca303df0f29.jpg"},{"id":49440543,"identity":"01c22e12-5085-46a1-a608-5583e137c22d","added_by":"auto","created_at":"2024-01-10 21:54:40","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4207565,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCCN1 induces NETs extrusion in mouse retina.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-B) Representative hematoxylin and eosin (H\u0026amp;E) staining of the retina (A) and the number of cells in the vitreous cavity per section (B) day 30 post lentivirus injection (n = 3). (C-E) Representative images of CD31, Ly6G, MPO staining in retina flat mount day 30 post lentivirus injection and positive fluorescence area quantification of MPO (D) and Ly6G (E) (n=3). (F-H) Representative immunoblots (F) and densitometry quantification of MPO (G) and CCN1 (H) of retina tissue dissected on day 30 post lentivirus injection. (I-K) Representative images of CD31, Ly6G, MPO staining on retina frozen section day 60 post lentivirus injection and positive fluorescence area quantification of MPO (J) and Ly6G (K) (n=3). (L-N) Representative images of CD31, Cit-H3, MPO staining in retina frozen section day 60 post lentivirus injection and positive fluorescence area quantification of MPO (M) and Cit-H3 (N) (n=3). (O-S) Representative images of CD31, Ly6G, MPO staining (O) and CD31, Ly6G, NE staining (P) on retina flat mount day 7 post rCCN1 injection and positive fluorescence area quantification of MPO (Q) (n=6), NE (R) and Ly6G (S) (n=3). (T-U) Representative images of CD31, Ly6G, CCN1 staining in retina frozen section of STZ-DM mice retinas (T) and MFI of Ly6G (U) (n=4). IPL: Inner Plexiform Layer; OPL: Outer Plexiform Layer. *: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, ****: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001. Data are shown as mean ± SEM. Statistical differences were examined by unpaired, 2-tailed Student’s t-test in data.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3845429/v1/9bc95cd794064709ea616b28.jpg"},{"id":49440935,"identity":"22a77475-7ec7-4e96-aa52-fec29c860604","added_by":"auto","created_at":"2024-01-10 22:02:40","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3771728,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCCN1 mediates adherence, migration, and NETs extrusion of neutrophils \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic diagram of co-culture of neutrophils with HRVEC overexpressed CCN1 (HRVEC\u003csup\u003eCCN1 OE\u003c/sup\u003e) or vehicle (HRVEC\u003csup\u003eVeh\u003c/sup\u003e). (B-C) Visual assessment of human primary neutrophil adherence to HRVEC after 4 hours’ co-culture (B) and quantification of the number of adherent neutrophils per field (C), assessed by crystal violet staining (adherent neutrophils appear dark purple) (n=3). (D) Schematic diagram of co-culture of neutrophils with conditioned medium (CM) derived from HRVEC\u003csup\u003eCCN1 OE\u003c/sup\u003e or HRVEC\u003csup\u003eVeh\u003c/sup\u003e. (E-F) Visual assessment of human primary neutrophil adherence to the plate after 4-hour co-culture with CM and the quantification of the number of adherent neutrophils per field (E), assessed by crystal violet staining (F, n=3). (G) Schematic diagram of co-culture of human primary neutrophils with HRVEC or CM in transwell. (H-I) The number of migrated neutrophils in the lower chamber with HRVEC co-culture (H) and CM treatment for 4 hours (I). (J) Schematic diagram of treating human primary neutrophils with rCCN1 or concentrated CM. (K-L) Representative images and 3D construction of human primary neutrophils stained with Cit-H3 and DNA (DAPI) (K), and quantification of Cit-H3\u003csup\u003e+\u003c/sup\u003e area (L, n=4). (M) Schematic diagram of the CM concentration process; highlighting the pre-concentrated medium, post-concentrated medium, and the depleted part were preserved for experiments. (N-Q) Representative images of human primary neutrophils treated with pre-, post-, and depleted CM for 2.5 h (N), and quantification of DNA\u003csup\u003e+ \u003c/sup\u003e(O), PAD4\u003csup\u003e+\u003c/sup\u003e (P), Cit-H3\u003csup\u003e+\u003c/sup\u003e (Q) area (n=3). *: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, ****: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001. Data are shown as mean ± SEM. Statistical differences were examined by unpaired, 2-tailed Student’s t-test.\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3845429/v1/bd5923b3cdf3dc098d17ab02.jpg"},{"id":49440552,"identity":"25dcf844-96aa-426d-a6ac-f62927e7fff2","added_by":"auto","created_at":"2024-01-10 21:54:40","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4225229,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKnockdown of CCN1 reduces NETs extrusion and retinal leakage.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic of lentivirus injection in diabetic mouse experiment workflow. (B-C) Representative retinal flat mount images with Evans blue dye (B) and quantification of Evans blue extravasation per retina 1 month after intravitreal LV-siCCN1 or LV-Con injection in STZ-DM or Non-DM mice (C, n=8). ­(D-E) Representative retinal flat mount images stained with CD31 and Col IV (D) and the number of empty sleeves (E) in retinal deep layer vessels plexus (n=4), the white arrow indicates one classical empty sleeve on the image. (F-G) Representative retinal flat mount images stained with CD31 and Ly6G (F) and the quantification of Ly6G\u003csup\u003e+\u003c/sup\u003e area (G, n=10). *: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, ****: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001. Data are shown as mean ± SEM. Statistical differences were examined by unpaired, 2-tailed Student’s t-test.\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3845429/v1/f7af96b7d26610a2f1ac775f.jpg"},{"id":49440553,"identity":"6ed47435-2280-43d2-b52b-8b665715141b","added_by":"auto","created_at":"2024-01-10 21:54:41","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":5626586,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eClearance of NETs with DNase I alleviated retinal leakage.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) DNase I and rCCN1 treatment workflow schematic. (B-C) Representative retinal flat mount images with Evans blue dye (B) and quantification of Evans blue extravasation per retina (C) one week after the second DNase I injection (n=6). (D-E) Representative retinal flat mount images stained with CD31 and Col IV (D) and the number of empty sleeves (E) in retinal deep layer vessels plexus (n=4), the white arrow indicates classical empty sleeve on the image. (F-G) Representative images of CD31, GFAP, and Col IV on retina flat mount (F) and the percentage of astrocytes’ endfeet coverage around retinal blood vessels (G, n=4). (H) Schematic of DNase I treatment workflow on STZ-DM or Non-DM mice. (I-J) Representative retinal flat mount images with Evans blue dye (I) and quantification of Evans blue extravasation per retina one week after last injection (J, n=6). (K-L) Representative retinal flat mount images stained with CD31 and Col IV (K) and the number of empty sleeves (L) in retinal deep layer vessels plexus (n=4), the white arrow indicates one classical empty sleeve on the image. (M-N) Representative images of CD31 and GFAP on retina flat mount (M) and the percentage of astrocytes’ endfeet coverage around retinal blood vessels (N, n=4). (O-Q) Representative retinal flat mount images stained with MPO, Ly6G, and CD31 (O) and the quantification of Ly6G\u003csup\u003e+\u003c/sup\u003e area (P) and MPO\u003csup\u003e+\u003c/sup\u003e area (Q, n=7). *: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, ****: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001. Data are shown as mean ± SEM. Statistical differences were examined by unpaired, 2-tailed Student’s t-test.\u003c/p\u003e","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3845429/v1/b8423dc47fdb24de81c0d04f.jpg"},{"id":49440549,"identity":"182df5a4-4765-496f-b728-d4feb3da8861","added_by":"auto","created_at":"2024-01-10 21:54:40","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1655104,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCirculating CCN1 and NE are potentially valuable markers for the assessment of DR.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Circulating NE protein level in peripheral blood of Non-DM, DM, and DR group. (B-D) Linear correlation among circulating CCN1 and circulating NE (B), NLR (C), and neutrophils (D) in the whole study population (\u003cem\u003eN\u003c/em\u003e=88). (E-H) Linear correlation between circulating NE and HbA1c (E), FBG (F), DM duration (G), and TG (H) in the whole study population (\u003cem\u003eN\u003c/em\u003e=88). (I) ROC analysis was performed to evaluate the performance of CCN1+NE, CCN1, NE, and HbA1C in distinguishing DM (including DR) from Non-DM (Non-DM, \u003cem\u003eN\u003c/em\u003e=12; DM, \u003cem\u003eN\u003c/em\u003e=76). (J) ROC analysis was performed to evaluate the performance of CCN1+NE, CCN1, NE, and HbA1C in distinguishing DR from DM (DM, \u003cem\u003eN\u003c/em\u003e=49; DR, n=27). ***: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, ****: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001. The Spearman correlation test was conducted in data b-h. Data are shown as mean ± SEM. Statistical differences were examined by unpaired, 2-tailed Student’s t-test.\u003c/p\u003e","description":"","filename":"Fig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3845429/v1/0e5098647118731d39e77178.jpg"},{"id":49440937,"identity":"cc01bf68-9620-4569-91cd-b3c976314cf0","added_by":"auto","created_at":"2024-01-10 22:02:40","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":787658,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic outlining the process of CCN1 attracting neutrophils and inducing NETs in diabetic retinas.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUnder diabetic conditions, there is an upregulation of CCN1 within the retinal microenvironment, fostering heightened attraction and subsequent accumulation of neutrophils within retinal vessels. Consequently, neutrophils adhere to the endothelium and undergo migration beyond the vascular barrier. Augmented levels of CCN1 secretion activate neutrophils, precipitating the extrusion of NETs, thereby contributing to the progression of DR. The illustrative depiction was generated using the Biorender website (\u003ca href=\"https://app.biorender.com/\"\u003ehttps://app.biorender.com\u003c/a\u003e).\u003c/p\u003e","description":"","filename":"Fig8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3845429/v1/5345f9902e888695592a7721.jpg"},{"id":49486153,"identity":"2aa267d0-5945-4294-b564-50d393c87405","added_by":"auto","created_at":"2024-01-11 16:22:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2019829,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3845429/v1/50d82d8b-e353-48aa-8c6d-0da33fc301b1.pdf"},{"id":49441138,"identity":"a2ece006-2802-4ce9-a285-cdd430532dc8","added_by":"auto","created_at":"2024-01-10 22:10:40","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1754255,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Figure 1. The expression pattern of CCN1 in the retina.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-B) Representative images (A) and MFI quantification (B) of CCN1 and IB4 on retinas frozen section for NOD mice (n=4). (C-D) Representative immunoblots (C) and quantification of CCN1 (D) in retina tissue of Non-DM and STZ-DM mice (n = 3). (E) Violin plots illustrating CCN1 gene expression across various cell types in retina single-cell RNA sequencing data from STZ-treated diabetic mice and control mice (GSE178121). (F) Violin plots displaying CCN1 gene expression in different cell types based on retina single-cell RNA sequencing data from rats fed with high-fat diets and STZ-treated diabetic rats, as compared to control rats (GSE209872). CD: Chow Diets; HFD: High-Fat Diets. (G) Violin plots representing CCN1 gene expression in various cell types as observed in retina single-cell RNA sequencing data from mice with oxygen-induced retinopathy (OIR) and normoxia control (NORM) mice at postnatal day 14 (p14) and p17 (GSE150703). *: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01. Data are shown as mean ± SEM. Statistical differences were assessed using unpaired, 2-tailed Student’s t-test.\u003c/p\u003e","description":"","filename":"FigS1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3845429/v1/5d0cb8449fc087dadeef64f5.jpg"},{"id":49440932,"identity":"18b86b9d-4353-4061-9add-e92ffe6cc9ed","added_by":"auto","created_at":"2024-01-10 22:02:40","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":25,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Figure 2. Exogenous CCN1 does not impact retinal vasculature.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-E) Vasculature representative images (A) of deep (red), intermedium (green), and superficial (blue) layers day 14 post rCCN1 injection (n=3 per condition), the total vessel length (B), the average vessel length (C), the number of vessel endpoints (D), and the number of junctions (E). (F-G) Representative images (F) and the number of empty sleeves (G) in retinal intermedium layers day 14 post rCCN1 injection (n=3 per condition). (H) Representative confocal imaging and 3D reconstruction of retina flat mounts showing a view of the inner retina surface and a z section of the retina stained with CCN1 and CD31 on day 7 post lentivirus injection. (I-M) Representative images (I) and quantification of the total vessel length (J), the average vessel length (K), the number of vessel endpoints (L), and the number of junctions (M). *: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05. Data are shown as mean ± SEM. Statistical differences were examined by unpaired, 2-tailed Student’s t-test in data.\u003c/p\u003e","description":"","filename":"FigS2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3845429/v1/104f1531a190164c2d9c590d.jpg"},{"id":49440539,"identity":"fefdd884-7673-4b3c-96f7-3272435fc995","added_by":"auto","created_at":"2024-01-10 21:54:40","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":25,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Figure 2. Exogenous CCN1 does not impact retinal vasculature.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-E) Vasculature representative images (A) of deep (red), intermedium (green), and superficial (blue) layers day 14 post rCCN1 injection (n=3 per condition), the total vessel length (B), the average vessel length (C), the number of vessel endpoints (D), and the number of junctions (E). (F-G) Representative images (F) and the number of empty sleeves (G) in retinal intermedium layers day 14 post rCCN1 injection (n=3 per condition). (H) Representative confocal imaging and 3D reconstruction of retina flat mounts showing a view of the inner retina surface and a z section of the retina stained with CCN1 and CD31 on day 7 post lentivirus injection. (I-M) Representative images (I) and quantification of the total vessel length (J), the average vessel length (K), the number of vessel endpoints (L), and the number of junctions (M). *: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05. Data are shown as mean ± SEM. Statistical differences were examined by unpaired, 2-tailed Student’s t-test in data.\u003c/p\u003e","description":"","filename":"FigS2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3845429/v1/c49e90c09553eaabea221ddf.jpg"},{"id":49440542,"identity":"5f3b4720-f56b-4f26-b20f-8ce8bbe5ec5d","added_by":"auto","created_at":"2024-01-10 21:54:40","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":2886982,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Figure 4. CCN1 mediates the adherence, migration, and NETs extrusion.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-B) Visual assessment of dHL60s adherence to the plate after 4-hour co-culture with CM (A) and the number of adherent neutrophils per field (B) was assessed by crystal violet staining (n=5). (C-E) Representative images of Hoechst33342 and SYTOX Green (C), and SYTOX Green fluorescence measurement (D) and quantification of SYTOX Green+ area (E) for human primary neutrophils treated with PBS, rCCN1 or Ionomycin (Iono) for 2.5 h (n=5), PBS served as a negative control, and Iono served as a positive control. (F) The SYTOX Green fluorescence measurement for mouse bone marrow neutrophils treated with PBS, rCCN1, or ionomycin for 2.5 h (n=6). (G) ROS production of human primary neutrophils treated with PBS, rCCN1, or Ionomycin (Iono) for 2.5 h (n=4). (H-K) Representative images of mouse bone marrow primary neutrophils stained with Cit-H3 and DNA (SYTOX Green) (H), and quantification of Cit-H3\u003csup\u003e+\u003c/sup\u003e area (I, n=3). (J) The SYTOX Green fluorescence measurement for mouse bone marrow neutrophils treated with PBS, rCCN1, or phorbol myristate acetate (PMA) for 2.5 h (n=5). (K) ROS production of mouse bone marrow neutrophils treated with PBS, rCCN1, or PMA for 2.5 h (n=4). **: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, ****: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001. Data are shown as mean ± SEM. Statistical differences were assessed using unpaired, 2-tailed Student’s t-test.\u003c/p\u003e","description":"","filename":"FigS4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3845429/v1/62d0eb9a826450d79bd8ed1e.jpg"},{"id":49440550,"identity":"9d6ad65d-4f52-4579-9a3d-db00fa741781","added_by":"auto","created_at":"2024-01-10 21:54:40","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":5166062,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Figure 5. CCN1 knockdown in diabetic retina does not cause vessel change in the short term.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The random blood glucose of STZ-modeling mice. (B-C) Representative retinal flat mount images stained with CCN1 (B) and MFI of CCN1 1 month after lentivirus injection (C, n=3 per condition). (D-H) Retinal vasculature representative images (D) of deep (red), intermedium (green), and superficial (blue) layers 1 month after lentivirus injection (n=3 per condition), the total vessel length (E), the average vessel length (F), the number of vessel endpoints (G), and the number of junctions (H), n=3 per condition. (I-J) Representative images of CD31 and GFAP stained retina flat mount (I) and the percentage of astrocytes’ endfeet coverage around retinal blood vessels (J), n=4 per condition. *: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, ****: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001. Data are shown as mean ± SEM. Statistical differences were assessed using unpaired, 2-tailed Student’s t-test.\u003c/p\u003e","description":"","filename":"FigS5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3845429/v1/316b6bad00dbe9f309d682ce.jpg"},{"id":49441139,"identity":"ab265973-c0b1-412c-ab98-cd4d3bb60f0b","added_by":"auto","created_at":"2024-01-10 22:10:40","extension":"jpg","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":617787,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Figure 6. Correlation of each index.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Heatmap of Spearman’s correlation between each test outcome. Red represents the correlation coefficient closer to 1 and blue represents the correlation coefficient closer to -1. *: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001. The Spearman correlation test was conducted.\u003c/p\u003e","description":"","filename":"FigS6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3845429/v1/ad3f8c6973196dd57d692c60.jpg"},{"id":49440545,"identity":"046da241-2559-47c6-8def-9e31b4d0f850","added_by":"auto","created_at":"2024-01-10 21:54:40","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":21882,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalTables.docx","url":"https://assets-eu.researchsquare.com/files/rs-3845429/v1/386cd08f5cbdc3e203a04e7c.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Cellular communication network factor 1 promotes retinal leakage in diabetic retinopathy via inducing neutrophil stasis and neutrophil extracellular traps extrusion","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDiabetic retinopathy (DR) stands as one of the most prevalent complications of diabetes mellitus (DM) and poses a significant threat to vision[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In 2020, DR was reported as the fifth most prevalent cause of blindness among individuals aged 50 years and older[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Pathologically, the increased infiltration of neutrophils and heightened adhesion between neutrophils and the endothelium result in retinal inflammation and vessel leukostasis, contributing to the progression of DR[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. To date, studies have confirmed a positive correlation between diabetic retinopathy and both the neutrophil-to-lymphocyte ratio (NLR) and the neutrophil percentage-to-albumin ratio (NAR) in peripheral blood[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. NETosis is a specific cell death process of neutrophils[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] and is characterized by the release of neutrophil extracellular traps (NETs), which are extracellular structures composed of chromatin and proteins such as myeloperoxidase (MPO), citrullinated histone H3 (Cit-H3), and neutrophil elastase (NE)[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. NETs hallmarks including NE and DNA-histone complexes were identified as independent risk factors for DR[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Beyond peripheral blood, NETs have been detected in both vitreous bodies[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] and retina[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] of DR patients. The components of NETs cause vascular dysfunction by promoting retinal cells\u0026rsquo; oxidative stress, senescence[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], apoptosis[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], thrombosis[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], inflammation pathway activation[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], and wrecked cell-to-cell integrity[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. As a result, the increased trafficking of leukocytes in capillaries and the presence of NETs ultimately lead to the disruption of the blood-retinal barrier (BRB)[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. These findings underscore the importance of identifying the key molecule that precisely modulates NETs extrusion for the treatment of DR at different disease stages.\u003c/p\u003e \u003cp\u003eCellular communication network factor 1 (CCN1), also known as cysteine-rich protein 61, is a matricellular protein secreted by various cell types including endothelial cells[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. CCN1 is recognized for its involvement in a multitude of cellular processes, encompassing proliferation, differentiation, angiogenesis, apoptosis, and the formation of the extracellular matrix[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. A previous study has unveiled elevated CCN1 levels in the vitreous humor of DR patients when compared to individuals without diabetes-related ocular conditions[\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The upregulation of CCN1 in the retinas was also reported in diabetic mice[\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] and rats[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Notably, intravitreal injection of anti-CCN1 antibody has been confirmed to reduce retinal neovascularization[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Beyond its role in promoting angiogenesis, CCN1 is now emerging as a pivotal molecular player in the modulation of inflammation under pathological conditions[\u003cspan additionalcitationids=\"CR25 CR26\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. A growing body of research considers CCN1 as a regulator in immune cell trafficking, which attracts and locally immobilizes immune cells including monocytes[\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], macrophages[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], leukocytes[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] and lymphocytes[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Moreover, CCN1 has been reported to stimulate the production of reactive oxygen species (ROS) through the activation of Rac1 and NADPH oxidase (NOX) 2[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In line with this, our prior research has demonstrated that CCN1 stimulates ROS production by activating NOX4 in diabetic retinopathy[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Considering the well-established critical role of NOX/ROS activation in NETs extrusion[\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], the aforementioned evidence suggests a potential connection between CCN1 and neutrophils. Yet, the mechanism underlying the interaction between CCN1 and neutrophils remains largely unclear and necessitates further investigation.\u003c/p\u003e \u003cp\u003eIn this study, we delved into the intricate interplay between CCN1 and neutrophils and examined their roles in exacerbating retinal leakage in the context of diabetes. Our study cohort unveiled a marked elevation of circulating CCN1 in DR patients. Notably, circulating CCN1 was positively correlated with several key clinical parameters, including the absolute neutrophil count, NLR, NE, and the duration of diabetes. Mechanistically, CCN1 promoted neutrophil stasis within microvasculature by enhancing the adhesion, migration, and NETs extrusion of neutrophils. Significantly, CCN1 knockdown effectively counteracted the retinal leakage of diabetic mice. In summary, our study uncovers the substantial role played by CCN1 in retinal inflammation and retinal leakage by promoting neutrophil stasis and NETs extrusion during the progression of DR.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy participants and blood sample collection\u003c/h2\u003e \u003cp\u003eThis study was approved by the Third Affiliated Hospital of Sun Yat-sen University Network Ethics Committee following the principles of the Helsinki Declaration. Individuals with diabetes or healthy adults were respectively recruited from the Department of Endocrinology and Metabolism and Physical Examination Center of the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China, in 2023. In addition, healthy adults were enrolled with written informed consent.\u003c/p\u003e \u003cp\u003eThe study population comprised 77 consecutive patients with diabetes and 12 volunteers without chronic disease history between 18 and 70 years old. Of 77 patients with diabetes, 17 patients with diabetic retinopathy, and 50 patients without retinopathy were enrolled in the study. The diagnosis of diabetes is based on the 1999 World Health Organization (WHO) criteria[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The 7-field color fundus photographs were taken by a trained ophthalmic technician using VISUCAM Lite Digital Fundus Camera (Carl Zeiss Medite, AG, Jena, Germany) to diagnose diabetic retinopathy. Patients were excluded from this study if they had a history of renal failure with estimated glomerular filtration rate (eGFR)\u0026thinsp;\u0026lt;\u0026thinsp;30 ml/min; acute infectious disease at the time of evaluation; a history of malignancy, mental disorders, autoimmune diseases, or severe heart or liver dysfunction; and history of solid or hematological neoplasia or active neoplasia. Those who had a history of eye diseases were also excluded. For each patient, a personal interview was conducted to collect basic demographic data regarding age, sex, height, and body weight. Information about medical history, duration of diabetes (years), diabetes treatment, and chemobiological examination results were also collected. Peripheral blood was collected in sodium citrate tubes (Becton Dickinson, San Jose, CA, USA). Whole blood samples were centrifuged for 15 min at 1550 g to isolate plasma for further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eHuman peripheral blood and mouse neutrophil isolation\u003c/h2\u003e \u003cp\u003eIsolation of neutrophils from human whole blood and mouse bone marrow was performed as MojoSort Human Neutrophils Isolation Kit (Biolegend, California, United States) and MojoSort Mouse Neutrophils Isolation Kit (Biolegend, California, United States) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eMice and diabetes modeling\u003c/h2\u003e \u003cp\u003eMice Conventional Specific Pathogen Free (SPF) NOD/ShiLtJ mice and wild-type C57BL/6J mice were purchased from Guangdong Province Medical Experimental Animal Center. All mice were bred and maintained at the animal facility of Sun Yat-sen University under specific pathogen-free conditions. Mice were group housed in a controlled environment under the 12-h cycles of light-darkness, with free access to water and a standard chow diet. Mice were randomly assigned to each experimental group. The animal experiment was approved by the Animal Care Committee assigned by Sun Yat-sen University.\u003c/p\u003e \u003cp\u003eFor NOD/ShiLtJ mice, 25-week-old female mice with random blood glucose levels above 16.7 mmol/L were assigned to the experiment group, and the age-matched female mice (with random blood glucose levels below 16.7 mmol/L) were assigned to the control group. As for C57BL/6J mice, 6-week-old male mice were used in animal experiments. For the modeling of diabetes, multiple low doses of STZ (55mg/kg, Sigma-Aldrich, Steinheim, Germany), dissolved in citrate buffer (0.1M, pH\u0026thinsp;=\u0026thinsp;4.7), were intraperitoneally injected into animals (STZ-group) for continues 5 days. Meanwhile, the control group received an equal 0.1M citrate buffer volume. Random blood glucose levels above 16.7 mmol/L were considered as diabetes one week after a 5-day injection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eIntravitreal injection\u003c/h2\u003e \u003cp\u003eThe intravitreal injection was operated as reported[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In our study, recombinant CCN1 protein (MedchemExpress, NJ, USA) was expressed in HEK 293 cells system. In intravitreal injection, 2ul volume of rCCN1 (10ug/ml) or vehicle control (PBS) was applied. Injection of rCCN1 was operated on day 1 and day 4, and the retina was dissected on day 7 and day 14 respectively.\u003c/p\u003e \u003cp\u003eLentivirus overexpressed CCN1 (LV-CCN1, pSLenti-CMV-CCN1-3xFLAG-PGK-Puro-WPRE) was synthesized and purified by OBIO Technology (Shanghai, China) Corp.Ltd. and the titer was 4.57E\u0026thinsp;+\u0026thinsp;08/ml. In intravitreal injection, 2ul of virus was applied to overexpress CCN1 in the mouse eye. The overexpression efficiency was confirmed on day 14. Further experiments were performed on day 30 and day 60. To knock down CCN1 in the mouse eyes, lentivirus-silenced CCN1 (LV-siCCN1, target sequence: CTTCTACAGGCTGTTCAAT) and vehicle (LV-Con, target sequence: TTCTCCGAACGTGTCACGT) were synthesized by GENECHEM Technology (Shanghai, China) Corp.Ltd. The titer was 2E\u0026thinsp;+\u0026thinsp;8/ml and the volume used in intravitreal injection was 2ul per eye. Intravitreal injection was performed at 12 weeks after diabetes modeling. The left eye of the mice accepted LV-Con injection, and the right eye accepted LV-siCCN1 injection. Mice were sacrificed one month after the virus interfered.\u003c/p\u003e \u003cp\u003eFor the DNase I treatment experiment, 2ul of DNase I (Thermo, Massachusetts, USA) or vehicle control (PBS) was intravenously administered to mice 30 minutes before rCCN1 treatment on day 1 and day 7. Mice were sacrificed on day 14 and the retina was collected. In STZ-DM mice, 2ul of DNase I was administered to the left eye, and an equal volume was administered to the right eye 8 weeks after diabetes modeling. The intravitreal injection was performed once per week for continuously 4 weeks, and the mice were sacrificed 1 week after the 4th injection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eEvans Blue Permeability Assay\u003c/h2\u003e \u003cp\u003eEvans Blue (EB, 45 mg/kg, Sigma-Aldrich, Missouri, USA) was injected intravenously and then mice were placed on thermal blankets for 2h. For visualization of EB leakage, eyes were then enucleated, fixed for 1 h in 4% PFA, and dissected to collect the retina for flat-mount. To quantify EB leakage, the mice were perfused with 20 ml of PBS to clear EB in vessels after 2h-circulation on thermal blankets. Retinas were dissected and weighed. Then retinal tissues were homogenized and sonicated in formamide, followed by a 65\u0026deg;C-metal bath for 18h. The lysates were centrifuged, and the supernatant was used for EB quantification with a BioTek Synergy H1(excitation at 620 nm, emission at 680 nm).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence staining\u003c/h2\u003e \u003cp\u003eFor the retina paraffin sections, samples were dewaxed and antigen-retrieved before staining. For the retina frozen sections, samples were restored to room temperature before staining. For retina flat-mount, retinas were treated as reported[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Briefly, eyeballs were enucleated from mice and then fixed in 4% PFA made up in 2x PBS at room temperature for 20 min. Retinas were dissected, flattened, and fixed again by cold methanol for 20 min at -20\u0026deg;C. Samples were blocked and permeabilized in 10% goat serum and 0.1% triton-X for 1 hour at room temperature and then incubated with primary antibodies at 4◦C overnight. Secondary antibodies conjugated fluorescence were used to incubate samples for 1 hour at room temperature. At last, samples were applied with mounting medium with DAPI (Abcam, MA, USA). Between each step, samples were washed with PBS 3 times. Slides were photographed under confocal microscopy (Leica TCS SP5, Wetzlar, Germany). Quantification was determined from 3 or 4 non-overlapping fields per sample and the average was used for statistics in every experiment. Both fluorescence intensity and related areas were analyzed with ImageJ software.\u003c/p\u003e \u003cp\u003eThe antibodies used for immunofluorescence staining were listed as follows: Isolectin GS-IB4 (1:200, Invitrogen#I21411, California, USA), CCN1 (Abcam#24448, 1:200), Ly6G (Biolegend#108401, 1:400), IL-1b (Cell Signaling Technology#12242S, 1:500), MPO (Thermo Fisher Scientific#MA1-34067, 1:400), MPO (Cell Signaling Technology#79623, 1:400), NE (Cell Signaling Technology#89241; 1:200), Collagen IV (Sigma Aldrich#SAB4500375, 1:500), CD31 (Cell Signaling Technology#3528S, 1:800), GFAP (Cell Signaling Technology#3670, 1:1000), Histone H3 (citrulline R2\u0026thinsp;+\u0026thinsp;R8\u0026thinsp;+\u0026thinsp;R17) (Abcam#5103, 1:400), PAD4 (Thermo Fisher Scientific# PA5-18318,1:200), Iba1 (Abcam#5067, 1:800), goat anti-mouse conjugated with Alexa Fluor\u0026reg;555 (Cell Signaling Technology#4409, 1:1000), goat anti-mouse conjugated with Alexa Fluor\u0026reg;647 (Abcam#150115, 1:1000), goat anti-rabbit conjugated with Alexa Fluor\u0026reg;555 (Cell Signaling Technology#4413, 1:1000), goat anti-mouse conjugated with Alexa Fluor\u0026reg;488 (Abcam#150077, 1:1000).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eELISA\u003c/h2\u003e \u003cp\u003eThe concentrations of CCN1 (#EK10933) and NE (#EK1447) in the plasma of human peripheral blood were assayed with ELISA kits (Signalway Antibody, Maryland, USA) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot\u003c/h2\u003e \u003cp\u003eWestern Blot was performed according to our previous study[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Briefly, retinal tissues were homogenized and sonicated and lysates were analyzed by SDS-PAGE (10% acrylamide). Specific primary antibodies were used to incubate the membranes overnight at 4\u0026deg;C. Secondary antibodies were used to incubate the membranes for 1 hour at room temperature. Band density was then photographed by the ChemiDOC XRS\u0026thinsp;+\u0026thinsp;system (Biorad) and quantified using the Image Lab software. The detailed antibodies information for Western Blot was listed as follows: CCN1 (Abcam#24448, 1:1000), IL-1b (Cell Signaling Technology#12242S, 1:2000), MPO (Thermo Fisher Scientific#MA1-34067, 1:1000), HSP90 (Cell Signaling Technology#14793, 1:2000), HRP conjugated goat anti-mouse antibody (Biorad#1706516, 1:5000), HRP conjugated goat anti-rabbit secondary (Biorad#1706515, 1:5000).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eHuman retinal vascular endothelial cell (HRVEC), 293T, and HL60 cell line were obtained from Otwo Biotech (Shanghai, China) and cultured at 37◦C with 5% CO2. Both HRVEC and 293T were maintained in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (Corning#10-014-CVR) containing 10% fetal bovine serum (Hyclone#SH30406.05) and 1% penicillin/streptomycin (Hyclone#SV30010-5). HL60 were cultured in RPMI 1640 (Gibco#C11875500CP-10) with L-glutamine (Gibco #25030081) with 25 mM HEPES (HyClone#SH30237.01), 1% penicillin/streptomycin and 10% fetal bovine serum. To differentiate HL60 cells into granulocyte-like cells, the cells were incubated for 5 days with DMSO (1.3%). Cells in passages 3\u0026ndash;6 were used to perform experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAdherence assay\u003c/h2\u003e \u003cp\u003eHRVEC were plated in 48-well plates and cultured for 48h to 80% confluence. To evaluate the adherence of neutrophils to HRVEC, neutrophils were isolated as previously described and then co-cultured with HRVEC for 4h. Medium and non-adherent neutrophils were then removed, and adherent cells were washed 3 times with PBS. The number of adherent neutrophils to HRVEC was counted under the microscope after fixation and 0.5% crystal violet staining.\u003c/p\u003e \u003cp\u003eDirect adherence of neutrophils to wells was evaluated using 48-well plates. Conditioned medium to suspend neutrophils was collected from HRVEC\u003csup\u003eVeh\u003c/sup\u003e and HRVEC\u003csup\u003eCCN1 OE\u003c/sup\u003e. Neutrophils in different mediums were then seeded into each well and incubated for 4h. After the removal of non-adherent cells, the wells were fixed and stained with crystal violet, and the adherence cell number was counted under the microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eTranswell chemotaxis assay\u003c/h2\u003e \u003cp\u003eHRVEC were seeded on the bottom chamber of a transwell with 3.0 \u0026micro;m Pore Polycarbonate Membrane (Corning, NY, USA) for 48 h. Neutrophils were placed in the top chamber and incubated for 2h. Neutrophils migrated into the bottom chambers were harvested with PBS containing 5 mM EDTA and their absolute numbers were determined by counter. In the conditioned medium-treated experiment, the conditioned medium was collected as described previously and added into the bottom chamber instead of HRVEC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eConcentration of supernatant\u003c/h2\u003e \u003cp\u003eThe supernatant derived from HRVEC\u003csup\u003eVeh\u003c/sup\u003e and HRVEC\u003csup\u003eCCN1 OE\u003c/sup\u003e was concentrated via Amicon\u0026reg; Ultra-2 centrifugal filter devices (MilliporeSigma#1145U09, Darmstadt, Germany) according to the manufacturer\u0026rsquo;s instructions. The supernatant pre- and post-concentrated, as well as the depleted part, were used in our experiment with 1:50 dilution in RPMI-1640 medium for further treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eNETs assay\u003c/h2\u003e \u003cp\u003eNeutrophils were plated at 20,000 cells per well in 48-well plates. After incubation in the RPMI 1640 for 1 h, cells were pre-treated with SYTOX Green (Invitrogen#S7020, 1 \u0026micro;M) and then stimulated with rCCN1 (4 ug/ml), ionomycin (4 \u0026micro;M), or an equal volume of PBS for 2.5 h. For experiments that involve CM treatment, CM was added to the wells with 1:50 dilution for 2.5 h. Extracellular DNA release was examined by measuring the green fluorescence with a microplate fluorescence reader (excitation: 485 nm, emission: 527 nm). Neutrophils were then fixed in 4% PFA, followed by Hoechst 33342 (Invitrogen#H3570,1:10,000) staining for 20 min. The quantification of NETs was performed as reported[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Briefly, the NETs area was quantified by subtracting the Hoechst 33342 signal from the SYTOX Green signal to remove the nucleus in the quantification.\u003c/p\u003e \u003cp\u003eIn immunocytochemistry experiments, the medium was removed, and cells were washed with PBS 3 times after 2.5 h treatment. Cells were then fixed with 4% PFA instantly for 20 min. After fixation, cells were blocked and permeabilized in 10% goat serum and 0.1% triton-X for 1 hour at room temperature and then incubated with primary antibodies at 4◦C overnight. Secondary antibodies conjugated fluorescence were used to incubate samples for 1 hour at room temperature. At last, samples were applied with mounting medium with DAPI (Abcam#104139). Slides were photographed under confocal microscopy (Leica TCS SP5, Wetzlar, Germany). Quantification was determined from 3 or 4 non-overlapping fields per sample and the average was used for statistics in every experiment. Both fluorescence intensity and related areas were analyzed with ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eClinical data analyses were performed using R (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://www.Rproject.org\" target=\"_blank\"\u003ewww.Rproject.org\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.Rproject.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) or Prism (Version 9.5.0, GraphPad Software, LLC.) software, and we considered \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 to be statistically significant. The Chi-square test or Fisher's exact test was used for the analysis of categorical variables. Student's t-test was used for comparing continuous variables. Logistic regression analysis was used to identify significant risk factors for diabetic retinopathy.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eCCN1 is positively related to the progression of DR.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo gain insight into the role of CCN1 in DR, we first enrolled participants including control participants without diabetes (Non-DM), diabetes participants without diabetic retinopathy (DM), and diabetes participants with retinopathy complication (DR) to examine the CCN1 protein level change in peripheral blood. 88 participants participated in the study including 12 Non-DM, 49 DM, and 27 DR (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The mean age of Non-DM, DM, and DR was 54.0, 53.0, and 54.0 years respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). According to the previous report[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], insulin treatment, elevated FBG levels, and higher HbA1c concentration were considered risk factors for a higher prevalence of DR in people with DM. In our study cohort, the percentage of HbA1c and fasting plasma glucose (FBG) rose in diabetic participants as expected (both \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), although no significant differences were found between the DR and DM groups. Among these groups, the level of uric acid (UA), estimated glomerular filtration rate (eGFR), Creatinine (Cr) and Serum cystatin C (CysC) was higher in DR than DM (Table S1), indicating poor kidney function in diabetic patients with retinopathy. Medication in DM and DR groups has no statistical difference in our study cohort (Table S2).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCharacteristics of participants by the presence of DM or DR\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariables\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNon-DM(\u003cem\u003eN\u0026thinsp;=\u0026thinsp;12\u003c/em\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDM(\u003cem\u003eN\u0026thinsp;=\u0026thinsp;49\u003c/em\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDR(\u003cem\u003eN\u0026thinsp;=\u0026thinsp;27\u003c/em\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e overall\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSex (Female)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6 (50.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18 (36.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15 (55.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.262\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.045*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge (years)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e54.0 [49.5;59.2]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e53.0 [47.0;59.0]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e54.0 [50.5;60.5]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.750\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.457\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBMI (kg/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25.6 [23.5;26.6]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25.1 [22.9;26.7]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24.8 [23.1;26.0]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.530\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.931\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDiabetes duration (years)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.00 [0.00;0.00]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.00 [0.50;9.00]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.0 [6.75;17.1]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHbA1c (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.80 [5.60;6.40]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.30 [7.00;10.3]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.30 [7.20;9.30]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.987\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFBG (mmol/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.81 [5.14;5.87]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.86 [6.77;9.17]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.55 [5.89;10.1]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.353\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCCN1 (pg/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e124 [116;133]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e134 [116;169]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e160 [128;247]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.007*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.036*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003cem\u003eP\u003c/em\u003e values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 indicates the statistical significance and are shown with an asterisk. Results were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD for continuous normally distributed variables and or medians (quartile 1, quartile 3) for not normally distributed variables, or n (%) for categorical variables. Abbreviations: BMI: body mass index; FBG: fasting plasma glucose; HbA1c: glycated hemoglobin A1c; CCN1: cellular communication network factor 1; NE: neutrophil elastase. \u003cem\u003eP\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e: statistical significance between DM and DR group.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eWe then performed an ELISA assay to detect whether the circulating level of CCN1 differed from each group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The circulating level of CCN1 in the plasma of DR was higher than that of DM or Non-DM (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). This was also confirmed by the Western blot (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-E). To delve further into the relationship between CCN1 and the progression of diabetic retinopathy, we performed linear correlation analysis, revealing a positive correlation between CCN1 and the duration of diabetes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). This suggests that increased CCN1 levels are associated with the prevalence of DR. We further investigate whether CCN1 was involved in the progress of DR. No elevated circulating CCN1 level was observed in DR patients in proliferation stage or with macular edema or microaneurysms (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF-H). However, the level of circulating CCN1 in DR patients with hard exudates was higher than those without hard exudates (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI), suggesting that CCN1 levels begin to rise in the early stages of DR, and that there is a positive association between CCN1 and active DR.\u003c/p\u003e \u003cp\u003eBuilding on this, we turned our attention to the retinas, the primary site of DR lesions, to investigate whether CCN1 is similarly upregulated in this critical context (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ). Non-obese diabetic (NOD) mouse is an experimental model for type 1 diabetes mellitus that spontaneously develops diabetes in a manner akin to humans[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Our immunofluorescence analysis revealed that CCN1 expression was notably upregulated in the retinas of NOD mice that had spontaneously progressed to diabetes (random blood glucose levels exceeding 16.7 mmol/L) when compared to their diabetes-free littermates (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK-L, Fig S1A-B). This trend was also observed in the retinas of streptozocin (STZ)-induced diabetic mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eM-N). Notably, the CCN1 fluorescence is highly co-localized with isolectin B4 (IB4)-positive and CD31-positive endothelial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK\u0026amp;M, Fig S1A). Furthermore, we validated the expression of CCN1 in publicly available single-cell sequencing (scRNA-Seq) data of retinas from various diabetes models. In the retinas of STZ-induced diabetic mice, CCN1 was predominantly expressed in M\u0026uuml;ller cells and endothelial cells (Fig S1E). In STZ and high-fat diet (HFD)-treated rats, CCN1 was primarily expressed in endothelial cells (Fig S1F). It is noteworthy that in both diabetic conditions, there was a discernible trend of increased CCN1 expression, particularly within the endothelial cell population (Fig S1E-F). Additionally, in scRNA-Seq data from an oxygen-induced retinopathy (OIR) model, which is commonly used to mimic ischemic retinopathies such as retinopathy of prematurity and proliferative diabetic retinopathy[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], we observed CCN1 highly expressed in M\u0026uuml;ller cells, pericytes, endothelial cells, neural stem cells, and astrocytes (Fig S1G). Under normoxia conditions, the retinal vasculature of neonatal mice matures within 14 days. As an angiogenic factor, CCN1 was highly expressed at postnatal day 14 (P14) and was rarely detected at P17 in the endothelial cells of retinas in normoxia-exposed mice (NORM) (Fig S1C). In the OIR model, pathological neovasculature proliferates until P17 and then begins to regress[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Intriguingly, the pattern of CCN1 mRNA expression was entirely reversed in OIR retinas; it exhibited a robust upregulation at P17 compared to P14 (Fig S1G). This result indicated that under physiological conditions CCN1 primarily participates in retinal vasculature development, while under OIR conditions, it may play a role in the regression of pathological vasculature. In summary, our findings indicate that CCN1 is expressed in various cell types during different stages of retinal development but is primarily found in M\u0026uuml;ller cells and endothelial cells in the mature retinas of mice. Collectively, these results suggest that CCN1 is upregulated in both retinas and peripheral blood under diabetic conditions, and the elevated levels of CCN1 may contribute to the progression of diabetic retinopathy.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCCN1 promotes retinal leakage.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo evaluate the contribution of CCN1 to DR pathogenesis, we then administered rCCN1 directly into the eyes of mice via intravitreal injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Elevated CCN1 within the vitreous cavity led to a trend of increased vascular permeability on day 7 following the first rCCN1 injection but was not statistically significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-C). However, by day 14, there was a significant increase in vascular permeability in the retinas of mice treated with rCCN1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). To further explore the impact of CCN1 on retinal vasculature, we examined various vessel parameters. Interestingly, rCCN1 injection did not result in changes to the total vessel length or the number of junctions or endpoints of the retinal vasculature (Fig S2A, B-E). The average vessel length appeared to be longer in the retinas treated with rCCN1 (Fig S2C). Astrocytes play a crucial role in BRB, and their support is essential for maintaining retinal vascular integrity. Our data revealed that the number of astrocytes per field and the extent of astrocyte coverage were not significantly altered by the rCCN1 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-G). Of particular interest, we investigated non-functional empty basement membrane sleeves, which are acellular and express collagen IV but not IB4 and are associated with limited flow[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. An increased number of these acellular empty sleeves suggests heightened capillary degeneration, which is a significant contributor to the progression of DR[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Strikingly, we observed an elevated number of acellular empty sleeves in the deep vessel plexus (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH-I), but not in the intermediate vessel plexus (Fig S2F-G), of the retinas that had been injected with rCCN1 on day 14, suggesting the role of CCN1 in promoting capillary degeneration.\u003c/p\u003e \u003cp\u003eConsidering the limited duration of recombinant protein administration, we established a model of continuous CCN1 overexpression within the eye by intravitreal injection of lentivirus (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ). The successful overexpression of CCN1 was confirmed through immunofluorescent staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK-L, S2H-I) and western blot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, H) on day 14. Our findings revealed a significant increase in vascular permeability in the retinas with CCN1 overexpression on day 30, and this effect was sustained through day 60 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eM-O). Importantly, the total vessel length, average vessel length, and the number of junctions or endpoints of the retinal vasculature remained unaltered following lentivirus-mediated CCN1 overexpression (Fig S2I-M). However, we observed a reduction in astrocyte coverage after CCN1 overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eP-Q), even though the number of astrocytes remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eR). These results collectively support the role of CCN1 in promoting retinal leakage through the induction of capillary degeneration.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCCN1 boosts neutrophil stasis and NETs extrusion.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOur previous findings have shown that elevated levels of CCN1 in the retina resulted in capillary degeneration and increased retinal leakage. To further elucidate the role of CCN1 in DR, we sought to determine whether CCN1 overexpression also leads to retinal neurodegeneration. However, CCN1 overexpression did not alter the number of neuron cells or the thickness of retinal layers (Fig S3F-H). This suggests that the increase in CCN1 primarily contributes to the augmentation of retinal leakage rather than neuronal degeneration in DR. Microvessels, especially capillaries, are the sites most susceptible to neutrophil stasis and represent the severest sites of leakage in the progression of DR[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Therefore, we proceeded to investigate whether the overexpression of CCN1 in retinas leads to an upregulation of neutrophil stasis within capillaries. Upon examination of the entire eye section, we observed a significant accumulation of cells, including neutrophils, in the vitreous cavity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B). Furthermore, aggregated neutrophils were observed at the branch points of vessels, and NETs appeared around vessels on day 30 following LV-CCN1 injection, as indicated by the increased MPO and Ly6G positive area on retina flat-mount (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-E). Consistently, MPO levels were markedly increased in parallel with the elevation of CCN1 throughout the whole retina (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF-H). To gain further insight into the location of NETs within the retina, we performed immunofluorescent staining of retinal frozen sections on day 60 after injection. Our observations revealed an accumulation of immune cells on the surface of the inner retina, and we noted that NETs were present on retinas overexpressed CCN1, as indicated by the colocalization of MPO and Ly6G (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI-K) or MPO and Cit-H3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL-N). This suggests that CCN1 modulates the adhesion, migration, and NETs extrusion by neutrophils. In accordance with CCN1 overexpression by lentivirus, the supplementation of rCCN1 also led to neutrophil stasis and NETs formation on day 7, characterized by the colocalization of MPO and Ly6G (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eO, Q, S) or MPO and NE (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eP, R, S). However, these effects diminished on day 14 (Fig S3D-E). Remarkably, despite the reduction in NETs, the vascular permeability on day 14 was significantly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). As previously reported, NETs disrupt endothelial integrity and cause vascular leakage[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Hence, the abnormal neutrophil stasis and NETs in the retina appear to be resolved over time, while the disruption of retinal vasculature can persist for a longer duration. In the retinas of STZ-DM mice, more Ly6G\u003csup\u003e+\u003c/sup\u003e fluorescence was evident and colocalized with CCN1 and IB4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eT-U), suggesting increasing neutrophil adherence to retinal vessels under diabetic conditions. In summary, these findings collectively support the role of CCN1 in modulating neutrophil stasis and the NETs extrusion in the context of DR.\u003c/p\u003e \u003cp\u003eNeutrophil stasis and subsequent inflammation are significant contributors to the pathogenesis of DR, and this complex process involves various cell types, including microglia, M\u0026uuml;ller cells, and leukocytes[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. In our study, we observed a substantial increase in the disorganized IB4\u003csup\u003e+\u003c/sup\u003e area in retinas treated with rCCN1 supplementation (Fig S3A). This area extended from the inner retina to the outer retina, in contrast to the control retinas where the IB4\u003csup\u003e+\u003c/sup\u003e area was primarily confined to the inner retina (Fig S3A). It\u0026rsquo;s worth noting that while IB4 is often used as a vascular indicator, it can also be utilized to label microglia and macrophages[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Furthermore, microglial cells predominantly reside in the inner plexiform layer (IPL) and outer plexiform layer (OPL) of the retina and remain in a quiescent or resting state under normal conditions. However, they become activated and migrate toward the outer retina in response to diabetic conditions[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Hence, rCCN1 supplementation may trigger an inflammatory cascade and gliosis in the retina, as confirmed by the co-staining of IB4 and CD31 (Fig S3B-C). This observation was further corroborated by co-staining with IBA1, a marker for microglia cells in the retinas (Fig S3I). Consistent with the activation of microglia cells in rCCN1-injected retinas, we also observed an increase in both IB4\u003csup\u003e+\u003c/sup\u003e and IBA1\u003csup\u003e+\u003c/sup\u003e areas in the retinas injected with LV-CCN1 (Fig S3I-K). On day 60 after LV-CCN1 injection, the IBA1\u003csup\u003e+\u003c/sup\u003e area was notably expanded in the IPL and OPL (Fig S3I), indicating that the elevated CCN1 in the retina induced both NETs extrusion and microglial cell activation. In summary, our findings suggest that CCN1 is also a catalyst for microglial cell activation in retinas.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCCN1 enhances the adhesion and migration of neutrophils.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo further explore the potential involvement of CCN1 in modulating neutrophil behavior, we delved into the underlying mechanisms of how CCN1 interacts with neutrophils \u003cem\u003ein vitro\u003c/em\u003e. We first established an HRVECs cell line overexpressing CCN1 (HRVEC\u003csup\u003eCCN1 OE\u003c/sup\u003e) and isolated human primary neutrophils from whole peripheral blood for adhesion assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Our cell-cell adhesion assay revealed that a greater number of neutrophils adhered to HRVEC\u003csup\u003eCCN1 OE\u003c/sup\u003e when compared to HRVEC\u003csup\u003eVeh\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-C), indicating that CCN1 mediated neutrophils-to-endothelial cell adhesion. Beyond its role in modulating cell-cell adhesion, we found that pre-treatment with a conditioned medium enriched with CCN1 (CCN1-CM) increased the number of adherent neutrophils (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-F) and dHL60s (Fig S4A-B), which indicated that CCN1 also directly supported cell-matrix adhesion. To assess the impact of CCN1 on neutrophil migration, we conducted migration assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). In both co-culture with HRVECs\u003csup\u003eCCN1 OE\u003c/sup\u003e and treatment with CCN1-CM, the number of migrated neutrophils exhibited a significant increase compared to the control conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH-I). Therefore, the above results collectively suggest that CCN1 promotes the adhesion and migration of neutrophils and potentially captures neutrophils within retinas.\u003c/p\u003e \u003cp\u003eTo further confirm that CCN1 contributes to NETs extrusion, we utilized rCCN1 and concentrated conditioned medium to treat human primary neutrophils and mouse bone marrow neutrophils (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ) \u003cem\u003ein vitro\u003c/em\u003e. Under rCCN1 treatment, NETs was detected in both human (Fig S4C-E) and mouse neutrophils (Fig S4F) indicated by increased SYTOX Green fluorescence or area. Moreover, co-staining of DNA and Cit-H3 further confirmed that CCN1 contributed to NETs extrusion (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK-L, S4H-J). Additionally, ROS production was induced by rCCN1 treatment in both human (Fig S4G) and mouse neutrophils (Fig S4K). This further supports the idea that CCN1 activates neutrophils and leads to NETs extrusion. Furthermore, concentrated CCN1-CM also strongly induced NETs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eN-Q). In conclusion, our experimental evidence underscores the role of CCN1 in modulating neutrophil adherence, migration, and NETs extrusion.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSuppressing CCN1 attenuates retinal leakage in diabetic mice.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBased on the accumulating evidence, CCN1 might increase retinal leakage by promoting neutrophil stasis and triggering NETs extrusion. Therefore, targeting CCN1 offers a potential strategy to alleviate retinal leakage under diabetic conditions. To further evaluate whether reducing CCN1 could diminish neutrophil stasis and consequently ameliorate retinal leakage, we conducted intravitreal injections to knock down CCN1 expression in STZ-DM mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). It\u0026rsquo;s worth noting that the intravitreal injection did not impact blood glucose levels (Fig S5A). The LV-siCCN1 treatment led to a significant decrease in CCN1 expression in the retinas of STZ-DM mice (Fig S5B-C). Consistent with CCN1 reduction, LV-siCCN1 effectively mitigated retinal leakage in STZ-DM mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-C). However, LV-siCCN1 did not elicit alterations in the vasculature parameters (Fig S5D-H) and astrocyte coverage (Fig S5I-J). We also observed a significant increase in pathological empty sleeves in the retinas of STZ-DM mice, which declined after CCN1 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-E). As expected, CCN1 knockdown also resulted in a reduction of neutrophil stasis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF-G), strongly suggesting that CCN1 is responsible for neutrophil stasis, capillary degeneration, and retinal leakage in DR. This finding further underscores the pivotal role of CCN1 in facilitating the progression of DR.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDNase I alleviates CCN1-dependent retinal leakage.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo further investigate the contribution of NETs in CCN1-dependent retinal leakage, we injected DNase I into the vitreous humor 30 minutes before rCCN1 injection to determine if DNase I pretreatment would abrogate the negative effects caused by rCCN1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). As a result, DNase I pretreatment significantly improved retinal leakage (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-C) and reduced the number of empty sleeves (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD-E), although it did not affect astrocyte coverage (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF-G). We further explored the effects of DNase I treatment in diabetic retinas to observe whether DNase I could improve retinal leakage (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). Consequently, the inhibition of NETs through DNase I significantly ameliorated retinal leakage (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI-J) and decreased the number of empty sleeves (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK-L). Interestingly, DNase I treatment also significantly increased astrocyte coverage in the retinas of STZ-DM mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eM-N). Notably, DNase I treatment promoted the degradation of NETs and ameliorated retinal inflammation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eO-Q). In summary, these comprehensive findings suggest that CCN1 induces retinal leakage by promoting neutrophil stasis within the retinal microvasculature and modulating NETs extrusion. By suppressing CCN1 or abolishing NETs, retinal leakage can be significantly reduced, providing a novel insight into the treatment of diabetic retinopathy.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCirculating CCN1 and NE are potentially valuable markers for the assessment of DR.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFinally, we investigated the circulating level of NE in our study cohort. Our findings indicated that the circulating level of NE was increased in both DM and DR patients when compared to Non-DM (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). However, there were no significant differences between DR and DM patients. Nevertheless, NE was found to be positively correlated with CCN1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Additionally, the NLR is a marker of systemic inflammation and has previously been reported to positively correlate with DR[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], as is NAR[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. We also observed a positive correlation between circulating CCN1 and NLR (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC), but no statistically significant correlation was identified between circulating CCN1 and NAR in our study population (Fig S6A). CCN1 also exhibited a positive correlation with the absolute number of neutrophils in the peripheral blood (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Circulating NE was positively correlated with HbA1c, FBG, DM duration, and TG (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE-H), thereby supporting the potential role of NE in the pathology of diabetes. Consequently, we constructed receiver operating characteristic (ROC) curves to determine whether circulating CCN1 or NE could better predict the presence of DR and DM. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI, the area under the curve (AUC) of a combination of CCN1 and NE was higher than that of HbA1c, suggesting that combining CCN1 and NE can effectively identify the presence of DM. To predict DR, CCN1 outperformed NE and HbA1c (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ). In conclusion, these findings lead us to conclude that CCN1 is intricately involved in the pathogenesis of DR, and it may play a role in modulating the physiological behavior of circulating neutrophils, making it a potentially valuable marker for the assessment of DR (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eInitially, CCN1 was primarily characterized as an angiogenesis factor[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] and was found to exert influence over pathological angiogenesis in patients with PDR[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] and within the retinas of diabetic animal models[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Furthermore, previous research had established a positive correlation between CCN1 levels and the extent of neutrophil infiltration within lesion tissues[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. This observation prompted us to embark on an exploration of the potential link between CCN1 and neutrophils. In our previous study, we unveiled the capacity of CCN1 to induce injury in endothelial cells[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In this present investigation, we not only observed an elevation in CCN1 levels in the peripheral blood of patients with DR and in the retinas of diabetic mice, but provided conclusive evidence demonstrating that CCN1 instigated the adhesion, migration, and NETs extrusion of neutrophils. Additionally, treatment with LV-siCCN1 or DNase I yields significant amelioration of neutrophil adhesion to the endothelial lining, consequently reversing the diabetes-induced retinal permeability. Collectively, these findings unequivocally validate the pivotal role of CCN1 in shaping the biological behavior of neutrophils, expanding its functional repertoire beyond its previously reported functions. More importantly, it stands to reason that the targeting of CCN1 may hold considerable promise in curtailing neutrophil stasis and, by extension, halting the progression of DR.\u003c/p\u003e \u003cp\u003eThe influence of CCN1 on BRB disruption is discernible across all the constituent cell types comprising the BRB. Firstly, CCN1 exhibits the capacity to incite sterile inflammation, even in the absence of infection[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], therefore causing endothelial cell injury. Secondly, CCN1 triggers pericyte apoptosis, exacerbating the compromise of BRB integrity[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Thirdly, our investigation reveals that CCN1 overexpression corresponds to a reduction in astrocyte coverage. Lastly, we observe that CCN1 overexpression induces gliosis and activates microglial cells. Contrary to our findings, Lulu Yan et al. have reported that the loss of CCN1 led to microglial cell activation in the retinas of the OIR model[\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Given that microglial cells function as the resident macrophages of the retina and are responsive to inflammatory cues[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e], we posit that the activation of microglial cells detected in our study is more likely attributed to inflammation rather than a direct consequence of CCN1. Furthermore, the dynamic nature of CCN1 expression in various cell types within the retina, as previously described, may account for the divergent conclusions stemming from its differential roles at distinct life stages. The elevation of CCN1 in the postnatal retina appears to play a pivotal role in maintaining retinal homeostasis during vasculature development. However, within the context of the adult retina under diabetic conditions, CCN1 assumes a contradictory role. Last but not least, despite the anticipated damaging effects of a pronounced inflammatory cascade upon neurons[\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e], our study does not reveal any significant alterations in neuron numbers following CCN1 treatment. This intriguing phenomenon may be attributable to CCN1\u0026rsquo;s multifunctionality, as it has been reported to exhibit neuroprotective properties within retinal tissue[\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Consequently, the role of CCN1 in DR is indisputably multifaceted, rather than singular. CCN1 can be likened to a double-edged sword, capable of inflicting catastrophe upon the BRB while simultaneously conferring neuroprotective benefits.\u003c/p\u003e \u003cp\u003eOur data demonstrated a noteworthy correlation between CCN1 and the duration of diabetes, in keeping with findings from the study by Bin Feng et al.[\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e] and Zhao-Yu Xiang et al.[\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Notably, we have provided new and compelling evidence, showcasing elevated levels of circulating CCN1 in DR patients presenting with hard exudates. This newfound insight further substantiates the role of CCN1 in promoting retinal leakage, shedding light on its multifaceted impact on DR progression. Additionally, a multitude of studies have consistently demonstrated the elevation of circulating NETs markers in DR patients when compared to diabetic patients without DR[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Among these markers, circulating NE has been identified as an independent risk factor of DR[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, no significant variance was detected between the DM and DR groups in our study. This observation may be attributed, in part, to the relatively small number of participants in our study. Additionally, it is worth noting that elevated circulating NE is not exclusive to retinopathy complications; it is also observed in other microvascular and macrovascular complications, such as atherosclerosis, nephropathy, and neuropathy[\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Importantly, we did not exclude patients with these complications in the DM group, potentially contributing to the higher levels of circulating NE. Moreover, our findings reveal a positive correlation between circulating NE and diabetes duration, FBG, and HbA1c, in line with previous research[\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Given the associations of circulating NE with multiple clinical indicators and its link to various diabetic complications, the absence of a difference in NE levels between the DR and DM groups may, at least in part, be attributed to these confounding factors. Consequently, circulating CCN1, rather than NE, emerges as a potential risk factor and biomarker for DR. However, it is noteworthy that our results highlight that the combination of circulating CCN1 and NE yields higher diagnostic accuracy for DM than HbA1c. This suggests that the combined assessment of circulating CCN1 and NE may also serve as a risk factor and biomarker for DM.\u003c/p\u003e \u003cp\u003eHowever, the precise mechanism by which CCN1 activates neutrophils and triggers NETs extrusion remained unelucidated in our present study. The mechanisms of NETosis (the process of NETs extrusion) are still controversial and warrant further investigation. To date, three distinct mechanisms have been identified in NETosis, categorized as suicidal NETosis, vital NETosis, and mitochondrial NETosis[\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. Suicidal NETosis is dependent on NOX and culminates in neutrophil death[\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. In contrast, vital NETosis and mitochondrial NETosis are believed to result in NETs release without causing neutrophil death[\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. In DR, NETs triggered by hyperglycemia are dependent on NOX and ROS production, signifying that neutrophils confined within the retinal microvasculature undergo suicidal NETosis[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Additionally, activated endothelial cells can enhance NETs formation by releasing cytokines like IL-1β and ROS, while NETs can induce endothelial cell activation through protease components like histones, creating a positive feedback loop[\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. Notably, our prior research has revealed that CCN1 promotes NOX4 activation and increases ROS production in human retinal vascular endothelial cells[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In this study, heightened ROS production was also observed in neutrophils after rCCN1 treatment. Therefore, CCN1 may induce suicidal NETosis by activating the NOX/ROS signaling pathway. As a secreted protein, CCN1 initiates intracellular signaling pathways through interactions with cell membrane receptors, such as integrins[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. Furthermore, it has been reported that CCN1 directly binds to Toll-like receptors to activate neutrophil mobilization[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Hence, CCN1 may potentially stimulate NETs extrusion in a NOX-dependent manner via interaction with surface receptors on neutrophils, although this hypothesis necessitates further validation. Moreover, prior research has recognized CCN1 as a mediator of leukocyte migration and vascular inflammation[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Our data reinforces this concept by confirming that CCN1 can attract and locally immobilize neutrophils, thereby expanding the conventional notion of CCN1 as a cell-matrix adhesion molecule.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eHere, we present evidence establishing a pivotal link between CCN1 and NETs in the progression of DR. By orchestrating the adhesion, migration, and NETs extrusion by neutrophils, CCN1 promotes vascular occlusion, ultimately culminating in capillary degeneration and subsequent retinal leakage (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). These findings not only shed light on the etiology of leukostasis in the early stages of DR but also underscore the potential therapeutic benefits of CCN1 knockdown or the prevention of NETs extrusion in ameliorating retinal leakage in the context of diabetes.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCCN1: Cellular Communication Network Factor 1; DR: Diabetic Retinopathy; DM: Diabetes Mellitus; NPDR: Non-Proliferative Diabetic Retinopathy; PDR: Proliferative Diabetic Retinopathy; NETs: Neutrophil Extracellular Traps; MPO: Myeloperoxidase; Cit-H3: Citrullinated Histone H3; NE: Neutrophil Elastase; BRB: Blood-Retinal Barrier; ELISA: Enzyme-Linked Immunosorbent Assay; IB4: Isolectin B4; STZ: Streptozocin; scRNA-Seq: Single-cell RNA Sequencing; OIR: Oxygen-Induced Retinopathy; rCCN1: Recombinant CCN1 protein; LV: Lentivirus; HRVECs: Human Retinal Vascular Endothelial Cells; LV-siCCN1: Lentivirus carrying small interfering RNA targeting CCN1; DNase I: Deoxyribonuclease I; AUC: Area Under the Curve; NOX: NADPH Oxidase; ROS: Reactive Oxygen Species; EB: Evans Blue; IL-1b: Interleukin-1 beta; SPF: Specific Pathogen Free; PFA: Paraformaldehyde; DMSO: Dimethyl Sulfoxide; OIR: Oxygen-Induced Retinopathy; NORM: Normoxia Control; GCL: ganglion cell layer; IPL: Inner Plexiform Layer; INL: inner nuclear layer; OPL: Outer Plexiform Layer; ONL: outer nuclear layer; RPE: retinal pigment epithelium; WBC: white blood cell; RBC: red blood cell; PLT: platelet; HGB: hemoglobin; NEUT: neutrophils; LYMPH: lymphocytes; EO: eosinophils; BASO: basophils; MONO: monocytes; NLR: neutrophil to lymphocyte ratio; NAR: neutrophil to albumin ratio; TC: total cholesterol; TG: triglycerides; LDL: low-density lipoprotein; HDL: high-density lipoprotein; AST: aspartate aminotransferase; ALT: alanine aminotransferase; ALB: albumin; BUN: blood urea nitrogen; Cr: creatinine; CysC: serum cystatin C; UA: uric acid; eGFR: estimated glomerular filtration rate.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Third Affiliated Hospital of Sun Yat-sen University Network Ethics Committee following the principles of the Helsinki Declaration. The animal experiment was approved by the Animal Care Committee assigned by Sun Yat-sen University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll participants consented to submit the manuscript to the journal.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData of this study could be accessed freely under reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare no potential conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the National Natural Science Foundation of China (No. 82270886, China), Clinical Research 5010 Program (No. 2023006, China), Sci-Tech Research Development Program of Guangzhou City (No. 202201020589, China), and Fundamental Research Funds for the Central Universities, Sun Yat-sen University to Y.C. This work was also supported by grants from the National Natural Science Foundation of China (No. 32000621, China), the Guangdong Basic and Applied Basic Research Foundation (No.s 2022A1515220129, 2023A1515010526, China) and the Science and Technology Projects in Guangzhou (No. 202102010338, China) to Y.L.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYan Lu and Yanming Chen conceived the study and acquitted funding. Yanming Chen, Yan Lu, Wenru Su, Guojun Shi, Xuemin He, Yanhua Zhu, and Shasha Li supervised this study. Ting Li designed the experiments and wrote the manuscript. Ting Li, Yixia Qian, and Tongtong Wang carried out the animal and cellular experiments. Haicheng Li, Wenru Su, Yuchan Wang, and Qi Jiang recruited the patients and collected clinical samples. Yixia Qian and Haicheng Li, blinded to the group information, assisted in analyzing the experiment data. All authors have reviewed and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Dr. Yu Tao and Dr. Mingqiang Li (The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China) for their technical assistance.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eNanegrungsunk O, Patikulsila D, Sadda SR. Ophthalmic imaging in diabetic retinopathy: A review. Clinical Exper Ophthalmology. 2022;50:1082\u0026ndash;96. \u003c/li\u003e\n\u003cli\u003eGBD 2019 Blindness and Vision Impairment Collaborators, Vision Loss Expert Group of the Global Burden of Disease Study. Causes of blindness and vision impairment in 2020 and trends over 30 years, and prevalence of avoidable blindness in relation to VISION 2020: the Right to Sight: an analysis for the Global Burden of Disease Study. 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Spontaneous Neutrophil Extracellular Traps Release Are Inflammatory Markers Associated with Hyperglycemia and Renal Failure on Diabetic Retinopathy. Biomedicines. 2023;11:1791. \u003c/li\u003e\n\u003cli\u003eEvaluation of Circulating Markers of Neutrophil Extracellular Trap (NET) Formation as Risk Factors for Diabetic Retinopathy in a Case-Control Association Study - PubMed. [cited 2023 May 14]; Available from: https://pubmed.ncbi.nlm.nih.gov/27420129/\u003c/li\u003e\n\u003cli\u003eShafqat A, Abdul Rab S, Ammar O, Al Salameh S, Alkhudairi A, Kashir J, et al. Emerging role of neutrophil extracellular traps in the complications of diabetes mellitus. Front Med. 2022;9:995993. \u003c/li\u003e\n\u003cli\u003eZhao Z, Pan Z, Zhang S, Ma G, Zhang W, Song J, et al. Neutrophil extracellular traps: A novel target for the treatment of stroke. Pharmacology \u0026amp; Therapeutics. 2023;241:108328. \u003c/li\u003e\n\u003cli\u003eBrinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS, et al. Neutrophil Extracellular Traps Kill Bacteria. Science. 2004;303:1532\u0026ndash;5. \u003c/li\u003e\n\u003cli\u003eTakishita Y, Yasuda H, Shimizu M, Matsuo A, Morita A, Tsutsumi T, et al. Formation of neutrophil extracellular traps in mitochondrial DNA-deficient cells. J Clin Biochem Nutr. 2020;66:15\u0026ndash;23. \u003c/li\u003e\n\u003cli\u003eYipp BG, Kubes P. NETosis: how vital is it? Blood. 2013;122:2784\u0026ndash;94. \u003c/li\u003e\n\u003cli\u003eGupta AK, Joshi MB, Philippova M, Erne P, Hasler P, Hahn S, et al. Activated endothelial cells induce neutrophil extracellular traps and are susceptible to NETosis‐mediated cell death. FEBS Letters. 2010;584:3193\u0026ndash;7. \u003c/li\u003e\n\u003cli\u003eSaffarzadeh M, Juenemann C, Queisser MA, Lochnit G, Barreto G, Galuska SP, et al. 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PLOS ONE. 2012;7:e32366. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Diabetic retinopathy, retinal inflammation, CCN1, Retinal leakage, Blood-retinal barrier, Neutrophils, Neutrophil extracellular traps","lastPublishedDoi":"10.21203/rs.3.rs-3845429/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3845429/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eDiabetic retinopathy (DR) is a major cause of blindness and is characterized by dysfunction of the retinal microvasculature. Neutrophil stasis, resulting in retinal inflammation and the occlusion of retinal microvessels, is a key mechanism driving DR. These plugging neutrophils subsequently release neutrophils extracellular traps (NETs), which further disrupts the retinal vasculature. Nevertheless, the primary catalyst for NETs extrusion in the retinal microenvironment under diabetic conditions remains unidentified. In recent studies, cellular communication network factor 1 (CCN1) has emerged as a central molecule modulating inflammation in pathological settings. Additionally, our previous research has shed light on the pathogenic role of CCN1 in maintaining endothelial integrity. However, the precise role of CCN1 in microvascular occlusion and its potential interaction with neutrophils in diabetic retinopathy have not yet been investigated.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe first examined the circulating level of CCN1 and NETs in our study cohort and analyzed related clinical parameters. To further evaluate the effects of CCN1 \u003cem\u003ein vivo\u003c/em\u003e, we used recombinant CCN1 protein and CCN1 overexpression for gain-of-function, and CCN1 knockdown for loss-of-function by intravitreal injection in diabetic mice. The underlying mechanisms were further validated on human and mouse primary neutrophils and dHL60 cells.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe detected increases in CCN1 and neutrophil elastase in the plasma of DR patients and the retinas of diabetic mice. CCN1 gain-of-function in the retina resulted in neutrophil stasis, NETs extrusion, capillary degeneration, and retinal leakage. Pre-treatment with DNase I to reduce NETs effectively eliminated CCN1-induced retinal leakage. Notably, both CCN1 knockdown and DNase I treatment rescued the retinal leakage in the context of diabetes. \u003cem\u003eIn vitro\u003c/em\u003e, CCN1 promoted adherence, migration, and NETs extrusion of neutrophils.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eIn this study, we uncover that CCN1 contributed to retinal inflammation, vessel occlusion and leakage by recruiting neutrophils and triggering NETs extrusion under diabetic conditions. Notably, manipulating CCN1 was able to hold therapeutic promise for the treatment of diabetic retinopathy.\u003c/p\u003e","manuscriptTitle":"Cellular communication network factor 1 promotes retinal leakage in diabetic retinopathy via inducing neutrophil stasis and neutrophil extracellular traps extrusion","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-10 21:54:35","doi":"10.21203/rs.3.rs-3845429/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e7727de4-25db-4f67-9197-128f7d766fdc","owner":[],"postedDate":"January 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-01-19T08:29:12+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-10 21:54:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3845429","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3845429","identity":"rs-3845429","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}