Endothelial protein C receptor promotes retinal neovascularization through heme catabolism

Endothelial protein C receptor promotes retinal neovascularization through heme catabolism | 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 Article Endothelial protein C receptor promotes retinal neovascularization through heme catabolism Hongyuan Song, Qing Li, Xiao Gui, Ziyu Fang, Wen Zhou, Mengzhu Wang, and 14 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4188758/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Feb, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Pathological retinal neovascularization (RNV) is one of the leading causes of blindness worldwide; however, its underlying mechanism remains unclear. Here, we found that the expression of endothelial protein C receptor (EPCR) was increased during RNV, and its ligand was elevated in the serum or vitreous body of patients with proliferative diabetic retinopathy. Deleting endothelial Epcr or using an EPCR neutralizing antibody ameliorated pathological retinal angiogenesis. EPCR promoted endothelial heme catabolism and carbon monoxide release through heme oxygenase 1 (HO-1). Inhibition of heme catabolism by deleting of endothelial Ho-1 or using an HO-1 inhibitor suppressed pathological angiogenesis in retinopathy. Conversely, supplementation with CO rescued the angiogenic defects after endothelial Epcr or Ho-1 deletion. Our results identified EPCR-dependent endothelial heme catabolism as an important contributor to pathological angiogenesis, which may serve as a potential target for treating vasoproliferative retinopathy. Health sciences/Diseases/Eye diseases/Retinal diseases Health sciences/Cardiology/Cardiovascular biology/Angiogenesis Biological sciences/Developmental biology/Angiogenesis Biological sciences/Physiology/Cardiovascular biology/Angiogenesis angiogenesis retinopathy diabetes complications endothelial protein C receptor heme catabolism carbon monoxide Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Pathological retinal neovascularization (RNV) is a major cause of vision loss worldwide. 1 , 2 The most common causes include diabetic retinopathy, retinopathy of prematurity (ROP), and retinal vein occlusion, which affect millions of people. 3 – 5 The aberrant vessel growth usually leads to macular edema, fibrotic scarring, and retinal detachment. 2 , 6 These conditions are the primary causes of blindness in working-age and pediatric populations in developed countries. 6 , 7 Endothelial cells (ECs) rarely proliferate and remain in quiescent state in adult organism, whereas uncontrolled proliferation of ECs is initiated and drives aberrant vessel growth under pathological stimuli. 8 Vascular endothelial growth factor (VEGF) is extensively studied in the regulation of EC proliferation and angiogenesis. 9 , 10 Anti-VEGF regents are first-line therapy for RNV, but resistance occurs in certain patients. 11 , 12 Hence, it is necessary to identify novel therapeutic targets of RNV. Endothelial protein C receptor (EPCR, also known as PROCR) is a single-pass transmembrane glycoprotein expressed in ECs and stem cells. 13 , 14 In addition to its anti-coagulation role, 15 EPCR mediates intracellular signaling of activated protein C (aPC). 16 Activation of downstream protein kinase B (AKT) promotes cell growth, improves cardiac function, and favors tumor progression. 16 – 20 Lineage tracing of EPCR-positive cells indicate that EPCR is specifically expressed in the ECs of the retinal vasculature. 21 EPCR transcription is activated in the retinal vessels during angiogenic expansion stage but not during the quiescent stage. 21 Ablation of EPCR-positive cells leads to delayed retinal vascular expansion in postnatal mice, suggesting a role for EPCR in retinal angiogenesis. 22 However, the role of EPCR in RNV pathology remains unclear. In this study, we found that EPCR transcription was activated in ECs during RNV. Conditional knockout of Epcr in ECs or the use of an EPCR-neutralizing antibody ameliorated pathological RNV. Further data showed that EPCR affected heme catabolism by regulating heme oxygenase 1 (HMOX1, also known as HO-1). Conditional knockout of Ho-1 in ECs or treatment with an HO-1 enzymatic inhibitor suppressed pathological angiogenesis in retinopathy. Carbon monoxide releasing molecule-3 (CORM3) partially rescued the reduced neovascularization caused by the deletion of endothelial Epcr and Ho-1 , indicating a role for carbon monoxide (CO). Clinically, levels of aPC, which is an EPCR ligand, were elevated in proliferative diabetic retinopathy (PDR) patients’ serum and vitreous body. These results indicate that EPCR-dependent endothelial heme catabolism is an important contributor to RNV. Results 1. EPCR is highly expressed in retinal ECs during RNV. The mouse model of oxygen-induced retinopathy (OIR) serves as a proxy for human pathological RNV, including PDR and ROP. 23 The neovascular response peaked on postnatal day 17 (P17) when the mice were returned to room air for five days (Fig. 1 A). To identify the potential genes involved in pathological neovascularization, we performed transcriptomic analyses of the retinas of P17 mice of OIR (GSE241239). To minimize the RNA-seq bias, we further analyzed transcriptomic data from two other independent studies (GSE194176 and GSE158799). 24 , 25 A combined analysis revealed 149 upregulated genes (Fig. 1 .B-C). Among these candidate genes, we decided to focus on Epcr because it was reported to be specifically expressed on the surface of vascular ECs and stem cells. 13 , 14 Further data confirmed that EPCR expression was upregulated in the retinas of P17 mice in the OIR (Fig. 1 .D-E). We sorted ECs from the mouse retina to evaluate the expression of EPCR in the retinal vasculature. Endothelial EPCR expression was much higher in the OIR group than that in the control group (Fig. 1 F-G). Next, we examined the expression pattern of EPCR in the retina. Immunofluorescence staining with an anti-EPCR antibody showed that EPCR was selectively expressed in the retinal ECs of P17 mice under physiological conditions (FigS1A). In the OIR model, the expression of EPCR was detected only in retinal ECs, and the fluorescence intensity of EPCR signals was much higher in the neovascular tufts (FigS1B-C). Epcr -CreER T 2 (also known as Procr -CreER T 2 ) mice were previously used to mark proliferative ECs in the retinal vasculature. 21 Here, we used Epcr -CreER T 2 and Rosa26 tdT mice to perform genetic lineage tracing of EPCR-positive cells in the retina of P17 mice. Tamoxifen (TAM) was administered for three consecutive days at P10, P11, and P12, and the retina was analyzed at P17 (FigS1D). Under physiological conditions, a very small proportion of EPCR + cells were observed (Fig. 1 H). While there was a large proportion of EPCR + cells in the retina of OIR mice, and EPCR + cells mainly colocalized with neovascular tufts (Fig. 1 H-I). These data indicated that hypoxia significantly increased the transcription of EPCR. Hypoxia is also known to play a vital role in the retinal vascular development process. 26 Therefore, the expression pattern of EPCR in the retinal vasculature of postnatal mice were then assessed. Immunofluorescence staining with anti-EPCR showed that the levels of EPCR were much higher in the margin of the retinal vasculature (FigS1E). Consistent data were acquired using Epcr -CreER T 2 and Rosa26 tdT mice (FigS1F-G). Collectively, these data suggest that EPCR is specifically expressed in ECs of the retina and is upregulated in neovascularization in the OIR model and during retinal vascular expansion. EPCR activates downstream signaling pathways by binding to its ligands. Thus, we assessed the concentrations of EPCR ligands in the serum of OIR mice. The data showed that the levels of aPC in the serum of OIR mice were higher than those in control mice (Fig. 1 J). To evaluate whether the findings in mice hold promise for human patients with RNV, we investigated the levels of aPC in the vitreous humor and serum in patients with PDR. The concentrations of aPC in the vitreous humor and serum were significantly higher in patients with PDR than in control with macular hole or idiopathic epiretinal membrane (Fig. 1 K-L). 2. EPCR contributes to RNV in vivo and in vitro. To determine the role of EPCR in RNV, we intercrossed the Cdh5 -CreER T 2 mouse with Epcr flox/flox mouse to generate EC-specific Epcr KO mice ( Epcr iΔEC ). The expression of EPCR was almost completely ablated in ECs sorted from Epcr iΔEC mice (FigS2A-B). To assess the role of EPCR in pathological RNV, we examined the changes in the retinal vasculature of Epcr iΔEC mice of OIR at P17. Our data showed that the area of neovascular tufts in the retina of Epcr iΔEC mice was smaller than that in Epcr WT mice (Fig. 2 A-B). In contrast, the avascular area in Epcr iΔEC mice’ retina was larger than that in Epcr WT mice (Fig. 2 A, 2 C). Furthermore, we used 5-ethynyl-2’-deoxyuridine (EdU) to label proliferative cells in the retina and used E26 transformation-specific (ETS)-related gene (ERG) as an ECs marker. The result showed that EdU + ECs in the retina decreased significantly in Epcr iΔEC mice compared to Epcr WT mice (Fig. 2 D-E). These in vivo results suggest that upregulated EPCR in pathological vasculature drives EC growth and angiogenesis in OIR. Next, we determined the effect of Epcr deletion on retinal vascular expansion in postnatal retinas. EC-specific deletion of Epcr led to a sparse vascular network and reduced radial expansion of the superficial retinal vascular plexus (FigS2C-D). Tip cells and branch points were decreased in the retina of Epcr KO mice (FigS2E-G). Meanwhile, ECs proliferation was also significantly inhibited in Epcr iΔEC mice (FigS2H-I). To further evaluate the impact of elevated EPCR on angiogenesis, we used an adenovirus to overexpress EPCR and assessed the angiogenic behavior of ECs in vitro . EPCR adenovirus treatment significantly increased the protein levels of EPCR in ECs, which promoted ECs proliferation in an EdU staining assay (Fig. 2 F-G, FigS2J). A three-dimensional endothelial spheroid assay showed the number and length of endothelial sprouts were increased in the EPCR adenovirus treatment group (Fig. 2 H-I). Moreover, a wound scratch assay revealed that EPCR adenovirus-treated ECs were more motile than control adenovirus treated ECs (Fig. 2 J-K). Furthermore, the knockdown of EPCR using siRNA suppressed ECs proliferation, migration and sprouting (Fig. 2 L-Q, FigS2K). Together with in vivo results, these data indicated that EPCR promotes vascular growth. 3. Pharmacological inhibition of EPCR ameliorates pathological retinal neovascularization. To evaluate the translational potential of targeting EPCR in anti-angiogenic therapy, we assessed the effects of a neutralizing antibody against EPCR on pathological angiogenesis in vitro and in vivo . We found that a single dose of EPCR antibody significantly reduced the area of neovascular tufts compared to the vehicle and anti-IgG groups (Fig. 3 A-B). Meanwhile, the avascular area increased in the EPCR antibody treatment group, which was consistent with that in Epcr KO mice (Fig. 3 A, C). Suppressed proliferation of ECs in retinal neovascular tufts was also observed after treatment with the EPCR antibody (Fig. 3 D-E). Similar results were obtained in vitro as EPCR antibody treatment significantly inhibited ECs proliferation, migration and sprouting (Fig. 3 F-L). These results were consistent with our findings using Epcr iΔEC mice, suggesting the translational potential of anti-EPCR in retinal neovascularization. 4. EPCR regulates the expression of HO-1 through Nrf2/Keap1 To reveal the possible molecular mechanisms underlying EPCR-regulated angiogenesis, we performed transcriptomic analyses of EPCR siRNA- or control siRNA-treated ECs (GSE249130, FigS3A). These data were then combined and analyzed with the mice retina results with OIR (FigS3B). 26 genes were upregulated in the retina of OIR mice and downregulated in EPCR-depleted ECs (Fig. 4 A). Among these genes, HO-1 was the most significantly affected (Fig. 4 A). Increased mRNA and protein levels of HO-1 were confirmed in the retinas of OIR mice (FigS3C-D). Further data showed that the expression of HO-1 was suppressed at the mRNA and protein levels in ECs treated with EPCR siRNA (Fig. 4 B-C). Additionally, the expression of HO-1 was upregulated at the mRNA and protein levels in ECs treated with EPCR adenovirus (Fig. 4 D-E). The expression of HO-1 in retinal ECs of OIR mice was then evaluated. We sorted ECs from the retina of OIR mice using flow cytometry. The results indicated that the expression of HO-1 in ECs was upregulated in the OIR group (FigS3E-F). This was further confirmed by immunofluorescence staining. The expression of HO-1 in physiological retina did not show apparent pattern, while it was highly expressed in neovascular tufts of OIR retinas (FigS3G-H). Further data showed that the expression of HO-1 in ECs sorted from the retina of Epcr iΔEC mice decreased compared with that in Epcr wt mice (Fig. 4 F-G). Collectively, these results suggested that HO-1 was a downstream molecule of EPCR in regulating angiogenesis. AKT is recognized as a target of EPCR signaling. 27 High EPCR expression or stimulation with aPC usually leads to AKT activation. 16 , 19 We found that overexpression of EPCR using an adenovirus promoted the activation of AKT, whereas silencing EPCR with siRNA inhibited AKT phosphorylation (Fig. 4 H-I). Nuclear factor erythroid 2-related factor 2 (NRF2) is a ubiquitous transcription factor directly regulating HO-1. 28 NRF2 is tightly regulated by Kelch-like ECH-associated protein 1 (KEAP1) through ubiquitination and proteasome-dependent degradation. 29 Therefore, we assessed the effects of EPCR on the expression of KEAP1 and NRF2. Overexpression of EPCR in ECs promoted the levels of NRF2 and HO-1 and inhibited the expression of KEAP1 (Fig. 4 I). Decreased expression of NRF2 and HO-1, and increased expression of KEAP1 were observed after EPCR knockdown in ECs (Fig. 4 H). Meanwhile, aPC treatment stimulated the activation of AKT, and increased the expression of NRF2 and HO-1 while suppressing the expression of KEAP1 (FigS3I). AKT activates NRF2 in multiple cells. 30 , 31 Our data showed that inactivating AKT with LY294002 (a PI3K inhibitor) blocked the effect of EPCR overexpression on NRF2, KEAP1, and HO-1 (Fig. 4 J). Consistent results were observed when ECs were treated with aPC (FigS3J). These results suggested that AKT plays an important role in mediating EPCR signaling. Furthermore, we observed that depleting the expression of KEAP1 using siRNA rescued the downregulation of NRF2 and HO-1 caused by EPCR siRNA (Fig. 4 K, FigS3K). Consistent results were obtained after treatment with Ki696 (a KEAP1 inhibitor) (Fig. 4 L). These data suggested that EPCR controlled HO-1 expression via the AKT-KEAP1-NRF2 pathway. 5. Genetic ablation of endothelial Ho-1 attenuates angiogenesis To evaluate the role of HO-1 in retinal angiogenesis, we bred Cdh5 -CreER T 2 mice with Ho-1 flox/flox mice to generate EC-specific Ho-1 KO mice ( Ho-1 iΔEC ). The deletion efficiency was determined using flow cytometry-sorted ECs, and the expression of HO-1 in ECs sorted from Ho-1 iΔEC mice was significantly decreased (FigS4A). The role of HO-1 in pathological retinal angiogenesis was then determined in the OIR mice. Our data showed that the area of neovascular tufts in the retina of Ho-1 iΔEC mice was smaller, while the avascular area in the retina of Ho-1 iΔEC mice was larger than that in Ho-1 WT mice (Fig. 5 A-C). Furthermore, our results showed that EdU + ECs decreased significantly in the retina of Ho-1 iΔEC mice than in Ho-1 WT mice (Fig. 5 D-E). Physiological retinal vascular development was also affected in Ho-1 iΔEC mice. Deleting endothelial Ho-1 reduced radial expansion of the superficial retinal vascular plexus (FigS4B, C). Meanwhile, the vascular density, tip cells and branch points were decreased in the retina of Ho-1 iΔEC mice (FigS4D-F). Additionally, EdU + ECs decreased significantly in the retina of Ho-1 iΔEC mice than in Ho-1 WT mice (FigS4G-H). The role of HO-1 in angiogenesis was further assessed in vitro . First, the knockdown efficiency of HO-1 siRNA was evaluated. The EdU assay showed that silencing HO-1 inhibited ECs proliferation induced by the EPCR adenovirus (Fig. 5 F-G). A three-dimensional endothelial spheroid assay was used to evaluate the angiogenic potential of ECs treated with EPCR adenovirus and HO-1 siRNAs. Fewer and shorter sprouts were observed in HO-1 siRNAs-treated ECs than EPCR adenovirus treated ECs (Fig. 5 H-I). Besides, ECs treated with HO-1 siRNAs exhibited impaired migration ability compared to the EPCR adenovirus treatment group (Fig. 5 J-K). We also assessed the effects of HO-1 knockdown on ECs. The results showed that silencing HO-1 suppressed ECs proliferation, migration, and sprouting (FigS4J-O). These results collectively revealed the vital role of HO-1 in developmental angiogenesis and RNV. 6. Endothelial heme catabolism impacts angiogenesis. HO-1 catalyzes heme degradation into CO, biliverdin, and iron. 32 Catabolism of cytotoxic labile heme and the generation of CO exert cytoprotective effects. 33 Thus, we further assessed whether the angiogenic role of HO-1 was mediated by its enzymatic function. Here, HO-1 enzymatic inhibitor, zinc protoporphyrin Ⅸ (ZnPPⅨ), was used to determine their effect on pathological RNV. The data showed that ZnPPⅨ treatment decreased the area of neovascular tufts in the retina and increased the avascular area (Fig. 6 A-C). Besides, ZnPPⅨ suppressed ECs proliferation as EdU + retinal ECs decreased significantly in mice treated with ZnPPⅨ (Fig. 6 D-E). Next, we determined the effect of ZnPPⅨ on retinal vascular expansion. ZnPPⅨ treatment led to a sparse vascular network and reduced radial expansion of the superficial retinal vascular plexus (FigS5A-B). The tip cells and branch points were decreased in the retina of mice treated with ZnPPⅨ (FigS5C-E). Meanwhile, ZnPPⅨ significantly suppressed ECs proliferation in the retina (FigS5F-G). Further data showed that EPCR-induced cell proliferation, migration and sprouting of ECs in vitro were inhibited by ZnPPⅨ (Fig. 6 F-K). Additionally, we evaluated the effect of ZnPPⅨ on ECs treated with HO-1 siRNA. The result showed that ZnPPⅨ did not affect ECs proliferation, migration, and sprouting after HO-1 knockdown (FigS5H-M). These data indicated that endothelial heme catabolism contributes to EPCR-mediated RNV. 7. Heme catabolism-derived CO contributes to pathological angiogenesis. Endogenous CO is a signaling molecule that promotes tumor growth and angiogenesis. 34 , 35 Heme catabolism in macrophages increases tumor angiogenesis through CO production, suggesting a role for CO in regulating angiogenesis. 33 Here, we showed that pathological retinal neovascular tufts in carbon monoxide releasing molecule-3 (CORM3) treated Ho-1 iΔEC mice significantly increased compared with that in Ho-1 iΔEC mice, while the avascular area was decreased (Fig. 7 A-C). Furthermore, the data showed that EdU + ECs also increased significantly in the CORM3-treated retina of Ho-1 iΔEC mice than that in Ho-1 WT mice (Fig. 7 D-E). Pathological retinal neovascular tufts in CORM3-treated Epcr iΔEC mice also significantly increased, and avascular area decreased compared with that in Epcr iΔEC mice (FigS6A-C). Consistent data were acquired for the number of EdU + ECs (FigS6D-E). We assessed the effect of CORM3 on postnatal retinal vasculature in Ho-1 iΔEC and Epcr iΔEC mice. Treatment with CORM3 could rescue the sparse vascular network and reduce the radial expansion of superficial retinal vascular plexus in Ho-1 iΔEC mice (FigS7A-B). Compared with Ho-1 iΔEC mice, tip cells and branch points increased in the retina treated with CORM3 (FigS7C-E). Consistent results were acquired in CORM3-treated Epcr iΔEC mice (FigS7F-J). Furthermore, CORM3 rescued HO-1 silence-caused inhibition of ECs proliferation, migration, and sprouting (Fig. 7 F-K). Additionally, the effect of CORM3 on EPCR-deleted ECs was evaluated. The results showed that EPCR silencing inhibited ECs proliferation, migration, and sprouting, which was partially reversed after treatment with CORM3 (FigS8A-F). These results suggested that CO was an important contributor to EPCR dependent endothelial heme catabolism-regulated angiogenesis. Discussion Therapeutic strategies for RNV are effective, but off-target effect and resistance limit their efficacy. 12 , 36 The reason is that these therapies cannot distinguish between healthy and pathological vessels. Our current study showed that EPCR was selectively expressed on ECs of retina and EPCR transcription was activated under angiogenic stimulus. Conditional deletion of Epcr in ECs or anti-EPCR neutralizing antibody treatment attenuated pathological retinopathy. Mechanistically, EPCR controlled endothelial heme catabolism through HO-1, and heme catabolism-derived CO plays a vital role. Notably, plasma levels of the EPCR ligand were elevated in the vitreous body or serum of patients with PDR. These observations revealed the potential therapeutic value of targeting EPCR for suppressing pathological RNV (Fig. 8 ). Here, we observed that the expression of EPCR was detected specifically in the retinal ECs and was upregulated in neovascular tufts using anti-EPCR staining. EPCR was not detected in retinal pigment epithelia cells, photoreceptor cells, or ganglion cells. Consistent with our previous work, 21 we found that EPCR transcription is activated during vessels expansion stage in the retina. Meanwhile, EPCR transcription was activated in ECs during RNV using Epcr -CreER T 2 ; Rosa26 tdT mice. These findings revealed that EPCR expression was selectively detected on retinal ECs and was upregulated under angiogenic stimulus. Vascular endothelial growth factor receptor 2 (VEGFR2) is the major receptor for VEGF guiding angiogenesis. 37 However, VEGFR2 is widely expressed in the retina, including ECs and neurons. 38 Sustained suppression of VEGF would damage the photoreceptors, and lead to loss of vision. 39 , 40 This is observed in clinic that retinal atrophy occurs in patients received anti-VEGF treatment. 41 The specific expression pattern of EPCR in retina indicates that targeting EPCR is unlikely to induce off-target effects. It is reported that EPCR + ECs are highly proliferative and are the major contributor toward vessel development. 21 Conditional expression of diphtheria toxin (DTA) in EPCR + cells delay retinal vessel extension in postnatal mice, indicating a role for EPCR in angiogenesis. 22 Consistent data was acquired in our study that conditional deletion of EPCR in ECs suppressed the expansion of retinal vascular plexus. Using OIR mice model, we showed that endothelial deletion of EPCR or EPCR neutralization antibody reduced ECs proliferation and attenuated pathological RNV. The angiogenic role of EPCR in neovascularization is also observed in hindlimb ischemia mice. 42 Deletion of EPCR in ECs using Tie2 -Cre; Epcr flox/flox mice suppresses new vessel formation. 42 Given that EPCR transcription is specifically activated in proliferating ECs, 21 EPCR blockade could be a novel option for treating RNV with minimal effects on quiescent ECs. The mechanism by which EPCR contributes to RNV remain to be revealed. We found that deletion of EPCR in ECs reduced the expression of HO-1 through AKT/KEAP1/NRF2 pathway. Whereas EPCR ligand (aPC) treatment upregulated the expression of HO-1 via AKT/KEAP1/NRF2. The data were consistent with studies showing that EPCR activate AKT signaling in heart and multiple cells. 16 – 19 The levels of the EPCR ligand were elevated in the serum or vitreous body of patients with PDR, suggesting that it activates EPCR signaling to favor retinal neovascularization. It is well established that KEAP1/NRF2 directly regulates the expression of HO-1 during tumor growth and metastasis. 28 KEAP1 is a negative regulator of NRF2, affecting the expression of NRF2 in ECs. 43 Endothelial ablation of KEAP1 favors retinal vascular expansion, increases vascular density, and promotes ECs proliferation. 44 Whereas endothelial deletion of NRF2 delays vascular expansion and decreases vascular density. 44 Here, we showed that ECs-specific deletion of Ho-1 suppressed retinal neovascularization in the retina of OIR mice and postnatal mice. The results were consistent with that observed in ECs-specific Keap1 or Nrf2 knockout mice. 44 HO-1 catalyzes the degradation of heme to CO, ferrous iron, and biliverdin. 45 While initially considered to be a waste product, CO is increasingly recognized as a cytoprotective and homeostatic molecule. 46 Heme catabolism-derived CO is a major source of endogenous CO, 47 which contributes to tumor metastasis and angiogenesis. 33 In ECs, CO is reported to increase cell growth in vitro , whereas its role in vivo is unknown. 34 Here, our data indicate that heme catabolism-derived CO promoted retinal angiogenesis, revealing a vital role of CO in angiogenesis. Clinically, the levels of exhaled CO significantly increase in patients with diabetes and correlate with blood glucose levels and duration of the disease. 48 – 49 PDR, characterized by uncontrolled RNV, is a late-stage microvascular complication of diabetes. 3 Increased levels of aPC in the serum or vitreous body of patients with PDR imply upregulated EPCR-dependent heme catabolism during RNV. These findings indicate that CO derived from heme catabolism plays a vital role in RNV. In summary, we determined that EPCR is highly expressed in retinal ECs and that blocking EPCR attenuates pathological retinal angiogenesis. Mechanistically, EPCR controls heme catabolism via the KEAP1/NRF2/HO-1 pathway, and CO derived from heme catabolism plays a vital role in RNV. Hence, our findings elucidate a novel mechanism of RNV and suggest that EPCR may be a promising therapeutic target for translational application. However, these mouse models do not accurately represent the pathological progression of retinopathy in primates. Therefore, determining the therapeutic efficiency of anti-EPCR for pathological retinal angiogenesis in non-human primates is indispensable in the future. Moreover, the expression pattern of EPCR in human retina is unknown. For translational significance, it is important to determine the expression pattern of EPCR in post-mortem retinas under diverse pathophysiological conditions. Methods Patient samples The patients diagnosed with proliferative diabetic retinopathy (PDR), idiopathic epiretinal membrane (ERM) and idiopathic macular hole (MH) were included. Vitreous samples were obtained from patients who underwent vitrectomy surgery by the same surgeon. Details of the patient information was showed in Supplementary Table S1 . The collected vitreous samples were placed on ice immediately and centrifuged to exclude the debris. The supernatants were aliquoted into sterile tubes and stored in liquid nitrogen. Serum samples were obtained from patients who were diagnosed with PDR. The control serum samples were obtained from patients who were diagnosed with ERM and MH. Details of the patient information was showed in Supplementary Table S2. The obtained serum samples were centrifuged at 4 ℃ and the supernatants were stored at -80 ℃. All surgeries were performed, and human samples were harvested in accordance with the principles in the Declaration of Helsinki. Informed consent was obtained from ethics committee of Shanghai General Hospital ([2022]-109). Mice All the experimental animals were accommodated in animal facilities, where they were subjected to 12-hour cycles of light and darkness. These animals were provided with unrestricted access to standard chow and water. The C57BL/6J mice were purchased from Jihui Laboratory Animal Care Co., Ltd. Epcr flox/flox mice (the Cyagen Biotechnology Co., Ltd.) and Ho-1 flox/flox mice (the Cyagen Biotechnology Co., Ltd.) were crossed with Cdh5 CreER T 2 mice 10 respectively to generate conditional knockout mice. For the lineage tracing of Epcr + cells, the Epcr CreER T 2 mice 21 were crossed to Rosa26 tdTomato reporter mice (the Cyagen Biotechnology Co., Ltd.) All animal studies were approved by the Institutional Animal Care and Use Committee at Shanghai Changhai Hospital (CHEC(A.E)2023-024). Oxygen induced retinopathy (OIR) Oxygen-induced retinopathy (OIR) was performed as previously reported. 25 In brief, the pups were exposed to 75% oxygen with their nursing mothers for five days from P7 to P12. Then the pups were returned to room air at P12. When the mice were return to room air, the hypoxia-induced neovascularization is initiated and peaked on P17. Animals were euthanized and the retinas were harvested at P17. To activate CreER T 2 , 50µl of tamoxifen (2mg/ml) was injected intraperitoneally from P10 to P12. To evaluate the anti-angiogenic role of EPCR neutralizing antibody, mice were randomly divided into three groups and injected intravitreally with 1 µl anti-EPCR antibody, PBS and anti-IgG respectively at P12. ZnPPIX (25mg/kg) and CORM3 (40mg/kg) were injected intraperitoneally twice at P12 and P13. To detect proliferating cells, EdU (50mg/kg) were administered intraperitoneally 6 h before euthanasia. RNA-Seq sample preparation and sequencing Total RNA from retinas and cells were extracted with RNA Easy Fast Tissue/Cell Kit (TIANGEN). Agilent 2100 Bioanalyzer and RNA nano 6000 assay kit (Agilent Technologies) were used to evaluate the quality of RNA samples. The transcriptome sequencing library was constructed through 1 ng/µl RNA randomly fragmentation, cDNA strand 1 / strand 2 synthesis, end repair, A-tailing, ligation of sequencing adapters, size selection and library PCR enrichment. The integrity of cDNA was evaluated by Agilent 2100 Bioanalyzer. The library preparations were sequenced on an Illumina HiSeq 2500 platform (Illumina, USA). The raw data of sequences were uploaded in the NCBI Sequence Read Archive (SRA) database (GSE241239 for the retinas of OIR mice; GSE249130 for HUVECs with EPCR deletion). Isolation of retinal ECs Isolation of retinal ECs was performed according to protocols we previously described with some modifications. 21 The retinas were separated from eyeballs then minced into fine fragments and digested in Iscove’s Modified Dulbecco’s MediumIs (IMDM, LI1090-500,BioAgrio) with 1.5mg/ml Collagenase H(11087789001, Roche), 1.25 mM CaCl 2 , 0.4 mM MgCl 2 , 1% P/S and 3.5 µg/ml DNaseI (D4263, Sigma) at 37°C for 45min. The samples were pipetted every 10min using 1ml pipette tip to ensure even digestion. The digestion mix was passed through a 40µm nylon mesh to prepare single-cell suspensions. Cells were then incubated for 20min with FITC-conjugated anti -CD31(11-0317-82, Invitrogen, diluted 1:100) and PE-Cy7 conjugated anti-CD45 (25-0451-82, Invitrogen, diluted 1:200) in PBS with 5% FBS. Flow cytometry and cell sorting was performed using Then the single cell suspensions were subjected to FACS using SONY ID7000 or LSR Fortessa (BD Biosciences). FACS data were analyzed by FlowJo software. The purity of sorted ECs was routinely checked and ensured to be more than 95%. Retinal dissection and whole-mount staining Eyes were fixed in 4% paraformaldehyde (PFA) for 0.5h at RT. After dissection, retinas were blocked in blocking buffer (1%BSA, 5%FBS and 0.5% Triton-X-100 in PBS) for 1h at RT. Primary antibodies were incubated in blocking buffer at 4℃ overnight. After washed twice with PBS, retinas were incubated with secondary antibodies at RT for 2h. Then, the retinas were washed and flat-mounted with Fluoromount-G (SouthernBiotech). For the labeling of EdU, an additional step was performed to detect EdU-labeled proliferative cells using the Click-It EdU kit (C10338, Invitrogen) prior to mounting. Images were acquired with a Mica confocal microscope (Leica Microsystems). Quantitative analysis of retinal vasculature was performed as described. 23 Quantification and statistical analysis All calculations were performed using GraphPad Prism (GraphPad software 9.0, GraphPad, Bethesda, MD, USA). Statistical analysis was performed using the two-tailed Student’s t-test and one-way analysis of variance (ANOVA), where appropriate, to compare different groups. For all bar graphs, data were presented as means ± SD. All experiments were repeated at least three times and P values of < 0.05 were considered as statistically significant. Declarations Acknowledgments Thanks to Yi Arial Zeng (State Key Laboratory of Cell Biology, Chinese Academy of Sciences) for kindly providing Epcr -CreER T2 mice and technical assistance; to Youheng Wei (State Key Laboratory of Genetic Engineering, Fudan University, Shanghai, China) for providing technical assistance. This work was supported by grants from National Natural Science Foundation of China (82171081, 82271106, 82171076, U22A20311, 82388101), National Key R&D Program (2022YFC2502800), Shanghai Municipal Education Commission (2023ZKZD18), Shanghai Science and Technology committee (22ZR1478200), Shanghai Changhai Hospital excellent top-notch project (2023YQ01). Author contributions: Conceptualization, X.S., H.S., W.S., H.Z. and F.L.; Methodology, Q.L., X.S., and A.G.; investigation, H.S., Q.L., X.G., Z.F., W.Z., M.W., Y.L., H.Z., Z.N., L.Z., H.Z., and Y.J.; Funding acquisition, X.S., H.S. and W.S.; Writing-original draft, H.S and Q.L.; Writing-review & editing, X.S., F.Z., F.L., and X.L.; Supervision, X.S., X.L. and F.Z. All authors approved the final version of the manuscript. Supplemental information Supplemental information can be found online. Declaration of interests The authors declare no competing interests. References Binet, F., Cagnone, G., Crespo-Garcia, S., Hata, M., Neault, M., Dejda, A., Wilson, A.M., Buscarlet, M., Mawambo, G.T., Howard, J.P., et al. (2020). Neutrophil extracellular traps target senescent vasculature for tissue remodeling in retinopathy. Science 369 , eaay5356. 10.1126/science.aay5356. Campochiaro, P.A. (2015). Molecular pathogenesis of retinal and choroidal vascular diseases. Prog. Retinal Eye Res. 49 , 67-81. 10.1016/j.preteyeres.2015.06.002. Antoszyk, A.N., Glassman, A.R., Beaulieu, W.T., Jampol, L.M., Jhaveri, C.D., Punjabi, O.S., Salehi-Had, H., Wells, J.A., 3rd, Maguire, M.G., Stockdale, C.R., et al. (2020). Effect of Intravitreous Aflibercept vs Vitrectomy With Panretinal Photocoagulation on Visual Acuity in Patients With Vitreous Hemorrhage From Proliferative Diabetic Retinopathy: A Randomized Clinical Trial. JAMA 324 , 2383-2395. 10.1001/jama.2020.23027. Stahl, A., Sukgen, E.A., Wu, W.C., Lepore, D., Nakanishi, H., Mazela, J., Moshfeghi, D.M., Vitti, R., Athanikar, A., Chu, K., et al. (2022). Effect of Intravitreal Aflibercept vs Laser Photocoagulation on Treatment Success of Retinopathy of Prematurity: The FIREFLEYE Randomized Clinical Trial. JAMA 328 , 348-359. 10.1001/jama.2022.10564. Yeh, S., Kim, S.J., Ho, A.C., Schoenberger, S.D., Bakri, S.J., Ehlers, J.P., and Thorne, J.E. (2015). Therapies for macular edema associated with central retinal vein occlusion: a report by the American Academy of Ophthalmology. Ophthalmology 122 , 769-778. 10.1016/j.ophtha.2014.10.013. Gariano, R.F., and Gardner, T.W. (2005). Retinal angiogenesis in development and disease. Nature 438 , 960-966. 10.1038/nature04482. Selvam, S., Kumar, T., and Fruttiger, M. (2018). Retinal vasculature development in health and disease. Prog. Retinal Eye Res. 63 , 1-19. 10.1016/j.preteyeres.2017.11.001. Eelen, G., Treps, L., Li, X., and Carmeliet, P. (2020). Basic and Therapeutic Aspects of Angiogenesis Updated. Circ. Res. 127 , 310-329. 10.1161/circresaha.120.316851. Apte, R.S., Chen, D.S., and Ferrara, N. (2019). VEGF in Signaling and Disease: Beyond Discovery and Development. Cell 176 , 1248-1264. 10.1016/j.cell.2019.01.021. Simons, M., Gordon, E., and Claesson-Welsh, L. (2016). Mechanisms and regulation of endothelial VEGF receptor signalling. Nat. Rev. Mol. Cell Biol. 17 , 611-625. 10.1038/nrm.2016.87. Arima, M., Nakao, S., Yamaguchi, M., Feng, H., Fujii, Y., Shibata, K., Wada, I., Kaizu, Y., Ahmadieh, H., Ishibashi, T., et al. (2020). Claudin-5 Redistribution Induced by Inflammation Leads to Anti-VEGF-Resistant Diabetic Macular Edema. Diabetes 69 , 981-999. 10.2337/db19-1121. Wallsh, J.O., and Gallemore, R.P. (2021). Anti-VEGF-Resistant Retinal Diseases: A Review of the Latest Treatment Options. Cells 10 , 1049. 10.3390/cells10051049. Wang, D., Wang, J., Bai, L., Pan, H., Feng, H., Clevers, H., and Zeng, Y.A. (2020). Long-Term Expansion of Pancreatic Islet Organoids from Resident Procr(+) Progenitors. Cell 180 , 1198-1211.e19. 10.1016/j.cell.2020.02.048. Mohan Rao, L.V., Esmon, C.T., and Pendurthi, U.R. (2014). Endothelial cell protein C receptor: a multiliganded and multifunctional receptor. Blood 124 , 1553-1562. 10.1182/blood-2014-05-578328. Magisetty, J., Kondreddy, V., Keshava, S., Das, K., Esmon, C.T., Pendurthi, U.R., and Rao, L.V.M. (2022). Selective inhibition of activated protein C anticoagulant activity protects against hemophilic arthropathy in mice. Blood 139 , 2830-2841. 10.1182/blood.2021013119. Ren, D., Fedorova, J., Davitt, K., Van Le, T.N., Griffin, J.H., Liaw, P.C., Esmon, C.T., Rezaie, A.R., and Li, J. (2022). Activated Protein C Strengthens Cardiac Tolerance to Ischemic Insults in Aging. Circ. Res. 130 , 252-272. 10.1161/circresaha.121.319044. Yang, X.V., Banerjee, Y., Fernández, J.A., Deguchi, H., Xu, X., Mosnier, L.O., Urbanus, R.T., de Groot, P.G., White-Adams, T.C., McCarty, O.J., and Griffin, J.H. (2009). Activated protein C ligation of ApoER2 (LRP8) causes Dab1-dependent signaling in U937 cells. Proc. Natl. Acad. Sci. U. S. A. 106 , 274-279. 10.1073/pnas.0807594106. Sinha, R.K., Yang, X.V., Fernández, J.A., Xu, X., Mosnier, L.O., and Griffin, J.H. (2016). Apolipoprotein E Receptor 2 Mediates Activated Protein C-Induced Endothelial Akt Activation and Endothelial Barrier Stabilization. Arterioscler., Thromb., Vasc. Biol. 36 , 518-524. 10.1161/atvbaha.115.306795. Wang, D., Liu, C., Wang, J., Jia, Y., Hu, X., Jiang, H., Shao, Z.M., and Zeng, Y.A. (2018). Protein C receptor stimulates multiple signaling pathways in breast cancer cells. J. Biol. Chem. 293 , 1413-1424. 10.1074/jbc.M117.814046. Wang, D., Hu, X., Liu, C., Jia, Y., Bai, Y., Cai, C., Wang, J., Bai, L., Yang, R., Lin, C., et al. (2019). Protein C receptor is a therapeutic stem cell target in a distinct group of breast cancers. Cell Res. 29 , 832-845. 10.1038/s41422-019-0225-9. Yu, Q.C., Geng, A., Preusch, C.B., Chen, Y., Peng, G., Xu, Y., Jia, Y., Miao, Y., Xue, H., Gao, D., et al. (2022). Activation of Wnt/β-catenin signaling by Zeb1 in endothelial progenitors induces vascular quiescence entry. Cell Rep. 41 , 111694. 10.1016/j.celrep.2022.111694. Yu, Q.C., Song, W., Wang, D., and Zeng, Y.A. (2016). Identification of blood vascular endothelial stem cells by the expression of protein C receptor. Cell Res. 26 , 1079-1098. 10.1038/cr.2016.85. Connor, K.M., Krah, N.M., Dennison, R.J., Aderman, C.M., Chen, J., Guerin, K.I., Sapieha, P., Stahl, A., Willett, K.L., and Smith, L.E. (2009). Quantification of oxygen-induced retinopathy in the mouse: a model of vessel loss, vessel regrowth and pathological angiogenesis. Nat. Protoc. 4 , 1565-1573. 10.1038/nprot.2009.187. Zou, J., Tan, W., Li, B., Wang, Z., Li, Y., Zeng, J., Jiang, B., Yoshida, S., and Zhou, Y. (2022). Interleukin-19 Promotes Retinal Neovascularization in a Mouse Model of Oxygen-Induced Retinopathy. Invest. Ophthalmol. Visual Sci. 63 , 9. 10.1167/iovs.63.8.9. Crespo-Garcia, S., Tsuruda, P.R., Dejda, A., Ryan, R.D., Fournier, F., Chaney, S.Y., Pilon, F., Dogan, T., Cagnone, G., Patel, P., et al. (2021). Pathological angiogenesis in retinopathy engages cellular senescence and is amenable to therapeutic elimination via BCL-xL inhibition. Cell Metab. 33 , 818-832.e7. 10.1016/j.cmet.2021.01.011. Caprara, C., and Grimm, C. (2012). From oxygen to erythropoietin: relevance of hypoxia for retinal development, health and disease. Prog. Retinal Eye Res. 31 , 89-119. 10.1016/j.preteyeres.2011.11.003. Liu, C., Lin, C., Wang, D., Wang, J., Tao, Y., Li, Y., Chen, X., Bai, L., Jia, Y., Chen, J., and Zeng, Y.A. (2022). Procr functions as a signaling receptor and is essential for the maintenance and self-renewal of mammary stem cells. Cell Rep. 38 , 110548. 10.1016/j.celrep.2022.110548. Lignitto, L., LeBoeuf, S.E., Homer, H., Jiang, S., Askenazi, M., Karakousi, T.R., Pass, H.I., Bhutkar, A.J., Tsirigos, A., Ueberheide, B., et al. (2019). Nrf2 Activation Promotes Lung Cancer Metastasis by Inhibiting the Degradation of Bach1. Cell 178 , 316-329.e18. 10.1016/j.cell.2019.06.003. Mills, E.L., Ryan, D.G., Prag, H.A., Dikovskaya, D., Menon, D., Zaslona, Z., Jedrychowski, M.P., Costa, A.S.H., Higgins, M., Hams, E., et al. (2018). Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1. Nature 556 , 113-117. 10.1038/nature25986. Dai, X., Yan, X., Zeng, J., Chen, J., Wang, Y., Chen, J., Li, Y., Barati, M.T., Wintergerst, K.A., Pan, K., et al. (2017). Elevating CXCR7 Improves Angiogenic Function of EPCs via Akt/GSK-3β/Fyn-Mediated Nrf2 Activation in Diabetic Limb Ischemia. Circ Res. 120 , e7-e23. 10.1161/circresaha.117.310619. Lien, E.C., Lyssiotis, C.A., Juvekar, A., Hu, H., Asara, J.M., Cantley, L.C., and Toker, A. (2016). Glutathione biosynthesis is a metabolic vulnerability in PI(3)K/Akt-driven breast cancer. Nat. Cell Biol. 18 , 572-578. 10.1038/ncb3341. Otterbein, L.E., Soares, M.P., Yamashita, K., and Bach, F.H. (2003). Heme oxygenase-1: unleashing the protective properties of heme. Trends Immunol. 24 , 449-455. 10.1016/s1471-4906(03)00181-9. Consonni, F.M., Bleve, A., Totaro, M.G., Storto, M., Kunderfranco, P., Termanini, A., Pasqualini, F., Alì, C., Pandolfo, C., Sgambelluri, F., et al. (2021). Heme catabolism by tumor-associated macrophages controls metastasis formation. Nat. Immunol. 22 , 595-606. 10.1038/s41590-021-00921-5. Dulak, J., Deshane, J., Jozkowicz, A., and Agarwal, A. (2008). Heme oxygenase-1 and carbon monoxide in vascular pathobiology: focus on angiogenesis. Circulation 117 , 231-241. 10.1161/circulationaha.107.698316. Loboda, A., Jozkowicz, A., and Dulak, J. (2015). HO-1/CO system in tumor growth, angiogenesis and metabolism - Targeting HO-1 as an anti-tumor therapy. Vasc. Pharmacol. 74 , 11-22. 10.1016/j.vph.2015.09.004. Fogli, S., Del Re, M., Rofi, E., Posarelli, C., Figus, M., and Danesi, R. (2018). Clinical pharmacology of intravitreal anti-VEGF drugs. Eye (Lond) 32 , 1010-1020. 10.1038/s41433-018-0021-7. Pérez-Gutiérrez, L., and Ferrara, N. (2023). Biology and therapeutic targeting of vascular endothelial growth factor A. Nat. Rev. Mol. Cell Biol. 24 , 816-834. 10.1038/s41580-023-00631-w. Okabe, K., Kobayashi, S., Yamada, T., Kurihara, T., Tai-Nagara, I., Miyamoto, T., Mukouyama, Y.S., Sato, T.N., Suda, T., Ema, M., and Kubota, Y. (2014). Neurons limit angiogenesis by titrating VEGF in retina. Cell 159 , 584-596. 10.1016/j.cell.2014.09.025. Usui, Y., Westenskow, P.D., Kurihara, T., Aguilar, E., Sakimoto, S., Paris, L.P., Wittgrove, C., Feitelberg, D., Friedlander, M.S., Moreno, S.K., et al. (2015). Neurovascular crosstalk between interneurons and capillaries is required for vision. J. Clin. Invest. 125 , 2335-2346. 10.1172/jci80297. Bucher, F., Zhang, D., Aguilar, E., Sakimoto, S., Diaz-Aguilar, S., Rosenfeld, M., Zha, Z., Zhang, H., Friedlander, M., and Yea, K. (2017). Antibody-Mediated Inhibition of Tspan12 Ameliorates Vasoproliferative Retinopathy Through Suppression of β-Catenin Signaling. Circulation 136 , 180-195. 10.1161/circulationaha.116.025604. Sadda, S.R., Guymer, R., Monés, J.M., Tufail, A., and Jaffe, G.J. (2020). Anti-Vascular Endothelial Growth Factor Use and Atrophy in Neovascular Age-Related Macular Degeneration: Systematic Literature Review and Expert Opinion. Ophthalmology 127 , 648-659. 10.1016/j.ophtha.2019.11.010. Bochenek, M.L., Gogiraju, R., Großmann, S., Krug, J., Orth, J., Reyda, S., Georgiadis, G.S., Spronk, H.M., Konstantinides, S., Münzel, T., et al. (2022). EPCR-PAR1 biased signaling regulates perfusion recovery and neovascularization in peripheral ischemia. JCI Insight. 7 , e157701. 10.1172/jci.insight.157701. Kopacz, A., Kloska, D., Targosz-Korecka, M., Zapotoczny, B., Cysewski, D., Personnic, N., Werner, E., Hajduk, K., Jozkowicz, A., and Grochot-Przeczek, A. (2020). Keap1 governs ageing-induced protein aggregation in endothelial cells. Redox Biol. 34 , 101572. 10.1016/j.redox.2020.101572. Wei, Y., Gong, J., Thimmulappa, R.K., Kosmider, B., Biswal, S., and Duh, E.J. (2013). Nrf2 acts cell-autonomously in endothelium to regulate tip cell formation and vascular branching. Proc. Natl. Acad. Sci. U. S. A. 110 , E3910-3918. 10.1073/pnas.1309276110. Ayer, A., Zarjou, A., Agarwal, A., and Stocker, R. (2016). Heme Oxygenases in Cardiovascular Health and Disease. Physiol. Rev. 96 , 1449-1508. 10.1152/physrev.00003.2016. Yuan, Z., De La Cruz, L.K., Yang, X., and Wang, B. (2022). Carbon Monoxide Signaling: Examining Its Engagement with Various Molecular Targets in the Context of Binding Affinity, Concentration, and Biologic Response. Pharmacol. Rev. 74 , 823-873. 10.1124/pharmrev.121.000564. Otterbein, L.E., Foresti, R., and Motterlini, R. (2016). Heme Oxygenase-1 and Carbon Monoxide in the Heart: The Balancing Act Between Danger Signaling and Pro-Survival. Circ Res. 118 , 1940-1959. 10.1161/circresaha.116.306588. Paredi, P., Biernacki, W., Invernizzi, G., Kharitonov, S.A., and Barnes, P.J. (1999). Exhaled carbon monoxide levels elevated in diabetes and correlated with glucose concentration in blood: a new test for monitoring the disease? Chest 116, 1007-1011. 10.1378/chest.116.4.1007. Cheng, S., Lyass, A., Massaro, J.M., O'Connor, G.T., Keaney, J.F., Jr., and Vasan, R.S. (2010). Exhaled carbon monoxide and risk of metabolic syndrome and cardiovascular disease in the community. Circulation 122, 1470-1477. 10.1161/circulationaha.110.941013. Additional Declarations There is NO Competing Interest. 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OIR and different phases of retinopathy. (B) Combined analysis of upregulated genes of three independent RNA-seq data in the retina of OIR mice. GSE241239 was performed in the present study. (C) Volcano plot of Log2 fold-changed genes of three RNA-seq data. (D) The protein levels of EPCR in the retina throughout the progression of OIR. (E) The mRNA levels of EPCR of the retina determined by qRT-PCR throughout the progression of OIR. (F) FACS-isolated retinal endothelial cells from OIR mice at P17 showed increased expression of mRNA levels of EPCR. (G) The representative protein levels of EPCR in FACS-isolated retinal endothelial cells from OIR mice. (H) Representative images of lineage tracing of EPCR positive cells in P17 mice using \u003cem\u003eEpcr\u003c/em\u003e-CreER\u003csup\u003eT2\u003c/sup\u003e; \u003cem\u003eRosa26\u003c/em\u003e\u003csup\u003etdT\u003c/sup\u003e mice. Scale bar 500 μm. (I) Quantification of the fluorescent intensity of EPCR positive cells. (J) OIR Mice serum aPC levels were determined using ELISA. (K) Human serum aPC levels were determined using ELISA. (L) aPC levels in human vitreous body were determined using ELISA. ∗∗∗p \u0026lt; 0.001, ∗∗p \u0026lt; 0.01, ∗p \u0026lt; 0.05. Data are represented as mean ± SD.\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4188758/v1/a92ac85a32b4fc9a738beed4.png"},{"id":58314516,"identity":"ac005f1d-ab61-4d1a-b928-6a07be4708e1","added_by":"auto","created_at":"2024-06-13 20:48:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":294402,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLoss of endothelial EPCR suppresses pathological RNV.\u003c/strong\u003e (A) Representative images of retinal vasculature stained with IB4 in \u003cem\u003eEpcr\u003c/em\u003e\u003csup\u003eWT\u003c/sup\u003e and \u003cem\u003eEpcr\u003c/em\u003e\u003csup\u003eiΔEC\u003c/sup\u003e mice at P17 using the OIR mice model. The orange space indicates the avascular area for two left images and neovascular tufts for two right images. Scale bar 1000 μm and 500 μm. (B) Quantification of the area of neovascular tufts in \u003cem\u003eEpcr\u003c/em\u003e\u003csup\u003eWT\u003c/sup\u003e and \u003cem\u003eEpcr\u003c/em\u003e\u003csup\u003eiΔEC\u003c/sup\u003e mice. (C) Quantification of the avascular area in \u003cem\u003eEpcr\u003c/em\u003e\u003csup\u003eWT\u003c/sup\u003e and \u003cem\u003eEpcr\u003c/em\u003e\u003csup\u003eiΔEC\u003c/sup\u003e mice. (D) Immunofluorescence staining for IB4 (blue), ERG (red), and EdU (green) in the retina of \u003cem\u003eEpcr\u003c/em\u003e\u003csup\u003eWT\u003c/sup\u003e and \u003cem\u003eEpcr\u003c/em\u003e\u003csup\u003eiΔEC\u003c/sup\u003e mice. Proliferating (ERG and EdU double positive) ECs are shown in yellow. Scale bar 50μm. (E) Quantification of the proliferating ECs in the retina of \u003cem\u003eEpcr\u003c/em\u003e\u003csup\u003eWT\u003c/sup\u003e and \u003cem\u003eEpcr\u003c/em\u003e\u003csup\u003eiΔEC\u003c/sup\u003e mice. (F) EdU-incorporation in EPCR-overexpressed HUVECs. DAPI (blue) was used to identify ECs nuclei. EdU (red) staining indicates proliferating ECs. Scale bar 200 μm. (G) Quantification of EdU-incorporation in EPCR-overexpressed HUVECs. (H) Representative images of HUVECs sprouting. Scale bar 50 μm. (I) Quantification of sprouts length in EPCR-overexpressed HUVECs. (J) Wound healing of EPCR-overexpressed HUVECs. Scale bar 100 μm. (K) Quantification of wound healing ability in EPCR-overexpressed HUVECs. (L) EdU-incorporation in EPCR-depleted HUVECs. DAPI (blue) is used to identify ECs nuclei. EdU (red) staining indicates proliferating ECs. Scale bar 200μm. (M) Quantification of EdU-incorporation in EPCR-depleted HUVECs. (N) Representative images of sprouting in EPCR-depleted HUVECs. Scale bar 50 μm. (O) Quantification of sprouts length in EPCR-depleted HUVECs. (P) Wound healing of EPCR-depleted HUVECs. Scale bar 100 μm. (Q) Quantification of wound healing ability in EPCR-depleted HUVECs. ∗∗∗∗p \u0026lt; 0.0001, ∗∗∗p \u0026lt; 0.001, ∗∗p \u0026lt; 0.01, ∗p \u0026lt; 0.05. Data are represented as mean ± SD.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4188758/v1/7fd39ecd8dc10b1648cfe177.png"},{"id":58314139,"identity":"cb99bfd2-406d-4405-a07d-14f6cdc866ec","added_by":"auto","created_at":"2024-06-13 20:40:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":345397,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePharmacological inhibition of EPCR attenuates pathological RNV.\u003c/strong\u003e (A) Representative images of retinal vasculature stained with IB4 in anti-EPCR treated mice at P17 using the OIR mice model. The orange space indicates the avascular area for three lower images and neovascular tufts for three upper images. Scale bar 1000 μm and 500 μm. (B) Quantification of the area of neovascular tufts in anti-EPCR treated mice. (C) Quantification of the avascular area in anti-EPCR treated mice. (D) Immunofluorescence staining for IB4 (blue), ERG (red), and EdU (green) in anti-EPCR treated mice. Proliferating (ERG and EdU double positive) ECs are shown in yellow. Scale bar 100 μm. (E) Quantification of the proliferating ECs in anti-EPCR treated mice. (F) EdU-incorporation in anti-EPCR treated ECs. DAPI (blue) is used to identify ECs nuclei. EdU (red) staining indicates proliferating ECs. Scale bar 200 μm. (G) Quantification of EdU-incorporation in anti-EPCR treated ECs. (H) Representative images of anti-EPCR-treated ECs sprouting. Scale bar 50 μm. (I) Quantification of sprouts length in anti-EPCR treated ECs. (J) Wound healing of anti-EPCR-treated ECs. Scale bar 100 μm. (K) Quantification of wound healing ability in anti-EPCR-treated ECs. ∗∗∗∗p \u0026lt; 0.0001, ∗∗∗p \u0026lt; 0.001, ∗∗p \u0026lt; 0.01, ∗p \u0026lt; 0.05. Data are represented as mean ± SD.\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4188758/v1/78024aca2cfe96032d0acf7f.png"},{"id":58314138,"identity":"c3a86131-4d40-4bec-9aa9-ebb8b1b959d9","added_by":"auto","created_at":"2024-06-13 20:40:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":245802,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEPCR regulate the expression of HO-1 through Nrf2/Keap1.\u003c/strong\u003e (A) Heatmap of genes (\u003cem\u003ep\u003c/em\u003e-value) downregulated in RNA-seq data of EPCR-depleted HUVECs and upregulated in the retinas of OIR mice. (B) Representative immunoblots and quantification for HO-1 levels in EPCR-depleted HUVECs. (C) The mRNA levels of HO-1 in EPCR-depleted HUVECs determined by qRT-PCR. (D) Representative immunoblots and quantification for HO-1 levels of EPCR adenovirus treated HUVECs. (E) The mRNA levels of HO-1 in EPCR adenovirus-treated HUVECs determined by qRT-PCR. (F) Representative immunoblots and quantification for HO-1 levels in ECs sorted from the retina of \u003cem\u003eEpcr\u003c/em\u003e\u003csup\u003eiΔEC \u003c/sup\u003emice. (G) The mRNA levels of HO-1 in ECs sorted from the retina of \u003cem\u003eEpcr\u003c/em\u003e\u003csup\u003eiΔEC \u003c/sup\u003emice. (H) Representative immunoblots and quantification for HO-1, NRF2, KEAP1, p-AKT(S473), AKT, and EPCR levels in EPCR-depleted HUVECs. (I) Representative immunoblots and quantification for HO-1, NRF2, KEAP1, p-AKT(S473), AKT, and EPCR levels in EPCR adenovirus-treated HUVECs. (J) Representative immunoblots and quantification for HO-1, NRF2, KEAP1, p-AKT(S473), AKT, and EPCR levels in EPCR adenovirus and LY294002 treated HUVECs. (K) Representative immunoblots and quantification for HO-1, NRF2 and KEAP1 levels in EPCR-depleted and KEAP1-depleted HUVECs. (L) Representative immunoblots and quantification for HO-1, NRF2 and KEAP1 levels in EPCR-depleted and KEAP1 inhibitor treated HUVECs. ∗∗∗∗p \u0026lt; 0.0001, ∗∗∗p \u0026lt; 0.001, ∗∗p \u0026lt; 0.01, ∗p \u0026lt; 0.05. Data are represented as mean ± SD.\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4188758/v1/5d317d3c4ef5e76a312cdaf3.png"},{"id":58314523,"identity":"36d68709-8fe1-4044-b6e3-28e6b4b5499d","added_by":"auto","created_at":"2024-06-13 20:48:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":241985,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEndothelial deletion of HO-1 inhibits pathological RNV.\u003c/strong\u003e (A) Representative images of retinal vasculature stained with IB4 in the retina of \u003cem\u003eHo-1\u003c/em\u003e\u003csup\u003eWT\u003c/sup\u003e and \u003cem\u003eHo-1\u003c/em\u003e\u003csup\u003eiΔEC\u003c/sup\u003e mice at P17 using the OIR mice model. The orange space indicates the avascular area for two left images and neovascular tufts for two right images. Scale bar 1000 μm and 500 μm. (B) Quantification of the area of neovascular tufts in\u003cem\u003e Ho-1\u003c/em\u003e\u003csup\u003eWT\u003c/sup\u003e and \u003cem\u003eHo-1\u003c/em\u003e\u003csup\u003eiΔEC\u003c/sup\u003e mice. (C) Quantification of the avascular area in \u003cem\u003eHo-1\u003c/em\u003e\u003csup\u003eWT\u003c/sup\u003e and \u003cem\u003eHo-1\u003c/em\u003e\u003csup\u003eiΔEC\u003c/sup\u003e mice. (D) Immunofluorescence staining for IB4 (blue), ERG (red), and EdU (green) in the retina of \u003cem\u003eHo-1\u003c/em\u003e\u003csup\u003eWT\u003c/sup\u003e and \u003cem\u003eHo-1\u003c/em\u003e\u003csup\u003eiΔEC\u003c/sup\u003e mice. Proliferating (ERG and EdU double positive) ECs are shown in yellow. Scale bar 50 μm. (E) Quantification of the proliferating ECs in the retina\u003cem\u003e Ho-1\u003c/em\u003e\u003csup\u003eWT\u003c/sup\u003e and \u003cem\u003eHo-1\u003c/em\u003e\u003csup\u003eiΔEC\u003c/sup\u003e mice. (F) EdU-incorporation in HUVECs treated with EPCR adenovirus and HO-1 siRNA. DAPI (blue) identifies ECs nuclei. EdU (red) staining indicates proliferating ECs. Scale bar 200 μm. (G) Quantification of EdU-incorporation in HUVECs treated with EPCR adenovirus and HO-1 siRNA. (H) Representative sprouting images in HUVECs treated with EPCR adenovirus and HO-1 siRNA. Scale bar 50 μm. (I) Quantification of sprouts length in in HUVECs treated with EPCR adenovirus and HO-1 siRNA. (J) Representative wound healing images in HUVECs treated with EPCR adenovirus and HO-1 siRNA. Scale bar 100 μm. (K) Quantification of wound healing ability in HUVECs treated with EPCR adenovirus and HO-1 siRNA. ∗∗∗p \u0026lt; 0.001, ∗∗p \u0026lt; 0.01, ∗p \u0026lt; 0.05. Data are represented as mean ± SD.\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4188758/v1/aba7672fe6a113eb83ac970b.png"},{"id":58314141,"identity":"160aa2b8-890a-4028-a02c-1b75a4b98f2c","added_by":"auto","created_at":"2024-06-13 20:40:49","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":234671,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEndothelial heme catabolism inhibition ameliorates RNV.\u003c/strong\u003e (A) Representative images of retinal vasculature stained with IB4 in DMSO and ZnPPIX treated mice at P17 using the OIR mice model. The orange space indicates the avascular area for two left images and neovascular tufts for two right images. Scale bar 1000 μm and 500 μm. (B) Quantification of the area of neovascular tufts in DMSO and ZnPPIX treated mice. (C) Quantification of the avascular area in the retina of DMSO and ZnPPIX treated mice. (D) Immunofluorescence staining for IB4 (blue), ERG (red), and EdU (green) in the retina of DMSO and ZnPPIX treated mice. Proliferating (ERG and EdU double positive) ECs are shown in yellow. Scale bar 50 μm. (E) Quantification of the proliferating ECs in the retina of DMSO and ZnPPIX treated mice. (F) EdU-incorporation in EPCR adenovirus and ZnPPIX treated HUVECs. DAPI (blue) identifies ECs nuclei. EdU (red) staining indicates proliferating ECs. Scale bar 200 μm. (G) Quantification of EdU-incorporation in EPCR adenovirus and ZnPPIX treated HUVECs. (H) Representative sprouting images of EPCR adenovirus and ZnPPIX treated HUVECs. Scale bar 50 μm. (I) Quantification of sprouts length in EPCR adenovirus and ZnPPIX treated HUVECs. (J) Wound healing of EPCR adenovirus and ZnPPIX treated HUVECs. Scale bar 100 μm. (K) Quantification of wound healing ability in EPCR adenovirus and ZnPPIX treated HUVECs. ∗∗∗∗p \u0026lt; 0.0001, ∗∗∗p \u0026lt; 0.001, ∗∗p \u0026lt; 0.01, ∗p \u0026lt; 0.05. Data are represented as mean ± SD.\u003c/p\u003e","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4188758/v1/8118325015f42574be10c07d.png"},{"id":58314519,"identity":"711b84ff-ac46-4a77-b820-5c0e44186b14","added_by":"auto","created_at":"2024-06-13 20:48:49","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":297816,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCO rescued the phenotype of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eHo-1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e deletion \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e in vivo.\u003c/strong\u003e\u003c/em\u003e (A) Representative images of retinal vasculature stained with IB4 in \u003cem\u003eHo-1\u003c/em\u003e\u003csup\u003eiΔEC\u003c/sup\u003e and CORM3 treated mice at P17 using the OIR mice model. The orange space indicates the avascular area for three lower images and neovascular tufts for three upper images. Scale bar 1000 μm and 500 μm. (B) Quantification of the area of neovascular tufts in the retina of \u003cem\u003eHo-1\u003c/em\u003e\u003csup\u003eiΔEC\u003c/sup\u003e and CORM3 treated mice. (C) Quantification of the avascular area in the retina of \u003cem\u003eHo-1\u003c/em\u003e\u003csup\u003eiΔEC\u003c/sup\u003e and CORM3 treated mice. (D) Immunofluorescence staining for IB4 (blue), ERG (red), and EdU (green) in the retina of \u003cem\u003eHo-1\u003c/em\u003e\u003csup\u003eiΔEC\u003c/sup\u003e and CORM3 treated mice. Proliferating (ERG and EdU double positive) ECs are shown in yellow. Scale bar 50 μm. (E) Quantification of the proliferating ECs in the retina of\u003cem\u003e Ho-1\u003c/em\u003e\u003csup\u003eiΔEC\u003c/sup\u003e and CORM3 treated mice. (F) EdU-incorporation in HO-1-depleted and CORM3 treated HUVECs. DAPI (blue) identifies ECs nuclei. EdU (red) staining indicated proliferating ECs. Scale bar 200 μm. (G) Quantification of EdU-incorporation in HO-1-depleted and CORM3 treated HUVECs. (H) Representative images of HO-1-depleted and CORM3 treated HUVECs sprouting. Scale bar 50 μm. (I) Quantification of sprouts length in HO-1-depleted and CORM3 treated HUVECs. (J) Wound healing of HO-1-depleted and CORM3 treated HUVECs. Scale bar 100 μm. (K) Quantification of wound healing ability in HO-1-depleted and CORM3 treated HUVECs. ∗∗∗∗p \u0026lt; 0.0001, ∗∗∗p \u0026lt; 0.001, ∗∗p \u0026lt; 0.01, ∗p \u0026lt; 0.05. Data are represented as mean ± SD.\u003c/p\u003e","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4188758/v1/526718ef7e064b8a998918f8.png"},{"id":58314140,"identity":"dc0f22d1-69f9-47cc-8296-56699ceb3a0c","added_by":"auto","created_at":"2024-06-13 20:40:49","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":258534,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIllustration showing the expression and role of EPCR during RNV.\u003c/strong\u003e (A) EPCR is specifically upregulated in retinal endothelial cells during RNV, and endothelial deletion of \u003cem\u003eEpcr\u003c/em\u003e ameliorates RNV. (B) EPCR regulated the expression of HO-1 and heme catabolism-derived CO through AKT/KEAP1/NRF2 signaling. EPCR blockade decreases CO levels and inhibits retinal angiogenesis.\u003c/p\u003e","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-4188758/v1/03d6166e9b282886e8746f69.png"},{"id":76267861,"identity":"fd1df739-c878-458a-9c7b-2369ed46aad0","added_by":"auto","created_at":"2025-02-14 08:05:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3984187,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4188758/v1/17e5517d-db9c-4568-ac16-b89c5958828a.pdf"},{"id":58314135,"identity":"5b00a107-645f-409a-b155-071c42f8c93d","added_by":"auto","created_at":"2024-06-13 20:40:49","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":9195804,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementalinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-4188758/v1/17646acec32b33a661b2d547.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Endothelial protein C receptor promotes retinal neovascularization through heme catabolism","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePathological retinal neovascularization (RNV) is a major cause of vision loss worldwide.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e The most common causes include diabetic retinopathy, retinopathy of prematurity (ROP), and retinal vein occlusion, which affect millions of people.\u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e The aberrant vessel growth usually leads to macular edema, fibrotic scarring, and retinal detachment.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e These conditions are the primary causes of blindness in working-age and pediatric populations in developed countries.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e Endothelial cells (ECs) rarely proliferate and remain in quiescent state in adult organism, whereas uncontrolled proliferation of ECs is initiated and drives aberrant vessel growth under pathological stimuli.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e Vascular endothelial growth factor (VEGF) is extensively studied in the regulation of EC proliferation and angiogenesis.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e Anti-VEGF regents are first-line therapy for RNV, but resistance occurs in certain patients.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e Hence, it is necessary to identify novel therapeutic targets of RNV.\u003c/p\u003e \u003cp\u003eEndothelial protein C receptor (EPCR, also known as PROCR) is a single-pass transmembrane glycoprotein expressed in ECs and stem cells.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e In addition to its anti-coagulation role,\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e EPCR mediates intracellular signaling of activated protein C (aPC).\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e Activation of downstream protein kinase B (AKT) promotes cell growth, improves cardiac function, and favors tumor progression.\u003csup\u003e\u003cspan additionalcitationids=\"CR17 CR18 CR19\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e Lineage tracing of EPCR-positive cells indicate that EPCR is specifically expressed in the ECs of the retinal vasculature.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e EPCR transcription is activated in the retinal vessels during angiogenic expansion stage but not during the quiescent stage.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e Ablation of EPCR-positive cells leads to delayed retinal vascular expansion in postnatal mice, suggesting a role for EPCR in retinal angiogenesis.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e However, the role of EPCR in RNV pathology remains unclear.\u003c/p\u003e \u003cp\u003eIn this study, we found that EPCR transcription was activated in ECs during RNV. Conditional knockout of \u003cem\u003eEpcr\u003c/em\u003e in ECs or the use of an EPCR-neutralizing antibody ameliorated pathological RNV. Further data showed that EPCR affected heme catabolism by regulating heme oxygenase 1 (HMOX1, also known as HO-1). Conditional knockout of \u003cem\u003eHo-1\u003c/em\u003e in ECs or treatment with an HO-1 enzymatic inhibitor suppressed pathological angiogenesis in retinopathy. Carbon monoxide releasing molecule-3 (CORM3) partially rescued the reduced neovascularization caused by the deletion of endothelial \u003cem\u003eEpcr\u003c/em\u003e and \u003cem\u003eHo-1\u003c/em\u003e, indicating a role for carbon monoxide (CO). Clinically, levels of aPC, which is an EPCR ligand, were elevated in proliferative diabetic retinopathy (PDR) patients\u0026rsquo; serum and vitreous body. These results indicate that EPCR-dependent endothelial heme catabolism is an important contributor to RNV.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003e1. EPCR is highly expressed in retinal ECs during RNV.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe mouse model of oxygen-induced retinopathy (OIR) serves as a proxy for human pathological RNV, including PDR and ROP.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e The neovascular response peaked on postnatal day 17 (P17) when the mice were returned to room air for five days (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). To identify the potential genes involved in pathological neovascularization, we performed transcriptomic analyses of the retinas of P17 mice of OIR (GSE241239). To minimize the RNA-seq bias, we further analyzed transcriptomic data from two other independent studies (GSE194176 and GSE158799).\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e A combined analysis revealed 149 upregulated genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.B-C). Among these candidate genes, we decided to focus on \u003cem\u003eEpcr\u003c/em\u003e because it was reported to be specifically expressed on the surface of vascular ECs and stem cells.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e Further data confirmed that EPCR expression was upregulated in the retinas of P17 mice in the OIR (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.D-E). We sorted ECs from the mouse retina to evaluate the expression of EPCR in the retinal vasculature. Endothelial EPCR expression was much higher in the OIR group than that in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF-G).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we examined the expression pattern of EPCR in the retina. Immunofluorescence staining with an anti-EPCR antibody showed that EPCR was selectively expressed in the retinal ECs of P17 mice under physiological conditions (FigS1A). In the OIR model, the expression of EPCR was detected only in retinal ECs, and the fluorescence intensity of EPCR signals was much higher in the neovascular tufts (FigS1B-C). \u003cem\u003eEpcr\u003c/em\u003e-CreER\u003csup\u003eT\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e (also known as \u003cem\u003eProcr\u003c/em\u003e-CreER\u003csup\u003eT\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e) mice were previously used to mark proliferative ECs in the retinal vasculature.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e Here, we used \u003cem\u003eEpcr\u003c/em\u003e-CreER\u003csup\u003eT\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e and \u003cem\u003eRosa26\u003c/em\u003e\u003csup\u003etdT\u003c/sup\u003e mice to perform genetic lineage tracing of EPCR-positive cells in the retina of P17 mice. Tamoxifen (TAM) was administered for three consecutive days at P10, P11, and P12, and the retina was analyzed at P17 (FigS1D). Under physiological conditions, a very small proportion of EPCR\u0026thinsp;+\u0026thinsp;cells were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). While there was a large proportion of EPCR\u0026thinsp;+\u0026thinsp;cells in the retina of OIR mice, and EPCR\u0026thinsp;+\u0026thinsp;cells mainly colocalized with neovascular tufts (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH-I). These data indicated that hypoxia significantly increased the transcription of EPCR. Hypoxia is also known to play a vital role in the retinal vascular development process.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e Therefore, the expression pattern of EPCR in the retinal vasculature of postnatal mice were then assessed. Immunofluorescence staining with anti-EPCR showed that the levels of EPCR were much higher in the margin of the retinal vasculature (FigS1E). Consistent data were acquired using \u003cem\u003eEpcr\u003c/em\u003e-CreER\u003csup\u003eT\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e and \u003cem\u003eRosa26\u003c/em\u003e\u003csup\u003etdT\u003c/sup\u003e mice (FigS1F-G). Collectively, these data suggest that EPCR is specifically expressed in ECs of the retina and is upregulated in neovascularization in the OIR model and during retinal vascular expansion.\u003c/p\u003e \u003cp\u003eEPCR activates downstream signaling pathways by binding to its ligands. Thus, we assessed the concentrations of EPCR ligands in the serum of OIR mice. The data showed that the levels of aPC in the serum of OIR mice were higher than those in control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ). To evaluate whether the findings in mice hold promise for human patients with RNV, we investigated the levels of aPC in the vitreous humor and serum in patients with PDR. The concentrations of aPC in the vitreous humor and serum were significantly higher in patients with PDR than in control with macular hole or idiopathic epiretinal membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK-L).\u003c/p\u003e \u003cp\u003e \u003cb\u003e2. EPCR contributes to RNV\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003ein vitro.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo determine the role of EPCR in RNV, we intercrossed the \u003cem\u003eCdh5\u003c/em\u003e-CreER\u003csup\u003eT\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e mouse with \u003cem\u003eEpcr\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e mouse to generate EC-specific \u003cem\u003eEpcr\u003c/em\u003e KO mice (\u003cem\u003eEpcr\u003c/em\u003e\u003csup\u003eiΔEC\u003c/sup\u003e). The expression of EPCR was almost completely ablated in ECs sorted from \u003cem\u003eEpcr\u003c/em\u003e\u003csup\u003eiΔEC\u003c/sup\u003e mice (FigS2A-B). To assess the role of EPCR in pathological RNV, we examined the changes in the retinal vasculature of \u003cem\u003eEpcr\u003c/em\u003e\u003csup\u003eiΔEC\u003c/sup\u003e mice of OIR at P17. Our data showed that the area of neovascular tufts in the retina of \u003cem\u003eEpcr\u003c/em\u003e\u003csup\u003eiΔEC\u003c/sup\u003e mice was smaller than that in \u003cem\u003eEpcr\u003c/em\u003e\u003csup\u003eWT\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B). In contrast, the avascular area in \u003cem\u003eEpcr\u003c/em\u003e\u003csup\u003eiΔEC\u003c/sup\u003e mice\u0026rsquo; retina was larger than that in \u003cem\u003eEpcr\u003c/em\u003e\u003csup\u003eWT\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Furthermore, we used 5-ethynyl-2\u0026rsquo;-deoxyuridine (EdU) to label proliferative cells in the retina and used E26 transformation-specific (ETS)-related gene (ERG) as an ECs marker. The result showed that EdU\u003csup\u003e+\u003c/sup\u003e ECs in the retina decreased significantly in \u003cem\u003eEpcr\u003c/em\u003e\u003csup\u003eiΔEC\u003c/sup\u003e mice compared to \u003cem\u003eEpcr\u003c/em\u003e\u003csup\u003eWT\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-E). These \u003cem\u003ein vivo\u003c/em\u003e results suggest that upregulated EPCR in pathological vasculature drives EC growth and angiogenesis in OIR.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we determined the effect of \u003cem\u003eEpcr\u003c/em\u003e deletion on retinal vascular expansion in postnatal retinas. EC-specific deletion of \u003cem\u003eEpcr\u003c/em\u003e led to a sparse vascular network and reduced radial expansion of the superficial retinal vascular plexus (FigS2C-D). Tip cells and branch points were decreased in the retina of \u003cem\u003eEpcr\u003c/em\u003e KO mice (FigS2E-G). Meanwhile, ECs proliferation was also significantly inhibited in \u003cem\u003eEpcr\u003c/em\u003e\u003csup\u003eiΔEC\u003c/sup\u003e mice (FigS2H-I).\u003c/p\u003e \u003cp\u003eTo further evaluate the impact of elevated EPCR on angiogenesis, we used an adenovirus to overexpress EPCR and assessed the angiogenic behavior of ECs \u003cem\u003ein vitro\u003c/em\u003e. EPCR adenovirus treatment significantly increased the protein levels of EPCR in ECs, which promoted ECs proliferation in an EdU staining assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF-G, FigS2J). A three-dimensional endothelial spheroid assay showed the number and length of endothelial sprouts were increased in the EPCR adenovirus treatment group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH-I). Moreover, a wound scratch assay revealed that EPCR adenovirus-treated ECs were more motile than control adenovirus treated ECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ-K). Furthermore, the knockdown of EPCR using siRNA suppressed ECs proliferation, migration and sprouting (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL-Q, FigS2K). Together with \u003cem\u003ein vivo\u003c/em\u003e results, these data indicated that EPCR promotes vascular growth.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3. Pharmacological inhibition of EPCR ameliorates pathological retinal neovascularization.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo evaluate the translational potential of targeting EPCR in anti-angiogenic therapy, we assessed the effects of a neutralizing antibody against EPCR on pathological angiogenesis \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. We found that a single dose of EPCR antibody significantly reduced the area of neovascular tufts compared to the vehicle and anti-IgG groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B). Meanwhile, the avascular area increased in the EPCR antibody treatment group, which was consistent with that in \u003cem\u003eEpcr\u003c/em\u003e KO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, C). Suppressed proliferation of ECs in retinal neovascular tufts was also observed after treatment with the EPCR antibody (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-E). Similar results were obtained \u003cem\u003ein vitro\u003c/em\u003e as EPCR antibody treatment significantly inhibited ECs proliferation, migration and sprouting (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF-L). These results were consistent with our findings using \u003cem\u003eEpcr\u003c/em\u003e\u003csup\u003eiΔEC\u003c/sup\u003e mice, suggesting the translational potential of anti-EPCR in retinal neovascularization.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e4. EPCR regulates the expression of HO-1 through Nrf2/Keap1\u003c/h2\u003e \u003cp\u003eTo reveal the possible molecular mechanisms underlying EPCR-regulated angiogenesis, we performed transcriptomic analyses of EPCR siRNA- or control siRNA-treated ECs (GSE249130, FigS3A). These data were then combined and analyzed with the mice retina results with OIR (FigS3B). 26 genes were upregulated in the retina of OIR mice and downregulated in EPCR-depleted ECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Among these genes, HO-1 was the most significantly affected (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Increased mRNA and protein levels of HO-1 were confirmed in the retinas of OIR mice (FigS3C-D). Further data showed that the expression of HO-1 was suppressed at the mRNA and protein levels in ECs treated with EPCR siRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-C). Additionally, the expression of HO-1 was upregulated at the mRNA and protein levels in ECs treated with EPCR adenovirus (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-E). The expression of HO-1 in retinal ECs of OIR mice was then evaluated. We sorted ECs from the retina of OIR mice using flow cytometry. The results indicated that the expression of HO-1 in ECs was upregulated in the OIR group (FigS3E-F). This was further confirmed by immunofluorescence staining. The expression of HO-1 in physiological retina did not show apparent pattern, while it was highly expressed in neovascular tufts of OIR retinas (FigS3G-H). Further data showed that the expression of HO-1 in ECs sorted from the retina of \u003cem\u003eEpcr\u003c/em\u003e\u003csup\u003eiΔEC\u003c/sup\u003e mice decreased compared with that in \u003cem\u003eEpcr\u003c/em\u003e\u003csup\u003ewt\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF-G). Collectively, these results suggested that HO-1 was a downstream molecule of EPCR in regulating angiogenesis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAKT is recognized as a target of EPCR signaling.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e High EPCR expression or stimulation with aPC usually leads to AKT activation.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e We found that overexpression of EPCR using an adenovirus promoted the activation of AKT, whereas silencing EPCR with siRNA inhibited AKT phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH-I). Nuclear factor erythroid 2-related factor 2 (NRF2) is a ubiquitous transcription factor directly regulating HO-1.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e NRF2 is tightly regulated by Kelch-like ECH-associated protein 1 (KEAP1) through ubiquitination and proteasome-dependent degradation.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e Therefore, we assessed the effects of EPCR on the expression of KEAP1 and NRF2. Overexpression of EPCR in ECs promoted the levels of NRF2 and HO-1 and inhibited the expression of KEAP1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). Decreased expression of NRF2 and HO-1, and increased expression of KEAP1 were observed after EPCR knockdown in ECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). Meanwhile, aPC treatment stimulated the activation of AKT, and increased the expression of NRF2 and HO-1 while suppressing the expression of KEAP1 (FigS3I).\u003c/p\u003e \u003cp\u003eAKT activates NRF2 in multiple cells.\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e Our data showed that inactivating AKT with LY294002 (a PI3K inhibitor) blocked the effect of EPCR overexpression on NRF2, KEAP1, and HO-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ). Consistent results were observed when ECs were treated with aPC (FigS3J). These results suggested that AKT plays an important role in mediating EPCR signaling. Furthermore, we observed that depleting the expression of KEAP1 using siRNA rescued the downregulation of NRF2 and HO-1 caused by EPCR siRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK, FigS3K). Consistent results were obtained after treatment with Ki696 (a KEAP1 inhibitor) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eL). These data suggested that EPCR controlled HO-1 expression via the AKT-KEAP1-NRF2 pathway.\u003c/p\u003e \u003cp\u003e \u003cb\u003e5. Genetic ablation of endothelial\u003c/b\u003e \u003cb\u003eHo-1\u003c/b\u003e \u003cb\u003eattenuates angiogenesis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo evaluate the role of HO-1 in retinal angiogenesis, we bred \u003cem\u003eCdh5\u003c/em\u003e-CreER\u003csup\u003eT\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e mice with \u003cem\u003eHo-1\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e mice to generate EC-specific \u003cem\u003eHo-1\u003c/em\u003e KO mice (\u003cem\u003eHo-1\u003c/em\u003e\u003csup\u003eiΔEC\u003c/sup\u003e). The deletion efficiency was determined using flow cytometry-sorted ECs, and the expression of HO-1 in ECs sorted from \u003cem\u003eHo-1\u003c/em\u003e\u003csup\u003eiΔEC\u003c/sup\u003e mice was significantly decreased (FigS4A). The role of HO-1 in pathological retinal angiogenesis was then determined in the OIR mice. Our data showed that the area of neovascular tufts in the retina of \u003cem\u003eHo-1\u003c/em\u003e\u003csup\u003eiΔEC\u003c/sup\u003e mice was smaller, while the avascular area in the retina of \u003cem\u003eHo-1\u003c/em\u003e\u003csup\u003eiΔEC\u003c/sup\u003e mice was larger than that in \u003cem\u003eHo-1\u003c/em\u003e\u003csup\u003eWT\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-C). Furthermore, our results showed that EdU\u003csup\u003e+\u003c/sup\u003e ECs decreased significantly in the retina of \u003cem\u003eHo-1\u003c/em\u003e\u003csup\u003eiΔEC\u003c/sup\u003e mice than in \u003cem\u003eHo-1\u003c/em\u003e\u003csup\u003eWT\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-E).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePhysiological retinal vascular development was also affected in \u003cem\u003eHo-1\u003c/em\u003e\u003csup\u003eiΔEC\u003c/sup\u003e mice. Deleting endothelial \u003cem\u003eHo-1\u003c/em\u003e reduced radial expansion of the superficial retinal vascular plexus (FigS4B, C). Meanwhile, the vascular density, tip cells and branch points were decreased in the retina of \u003cem\u003eHo-1\u003c/em\u003e\u003csup\u003eiΔEC\u003c/sup\u003e mice (FigS4D-F). Additionally, EdU\u003csup\u003e+\u003c/sup\u003e ECs decreased significantly in the retina of \u003cem\u003eHo-1\u003c/em\u003e\u003csup\u003eiΔEC\u003c/sup\u003e mice than in \u003cem\u003eHo-1\u003c/em\u003e\u003csup\u003eWT\u003c/sup\u003e mice (FigS4G-H).\u003c/p\u003e \u003cp\u003eThe role of HO-1 in angiogenesis was further assessed \u003cem\u003ein vitro\u003c/em\u003e. First, the knockdown efficiency of HO-1 siRNA was evaluated. The EdU assay showed that silencing HO-1 inhibited ECs proliferation induced by the EPCR adenovirus (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF-G). A three-dimensional endothelial spheroid assay was used to evaluate the angiogenic potential of ECs treated with EPCR adenovirus and HO-1 siRNAs. Fewer and shorter sprouts were observed in HO-1 siRNAs-treated ECs than EPCR adenovirus treated ECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH-I). Besides, ECs treated with HO-1 siRNAs exhibited impaired migration ability compared to the EPCR adenovirus treatment group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ-K). We also assessed the effects of HO-1 knockdown on ECs. The results showed that silencing HO-1 suppressed ECs proliferation, migration, and sprouting (FigS4J-O). These results collectively revealed the vital role of HO-1 in developmental angiogenesis and RNV.\u003c/p\u003e \u003cp\u003e \u003cb\u003e6. Endothelial heme catabolism impacts angiogenesis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eHO-1 catalyzes heme degradation into CO, biliverdin, and iron.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e Catabolism of cytotoxic labile heme and the generation of CO exert cytoprotective effects.\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e Thus, we further assessed whether the angiogenic role of HO-1 was mediated by its enzymatic function. Here, HO-1 enzymatic inhibitor, zinc protoporphyrin Ⅸ (ZnPPⅨ), was used to determine their effect on pathological RNV. The data showed that ZnPPⅨ treatment decreased the area of neovascular tufts in the retina and increased the avascular area (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-C). Besides, ZnPPⅨ suppressed ECs proliferation as EdU\u0026thinsp;+\u0026thinsp;retinal ECs decreased significantly in mice treated with ZnPPⅨ (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD-E).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we determined the effect of ZnPPⅨ on retinal vascular expansion. ZnPPⅨ treatment led to a sparse vascular network and reduced radial expansion of the superficial retinal vascular plexus (FigS5A-B). The tip cells and branch points were decreased in the retina of mice treated with ZnPPⅨ (FigS5C-E). Meanwhile, ZnPPⅨ significantly suppressed ECs proliferation in the retina (FigS5F-G).\u003c/p\u003e \u003cp\u003eFurther data showed that EPCR-induced cell proliferation, migration and sprouting of ECs \u003cem\u003ein vitro\u003c/em\u003e were inhibited by ZnPPⅨ (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF-K). Additionally, we evaluated the effect of ZnPPⅨ on ECs treated with HO-1 siRNA. The result showed that ZnPPⅨ did not affect ECs proliferation, migration, and sprouting after HO-1 knockdown (FigS5H-M). These data indicated that endothelial heme catabolism contributes to EPCR-mediated RNV.\u003c/p\u003e \u003cp\u003e \u003cb\u003e7. Heme catabolism-derived CO contributes to pathological angiogenesis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eEndogenous CO is a signaling molecule that promotes tumor growth and angiogenesis.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e Heme catabolism in macrophages increases tumor angiogenesis through CO production, suggesting a role for CO in regulating angiogenesis.\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e Here, we showed that pathological retinal neovascular tufts in carbon monoxide releasing molecule-3 (CORM3) treated \u003cem\u003eHo-1\u003c/em\u003e\u003csup\u003eiΔEC\u003c/sup\u003e mice significantly increased compared with that in \u003cem\u003eHo-1\u003c/em\u003e\u003csup\u003eiΔEC\u003c/sup\u003e mice, while the avascular area was decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-C). Furthermore, the data showed that EdU\u003csup\u003e+\u003c/sup\u003e ECs also increased significantly in the CORM3-treated retina of \u003cem\u003eHo-1\u003c/em\u003e\u003csup\u003eiΔEC\u003c/sup\u003e mice than that in \u003cem\u003eHo-1\u003c/em\u003e\u003csup\u003eWT\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD-E). Pathological retinal neovascular tufts in CORM3-treated \u003cem\u003eEpcr\u003c/em\u003e\u003csup\u003eiΔEC\u003c/sup\u003e mice also significantly increased, and avascular area decreased compared with that in \u003cem\u003eEpcr\u003c/em\u003e\u003csup\u003eiΔEC\u003c/sup\u003e mice (FigS6A-C). Consistent data were acquired for the number of EdU\u003csup\u003e+\u003c/sup\u003e ECs (FigS6D-E).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe assessed the effect of CORM3 on postnatal retinal vasculature in \u003cem\u003eHo-1\u003c/em\u003e\u003csup\u003eiΔEC\u003c/sup\u003e and \u003cem\u003eEpcr\u003c/em\u003e\u003csup\u003eiΔEC\u003c/sup\u003e mice. Treatment with CORM3 could rescue the sparse vascular network and reduce the radial expansion of superficial retinal vascular plexus in \u003cem\u003eHo-1\u003c/em\u003e\u003csup\u003eiΔEC\u003c/sup\u003e mice (FigS7A-B). Compared with \u003cem\u003eHo-1\u003c/em\u003e\u003csup\u003eiΔEC\u003c/sup\u003e mice, tip cells and branch points increased in the retina treated with CORM3 (FigS7C-E). Consistent results were acquired in CORM3-treated \u003cem\u003eEpcr\u003c/em\u003e\u003csup\u003eiΔEC\u003c/sup\u003e mice (FigS7F-J).\u003c/p\u003e \u003cp\u003eFurthermore, CORM3 rescued HO-1 silence-caused inhibition of ECs proliferation, migration, and sprouting (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF-K). Additionally, the effect of CORM3 on EPCR-deleted ECs was evaluated. The results showed that EPCR silencing inhibited ECs proliferation, migration, and sprouting, which was partially reversed after treatment with CORM3 (FigS8A-F). These results suggested that CO was an important contributor to EPCR dependent endothelial heme catabolism-regulated angiogenesis.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eTherapeutic strategies for RNV are effective, but off-target effect and resistance limit their efficacy.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e The reason is that these therapies cannot distinguish between healthy and pathological vessels. Our current study showed that EPCR was selectively expressed on ECs of retina and EPCR transcription was activated under angiogenic stimulus. Conditional deletion of \u003cem\u003eEpcr\u003c/em\u003e in ECs or anti-EPCR neutralizing antibody treatment attenuated pathological retinopathy. Mechanistically, EPCR controlled endothelial heme catabolism through HO-1, and heme catabolism-derived CO plays a vital role. Notably, plasma levels of the EPCR ligand were elevated in the vitreous body or serum of patients with PDR. These observations revealed the potential therapeutic value of targeting EPCR for suppressing pathological RNV (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHere, we observed that the expression of EPCR was detected specifically in the retinal ECs and was upregulated in neovascular tufts using anti-EPCR staining. EPCR was not detected in retinal pigment epithelia cells, photoreceptor cells, or ganglion cells. Consistent with our previous work,\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e we found that EPCR transcription is activated during vessels expansion stage in the retina. Meanwhile, EPCR transcription was activated in ECs during RNV using \u003cem\u003eEpcr\u003c/em\u003e-CreER\u003csup\u003eT\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e; \u003cem\u003eRosa26\u003c/em\u003e\u003csup\u003etdT\u003c/sup\u003e mice. These findings revealed that EPCR expression was selectively detected on retinal ECs and was upregulated under angiogenic stimulus. Vascular endothelial growth factor receptor 2 (VEGFR2) is the major receptor for VEGF guiding angiogenesis.\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e However, VEGFR2 is widely expressed in the retina, including ECs and neurons.\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e Sustained suppression of VEGF would damage the photoreceptors, and lead to loss of vision.\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e This is observed in clinic that retinal atrophy occurs in patients received anti-VEGF treatment.\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e The specific expression pattern of EPCR in retina indicates that targeting EPCR is unlikely to induce off-target effects.\u003c/p\u003e \u003cp\u003eIt is reported that EPCR\u0026thinsp;+\u0026thinsp;ECs are highly proliferative and are the major contributor toward vessel development.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e Conditional expression of diphtheria toxin (DTA) in EPCR\u0026thinsp;+\u0026thinsp;cells delay retinal vessel extension in postnatal mice, indicating a role for EPCR in angiogenesis.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e Consistent data was acquired in our study that conditional deletion of EPCR in ECs suppressed the expansion of retinal vascular plexus. Using OIR mice model, we showed that endothelial deletion of EPCR or EPCR neutralization antibody reduced ECs proliferation and attenuated pathological RNV. The angiogenic role of EPCR in neovascularization is also observed in hindlimb ischemia mice.\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e Deletion of EPCR in ECs using \u003cem\u003eTie2\u003c/em\u003e-Cre; \u003cem\u003eEpcr\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e mice suppresses new vessel formation.\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e Given that EPCR transcription is specifically activated in proliferating ECs,\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e EPCR blockade could be a novel option for treating RNV with minimal effects on quiescent ECs.\u003c/p\u003e \u003cp\u003eThe mechanism by which EPCR contributes to RNV remain to be revealed. We found that deletion of EPCR in ECs reduced the expression of HO-1 through AKT/KEAP1/NRF2 pathway. Whereas EPCR ligand (aPC) treatment upregulated the expression of HO-1 via AKT/KEAP1/NRF2. The data were consistent with studies showing that EPCR activate AKT signaling in heart and multiple cells.\u003csup\u003e\u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e The levels of the EPCR ligand were elevated in the serum or vitreous body of patients with PDR, suggesting that it activates EPCR signaling to favor retinal neovascularization. It is well established that KEAP1/NRF2 directly regulates the expression of HO-1 during tumor growth and metastasis.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e KEAP1 is a negative regulator of NRF2, affecting the expression of NRF2 in ECs.\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e Endothelial ablation of KEAP1 favors retinal vascular expansion, increases vascular density, and promotes ECs proliferation.\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e Whereas endothelial deletion of NRF2 delays vascular expansion and decreases vascular density.\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e Here, we showed that ECs-specific deletion of \u003cem\u003eHo-1\u003c/em\u003e suppressed retinal neovascularization in the retina of OIR mice and postnatal mice. The results were consistent with that observed in ECs-specific \u003cem\u003eKeap1\u003c/em\u003e or \u003cem\u003eNrf2\u003c/em\u003e knockout mice.\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eHO-1 catalyzes the degradation of heme to CO, ferrous iron, and biliverdin.\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e While initially considered to be a waste product, CO is increasingly recognized as a cytoprotective and homeostatic molecule.\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e Heme catabolism-derived CO is a major source of endogenous CO,\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e which contributes to tumor metastasis and angiogenesis.\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e In ECs, CO is reported to increase cell growth \u003cem\u003ein vitro\u003c/em\u003e, whereas its role \u003cem\u003ein vivo\u003c/em\u003e is unknown.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e Here, our data indicate that heme catabolism-derived CO promoted retinal angiogenesis, revealing a vital role of CO in angiogenesis. Clinically, the levels of exhaled CO significantly increase in patients with diabetes and correlate with blood glucose levels and duration of the disease.\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e PDR, characterized by uncontrolled RNV, is a late-stage microvascular complication of diabetes.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e Increased levels of aPC in the serum or vitreous body of patients with PDR imply upregulated EPCR-dependent heme catabolism during RNV. These findings indicate that CO derived from heme catabolism plays a vital role in RNV.\u003c/p\u003e \u003cp\u003eIn summary, we determined that EPCR is highly expressed in retinal ECs and that blocking EPCR attenuates pathological retinal angiogenesis. Mechanistically, EPCR controls heme catabolism via the KEAP1/NRF2/HO-1 pathway, and CO derived from heme catabolism plays a vital role in RNV. Hence, our findings elucidate a novel mechanism of RNV and suggest that EPCR may be a promising therapeutic target for translational application. However, these mouse models do not accurately represent the pathological progression of retinopathy in primates. Therefore, determining the therapeutic efficiency of anti-EPCR for pathological retinal angiogenesis in non-human primates is indispensable in the future. Moreover, the expression pattern of EPCR in human retina is unknown. For translational significance, it is important to determine the expression pattern of EPCR in post-mortem retinas under diverse pathophysiological conditions.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePatient samples\u003c/h2\u003e \u003cp\u003eThe patients diagnosed with proliferative diabetic retinopathy (PDR), idiopathic epiretinal membrane (ERM) and idiopathic macular hole (MH) were included. Vitreous samples were obtained from patients who underwent vitrectomy surgery by the same surgeon. Details of the patient information was showed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The collected vitreous samples were placed on ice immediately and centrifuged to exclude the debris. The supernatants were aliquoted into sterile tubes and stored in liquid nitrogen. Serum samples were obtained from patients who were diagnosed with PDR. The control serum samples were obtained from patients who were diagnosed with ERM and MH. Details of the patient information was showed in Supplementary Table S2. The obtained serum samples were centrifuged at 4 ℃ and the supernatants were stored at -80 ℃. All surgeries were performed, and human samples were harvested in accordance with the principles in the Declaration of Helsinki. Informed consent was obtained from ethics committee of Shanghai General Hospital ([2022]-109).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMice\u003c/h3\u003e\n\u003cp\u003eAll the experimental animals were accommodated in animal facilities, where they were subjected to 12-hour cycles of light and darkness. These animals were provided with unrestricted access to standard chow and water. The C57BL/6J mice were purchased from Jihui Laboratory Animal Care Co., Ltd. \u003cem\u003eEpcr\u003c/em\u003e \u003csup\u003eflox/flox\u003c/sup\u003e mice (the Cyagen Biotechnology Co., Ltd.) and \u003cem\u003eHo-1\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e mice (the Cyagen Biotechnology Co., Ltd.) were crossed with \u003cem\u003eCdh5\u003c/em\u003e CreER\u003csup\u003eT\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e mice\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e respectively to generate conditional knockout mice. For the lineage tracing of \u003cem\u003eEpcr\u003c/em\u003e\u0026thinsp;+\u0026thinsp;cells, the \u003cem\u003eEpcr\u003c/em\u003e CreER\u003csup\u003eT\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e mice\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e were crossed to \u003cem\u003eRosa26\u003c/em\u003e\u003csup\u003etdTomato\u003c/sup\u003e reporter mice (the Cyagen Biotechnology Co., Ltd.) All animal studies were approved by the Institutional Animal Care and Use Committee at Shanghai Changhai Hospital (CHEC(A.E)2023-024).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eOxygen induced retinopathy (OIR)\u003c/h2\u003e \u003cp\u003eOxygen-induced retinopathy (OIR) was performed as previously reported.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e In brief, the pups were exposed to 75% oxygen with their nursing mothers for five days from P7 to P12. Then the pups were returned to room air at P12. When the mice were return to room air, the hypoxia-induced neovascularization is initiated and peaked on P17. Animals were euthanized and the retinas were harvested at P17. To activate CreER\u003csup\u003eT\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, 50\u0026micro;l of tamoxifen (2mg/ml) was injected intraperitoneally from P10 to P12. To evaluate the anti-angiogenic role of EPCR neutralizing antibody, mice were randomly divided into three groups and injected intravitreally with 1 \u0026micro;l anti-EPCR antibody, PBS and anti-IgG respectively at P12. ZnPPIX (25mg/kg) and CORM3 (40mg/kg) were injected intraperitoneally twice at P12 and P13. To detect proliferating cells, EdU (50mg/kg) were administered intraperitoneally 6 h before euthanasia.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRNA-Seq sample preparation and sequencing\u003c/h3\u003e\n\u003cp\u003eTotal RNA from retinas and cells were extracted with RNA Easy Fast Tissue/Cell Kit (TIANGEN). Agilent 2100 Bioanalyzer and RNA nano 6000 assay kit (Agilent Technologies) were used to evaluate the quality of RNA samples. The transcriptome sequencing library was constructed through 1 ng/\u0026micro;l RNA randomly fragmentation, cDNA strand 1 / strand 2 synthesis, end repair, A-tailing, ligation of sequencing adapters, size selection and library PCR enrichment. The integrity of cDNA was evaluated by Agilent 2100 Bioanalyzer. The library preparations were sequenced on an Illumina HiSeq 2500 platform (Illumina, USA). The raw data of sequences were uploaded in the NCBI Sequence Read Archive (SRA) database (GSE241239 for the retinas of OIR mice; GSE249130 for HUVECs with EPCR deletion).\u003c/p\u003e\n\u003ch3\u003eIsolation of retinal ECs\u003c/h3\u003e\n\u003cp\u003eIsolation of retinal ECs was performed according to protocols we previously described with some modifications.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e The retinas were separated from eyeballs then minced into fine fragments and digested in Iscove\u0026rsquo;s Modified Dulbecco\u0026rsquo;s MediumIs (IMDM, LI1090-500,BioAgrio) with 1.5mg/ml Collagenase H(11087789001, Roche), 1.25 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 0.4 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 1% P/S and 3.5 \u0026micro;g/ml DNaseI (D4263, Sigma) at 37\u0026deg;C for 45min. The samples were pipetted every 10min using 1ml pipette tip to ensure even digestion. The digestion mix was passed through a 40\u0026micro;m nylon mesh to prepare single-cell suspensions. Cells were then incubated for 20min with FITC-conjugated anti -CD31(11-0317-82, Invitrogen, diluted 1:100) and PE-Cy7 conjugated anti-CD45 (25-0451-82, Invitrogen, diluted 1:200) in PBS with 5% FBS. Flow cytometry and cell sorting was performed using Then the single cell suspensions were subjected to FACS using SONY ID7000 or LSR Fortessa (BD Biosciences). FACS data were analyzed by FlowJo software. The purity of sorted ECs was routinely checked and ensured to be more than 95%.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eRetinal dissection and whole-mount staining\u003c/h2\u003e \u003cp\u003eEyes were fixed in 4% paraformaldehyde (PFA) for 0.5h at RT. After dissection, retinas were blocked in blocking buffer (1%BSA, 5%FBS and 0.5% Triton-X-100 in PBS) for 1h at RT. Primary antibodies were incubated in blocking buffer at 4℃ overnight. After washed twice with PBS, retinas were incubated with secondary antibodies at RT for 2h. Then, the retinas were washed and flat-mounted with Fluoromount-G (SouthernBiotech). For the labeling of EdU, an additional step was performed to detect EdU-labeled proliferative cells using the Click-It EdU kit (C10338, Invitrogen) prior to mounting. Images were acquired with a Mica confocal microscope (Leica Microsystems). Quantitative analysis of retinal vasculature was performed as described.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eQuantification and statistical analysis\u003c/h2\u003e \u003cp\u003eAll calculations were performed using GraphPad Prism (GraphPad software 9.0, GraphPad, Bethesda, MD, USA). Statistical analysis was performed using the two-tailed Student\u0026rsquo;s t-test and one-way analysis of variance (ANOVA), where appropriate, to compare different groups. For all bar graphs, data were presented as means\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;SD. All experiments were repeated at least three times and \u003cem\u003eP\u003c/em\u003e values of \u0026lt;\u0026thinsp;0.05 were considered as statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThanks to Yi Arial Zeng (State Key Laboratory of Cell Biology, Chinese Academy of Sciences) for kindly providing \u003cem\u003eEpcr\u003c/em\u003e-CreER\u003csup\u003eT2\u003c/sup\u003e mice and technical assistance; to Youheng Wei (State Key Laboratory of Genetic Engineering, Fudan University, Shanghai, China) for providing technical assistance. This work was supported by grants from National Natural Science Foundation of China (82171081, 82271106, 82171076, U22A20311, 82388101), National Key R\u0026amp;D Program (2022YFC2502800), Shanghai Municipal Education Commission (2023ZKZD18), Shanghai Science and Technology committee (22ZR1478200), Shanghai Changhai Hospital excellent top-notch project (2023YQ01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, X.S., H.S., W.S., H.Z. and F.L.; Methodology, Q.L., X.S., and A.G.; investigation, H.S., Q.L., X.G., Z.F., W.Z., M.W., Y.L., H.Z., Z.N., L.Z., H.Z., and Y.J.; Funding acquisition, X.S., H.S. and W.S.; Writing-original draft, H.S and Q.L.; Writing-review \u0026amp; editing, X.S., F.Z., F.L., and X.L.; Supervision, X.S., X.L. and F.Z. All authors approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplemental information can be found online.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBinet, F., Cagnone, G., Crespo-Garcia, S., Hata, M., Neault, M., Dejda, A., Wilson, A.M., Buscarlet, M., Mawambo, G.T., Howard, J.P., et al. (2020). Neutrophil extracellular traps target senescent vasculature for tissue remodeling in retinopathy. Science \u003cem\u003e369\u003c/em\u003e, eaay5356. 10.1126/science.aay5356.\u003c/li\u003e\n\u003cli\u003eCampochiaro, P.A. (2015). Molecular pathogenesis of retinal and choroidal vascular diseases. Prog. Retinal Eye Res. \u003cem\u003e49\u003c/em\u003e, 67-81. 10.1016/j.preteyeres.2015.06.002.\u003c/li\u003e\n\u003cli\u003eAntoszyk, A.N., Glassman, A.R., Beaulieu, W.T., Jampol, L.M., Jhaveri, C.D., Punjabi, O.S., Salehi-Had, H., Wells, J.A., 3rd, Maguire, M.G., Stockdale, C.R., et al. (2020). Effect of Intravitreous Aflibercept vs Vitrectomy With Panretinal Photocoagulation on Visual Acuity in Patients With Vitreous Hemorrhage From Proliferative Diabetic Retinopathy: A Randomized Clinical Trial. JAMA \u003cem\u003e324\u003c/em\u003e, 2383-2395. 10.1001/jama.2020.23027.\u003c/li\u003e\n\u003cli\u003eStahl, A., Sukgen, E.A., Wu, W.C., Lepore, D., Nakanishi, H., Mazela, J., Moshfeghi, D.M., Vitti, R., Athanikar, A., Chu, K., et al. (2022). Effect of Intravitreal Aflibercept vs Laser Photocoagulation on Treatment Success of Retinopathy of Prematurity: The FIREFLEYE Randomized Clinical Trial. JAMA \u003cem\u003e328\u003c/em\u003e, 348-359. 10.1001/jama.2022.10564.\u003c/li\u003e\n\u003cli\u003eYeh, S., Kim, S.J., Ho, A.C., Schoenberger, S.D., Bakri, S.J., Ehlers, J.P., and Thorne, J.E. (2015). Therapies for macular edema associated with central retinal vein occlusion: a report by the American Academy of Ophthalmology. Ophthalmology \u003cem\u003e122\u003c/em\u003e, 769-778. 10.1016/j.ophtha.2014.10.013.\u003c/li\u003e\n\u003cli\u003eGariano, R.F., and Gardner, T.W. (2005). Retinal angiogenesis in development and disease. Nature \u003cem\u003e438\u003c/em\u003e, 960-966. 10.1038/nature04482.\u003c/li\u003e\n\u003cli\u003eSelvam, S., Kumar, T., and Fruttiger, M. (2018). Retinal vasculature development in health and disease. Prog. Retinal Eye Res. \u003cem\u003e63\u003c/em\u003e, 1-19. 10.1016/j.preteyeres.2017.11.001.\u003c/li\u003e\n\u003cli\u003eEelen, G., Treps, L., Li, X., and Carmeliet, P. (2020). Basic and Therapeutic Aspects of Angiogenesis Updated. Circ. Res. \u003cem\u003e127\u003c/em\u003e, 310-329. 10.1161/circresaha.120.316851.\u003c/li\u003e\n\u003cli\u003eApte, R.S., Chen, D.S., and Ferrara, N. (2019). VEGF in Signaling and Disease: Beyond Discovery and Development. Cell \u003cem\u003e176\u003c/em\u003e, 1248-1264. 10.1016/j.cell.2019.01.021.\u003c/li\u003e\n\u003cli\u003eSimons, M., Gordon, E., and Claesson-Welsh, L. (2016). Mechanisms and regulation of endothelial VEGF receptor signalling. Nat. Rev. Mol. Cell Biol. \u003cem\u003e17\u003c/em\u003e, 611-625. 10.1038/nrm.2016.87.\u003c/li\u003e\n\u003cli\u003eArima, M., Nakao, S., Yamaguchi, M., Feng, H., Fujii, Y., Shibata, K., Wada, I., Kaizu, Y., Ahmadieh, H., Ishibashi, T., et al. (2020). Claudin-5 Redistribution Induced by Inflammation Leads to Anti-VEGF-Resistant Diabetic Macular Edema. Diabetes \u003cem\u003e69\u003c/em\u003e, 981-999. 10.2337/db19-1121.\u003c/li\u003e\n\u003cli\u003eWallsh, J.O., and Gallemore, R.P. (2021). Anti-VEGF-Resistant Retinal Diseases: A Review of the Latest Treatment Options. Cells \u003cem\u003e10\u003c/em\u003e, 1049. 10.3390/cells10051049.\u003c/li\u003e\n\u003cli\u003eWang, D., Wang, J., Bai, L., Pan, H., Feng, H., Clevers, H., and Zeng, Y.A. (2020). Long-Term Expansion of Pancreatic Islet Organoids from Resident Procr(+) Progenitors. Cell \u003cem\u003e180\u003c/em\u003e, 1198-1211.e19. 10.1016/j.cell.2020.02.048.\u003c/li\u003e\n\u003cli\u003eMohan Rao, L.V., Esmon, C.T., and Pendurthi, U.R. (2014). Endothelial cell protein C receptor: a multiliganded and multifunctional receptor. Blood \u003cem\u003e124\u003c/em\u003e, 1553-1562. 10.1182/blood-2014-05-578328.\u003c/li\u003e\n\u003cli\u003eMagisetty, J., Kondreddy, V., Keshava, S., Das, K., Esmon, C.T., Pendurthi, U.R., and Rao, L.V.M. (2022). Selective inhibition of activated protein C anticoagulant activity protects against hemophilic arthropathy in mice. Blood \u003cem\u003e139\u003c/em\u003e, 2830-2841. 10.1182/blood.2021013119.\u003c/li\u003e\n\u003cli\u003eRen, D., Fedorova, J., Davitt, K., Van Le, T.N., Griffin, J.H., Liaw, P.C., Esmon, C.T., Rezaie, A.R., and Li, J. (2022). Activated Protein C Strengthens Cardiac Tolerance to Ischemic Insults in Aging. Circ. Res. \u003cem\u003e130\u003c/em\u003e, 252-272. 10.1161/circresaha.121.319044.\u003c/li\u003e\n\u003cli\u003eYang, X.V., Banerjee, Y., Fern\u0026aacute;ndez, J.A., Deguchi, H., Xu, X., Mosnier, L.O., Urbanus, R.T., de Groot, P.G., White-Adams, T.C., McCarty, O.J., and Griffin, J.H. (2009). Activated protein C ligation of ApoER2 (LRP8) causes Dab1-dependent signaling in U937 cells. Proc. Natl. Acad. Sci. U. S. A. \u003cem\u003e106\u003c/em\u003e, 274-279. 10.1073/pnas.0807594106.\u003c/li\u003e\n\u003cli\u003eSinha, R.K., Yang, X.V., Fern\u0026aacute;ndez, J.A., Xu, X., Mosnier, L.O., and Griffin, J.H. (2016). Apolipoprotein E Receptor 2 Mediates Activated Protein C-Induced Endothelial Akt Activation and Endothelial Barrier Stabilization. Arterioscler., Thromb., Vasc. Biol. \u003cem\u003e36\u003c/em\u003e, 518-524. 10.1161/atvbaha.115.306795.\u003c/li\u003e\n\u003cli\u003eWang, D., Liu, C., Wang, J., Jia, Y., Hu, X., Jiang, H., Shao, Z.M., and Zeng, Y.A. (2018). Protein C receptor stimulates multiple signaling pathways in breast cancer cells. J. Biol. Chem. \u003cem\u003e293\u003c/em\u003e, 1413-1424. 10.1074/jbc.M117.814046.\u003c/li\u003e\n\u003cli\u003eWang, D., Hu, X., Liu, C., Jia, Y., Bai, Y., Cai, C., Wang, J., Bai, L., Yang, R., Lin, C., et al. (2019). Protein C receptor is a therapeutic stem cell target in a distinct group of breast cancers. Cell Res. \u003cem\u003e29\u003c/em\u003e, 832-845. 10.1038/s41422-019-0225-9.\u003c/li\u003e\n\u003cli\u003eYu, Q.C., Geng, A., Preusch, C.B., Chen, Y., Peng, G., Xu, Y., Jia, Y., Miao, Y., Xue, H., Gao, D., et al. (2022). Activation of Wnt/\u0026beta;-catenin signaling by Zeb1 in endothelial progenitors induces vascular quiescence entry. Cell Rep. \u003cem\u003e41\u003c/em\u003e, 111694. 10.1016/j.celrep.2022.111694.\u003c/li\u003e\n\u003cli\u003eYu, Q.C., Song, W., Wang, D., and Zeng, Y.A. (2016). Identification of blood vascular endothelial stem cells by the expression of protein C receptor. Cell Res. \u003cem\u003e26\u003c/em\u003e, 1079-1098. 10.1038/cr.2016.85.\u003c/li\u003e\n\u003cli\u003eConnor, K.M., Krah, N.M., Dennison, R.J., Aderman, C.M., Chen, J., Guerin, K.I., Sapieha, P., Stahl, A., Willett, K.L., and Smith, L.E. (2009). Quantification of oxygen-induced retinopathy in the mouse: a model of vessel loss, vessel regrowth and pathological angiogenesis. Nat. Protoc. \u003cem\u003e4\u003c/em\u003e, 1565-1573. 10.1038/nprot.2009.187.\u003c/li\u003e\n\u003cli\u003eZou, J., Tan, W., Li, B., Wang, Z., Li, Y., Zeng, J., Jiang, B., Yoshida, S., and Zhou, Y. (2022). Interleukin-19 Promotes Retinal Neovascularization in a Mouse Model of Oxygen-Induced Retinopathy. Invest. Ophthalmol. Visual Sci. \u003cem\u003e63\u003c/em\u003e, 9. 10.1167/iovs.63.8.9.\u003c/li\u003e\n\u003cli\u003eCrespo-Garcia, S., Tsuruda, P.R., Dejda, A., Ryan, R.D., Fournier, F., Chaney, S.Y., Pilon, F., Dogan, T., Cagnone, G., Patel, P., et al. (2021). Pathological angiogenesis in retinopathy engages cellular senescence and is amenable to therapeutic elimination via BCL-xL inhibition. Cell Metab. \u003cem\u003e33\u003c/em\u003e, 818-832.e7. 10.1016/j.cmet.2021.01.011.\u003c/li\u003e\n\u003cli\u003eCaprara, C., and Grimm, C. (2012). From oxygen to erythropoietin: relevance of hypoxia for retinal development, health and disease. Prog. Retinal Eye Res. \u003cem\u003e31\u003c/em\u003e, 89-119. 10.1016/j.preteyeres.2011.11.003.\u003c/li\u003e\n\u003cli\u003eLiu, C., Lin, C., Wang, D., Wang, J., Tao, Y., Li, Y., Chen, X., Bai, L., Jia, Y., Chen, J., and Zeng, Y.A. (2022). Procr functions as a signaling receptor and is essential for the maintenance and self-renewal of mammary stem cells. Cell Rep. \u003cem\u003e38\u003c/em\u003e, 110548. 10.1016/j.celrep.2022.110548.\u003c/li\u003e\n\u003cli\u003eLignitto, L., LeBoeuf, S.E., Homer, H., Jiang, S., Askenazi, M., Karakousi, T.R., Pass, H.I., Bhutkar, A.J., Tsirigos, A., Ueberheide, B., et al. (2019). Nrf2 Activation Promotes Lung Cancer Metastasis by Inhibiting the Degradation of Bach1. Cell \u003cem\u003e178\u003c/em\u003e, 316-329.e18. 10.1016/j.cell.2019.06.003.\u003c/li\u003e\n\u003cli\u003eMills, E.L., Ryan, D.G., Prag, H.A., Dikovskaya, D., Menon, D., Zaslona, Z., Jedrychowski, M.P., Costa, A.S.H., Higgins, M., Hams, E., et al. (2018). Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1. Nature \u003cem\u003e556\u003c/em\u003e, 113-117. 10.1038/nature25986.\u003c/li\u003e\n\u003cli\u003eDai, X., Yan, X., Zeng, J., Chen, J., Wang, Y., Chen, J., Li, Y., Barati, M.T., Wintergerst, K.A., Pan, K., et al. (2017). Elevating CXCR7 Improves Angiogenic Function of EPCs via Akt/GSK-3\u0026beta;/Fyn-Mediated Nrf2 Activation in Diabetic Limb Ischemia. Circ Res. \u003cem\u003e120\u003c/em\u003e, e7-e23. 10.1161/circresaha.117.310619.\u003c/li\u003e\n\u003cli\u003eLien, E.C., Lyssiotis, C.A., Juvekar, A., Hu, H., Asara, J.M., Cantley, L.C., and Toker, A. (2016). Glutathione biosynthesis is a metabolic vulnerability in PI(3)K/Akt-driven breast cancer. Nat. Cell Biol. \u003cem\u003e18\u003c/em\u003e, 572-578. 10.1038/ncb3341.\u003c/li\u003e\n\u003cli\u003eOtterbein, L.E., Soares, M.P., Yamashita, K., and Bach, F.H. (2003). Heme oxygenase-1: unleashing the protective properties of heme. Trends Immunol. \u003cem\u003e24\u003c/em\u003e, 449-455. 10.1016/s1471-4906(03)00181-9.\u003c/li\u003e\n\u003cli\u003eConsonni, F.M., Bleve, A., Totaro, M.G., Storto, M., Kunderfranco, P., Termanini, A., Pasqualini, F., Al\u0026igrave;, C., Pandolfo, C., Sgambelluri, F., et al. (2021). Heme catabolism by tumor-associated macrophages controls metastasis formation. Nat. Immunol. \u003cem\u003e22\u003c/em\u003e, 595-606. 10.1038/s41590-021-00921-5.\u003c/li\u003e\n\u003cli\u003eDulak, J., Deshane, J., Jozkowicz, A., and Agarwal, A. (2008). Heme oxygenase-1 and carbon monoxide in vascular pathobiology: focus on angiogenesis. Circulation \u003cem\u003e117\u003c/em\u003e, 231-241. 10.1161/circulationaha.107.698316.\u003c/li\u003e\n\u003cli\u003eLoboda, A., Jozkowicz, A., and Dulak, J. (2015). HO-1/CO system in tumor growth, angiogenesis and metabolism - Targeting HO-1 as an anti-tumor therapy. Vasc. Pharmacol. \u003cem\u003e74\u003c/em\u003e, 11-22. 10.1016/j.vph.2015.09.004.\u003c/li\u003e\n\u003cli\u003eFogli, S., Del Re, M., Rofi, E., Posarelli, C., Figus, M., and Danesi, R. (2018). Clinical pharmacology of intravitreal anti-VEGF drugs. Eye (Lond) \u003cem\u003e32\u003c/em\u003e, 1010-1020. 10.1038/s41433-018-0021-7.\u003c/li\u003e\n\u003cli\u003eP\u0026eacute;rez-Guti\u0026eacute;rrez, L., and Ferrara, N. (2023). Biology and therapeutic targeting of vascular endothelial growth factor A. Nat. Rev. Mol. Cell Biol. \u003cem\u003e24\u003c/em\u003e, 816-834. 10.1038/s41580-023-00631-w.\u003c/li\u003e\n\u003cli\u003eOkabe, K., Kobayashi, S., Yamada, T., Kurihara, T., Tai-Nagara, I., Miyamoto, T., Mukouyama, Y.S., Sato, T.N., Suda, T., Ema, M., and Kubota, Y. (2014). Neurons limit angiogenesis by titrating VEGF in retina. Cell \u003cem\u003e159\u003c/em\u003e, 584-596. 10.1016/j.cell.2014.09.025.\u003c/li\u003e\n\u003cli\u003eUsui, Y., Westenskow, P.D., Kurihara, T., Aguilar, E., Sakimoto, S., Paris, L.P., Wittgrove, C., Feitelberg, D., Friedlander, M.S., Moreno, S.K., et al. (2015). Neurovascular crosstalk between interneurons and capillaries is required for vision. J. Clin. Invest. \u003cem\u003e125\u003c/em\u003e, 2335-2346. 10.1172/jci80297.\u003c/li\u003e\n\u003cli\u003eBucher, F., Zhang, D., Aguilar, E., Sakimoto, S., Diaz-Aguilar, S., Rosenfeld, M., Zha, Z., Zhang, H., Friedlander, M., and Yea, K. (2017). Antibody-Mediated Inhibition of Tspan12 Ameliorates Vasoproliferative Retinopathy Through Suppression of \u0026beta;-Catenin Signaling. Circulation \u003cem\u003e136\u003c/em\u003e, 180-195. 10.1161/circulationaha.116.025604.\u003c/li\u003e\n\u003cli\u003eSadda, S.R., Guymer, R., Mon\u0026eacute;s, J.M., Tufail, A., and Jaffe, G.J. (2020). Anti-Vascular Endothelial Growth Factor Use and Atrophy in Neovascular Age-Related Macular Degeneration: Systematic Literature Review and Expert Opinion. Ophthalmology \u003cem\u003e127\u003c/em\u003e, 648-659. 10.1016/j.ophtha.2019.11.010.\u003c/li\u003e\n\u003cli\u003eBochenek, M.L., Gogiraju, R., Gro\u0026szlig;mann, S., Krug, J., Orth, J., Reyda, S., Georgiadis, G.S., Spronk, H.M., Konstantinides, S., M\u0026uuml;nzel, T., et al. (2022). EPCR-PAR1 biased signaling regulates perfusion recovery and neovascularization in peripheral ischemia. JCI Insight. \u003cem\u003e7\u003c/em\u003e, e157701. 10.1172/jci.insight.157701.\u003c/li\u003e\n\u003cli\u003eKopacz, A., Kloska, D., Targosz-Korecka, M., Zapotoczny, B., Cysewski, D., Personnic, N., Werner, E., Hajduk, K., Jozkowicz, A., and Grochot-Przeczek, A. (2020). Keap1 governs ageing-induced protein aggregation in endothelial cells. Redox Biol. \u003cem\u003e34\u003c/em\u003e, 101572. 10.1016/j.redox.2020.101572.\u003c/li\u003e\n\u003cli\u003eWei, Y., Gong, J., Thimmulappa, R.K., Kosmider, B., Biswal, S., and Duh, E.J. (2013). Nrf2 acts cell-autonomously in endothelium to regulate tip cell formation and vascular branching. Proc. Natl. Acad. Sci. U. S. A. \u003cem\u003e110\u003c/em\u003e, E3910-3918. 10.1073/pnas.1309276110.\u003c/li\u003e\n\u003cli\u003eAyer, A., Zarjou, A., Agarwal, A., and Stocker, R. (2016). Heme Oxygenases in Cardiovascular Health and Disease. Physiol. Rev. \u003cem\u003e96\u003c/em\u003e, 1449-1508. 10.1152/physrev.00003.2016.\u003c/li\u003e\n\u003cli\u003eYuan, Z., De La Cruz, L.K., Yang, X., and Wang, B. (2022). Carbon Monoxide Signaling: Examining Its Engagement with Various Molecular Targets in the Context of Binding Affinity, Concentration, and Biologic Response. Pharmacol. Rev. \u003cem\u003e74\u003c/em\u003e, 823-873. 10.1124/pharmrev.121.000564.\u003c/li\u003e\n\u003cli\u003eOtterbein, L.E., Foresti, R., and Motterlini, R. (2016). Heme Oxygenase-1 and Carbon Monoxide in the Heart: The Balancing Act Between Danger Signaling and Pro-Survival. Circ Res. \u003cem\u003e118\u003c/em\u003e, 1940-1959. 10.1161/circresaha.116.306588.\u003c/li\u003e\n\u003cli\u003eParedi, P., Biernacki, W., Invernizzi, G., Kharitonov, S.A., and Barnes, P.J. (1999). Exhaled carbon monoxide levels elevated in diabetes and correlated with glucose concentration in blood: a new test for monitoring the disease? Chest 116, 1007-1011. 10.1378/chest.116.4.1007.\u003c/li\u003e\n\u003cli\u003eCheng, S., Lyass, A., Massaro, J.M., O\u0026apos;Connor, G.T., Keaney, J.F., Jr., and Vasan, R.S. (2010). Exhaled carbon monoxide and risk of metabolic syndrome and cardiovascular disease in the community. Circulation 122, 1470-1477. 10.1161/circulationaha.110.941013.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"angiogenesis, retinopathy, diabetes complications, endothelial protein C receptor, heme catabolism, carbon monoxide ","lastPublishedDoi":"10.21203/rs.3.rs-4188758/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4188758/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePathological retinal neovascularization (RNV) is one of the leading causes of blindness worldwide; however, its underlying mechanism remains unclear. Here, we found that the expression of endothelial protein C receptor (EPCR) was increased during RNV, and its ligand was elevated in the serum or vitreous body of patients with proliferative diabetic retinopathy. Deleting endothelial \u003cem\u003eEpcr\u003c/em\u003e or using an EPCR neutralizing antibody ameliorated pathological retinal angiogenesis. EPCR promoted endothelial heme catabolism and carbon monoxide release through heme oxygenase 1 (HO-1). Inhibition of heme catabolism by deleting of endothelial \u003cem\u003eHo-1\u003c/em\u003eor using an HO-1 inhibitor suppressed pathological angiogenesis in retinopathy. Conversely, supplementation with CO rescued the angiogenic defects after endothelial \u003cem\u003eEpcr \u003c/em\u003eor \u003cem\u003eHo-1\u003c/em\u003e deletion. Our results identified EPCR-dependent endothelial heme catabolism as an important contributor to pathological angiogenesis, which may serve as a potential target for treating vasoproliferative retinopathy.\u003c/p\u003e","manuscriptTitle":"Endothelial protein C receptor promotes retinal neovascularization through heme catabolism","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-13 20:40:44","doi":"10.21203/rs.3.rs-4188758/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"2611fd0d-2615-4fbc-8c76-13ea4b28bcbb","owner":[],"postedDate":"June 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":32216792,"name":"Health sciences/Diseases/Eye diseases/Retinal diseases"},{"id":32216793,"name":"Health sciences/Cardiology/Cardiovascular biology/Angiogenesis"},{"id":32216794,"name":"Biological sciences/Developmental biology/Angiogenesis"},{"id":32216795,"name":"Biological sciences/Physiology/Cardiovascular biology/Angiogenesis"}],"tags":[],"updatedAt":"2025-02-14T08:05:31+00:00","versionOfRecord":{"articleIdentity":"rs-4188758","link":"https://doi.org/10.1038/s41467-025-56810-0","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-02-13 05:00:00","publishedOnDateReadable":"February 13th, 2025"},"versionCreatedAt":"2024-06-13 20:40:44","video":"","vorDoi":"10.1038/s41467-025-56810-0","vorDoiUrl":"https://doi.org/10.1038/s41467-025-56810-0","workflowStages":[]},"version":"v1","identity":"rs-4188758","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4188758","identity":"rs-4188758","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}