Mechanosensing Piezo1 mediates gut-vascular barrier dysfunction in portal hypertension and promotes deterioration of liver cirrhosis

Mechanosensing Piezo1 mediates gut-vascular barrier dysfunction in portal hypertension and promotes deterioration of liver cirrhosis | 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 Mechanosensing Piezo1 mediates gut-vascular barrier dysfunction in portal hypertension and promotes deterioration of liver cirrhosis Lei Zhang, Zhuanglong Xiao, Jun Song, Jing He, Wenjing Tian, li Du, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9418908/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Objectives Gut-vascular barrier (GVB) disruption in cirrhotic portal hypertension (PH) exacerbates hepatic and systemic damage via the gut-liver-organ axis, but the underlying mechanism is unclear. The mechanosensitive channel Piezo1, known to regulate endothelial homeostasis, may mediate GVB dysfunction, requiring validation. Methods Carbon tetrachloride (CCl 4 )-induced liver cirrhosis and partial portal vein ligation (PPVL)-induced pre-hepatic PH model were established in Piezo1 flox/flox and endothelial cells (ECs)-specific Piezo1-deficient (Piezo1 △EC ) mice. Portal pressure, GVB permeability, intestinal and systemic inflammation, and Piezo1 expression in mucosal ECs were measured. Cultured intestinal microvascular ECs were treated with the Piezo1 agonist Yoda1 or hydrostatic pressure to investigate Piezo1-mediated endothelial barrier regulation. Results PH induced significant GVB breakdown in CCl 4 and PPVL mice, presented as increased permeability from the gut-to-blood and also leakage from blood-to-gut, accompanied by increased Piezo1 and VEGFR2 expression in ECs, along with systemic and intestinal inflammation and leaky epithelium. These effects were reversed in Piezo1 △EC mice, and Piezo1 knockout in ECs reduced VEGFR2 expression which in turn inhibited intestinal leakage and inflammatory infiltration, and meanwhile improved systemic inflammation. Moreover, Yoda1 or hydrostatic pressure promoted VEGFR2 phosphorylation and ECs monolayer disruption, blocked by siRNA of Piezo1 or Vegfr2 . Conclusion Endothelial Piezo1 contributed to GVB dysfunction in PH via VEGFR2 pathway. The outward and inward hyper-permeability of GVB in PH may initiate the intestinal inflammation and trigger epithelial leakage, and further cause systemic inflammation and injury. Therefore, Piezo1-mediated GVB damage may be a novel explanation and potential therapeutic target for deterioration of liver cirrhosis with PH. Health sciences/Medical research/Experimental models of disease Health sciences/Diseases/Gastrointestinal diseases/Liver diseases/Portal hypertension Piezo1 Endothelial cells Gut-vascular barrier Portal hypertension Liver cirrhosis Figures Figure 1 Figure 2 Figure 3 Figure 5 Figure 6 Figure 7 Figure 8 INTRODUCTION Cirrhosis, the end stage of chronic liver disease caused by various reasons( 1 ), is a major global health concern with high morbidity and mortality rates worldwide( 2 ). Portal hypertension (PH) and declined liver function are the main features of progressive cirrhosis( 3 ). In particular, patients with cirrhosis are susceptible to microbiota translocation and entry of enterogenous toxins into the bloodstream, which may trigger systemic inflammation and injury( 4 ). These bacterial infections promote the progression and decompensation of cirrhosis, in some cases, can lead to life-threatening acute-on-chronic liver failure and even multi-organ failure via the gut-liver-organ axes( 5 – 7 ). Currently, targeting the gut-liver axis has become an important strategy and viewpoint for preventing further liver damage and related multi-organ injury in cirrhosis. Intestinal mucosal barrier dysfunction is commonly found in chronic liver disease and cirrhosis( 8 ), with destroyed integrity of mucus layer and capability of immune defense, and increased epithelial permeability( 9 ). Recently, beneath the epithelial barrier, a newly gut-vascular barrier (GVB) was characterized and proved important role in liver injury( 9 – 11 ), which is the structural and functional unit formed by microvascular endothelial cells (ECs), pericytes and enteric glial cells (Figure S1 )( 12 , 13 ). GVB acts as the last key guard between the intestine and liver, as well as the blood circulation, it defends against the dissemination of bacteria and luminal antigen that escaped from the epithelial barrier( 14 , 15 ). Most of the invaded pathogens from the intestinal epithelium are denied entry to the internal environment by GVB, and are finally phagocytose and clear by macrophages and other immunocytes( 13 ). Indubitably, GVB disruption may directly result in liver injury, and was confirmed in various intestinal inflammatory conditions( 15 ) and hepatic diseases, such as metabolic associated fatty liver disease (MAFLD), alcoholic liver disease (ALD) and cirrhosis( 11 , 16 , 17 ). However, the specific target and mechanism underlying GVB collapse is far from clear. Liver cirrhosis may disrupt the intestinal barrier integrity through gut dysbiosis and abnormal bile acid metabolism in enterohepatic circulation as reported in previous studies( 18 , 19 ). Even so, the direct mechanisms of liver cirrhosis on intestinal barrier, especially for the GVB, are still largely unclear. It is well-known that PH can cause direct damage to the gastrointestinal tract, so called portal hypertensive enteropathy( 20 ). The intestinal microvascular ECs may bear the first brunt from PH-induced rise of intravessel pressure and blood stasis, which is speculated to bring about GVB collapse, and intestinal edema and inflammation. To date, little is known about vascular mechanical sensing in inducing endothelial barrier disruption. Piezo1, a mechanosensitive ion channel expressed in ECs, is activated by elevated pressure and other mechanical stimuli( 21 ). Piezo1 was proven to play a crucial role in the endothelial homeostasis and angiogenesis, such as pulmonary artery( 22 , 23 ), coronary artery( 24 ), brain capillary( 25 – 27 ) and intestinal microvessel( 28 ). Piezo1 could sense disturbed blood flow and activated endothelial inflammatory signaling( 29 , 30 ). Upregulation of Piezo1 in ECs leads to vascular remodeling and pulmonary hypertension induced by high shear stress( 23 ). In particular, endothelial Piezo1 mediated pressure-induced lung vascular hyper-permeability via disruption of adherens junctions, while inhibition or deletion of Piezo1 in ECs markedly prevented capillary stress failure and lung edema( 22 ). Also, our preliminary findings indicated that endothelial Piezo1 might facilitate GVB disruption in response to elevated vascular pressures related to PH. Here, we addressed the possibility that Piezo1, as a mechanical sensor of high vascular pressure in the PH condition, is responsible for GVB breakdown in cirrhosis. In further, we explored the key role and underlying mechanism of Piezo1-mediated bi-directional GVB hyper-permeability in the intestinal and systemic inflammation that maybe the basis for the progress of liver cirrhosis. The study may provide new insights and potential targets for preventing the deterioration of liver cirrhosis with PH. MATERIALS AND METHODS Animals C57BL/6 mice were supplied by Shulaibao Biotech Co., Ltd (Wuhan, China). Piezo1 flox/flox mice (Cat. NO: NM-CKO-200275) were customized by Shanghai Model Organisms Center, Inc. (Shanghai, China). Cdh5-CreERT2 mice (Strain NO: T014691) were purchased from GemPharmatech Co., Ltd. (Nanjing, China). Piezo1 flox/flox mice were crossed with Cdh5-CreERT2 mice to generate ECs-specific Piezo1-deficient mice (Piezo1 △EC ) with the deletion of exon 4 and 5 of Piezo1 cDNA (NCBI ID: 234839). The Piezo1 △EC mice were intraperitoneally given 1 mg of tamoxifen (dissolved in corn oil; Sigma-Aldrich Chemistry) for 5 days to induce the complete deletion of Piezo1 specific on ECs. The Piezo1 flox/flox mice were used as controls (Piezo1 WT ). The mice were raised in the SPF environment (Animal Experimental Center, Tongji Medical College, HUST, China) with a constant temperature and humidity, and with the light / dark alternate time 12h/12h. All animal experiments were approved by the Institutional Animal Care and Use Committee of Tongji Medical College, HUST, China. CCl-induced Liver Cirrhosis Model Liver cirrhosis model was established through the administration of carbon tetrachloride (CCl 4 ) in mice( 31 ). CCl 4 (C822982, Macklin, China) was diluted at 5:5 (v/v) in olive oil (O815211, Macklin, China) for modeling. Male mice (8 weeks old) were injected subcutaneously with CCl 4 diluent at a dose of 3 ml/kg twice a week for 12 weeks. As a control, other mice administrated with 3 ml/kg olive oil only twice a week for 12 weeks. To further increase portal pressure, part of the CCl 4 mice were subjected partial portal vein ligation (26G) at the 8th week, while others conducted a sham operation as control. Partial Portal Vein Ligation (PPVL)-induced PH Model Mice model of pre-hepatic portal hypertension was induced by PPVL as previously described( 32 , 33 ). Briefly, mice were performed median laparotomy under anaesthesia, and the portal vein was carefully isolated. A blunt-tipped needle (26G or 27G) was placed on the portal vein, and a constricting ligature was conducted both on the needle and the portal vein with 7 − 0 silk. The needle was removed later on, leaving a calibrated stenosis (diameter about 0.46mm or 0.41mm) of the portal vein, and then the abdominal incision was sutured. A sham operation was performed in the control mice with portal vein isolated but not ligated. After a two-week recovery period following surgery, the mice were used for subsequent experimental testing. Portal Venous Pressure Measurement The portal pressure was measured via the water column method( 32 ). The mice were anesthetized with pentobarbital sodium, performed laparotomy incision and the portal vein was dissociated. An 26G infusion cannula was cannulated into the portal vein, and connected to a glass-tube manometer with water column (containing heparin sodium physiological saline). The portal pressure was recorded as the height of water column when the water column in the glass-tube manometer was stayed stationary. Assessment of Gut-Vascular Barrier Function The function of GVB was assessed by detecting the endothelial PV1 expression, and measuring the macromolecular permeability from the gut to blood and from blood to the gut. Endothelial PV1 expression . The PV1 distribution density was assessed via immunofluorescence, and present as the ratio of PV1 + /CD31 + area in the ileal and colonic slices. Permeability from the gut to blood . To assess the in-flux permeability of GVB, FITC-labeled dextran 70kDa (FD70, 2mg prepared in 2 ml physiological saline; Sigma-Aldrich Chemistry) was injected into a 4 cm-length intestinal loop (ligated at both ends with suture) through laparotomy incision under anesthesia( 34 ). One hour later, the blood samples were collected from the abdominal aorta, and the plasma FD70 level was detected via a fluorescence microplate reader (BioTek Instruments, USA). Meanwhile, the liver and spleen tissues were obtained under light-proof conditions, and fixed with 4% paraformaldehyde, made into 20 µm frozen sections, then imaged on a confocal fluorescence microscopy (Olympus Corporation, Japan). The density of FD70 particles in the liver and spleen were quantified via ImageJ software (National Institutes of Health, USA). Permeability from blood to the gut . To assess the out-flux permeability of GVB, Evans blue or FD70 was intravenous injected and the vascular leakage of these macromolecular was detected( 34 – 36 ). ( 1 ) Evans blue (30 mg/kg in 100µl normal saline; Sigma-Aldrich Chemistry) was injected into the tail vein of mice. The mice were killed 20 min later, the ileal and colonic tissues (the same segment for each mouse) were removed, and then cut along the sagittal direction, removed the faeces and blotted dry. Tissues were immersed in 1ml formamide overnight at 55°C to extract the Evans blue dye in the ileum and colon. The content of Evans blue was measured via spectrophotometry at 600nm, and the result expressed as µg/mg tissue. ( 2 ) 200 µl FD70 (2 mg/ml in normal saline; Sigma-Aldrich Chemistry) was injected into the tail vein of mice. After 2 h post injection, the mice were anesthetized and performed cardiac perfusion with physiological saline. The ileal and colonic tissues (the same segment for each mouse) were removed, and then fixed in 4% paraformaldehyde for 24 h, made into 20 µm frozen sections. The vascular ECs were labeled by immunofluorescent with anti-CD31 primary antibody and Alexa Fluor 594-conjugated secondary antibody. The sections were imaged on a confocal laser scanning microscope (Nikon, Japan), and the FD70 fluorescent extravasation was observed. The FITC positive area represented the vascular leakage of FD70 was quantified using ImageJ software. Assessment of Intestinal Epithelial Permeability The function of intestinal epithelial barrier was assessed via detecting the plasma endotoxin (LPS) on portal vein, and measuring the trans-epithelial cell resistance (TER) and the permeability to macromolecules in an Ussing Chamber method. Plasma endotoxin assay . Portal vein plasma were collected in endotoxin-free tubes and stored at − 80°C until assessment. LPS concentration was detected using a Mouse Endotoxin ELISA Kit (Bioswamp, China) following the manufacturer's instructions. In brief, 100 µl plasma sample was firstly reacted 100 µl limulus amebocyte lysate at 37°C for 5 min, incubated with 100 µl chromogenic substrate solution at 37°C for 5 min, and then terminated by reconstituted stop solution. The optical density (OD) was measured at 405 nm using a microplate reader (BIOBASE, China). Plasma endotoxin level was quantified against a standard curve. Ussing chamber analysis . The intestinal mucosal patches were prepared by stripping the seromuscular layer as previously described( 37 ). The intact mucosal patches were mounted on the sliders with a rectangular hole (opening area 0.15 cm 2 ) in the center, and the sliders were fixed on the U-type chambers filled with 37 ℃ oxygenated Krebs' solution at both the serosal and mucosal sides. Then, the U-type chambers were installed on an Ussing Chamber System (World Precision Instruments, USA). After a 20-min stability, the TER was recorded through the automatic voltage clamp model. To evaluate the mucosal-to-serosal flux of macromolecules, FITC-labeled dextran 4kDa (FD4, 1 mg/ml; Sigma-Aldrich Chemistry) was added into the mucosal side of the U-type chambers, and sampled from the serosal side every 30 min for a 2-h period. The FD4 concentration was further detected on a fluorescence microplate reader (BioTek Instruments, USA). The epithelial permeability was determined as the increase of FD4 intensity in the serosal side within 2 hours. Cell Culture of Intestinal Microvascular ECs ECs monolayer culture and interventions . Human intestinal microvascular endothelial cell (HIMECs; PriCells, China) were cultured in primary endothelial cell complete medium (MED-0002, PriCells) with growth supplement (SUP-0002, PriCells) of 5 ng/ml rh-VEGF, 5 ng/ml rh-EGF, 5 ng/ml rh-FGF, 15 ng/ml rh-IGF-1, 10 mM L-glutamine, 0.75 U/ml heparin sulfate, 1 µg/ml hydrocortisone and 50 µg/ml ascorbic acid at 37 ℃ in 5% CO 2 . As reached 80% confluence, the cells were rinsed twice with PBS, digested with 0.125% trypsin for 2 min, and passaged at a 1:2 dilution ratio, and cultured for no more than 3 generations. ECs were seeded on transwell membrane or coverslips in 24-well plates as the requirement of different experiments. Upon formation of stable intercellular connections, the cells were subjected interventions of Yoda1 (1 µM, 5µM) or hydrostatic pressure (2cm, 15cm, 30cmH 2 O) for 24 h for further detection, and preconditioned with siRNA transfection to silence Piezo1 or Vegfr2 when needed. Specially, the hydrostatic pressure was produced via an experimental apparatus (Fig. 6 A) as previously described ( 38 , 39 ). The culture chamber (a closed culture bottle) was filled with medium, where the cell-planted transwell membrane or coverslip was placed. The height of the medium reservoir was adjusted to maintain the pressure in the culture (2cm, 15cm, 30cm). The medium in the culture chamber and the medium reservoir was circulated by a peristaltic pump to ensure gas exchange and maintain stable oxygen tension. The entire device was placed in the incubator with 37 ℃ and 5% CO 2 . Transmembrane resistance and permeability assay. ECs were inoculated on the transwell membrane with 0.4µm pore size (Corning, USA) to form an endothelial monolayer. The transendothelial electrical resistance (TEER) was measured using an EVOM2 volt-ohmmeter (World Precision Instruments, USA), and calculated as Ω·cm 2 ( 40 ). A transwell with only culture medium was set as a blank control to measure the baseline membrane resistance. The TEER reached 180 Ω·cm 2 indicated formation of functional monolayer barrier, and then it could be used for further barrier studies. The transendothelial permeability was evaluated by apical-to-basolateral transmission of FITC-labeled bovine serum albumin (FITC-BSA; Solarbio Life Science). Briefly,10 mg/ml FITC-BSA was added into the upper compartment of the transwell chamber for 2 h, and then 100 µl medium was sampled at the lower compartment and detected FITC-BSA concentration via a Fluorescence Microplate Reader (BioTek Instruments, USA). The BSA throughput was calculated as the ratio between the fluorescence intensity in the lower chamber and upper chamber, and the data were presented as a percentage of the control( 41 , 42 ). Small Interfering RNA (siRNA) Transfection The gene silence of Piezo1 or Vegfr2 in ECs was achieved via specific siRNA. The siRNAs against human Piezo1, Vegfr2, and negative control were designed and chemically synthesized (Tsingke Biotech, China). The designed siRNAs were as follows: Piezo1-targeting siRNA (Piezo1-si) sequences were sense (5'-3') CUCAAGUACUUCAUCAACU(dT)(dT), antisense (5'-3') AGUUGAUGAAGUACUUGAG(dT)(dT), Gene ID: 9780; and Vegfr2-targeting siRNA (Vegfr2-si) sequences were sense (5'-3') GGAAAUCUCUUGCAAGCUA(dT)(dT), antisense (5'-3') UAGCUUGCAAGAGAUUUCC(dT)(dT), Gene ID: 3791. For cell transfection, ECs were seeded on BioFlex 6-well culture plates coated with type IV collagen. The cells were serum-starved for 6 hours, and transfected with 40 nM siRNA (Piezo1-si, Vegfr2-si, or control siRNA) via Lipofectamine™ 3000 reagent (L3000075, Invitrogen, Life Science) following the manufacturer’s instructions. The mRNA and protein expression was detected by RT-qPCR and Western blot at 48 h post transfection to confirm effect of knockdown. Data Availability The supporting data for this study are available from the corresponding authors upon reasonable request. The expanded methods are provided in the Supplementary Methods. Statistical Analysis All data were presented as the mean ± SEM. Statistical analysis and graphic production were performed using GraphPad Prism 10 (GraphPad Software, USA). The Shapiro-Wilk test and Brown-Forsythe test were employed to assess the normality of data distribution and the homogeneity of variances, respectively. Comparisons between groups were conducted performed using unpaired two-tailed Student's t-test (for two groups) or one-way analysis of variance (ANOVA) followed by least significant difference test (for multiple groups), if the data conformed to normal distribution and equal variance; otherwise, the non-parametric Mann-Whitney U test or Kruskal-Wallis test with Dunn's post hoc correction was applied accordingly. Pearson correlation was applied to assessed the correlations between parameters. A P < 0.05 was considered statistically significant. RESULTS Portal Hypertension Mediated GVB Disruption Promoted Intestinal and Systemic Inflammation in CCl 4 -induced Liver Cirrhosis Liver cirrhosis and related PH was induced by CCl4 administration (Fig. 1 A). CCl 4 induced significant liver injury and fibrosis, with diffuse inflammatory cell infiltration and vacuolar fat changes (Fig. 1 B), elevated serum ALT and AST levels (Fig. 1 D), increased areas of collagen (Sirius red positive, Fig. 1 E) and α-SMA expression (Fig. 1 F). The portal venous pressure markedly increased in CCl 4 mice, and further elevated in the CCl 4 mice with PPVL (Fig. 1 C). PH was associated with hepatic injury, manifesting as more severe inflammatory infiltration and elevation of serum ALT and AST in CCl 4 +PPVL mice compared with CCl 4 mice (Fig. 1 B, 1 D). There were obvious intestinal and systemic inflammation in CCl 4 -induced liver cirrhosis, and aggravate in the CCl 4 +PPVL mice. The levels of plasma endotoxin and circulating inflammatory cytokines, including TNF-α, IL-1β and IL-6, were increased in CCl 4 mice and CCl 4 +PPVL mice (Fig. 1 G, 1 H), which were positive correlated with the portal venous pressure (Figure S2 ). Furthermore, the microvascular dilation, inflammatory infiltration and the concentrations of inflammatory TNF-α, IL-1β and IL-6 were also markedly increased in ileum and colon of CCl 4 mice and CCl 4 +PPVL mice (Fig. 1 I-J; Figure S3). And the intestinal inflammation was accompanied by disruption of mucosal epithelial barrier, with reduced TER and increased FD4 permeability (Fig. 1 K-L). All above indicated that PH may contribute to the local inflammatory injury and leaky gut, and meanwhile promote the circulating inflammation, in status of liver cirrhosis. Importantly, it observed significantly GVB disruption in CCl 4 mice, and more serious GVB damage in CCl 4 +PPVL mice. As it shown, there was increased FD70 density detected in liver and spleen when administered through the intestine in CCl 4 mice (Fig. 1 M, 1 P-Q), which implied increased influx of enterogenous macromolecules into the bloodstream. The ileal and colonic PV1 expression, a marker of endothelial permeability, was also increased in CCl 4 mice which confirmed the GVB disruption in liver cirrhosis (Fig. 1 N, 1 R; Figure S4). On the other hand, the vascular leakage of circulating macromolecules into the gut was also increased in CCl 4 mice, with raised FD70 (Fig. 1 O, 1 S) and evans blue (Fig. 1 T) levels in the ileal and colonic mucosa when administered via tail-vein injection. Moreover, the GVB disruption was more remarkable in the CCl 4 +PPVL mice relative to the sham group. It further verified the inward and outward hyper-permeability of GVB in portal hypertension and liver cirrhosis. Impaired GVB Initiates Gut Inflammation and Leakage, and Led to Systemic Injury in PPVL-induced Portal Hypertension Prehepatic PH was induced via partial portal vein ligation with 26G and 27G needles (Fig. 2 A). PPVL led to significant increase in portal venous pressure (Fig. 2 C), and presented with local inflammatory cell infiltration and blood stagnation in the hepatic sinusoid (Fig. 2 B), but without elevation of serum ALT and AST levels (Fig. 2 D). Similarly, PPVL-induced PH appeared increased levels of plasma LPS and pro-inflammatory TNF-α, IL-1β and IL-6 (Fig. 2 E-F), as well as inflammatory infiltration and increased concentrations of TNF-α, IL-1β and IL-6 in ileum and colon (Fig. 2 G-H; Figure S5) with reduced TER and increased FD4 permeability (Fig. 2 I-J). The GVB dysfunction was also observed in PH mice induced by PPVL, including increased endothelial PV1 expression in the ileum and colon (Fig. 2 L, 2 P; Figure S6), increased FD70 permeability from the gut to blood (Fig. 2 K, 2 N-O), and increased FD70 and evans blue leakage from blood to the gut (Fig. 2 M, 2 Q-R). It again pointed out that PH destroyed the GVB may initiate the gut inflammation and leakage, and cause systemic inflammation and injury. Upregulation of Piezo1 in the Intestinal Microvascular ECs Under Portal Hypertension of CCl 4 and PPVL Mice Expression of mechanosensitive Piezo1 was up-regulated in the intestinal microvascular ECs at PH state. As it shown with immunofluorescence, Piezo1 intensity in CD31 + ECs of the ileum and colon was enhanced in both the CCl 4 and PPVL induced PH mice (Fig. 3 A). Additionally, we further identified the CD45 − CD90 − CD31 + vascular ECs in the ileum and colon via flow cytometry (Figure S7; Figure S8). The results revealed an increased average expression of Piezo1 in the intestinal vascular ECs from the CCl 4 and PPVL mice compared with the sham controls (Fig. 3 B-I). It indicated that Piezo1 may mediate the pressure sensation of intestinal vascular ECs in PH and liver cirrhosis. Endothelial Piezo1 Knockout Improved GVB Dysfunction and Leaky Gut Induced by Portal Hypertension Silence of endothelial Piezo1 effectively improved the GVB dysfunction, and relieved the intestinal and systemic inflammation in CCl 4 and PPVL induced PH mice. The PH-induced high expression of endothelial PV1 in the ileum and colon declined in the Piezo1 ΔEC mice compared to the Piezo1 WT mice (Fig. 4 L, 4 O; Figure S11). In Piezo1 ΔEC mice, PH-induced GVB hyper-permeability was obviously suppressed, which presented as decreased FD70 permeability from the gut to blood (Fig. 4 J, 4 M-N), and reduced FD70 and evans blue leakage from blood to the gut (Fig. 4 K, 4 P-Q). Piezo1 knockout relived the GVB function which was associated with declined circulating levels of LPS and pro-inflammatory cytokines TNF-α, IL-1β and IL-6 in CCl 4 and PPVL mice (Fig. 4 D-E), and as well reduced hepatic inflammatory infiltration and injury (Fig. 4 A-B; Figure S9). Moreover, the restored GVB function also improved the intestinal inflammation, reduced mucosal TNF-α, IL-1β and IL-6 secretion (Fig. 4 H-I; Figure S10), and recovered epithelial barrier and lessened gut leakage in the CCl 4 and PPVL mice (Fig. 4 F-G). Piezo1 Activation by Yoda1 and Hydrostatic Pressure Triggered the Increased Permeability of Intestinal Microvascular ECs Yoda1 activated Piezo1 channel and triggered Ca 2+ influx in intestinal microvascular ECs (Fig. 5 A-C). Piezo1 activation significantly increased the permeability of endothelial barrier, with decreased transendothelial cell resistance (TER) and increased passage of macromolecular FITC-BSA (Fig. 5 D-E), and also with increased PV1 levels and down-regulated expression of junctional proteins such as VE-cadherin, occludin, claudin-1, ZO-1 and JAM-A (Fig. 5 F-L). Additionally, the hydrostatic pressure (2cm, 15cm, and 30cmH 2 O) induced Piezo1 activation was further tested (Fig. 6 A). It indicated that elevated pressure stimulated Piezo1 expression and Ca 2+ influx in intestinal ECs, which could be obviously inhibited by Piezo1 silence with specific siRNA (Fig. 6 B-D). Also, there was marked reduce in TER and increase in FITC-BSA permeability in ECs as the pressure stimulus elevated (Fig. 6 E-F). High hydrostatic pressure promoted the expression of PV1, and disrupted the adhesion and tight junctions among ECs, with weak expression of VE-cadherin, occludin, claudin-1, ZO-1 and JAM-A (Fig. 6 G-L; Figure S14). The pressure-mediated hyper-permeability and junctional damage of ECs could be blocked by Piezo1-siRNA. Piezo1 Promoted Endothelial Hyperpermeability and GVB Dysfunction via Activating the VEGFR2 Signaling The differential genes were identified and exhibited via volcano plot based on the RNA sequencing data (Fig. 7 A), and the GSEA analysis illustrated the enrichment of the VEGF signaling pathway and the VEGFR regulation pathway in ECs treated with Yoda1 (Fig. 7 B-C; Figure S15). The RT-PCR assay further confirmed that Yoda1 and hydrostatic pressure significantly promoted the mRNA expression in ECs, which could be blocked by Piezo1-siRNA (Fig. 7 D, 7 N). In addition, Piezo1 activation by Yoda1 (1µM and 5µM) and hydrostatic pressure (15cm and 30cmH 2 O) facilitated the endothelial expression of VEGFR2 (Fig. 7 E-H), particularly promoted the phosphorylated level of VEGFR2 (Fig. 7 I-M). And hydrostatic pressure (30cmH 2 O) induced high expression of VEGFR2 and p-VEGFR2 was obviously inhibited by Piezo1-siRNA. In vivo study also indicated a remarkable increase of VEGFR2 expression in ileal and colonic vascular ECs in CCl 4 and PPVL mice, while it was repressed in the Piezo1 ΔEC mice (Fig. 7 O; Figure S16). The mRNA and protein expression of VEGFR2, and the phosphorylation level, in the gut of CCl 4 and PPVL mice were evidently inhibited in the Piezo1 knockout state (Fig. 7 P-T; Figure S16). All above suggested that endothelial Piezo1 may promote the GVB dysfunction via activating the VEGFR2 signaling. Silence of VEGFR2 Alleviated Piezo1-mediated Dysfunction and Hyper-permeability of Intestinal Microvascular ECs EC-model with silence of VEGFR2 was successfully established via specific siRNA (Fig. 8 A-E; Figure S17). It demonstrated that VEGFR2 knockdown ameliorated the dysfunction of endothelial barrier induced by Yoda1 (5µM) and hydrostatic pressure (30cmH 2 O), with increased TER and decreased FITC-BSA permeability (Fig. 8 G-H). Moreover, VEGFR2-siRNA inhibited the PV1 expression in ECs upon Piezo1 activation by Yoda1 and hydrostatic pressure, and it in turn restored the cell junctions by promoting the expression of VE-cadherin, occludin, claudin-1, ZO-1 and JAM-A (Fig. 8 F, 8 I-N). These further clarified the role of VEGFR2 activation in Piezo1-induced hyper-permeability of intestinal microvascular ECs and GVB dysfunction. DISCUSSION Gut dysfunction played an important role in the progression of chronic liver disease and cirrhosis, due to the closely anatomical and functional relations of the gut-liver axis( 8 , 43 ). The degeneration of multiple intestinal barriers, including the biochemical, immunological and physical barrier, has been identified in liver injury( 44 , 45 ). It was highly concerned but several questions are still open, especially for the newly defined GVB which is last key guard from the intestine to liver. Here, we focused on the GVB disruption, to elucidate its role and mechanism in the development of liver cirrhosis. GVB breakdown was remarkable in CCl 4 -induced cirrhosis mice, presented as upregulated endothelial PV1 expression, and increased permeability to macromolecular particles such as LPS, FD70 and Evans blue. The hyper-permeability of GVB was responsible for the dissemination of luminal bacteria in experimental cirrhosis( 10 , 11 ). Normally, the invaded pathogens and antigens escaped from the epithelial barrier are denied entry to the internal environment by GVB, and are finally clear by the mucosal immune system( 15 , 46 ). This highlighted the importance of GVB integrity in preventing the aggravation of liver cirrhosis and systemic inflammatory damages. As we shown, the GVB manifested as bi-directional hyper-permeability in CCl 4 mice, namely, increased passage from the gut to blood (inward) and also increased leakage from blood to the gut (outward). On the one aspect, the increased in-flux permeability of GVB directly resulted in bacterial translocation to the portal vein, which may further give rise to liver injury with increased ALT and AST levels, increased hepatic inflammation and fibrosis, and also systemic inflammation with increased levels of serum IL-1β, IL-6 and TNF-α. On the other hand, the raised out-flux permeability of GVB with microvascular leakage may initiate local inflammation in the intestine and trigger or aggravate epithelial leakage. It was true that GVB damage was associated with increased inflammatory infiltration, and then decreased TER and elevated epithelial permeability to FD4 in ileum and colon. This may also be one potential explanation for the cause of intestinal damage and leaky gut in liver cirrhosis. The epithelial leakage, in turn, leads to more entries of luminal antigens forming a vicious cycle of systemic damage. Portal hypertension was associated with GVB disruption in cirrhosis. Compared with the CCl 4 mice, the GVB damage was more evident in CCl 4 +PPVL mice, with more obvious intestinal and systemic inflammation and injury. Also, the extent of GVB damage was positive correlated with portal pressure in PPVL-induced prehepatic PH mice. As increasing portal pressure, the intestinal and systemic inflammation deteriorated in PH status, with raised plasma endotoxin and levels of local and serum cytokines such as IL-1β, IL-6 and TNF-α. These findings indicated the role of pressure-mediated GVB disruption in liver cirrhosis with PH. Similarly, it was reported that elevated microvessel pressure induced lung endothelial hyper-permeability and edema formation via disruption of adherens junction( 22 , 23 ). Intraluminal pressure remodeled the endothelial tissue, and regulated their barrier function via thinned junctions( 47 ). Our in vitro studies further revealed that elevated hydrostatic pressure destroyed the barrier function of intestinal microvascular ECs monolayer, with increased permeability to FITC-BSA and declined transendothelial resistance. Hydrostatic pressure facilitated endothelial barrier damage by increasing the expression of PV1, and breaking the endothelial adherens and tight junctions. Combined, PH-induced breakdown of endothelial integrity and increase of GVB permeability could be a potential explanation for the progress of liver cirrhosis and related systemic injury. The mechanism of endothelial sense to PH and subsequent GVB collapse was largely unknown. Notably, the mechanosensitive Piezo1 channel is distributed in ECs that directly sensing the mechanical stimuli, such as hydrostatic pressure, shear stress and substrate stiffness( 48 , 49 ). Piezo1 is crucial for the homeostasis and angiogenesis of various vascular endothelium( 22 , 24 , 27 ), including the intestinal microvessel ECs( 28 ). Endothelial Piezo1 stimulated angiogenesis to offer protection against intestinal ischemia-reperfusion injury( 28 ). Piezo1-mediated mechanotransduction contributes to endothelial inflammation activated by disturbed blood flow( 30 , 50 ). Deletion of endothelial Piezo1 effectively prevented pressure-induced vascular hyper-permeability and lung edema( 22 ). The expression of Piezo1 on intestinal vessel ECs was upregulated in CCl 4 - and PPVL-induced PH mice. And, specific knockout of Piezo1 in ECs improved the GVB function in PH mice, meanwhile inhibited the systemic and intestinal inflammation, and relieved the epithelial barrier injury. This further indicated the potential role of Piezo1 in PH-induced GVB impairment and related mucosal and circulating inflammation. It was further verified that Piezo1 activation by Yoda1 or hydrostatic pressure destroyed the barrier function of intestinal microvascular ECs monolayer, which could be alleviated by Piezo1 silence with specific siRNA. Also, activation of Piezo1 by ultrasonic stimulation promoted the hyper-permeability in human umbilical vein ECs (HUVECs), while the suppression of Piezo1 showed a decline in cell permeability( 51 ). Collectively, Piezo1 acted as a key sensor to PH and mediated the GVB impairment in liver cirrhosis, which could be a promising pharmacologic target to modulated GVB integrity. Piezo1 activation in ECs by Yoda1 upregulated the VEGF and downstream signaling pathway and increased the expression of VEGFR2, deriving from the transcriptome results. Endothelial Piezo1 triggered VEGF/myc signaling by enhancing Ca 2+ influx could be responsible for driving the angiogenesis( 52 ). Piezo1 promoted angiogenesis in ileal ECs through the activation of the Ca 2+ /HIF-1α/VEGF pathway during intestinal ischemia-reperfusion injury( 28 ). These data indicated the compact link of Piezo1 and VEGF signaling in endothelial function. We further confirmed that activation of Piezo1 by Yoda1 or hydrostatic pressure stimulated Ca 2+ influx, and promoted the expression and phosphorylation of VEGFR2 in ECs, which could be repressed by Piezo1-siRNA. Moreover, the high expression and activation of VEGFR2 in the ileum and colon of CCl 4 and PPVL mice were also inhibited in Piezo1 ΔEC mice. It also prompted that Piezo1/VEGFR2 collaboration may be involved in regulating the barrier function of ECs. The VEGFR2 kinase signaling is an important regulator of vascular permeability, however, the effect varies in different situations. For example, disrupt blood-retinal barrier function of diabetic retinopathy( 53 ), promote vascular leakage and edema in myocardial infarction( 54 ), strengthen retinal endothelial barrier via endothelial-pericyte interaction( 55 ), and restore pulmonary microvascular barrier following ischemia reperfusion injury( 56 ). Excessive VEGFR2 trafficking to the cell surface mediated vascular permeability, while inhibited this VEGFR2 recycling mitigated vascular leakage associated with inflammatory diseases( 57 ). In intestinal microvascular ECs, as we shown, inhibition of VEGFR2 with siRNA knockdown protected Yoda1 and hydrostatic pressure induced barrier disruption, through inhibiting the expression of PV1, enhancing the adherens junctions (VE-cadherin) and tight junctions (occludin, claudin-1, ZO-1 and JAM-A). Piezo1 is required in force-induced Ca 2+ -dependent remodelling of cell-cell junctions and associated cytoskeleton, especially for adherens junctions( 58 ). Piezo1 sensing of high vascular pressures at the endothelial surface may promote disassembly of adherens junctions( 22 , 59 ). These further clarified the role of VEGFR2 activation in Piezo1-induced hyper-permeability of intestinal microvascular ECs and GVB dysfunction. This study provided a preliminary image that endothelial Piezo1 mediated GVB dysfunction in PH and promoted deterioration of liver cirrhosis. Nevertheless, several limitations should be acknowledged, which also offered the possible directions for future research. Firstly, our study focused on the role of Piezo1 in ECs, but did not preclude the involvement of other mechanosensitive channels or receptors( 60 ) that may be responsible for pressure-induced endothelial hyperpermeability. Even so, the molecular pathway of Piezo1 in GVB impairment was currently limited, which should be multifaceted and requires further research to elucidate. Secondly, the actions of Piezo1 in modulating GVB function should be validated in PH patients with liver cirrhosis and related clinical manifestations of intestinal and systematic damage, for instance, through the study of gastrointestinal biopsy under endoscopy or surgery. And finally, the specific monoclonal antibody or fusion protein against Piezo1 is urgently needed, especially for the intestinal ECs-specific blocker with good in vivo safety and efficacy. In summary, we expounded that Piezo1 contributed to PH-induced damage of intestinal microvascular ECs and GVB dysfunction in cirrhosis via recruiting and activating the VEGFR2 signaling. Especially, the outward (from blood to the gut) and inward (from the gut to blood) hyper-permeability of GVB in PH status may initiate the intestinal inflammation, trigger or aggravate epithelial leakage, and in turn lead to more convenient bacterial translocation into the portal vein, facilitate liver and systemic damage, forming a vicious cycle. So that, Piezo1 mediated bi-directional hyper-permeability of the GVB should be a novel explanation and potential therapeutic target for preventing the exacerbation of liver cirrhosis with PH. Abbreviations CCl 4 , carbon tetrachloride; PPVL, partial portal vein ligation; PH, portal hypertension; ALT, alanine aminotransferase; AST, aspartate aminotransferase; GVB, gut-vascular barrier; ECs, endothelial cells; HIMECs, human intestinal microvascular endothelial cells; LPS, lipopolysaccharide; FD4, fluorescein isothiocyanate-labeled dextran 4kDa; FD70, fluorescein isothiocyanate-labeled dextran 70kDa; BSA, bovine serum albumin; TER, trans-epithelial/endothelial cell resistance; PV1, plasmalemma vesicle-associated protein 1; VEGFR2, vascular endothelial growth factor receptor 2. DEGs, differential expression genes. Declarations Acknowledgements We thank Dr. Liuying Chen (Division of Gastroenterology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology) for providing HIMECs and technical assistances. Author Contributions Xiao Z: methodology, investigation, validation, data curation, writing - original draft; He J: software, formal analysis; Tian W: investigation; Du L: investigation; Wang R: validation; Bai T: software, formal analysis; Qian W: methodology, resources; Song J: conceptualization, supervision, writing - review & editing; Hou X: project administration,funding acquisition, writing - review & editing; Zhang L: funding acquisition, conceptualization, methodology, investigation, data curation, writing - original draft. Ethical S tatement This study was approved by the Animal Care and Use Committee, Tongji Medical College, Huazhong University of Science and Technology, China (Approval NO:3372). Conflicts of Interest Statement The authors declare that they had full control over the data and complete access to the data, and no competing interest declared. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9418908","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":635879802,"identity":"2aa6486a-2f63-40a8-916a-6f02b036c915","order_by":0,"name":"Lei Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABD0lEQVRIie3QMUsDMRTA8RcO7pYnXXNcoV8hcHBSqpev0hDwllYEQTqmHOSWc6/YzyGOLQFd+gEKTl06OShIuU56ddIhoaNg/sODvOS3BMDn+4vRw7gC7ASkat7Vj6WbMMC4KlV81xI8lgBbPavk5BjSuy+3mw921oW1UOn5o+EcguULQn5pI2T+dJp22QWSmVByvDKihlAOEOS1jQR0mCWUGQyoUGaszRABswRhIZSFhLTYteQTQyqmZV8bjtDZOQnSURa/sQUiLsuAaENqwNBJKB3dJMAk0miqya0uRG3CtD9n0kp6s+IhbiY55ybawl4PeFSVm/XrJLeS7y/A38fDYI73baRx3/t8Pt9/7wuNQk28L4A5iAAAAABJRU5ErkJggg==","orcid":"","institution":"Wuhan Union Hospital","correspondingAuthor":true,"prefix":"","firstName":"Lei","middleName":"","lastName":"Zhang","suffix":""},{"id":635879803,"identity":"e9bb5991-3492-407a-87cc-1f887a97561b","order_by":1,"name":"Zhuanglong Xiao","email":"","orcid":"","institution":"Wuhan Union Hospital","correspondingAuthor":false,"prefix":"","firstName":"Zhuanglong","middleName":"","lastName":"Xiao","suffix":""},{"id":635879804,"identity":"74bf4036-f449-41ea-ad7d-c9c5e3e4516c","order_by":2,"name":"Jun Song","email":"","orcid":"","institution":"Union Hospital, Tongji Medical College Medical College, Huazhong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Song","suffix":""},{"id":635879805,"identity":"ad8d0e83-199b-4412-a064-d1a0a87a9112","order_by":3,"name":"Jing He","email":"","orcid":"","institution":"Wuhan Union Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"He","suffix":""},{"id":635879806,"identity":"19120148-15dc-4281-ad71-1f00a4734b52","order_by":4,"name":"Wenjing Tian","email":"","orcid":"","institution":"Wuhan Union Hospital","correspondingAuthor":false,"prefix":"","firstName":"Wenjing","middleName":"","lastName":"Tian","suffix":""},{"id":635879807,"identity":"42e4b964-736f-473e-9ebc-0fcdb448090b","order_by":5,"name":"li Du","email":"","orcid":"","institution":"Wuhan Union Hospital","correspondingAuthor":false,"prefix":"","firstName":"li","middleName":"","lastName":"Du","suffix":""},{"id":635879808,"identity":"1bb35d08-7333-4f37-a183-56a82ff6bded","order_by":6,"name":"Ruiyun Wang","email":"","orcid":"","institution":"Wuhan Union Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ruiyun","middleName":"","lastName":"Wang","suffix":""},{"id":635879809,"identity":"33ee0c8b-3717-401b-9578-a2c9d403b30b","order_by":7,"name":"Tao Bai","email":"","orcid":"","institution":"Union Hospital, Tongji Medical College, Huazhong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Tao","middleName":"","lastName":"Bai","suffix":""},{"id":635879810,"identity":"6a6c2423-9cab-409a-a393-acff57efe722","order_by":8,"name":"Wei Qian","email":"","orcid":"","institution":"Wuhan union hospital","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Qian","suffix":""},{"id":635879811,"identity":"3a288ace-94f2-440c-bbea-206d0f1c724a","order_by":9,"name":"Xiaohua Hou","email":"","orcid":"","institution":"Wuhan Union Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiaohua","middleName":"","lastName":"Hou","suffix":""}],"badges":[],"createdAt":"2026-04-14 18:50:58","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9418908/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9418908/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109337485,"identity":"d76a6c4a-ef03-4920-91b0-6b6305b5f450","added_by":"auto","created_at":"2026-05-15 17:39:23","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5901338,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePH mediated GVB disruption promoted intestinal and systemic inflammation in CCl\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e-induced liver cirrhosis. (A)\u003c/strong\u003e Modeling and grouping scheme, including the control group (olive oil with sham operation), CCl\u003csub\u003e4\u003c/sub\u003e group (CCl\u003csub\u003e4\u003c/sub\u003e with sham operation) and CCl\u003csub\u003e4\u003c/sub\u003e+PPVL group (CCl\u003csub\u003e4\u003c/sub\u003e with partial portal vein ligation). \u003cstrong\u003e(B)\u003c/strong\u003e Typical images of HE staining, Sirius red staining and immunohistochemical staining (α-SMA) of liver tissues.\u003cstrong\u003e (C)\u003c/strong\u003e Portal venous pressure measured via the water column method. n=6 to 12 for each group. \u003cstrong\u003e(D)\u003c/strong\u003e Activity of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST). n=6 to 12 for each group. \u003cstrong\u003e(E-F)\u003c/strong\u003e The fibrosis ratio was calculated as the Sirius red\u003csup\u003e+\u003c/sup\u003e and α-SMA\u003csup\u003e+ \u003c/sup\u003earea relative to total area.\u003cstrong\u003e \u003c/strong\u003en=5 for each group. \u003cstrong\u003e(G-H)\u003c/strong\u003e Levels of plasma endotoxin and serum cytokines of IL-1β, IL-6 and TNF-α. n=6 to 8 for each group. \u003cstrong\u003e(I-J)\u003c/strong\u003e HE staining images and levels of inflammatory cytokines of IL-1β, IL-6 and TNF-α in ileal tissues. n=6 to 8 for each group. \u003cstrong\u003e(K-L) \u003c/strong\u003eTransepithelial resistance (TER) and FD4 permeability of intestinal mucosa detected by Ussing chamber. n=6 to 8 for each group. \u003cstrong\u003e(M, P-Q)\u003c/strong\u003e The FD70 density detected in the liver and spleen while was injected into the intestinal loop. n=5 for each group.\u003cstrong\u003e (N, R)\u003c/strong\u003e The PV1 expression on vascular endothelium in the ileum, and calculated as the PV1\u003csup\u003e+\u003c/sup\u003e/CD31\u003csup\u003e+ \u003c/sup\u003earea. n=5 for each group. \u003cstrong\u003e(O, S)\u003c/strong\u003e The vascular leakage of FD70 into the ileal and colonic tissues while was injected from the tail vein.\u003cstrong\u003e \u003c/strong\u003e\u0026nbsp;\u003cstrong\u003e(T)\u003c/strong\u003e The vascular leakage of Evans blue into the ileum and colon while was intravenous injected. n=6 to 10 for each group. \u003csup\u003e*\u003c/sup\u003ep\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003ep\u0026lt;0.01, \u003csup\u003e***\u003c/sup\u003ep\u0026lt;0.001, \u003csup\u003e****\u003c/sup\u003ep\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9418908/v1/fa777e9b40b6fdf18e00c8ca.jpg"},{"id":109337371,"identity":"821fbc50-27bb-4282-b880-71827cba0e20","added_by":"auto","created_at":"2026-05-15 17:39:18","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4878191,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGVB collapse contributed to gut inflammation and leakage, and systemic injury in PPVL-induced prehepatic PH. (A)\u003c/strong\u003e Modeling and grouping scheme, including the sham group (with sham operation), PPVL-26G group (ligation with a 26G needle) and PPVL-27G group (ligation with a 27G needle).\u003cstrong\u003e (B)\u003c/strong\u003e Typical images of HE staining of liver tissues.\u003cstrong\u003e (C)\u003c/strong\u003e Portal venous pressure measured via the water column method. n=8 to 10 for each group. \u003cstrong\u003e(D)\u003c/strong\u003e Activity of serum ALT and AST. n=8 to 10 for each group. \u003cstrong\u003e(E-F)\u003c/strong\u003e Levels of plasma LPS and serum cytokines of IL-1β, IL-6 and TNF-α. n=6 to 8 for each group.\u003cstrong\u003e (G-H) \u003c/strong\u003eHE staining images and levels of inflammatory cytokines of IL-1β, IL-6 and TNF-α in ileal tissues. n=6 to 8 for each group.\u003cstrong\u003e (I-J) \u003c/strong\u003eTransepithelial resistance (TER) and FD4 permeability of intestinal mucosa detected by Ussing chamber. n=5 to 8 for each group. \u003cstrong\u003e(K, N-O)\u003c/strong\u003e The FD70 density detected in the liver and spleen while was injected into the intestinal loop. n=5 for each group. \u003cstrong\u003e(L, P) \u003c/strong\u003eThe PV1 expression on vascular endothelium in the ileum, and calculated as the PV1\u003csup\u003e+\u003c/sup\u003e/CD31\u003csup\u003e+ \u003c/sup\u003earea. n=5 for each group.\u003cstrong\u003e (M, Q) \u003c/strong\u003eThe vascular leakage of FD70 into the ileal and colonic tissues while was injected from the tail vein. \u003cstrong\u003e(R) \u003c/strong\u003eThe vascular leakage of Evans blue into the ileum and colon while was intravenous injected. n=6 to 10 for each group. \u003csup\u003e*\u003c/sup\u003ep\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003ep\u0026lt;0.01, \u003csup\u003e***\u003c/sup\u003ep\u0026lt;0.001, \u003csup\u003e****\u003c/sup\u003ep\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9418908/v1/cf1ca896beba2b18f807653f.jpg"},{"id":109337488,"identity":"6013bdff-a423-47ad-b092-ddb27ef664f5","added_by":"auto","created_at":"2026-05-15 17:39:23","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2162129,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUpregulated Piezo1 in the intestinal microvascular ECs under portal hypertension of CCl\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4 \u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eand PPVL Mice. (A) \u003c/strong\u003eThe expression intensity of Piezo1(green) in microvascular ECs (CD31, red) of the ileum (left panel) and colon (right panel) presented via immunofluorescence. DAPI (blue) displayed the cell nucleus.\u003cstrong\u003e (B-I) \u003c/strong\u003eThe average expression of Piezo1 (fluorescence intensity of Piezo1-FITC) in CD45\u003csup\u003e-\u003c/sup\u003eCD90\u003csup\u003e-\u003c/sup\u003eCD31\u003csup\u003e+\u003c/sup\u003e vascular ECs in the ileum and colon via flow cytometry, namely, B-C for the ileum of the CCl\u003csub\u003e4\u003c/sub\u003e and control mice, D-E for the ileum of the PPVL-26G, PPVL-27G and sham mice; while F-G for the colon of the CCl\u003csub\u003e4\u003c/sub\u003e and control mice, H-I for the colon of the PPVL-26G, PPVL-27G and sham mice. n=5 to 10 for each group. \u003csup\u003e*\u003c/sup\u003ep\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003ep\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9418908/v1/009ca2fe5b24e0cd0c05f2c7.jpg"},{"id":109337471,"identity":"44c38f1c-fc0c-4e1f-a46b-e6407aaffe67","added_by":"auto","created_at":"2026-05-15 17:39:20","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2582366,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePiezo1 activation by Yoda1 triggered the increased permeability of intestinal microvascular ECs.\u003c/strong\u003e \u003cstrong\u003e(A-C) \u003c/strong\u003eYoda1 activated Piezo1 channel and triggered Ca\u003csup\u003e2+\u003c/sup\u003e influx in intestinal microvascular ECs, with increased Fluo-4AM intensity but no significant changes in Piezo1 expression. \u003cstrong\u003e(D-E) \u003c/strong\u003eIncreased permeability of endothelial barrier, with decreased transendothelial cell resistance (TER) and increased passage of macromolecular FITC-BSA, upon Piezo1 activation by Yoda1. \u003cstrong\u003e(F) \u003c/strong\u003eYoda1 treatment increased the mRNA expression of PV1 and down-regulated the expression of junctional proteins in ECs.\u003cstrong\u003e (G-L) \u003c/strong\u003eThe typical images and expression intensity of PV1, VE-cadherin, occludin, claudin-1, ZO-1 and JAM-A in ECs treated by Yoda1. n=5 to 6 for each group. \u003csup\u003e*\u003c/sup\u003ep\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003ep\u0026lt;0.01, \u003csup\u003e***\u003c/sup\u003ep\u0026lt;0.001, \u003csup\u003e****\u003c/sup\u003ep\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9418908/v1/51323292b60398a837df5789.jpg"},{"id":109337349,"identity":"7f1f3ed3-e3c6-4d55-b857-b3cdba136883","added_by":"auto","created_at":"2026-05-15 17:39:15","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":4551544,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHydrostatic pressure activated endothelial Piezo1 and promoted junctional damage and hyper-permeability of ECs. (A) \u003c/strong\u003eThe schematic diagram of experimental apparatus for hydrostatic pressure. The culture chamber (a closed culture bottle) was filled with medium, where the cell-planted transwell membrane or coverslip was placed. The height of the medium reservoir was adjusted to maintain the pressure in the culture (2cm, 15cm, 30cm). The medium in the culture chamber and the medium reservoir was circulated by a peristaltic pump to ensure gas exchange and maintain stable oxygen tension.\u003cstrong\u003e (B-D)\u003c/strong\u003e The Piezo1 expression and intracellular Ca\u003csup\u003e2+ \u003c/sup\u003econcentration in ECs treated with hydrostatic pressure (2cm, 15cm, and 30cmH\u003csub\u003e2\u003c/sub\u003eO), as well as in the condition of Piezo1 silence with specific siRNA. \u003cstrong\u003e(E-F)\u003c/strong\u003e Reduced TER and increased FITC-BSA permeability in ECs monolayer was shown as the hydrostatic pressure elevated, which could be blocked by Piezo1-siRNA. \u003cstrong\u003e(G-L)\u003c/strong\u003e The typical images and expression intensity of PV1, and junctional proteins including VE-cadherin, occludin, claudin-1, ZO-1 and JAM-A in ECs treated with hydrostatic pressure and Piezo1-siRNA. n=5 for each group; \u003csup\u003e*\u003c/sup\u003ep\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003ep\u0026lt;0.01, \u003csup\u003e***\u003c/sup\u003ep\u0026lt;0.001, \u003csup\u003e****\u003c/sup\u003ep\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9418908/v1/f1bab68256ffdfd891674087.jpg"},{"id":109337480,"identity":"ccb934ed-dca0-4ce0-9403-1b6d32c53a99","added_by":"auto","created_at":"2026-05-15 17:39:22","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2781901,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePiezo1 promoted endothelial hyperpermeability and GVB dysfunction via activating the VEGFR2 signaling. (A) \u003c/strong\u003eThe differential genes were identified and exhibited via volcano plot based on the RNA sequencing data, and \u003cstrong\u003e(B-C)\u003c/strong\u003e the GSEA analysis illustrated the enrichment of the VEGF signaling pathway and the VEGFR regulation pathway in ECs treated with 1μM Yoda1. n=4 for each group. \u003cstrong\u003e(D-F) \u003c/strong\u003eThe expression of VEGFR2 in ECs treated with Yoda1 (1μM and 5μM) and DMSO quantified by RT-PCR and IF.\u003cstrong\u003e \u003c/strong\u003en=3 to 6 for each group. \u003cstrong\u003e(G-H, N) \u003c/strong\u003eThe expression of VEGFR2 in ECs treated with hydrostatic pressure (15cm and 30cmH\u003csub\u003e2\u003c/sub\u003eO) and preconditioned by Piezo1-siRNA detected by IF and RT-PCR. n=5 for each group. \u003cstrong\u003e\u0026nbsp;(I-M) \u003c/strong\u003eThe protein expression and phosphorylated level of VEGFR2 in ECs treated with Yoda1 or hydrostatic pressure detected by Western blot. n=3 to 4 for each group.\u003cstrong\u003e (O) \u003c/strong\u003eTypical images of VEGFR2 (green) expression in ileal vascular ECs (CD31, red) in CCl\u003csub\u003e4 \u003c/sub\u003eand PPVL mice with a Piezo1\u003csup\u003efl/fl\u003c/sup\u003e (WT) or Piezo1\u003csup\u003eΔEC\u003c/sup\u003e (KO) background. \u003cstrong\u003e(P) \u003c/strong\u003eThe mRNA expression, and\u003cstrong\u003e (Q-T) \u003c/strong\u003ethe protein expression and phosphorylated level of VEGFR2 in ileal tissues of CCl\u003csub\u003e4 \u003c/sub\u003eand PPVL mice and those with endothelial Piezo1 KO. n=3 for each group. \u003csup\u003e*\u003c/sup\u003ep\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003ep\u0026lt;0.01, \u003csup\u003e***\u003c/sup\u003ep\u0026lt;0.001, \u003csup\u003e****\u003c/sup\u003ep\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9418908/v1/2a690376ed761c37b818ce88.jpg"},{"id":109337503,"identity":"0d7088db-5b37-405a-ae92-85712ba70e61","added_by":"auto","created_at":"2026-05-15 17:39:29","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":4362631,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSilence of VEGFR2 alleviated Piezo1-mediated dysfunction and hyper-permeability of intestinal microvascular ECs. (A) \u003c/strong\u003eECs-model with silence of VEGFR2 was successfully established via specific siRNA, and \u003cstrong\u003e(B-E) \u003c/strong\u003ethe mRNA and protein expression of VEGFR2 were markedly inhibited by siRNA in ECs treated with hydrostatic pressure and Yoda1, detected by IF and RT-PCR.\u003cstrong\u003e (F)\u003c/strong\u003e VEGFR2 knockdown by siRNA improved the mRNA expression of PV1 and junctional proteins induced by Piezo1 activation in ECs, and\u003cstrong\u003e (G-H)\u003c/strong\u003e ameliorated the dysfunction of endothelial barrier induced by Yoda1 (5μM) or hydrostatic pressure (30cmH\u003csub\u003e2\u003c/sub\u003eO), with increased TER and decreased FITC-BSA permeability.\u003cstrong\u003e (I-N)\u003c/strong\u003e The typical images and expression intensity of PV1, VE-cadherin, occludin, claudin-1, ZO-1 and JAM-A in ECs treated with hydrostatic pressure and Yoda1, as well as VEGFR2-siRNA. n=5 to 6 for each group. \u003csup\u003e*\u003c/sup\u003ep\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003ep\u0026lt;0.01, \u003csup\u003e***\u003c/sup\u003ep\u0026lt;0.001, \u003csup\u003e****\u003c/sup\u003ep\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9418908/v1/c1890cfe23c8f0cb45a2802e.jpg"},{"id":109405816,"identity":"86ad9ce5-ec3b-49f0-b01e-e9ea67ebda2e","added_by":"auto","created_at":"2026-05-17 13:20:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":27564656,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9418908/v1/731409a7-7d93-496b-85b8-f78dee38ce64.pdf"},{"id":109337463,"identity":"8a422c2c-4739-4c2b-ab84-998d66b47431","added_by":"auto","created_at":"2026-05-15 17:39:19","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":13616,"visible":true,"origin":"","legend":"Table S1","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-9418908/v1/bdd7dbd72bf9fd83c970557c.docx"},{"id":109337354,"identity":"0c622017-c464-41bb-b575-a35d9b43ba5c","added_by":"auto","created_at":"2026-05-15 17:39:16","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":18747,"visible":true,"origin":"","legend":"Table S2","description":"","filename":"TableS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-9418908/v1/88defd3027ccf89710996bbf.docx"},{"id":109337292,"identity":"c400566e-a086-42ab-a6dd-aa15b29545f7","added_by":"auto","created_at":"2026-05-15 17:39:09","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":25842,"visible":true,"origin":"","legend":"Supplementary Methods","description":"","filename":"SupplementaryMethods.docx","url":"https://assets-eu.researchsquare.com/files/rs-9418908/v1/8e505fc59b4bb05655db9c3a.docx"},{"id":109337489,"identity":"6230312a-542b-45cf-8148-5babc972bb40","added_by":"auto","created_at":"2026-05-15 17:39:24","extension":"gif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":470970,"visible":true,"origin":"","legend":"Graphical abstract","description":"","filename":"Graphicalabstract.gif","url":"https://assets-eu.researchsquare.com/files/rs-9418908/v1/34cae419142bed7593c4c563.gif"},{"id":109337466,"identity":"8a6f750e-be61-4d46-b04a-468774078102","added_by":"auto","created_at":"2026-05-15 17:39:19","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":19109,"visible":true,"origin":"","legend":"Supplementary Figures","description":"","filename":"SupplementaryFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-9418908/v1/63a37ff02e2cd46583dc330d.docx"},{"id":109337469,"identity":"3483f406-cb4a-4bda-a05a-6f80f63663ee","added_by":"auto","created_at":"2026-05-15 17:39:19","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":25926,"visible":true,"origin":"","legend":"Supplementary Methods","description":"","filename":"SupplementaryMethods.docx","url":"https://assets-eu.researchsquare.com/files/rs-9418908/v1/9d182c6daacb3b7339c37803.docx"},{"id":109337357,"identity":"95c5789c-5bc8-4e77-a099-3e34f33cfc4d","added_by":"auto","created_at":"2026-05-15 17:39:17","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":4436067,"visible":true,"origin":"","legend":"Supplementary material","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-9418908/v1/2717c748064aab551e25cf9b.docx"}],"financialInterests":"There is no conflict of interest","formattedTitle":"Mechanosensing Piezo1 mediates gut-vascular barrier dysfunction in portal hypertension and promotes deterioration of liver cirrhosis","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eCirrhosis, the end stage of chronic liver disease caused by various reasons(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e), is a major global health concern with high morbidity and mortality rates worldwide(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Portal hypertension (PH) and declined liver function are the main features of progressive cirrhosis(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). In particular, patients with cirrhosis are susceptible to microbiota translocation and entry of enterogenous toxins into the bloodstream, which may trigger systemic inflammation and injury(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). These bacterial infections promote the progression and decompensation of cirrhosis, in some cases, can lead to life-threatening acute-on-chronic liver failure and even multi-organ failure via the gut-liver-organ axes(\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Currently, targeting the gut-liver axis has become an important strategy and viewpoint for preventing further liver damage and related multi-organ injury in cirrhosis.\u003c/p\u003e \u003cp\u003eIntestinal mucosal barrier dysfunction is commonly found in chronic liver disease and cirrhosis(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e), with destroyed integrity of mucus layer and capability of immune defense, and increased epithelial permeability(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Recently, beneath the epithelial barrier, a newly gut-vascular barrier (GVB) was characterized and proved important role in liver injury(\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e), which is the structural and functional unit formed by microvascular endothelial cells (ECs), pericytes and enteric glial cells (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e)(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). GVB acts as the last key guard between the intestine and liver, as well as the blood circulation, it defends against the dissemination of bacteria and luminal antigen that escaped from the epithelial barrier(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Most of the invaded pathogens from the intestinal epithelium are denied entry to the internal environment by GVB, and are finally phagocytose and clear by macrophages and other immunocytes(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Indubitably, GVB disruption may directly result in liver injury, and was confirmed in various intestinal inflammatory conditions(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e) and hepatic diseases, such as metabolic associated fatty liver disease (MAFLD), alcoholic liver disease (ALD) and cirrhosis(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). However, the specific target and mechanism underlying GVB collapse is far from clear.\u003c/p\u003e \u003cp\u003eLiver cirrhosis may disrupt the intestinal barrier integrity through gut dysbiosis and abnormal bile acid metabolism in enterohepatic circulation as reported in previous studies(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Even so, the direct mechanisms of liver cirrhosis on intestinal barrier, especially for the GVB, are still largely unclear. It is well-known that PH can cause direct damage to the gastrointestinal tract, so called portal hypertensive enteropathy(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). The intestinal microvascular ECs may bear the first brunt from PH-induced rise of intravessel pressure and blood stasis, which is speculated to bring about GVB collapse, and intestinal edema and inflammation. To date, little is known about vascular mechanical sensing in inducing endothelial barrier disruption.\u003c/p\u003e \u003cp\u003ePiezo1, a mechanosensitive ion channel expressed in ECs, is activated by elevated pressure and other mechanical stimuli(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Piezo1 was proven to play a crucial role in the endothelial homeostasis and angiogenesis, such as pulmonary artery(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e), coronary artery(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e), brain capillary(\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e) and intestinal microvessel(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Piezo1 could sense disturbed blood flow and activated endothelial inflammatory signaling(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Upregulation of Piezo1 in ECs leads to vascular remodeling and pulmonary hypertension induced by high shear stress(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). In particular, endothelial Piezo1 mediated pressure-induced lung vascular hyper-permeability via disruption of adherens junctions, while inhibition or deletion of Piezo1 in ECs markedly prevented capillary stress failure and lung edema(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Also, our preliminary findings indicated that endothelial Piezo1 might facilitate GVB disruption in response to elevated vascular pressures related to PH.\u003c/p\u003e \u003cp\u003eHere, we addressed the possibility that Piezo1, as a mechanical sensor of high vascular pressure in the PH condition, is responsible for GVB breakdown in cirrhosis. In further, we explored the key role and underlying mechanism of Piezo1-mediated bi-directional GVB hyper-permeability in the intestinal and systemic inflammation that maybe the basis for the progress of liver cirrhosis. The study may provide new insights and potential targets for preventing the deterioration of liver cirrhosis with PH.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eC57BL/6 mice were supplied by Shulaibao Biotech Co., Ltd (Wuhan, China). Piezo1\u003csup\u003eflox/flox\u003c/sup\u003e mice (Cat. NO: NM-CKO-200275) were customized by Shanghai Model Organisms Center, Inc. (Shanghai, China). Cdh5-CreERT2 mice (Strain NO: T014691) were purchased from GemPharmatech Co., Ltd. (Nanjing, China). Piezo1\u003csup\u003eflox/flox\u003c/sup\u003e mice were crossed with Cdh5-CreERT2 mice to generate ECs-specific Piezo1-deficient mice (Piezo1\u003csup\u003e△EC\u003c/sup\u003e) with the deletion of exon 4 and 5 of Piezo1 cDNA (NCBI ID: 234839). The Piezo1\u003csup\u003e△EC\u003c/sup\u003e mice were intraperitoneally given 1 mg of tamoxifen (dissolved in corn oil; Sigma-Aldrich Chemistry) for 5 days to induce the complete deletion of Piezo1 specific on ECs. The Piezo1\u003csup\u003eflox/flox\u003c/sup\u003e mice were used as controls (Piezo1\u003csup\u003eWT\u003c/sup\u003e). The mice were raised in the SPF environment (Animal Experimental Center, Tongji Medical College, HUST, China) with a constant temperature and humidity, and with the light / dark alternate time 12h/12h. All animal experiments were approved by the Institutional Animal Care and Use Committee of Tongji Medical College, HUST, China.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCCl-induced Liver Cirrhosis Model\u003c/h3\u003e\n\u003cp\u003eLiver cirrhosis model was established through the administration of carbon tetrachloride (CCl\u003csub\u003e4\u003c/sub\u003e) in mice(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). CCl\u003csub\u003e4\u003c/sub\u003e (C822982, Macklin, China) was diluted at 5:5 (v/v) in olive oil (O815211, Macklin, China) for modeling. Male mice (8 weeks old) were injected subcutaneously with CCl\u003csub\u003e4\u003c/sub\u003e diluent at a dose of 3 ml/kg twice a week for 12 weeks. As a control, other mice administrated with 3 ml/kg olive oil only twice a week for 12 weeks. To further increase portal pressure, part of the CCl\u003csub\u003e4\u003c/sub\u003e mice were subjected partial portal vein ligation (26G) at the 8th week, while others conducted a sham operation as control.\u003c/p\u003e\n\u003ch3\u003ePartial Portal Vein Ligation (PPVL)-induced PH Model\u003c/h3\u003e\n\u003cp\u003eMice model of pre-hepatic portal hypertension was induced by PPVL as previously described(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Briefly, mice were performed median laparotomy under anaesthesia, and the portal vein was carefully isolated. A blunt-tipped needle (26G or 27G) was placed on the portal vein, and a constricting ligature was conducted both on the needle and the portal vein with 7\u0026thinsp;\u0026minus;\u0026thinsp;0 silk. The needle was removed later on, leaving a calibrated stenosis (diameter about 0.46mm or 0.41mm) of the portal vein, and then the abdominal incision was sutured. A sham operation was performed in the control mice with portal vein isolated but not ligated. After a two-week recovery period following surgery, the mice were used for subsequent experimental testing.\u003c/p\u003e\n\u003ch3\u003ePortal Venous Pressure Measurement\u003c/h3\u003e\n\u003cp\u003eThe portal pressure was measured via the water column method(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). The mice were anesthetized with pentobarbital sodium, performed laparotomy incision and the portal vein was dissociated. An 26G infusion cannula was cannulated into the portal vein, and connected to a glass-tube manometer with water column (containing heparin sodium physiological saline). The portal pressure was recorded as the height of water column when the water column in the glass-tube manometer was stayed stationary.\u003c/p\u003e\n\u003ch3\u003eAssessment of Gut-Vascular Barrier Function\u003c/h3\u003e\n\u003cp\u003eThe function of GVB was assessed by detecting the endothelial PV1 expression, and measuring the macromolecular permeability from the gut to blood and from blood to the gut.\u003c/p\u003e \u003cp\u003e \u003cem\u003eEndothelial PV1 expression\u003c/em\u003e. The PV1 distribution density was assessed via immunofluorescence, and present as the ratio of PV1\u003csup\u003e+\u003c/sup\u003e/CD31\u003csup\u003e+\u003c/sup\u003e area in the ileal and colonic slices.\u003c/p\u003e \u003cp\u003e \u003cem\u003ePermeability from the gut to blood\u003c/em\u003e. To assess the in-flux permeability of GVB, FITC-labeled dextran 70kDa (FD70, 2mg prepared in 2 ml physiological saline; Sigma-Aldrich Chemistry) was injected into a 4 cm-length intestinal loop (ligated at both ends with suture) through laparotomy incision under anesthesia(\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). One hour later, the blood samples were collected from the abdominal aorta, and the plasma FD70 level was detected via a fluorescence microplate reader (BioTek Instruments, USA). Meanwhile, the liver and spleen tissues were obtained under light-proof conditions, and fixed with 4% paraformaldehyde, made into 20 \u0026micro;m frozen sections, then imaged on a confocal fluorescence microscopy (Olympus Corporation, Japan). The density of FD70 particles in the liver and spleen were quantified via ImageJ software (National Institutes of Health, USA).\u003c/p\u003e \u003cp\u003e \u003cem\u003ePermeability from blood to the gut\u003c/em\u003e. To assess the out-flux permeability of GVB, Evans blue or FD70 was intravenous injected and the vascular leakage of these macromolecular was detected(\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) Evans blue (30 mg/kg in 100\u0026micro;l normal saline; Sigma-Aldrich Chemistry) was injected into the tail vein of mice. The mice were killed 20 min later, the ileal and colonic tissues (the same segment for each mouse) were removed, and then cut along the sagittal direction, removed the faeces and blotted dry. Tissues were immersed in 1ml formamide overnight at 55\u0026deg;C to extract the Evans blue dye in the ileum and colon. The content of Evans blue was measured via spectrophotometry at 600nm, and the result expressed as \u0026micro;g/mg tissue. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) 200 \u0026micro;l FD70 (2 mg/ml in normal saline; Sigma-Aldrich Chemistry) was injected into the tail vein of mice. After 2 h post injection, the mice were anesthetized and performed cardiac perfusion with physiological saline. The ileal and colonic tissues (the same segment for each mouse) were removed, and then fixed in 4% paraformaldehyde for 24 h, made into 20 \u0026micro;m frozen sections. The vascular ECs were labeled by immunofluorescent with anti-CD31 primary antibody and Alexa Fluor 594-conjugated secondary antibody. The sections were imaged on a confocal laser scanning microscope (Nikon, Japan), and the FD70 fluorescent extravasation was observed. The FITC positive area represented the vascular leakage of FD70 was quantified using ImageJ software.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of Intestinal Epithelial Permeability\u003c/h2\u003e \u003cp\u003eThe function of intestinal epithelial barrier was assessed via detecting the plasma endotoxin (LPS) on portal vein, and measuring the trans-epithelial cell resistance (TER) and the permeability to macromolecules in an Ussing Chamber method.\u003c/p\u003e \u003cp\u003e \u003cem\u003ePlasma endotoxin assay\u003c/em\u003e. Portal vein plasma were collected in endotoxin-free tubes and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until assessment. LPS concentration was detected using a Mouse Endotoxin ELISA Kit (Bioswamp, China) following the manufacturer's instructions. In brief, 100 \u0026micro;l plasma sample was firstly reacted 100 \u0026micro;l limulus amebocyte lysate at 37\u0026deg;C for 5 min, incubated with 100 \u0026micro;l chromogenic substrate solution at 37\u0026deg;C for 5 min, and then terminated by reconstituted stop solution. The optical density (OD) was measured at 405 nm using a microplate reader (BIOBASE, China). Plasma endotoxin level was quantified against a standard curve.\u003c/p\u003e \u003cp\u003e \u003cem\u003eUssing chamber analysis\u003c/em\u003e. The intestinal mucosal patches were prepared by stripping the seromuscular layer as previously described(\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). The intact mucosal patches were mounted on the sliders with a rectangular hole (opening area 0.15 cm\u003csup\u003e2\u003c/sup\u003e) in the center, and the sliders were fixed on the U-type chambers filled with 37 ℃ oxygenated Krebs' solution at both the serosal and mucosal sides. Then, the U-type chambers were installed on an Ussing Chamber System (World Precision Instruments, USA). After a 20-min stability, the TER was recorded through the automatic voltage clamp model. To evaluate the mucosal-to-serosal flux of macromolecules, FITC-labeled dextran 4kDa (FD4, 1 mg/ml; Sigma-Aldrich Chemistry) was added into the mucosal side of the U-type chambers, and sampled from the serosal side every 30 min for a 2-h period. The FD4 concentration was further detected on a fluorescence microplate reader (BioTek Instruments, USA). The epithelial permeability was determined as the increase of FD4 intensity in the serosal side within 2 hours.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell Culture of Intestinal Microvascular ECs\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003eECs monolayer culture and interventions\u003c/em\u003e. Human intestinal microvascular endothelial cell (HIMECs; PriCells, China) were cultured in primary endothelial cell complete medium (MED-0002, PriCells) with growth supplement (SUP-0002, PriCells) of 5 ng/ml rh-VEGF, 5 ng/ml rh-EGF, 5 ng/ml rh-FGF, 15 ng/ml rh-IGF-1, 10 mM L-glutamine, 0.75 U/ml heparin sulfate, 1 \u0026micro;g/ml hydrocortisone and 50 \u0026micro;g/ml ascorbic acid at 37 ℃ in 5% CO\u003csub\u003e2\u003c/sub\u003e. As reached 80% confluence, the cells were rinsed twice with PBS, digested with 0.125% trypsin for 2 min, and passaged at a 1:2 dilution ratio, and cultured for no more than 3 generations. ECs were seeded on transwell membrane or coverslips in 24-well plates as the requirement of different experiments. Upon formation of stable intercellular connections, the cells were subjected interventions of Yoda1 (1 \u0026micro;M, 5\u0026micro;M) or hydrostatic pressure (2cm, 15cm, 30cmH\u003csub\u003e2\u003c/sub\u003eO) for 24 h for further detection, and preconditioned with siRNA transfection to silence Piezo1 or Vegfr2 when needed. Specially, the hydrostatic pressure was produced via an experimental apparatus (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e6\u003c/span\u003eA) as previously described (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). The culture chamber (a closed culture bottle) was filled with medium, where the cell-planted transwell membrane or coverslip was placed. The height of the medium reservoir was adjusted to maintain the pressure in the culture (2cm, 15cm, 30cm). The medium in the culture chamber and the medium reservoir was circulated by a peristaltic pump to ensure gas exchange and maintain stable oxygen tension. The entire device was placed in the incubator with 37 ℃ and 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eTransmembrane resistance and permeability assay.\u003c/em\u003e ECs were inoculated on the transwell membrane with 0.4\u0026micro;m pore size (Corning, USA) to form an endothelial monolayer. The transendothelial electrical resistance (TEER) was measured using an EVOM2 volt-ohmmeter (World Precision Instruments, USA), and calculated as Ω\u0026middot;cm\u003csup\u003e2\u003c/sup\u003e(\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). A transwell with only culture medium was set as a blank control to measure the baseline membrane resistance. The TEER reached 180 Ω\u0026middot;cm\u003csup\u003e2\u003c/sup\u003e indicated formation of functional monolayer barrier, and then it could be used for further barrier studies. The transendothelial permeability was evaluated by apical-to-basolateral transmission of FITC-labeled bovine serum albumin (FITC-BSA; Solarbio Life Science). Briefly,10 mg/ml FITC-BSA was added into the upper compartment of the transwell chamber for 2 h, and then 100 \u0026micro;l medium was sampled at the lower compartment and detected FITC-BSA concentration via a Fluorescence Microplate Reader (BioTek Instruments, USA). The BSA throughput was calculated as the ratio between the fluorescence intensity in the lower chamber and upper chamber, and the data were presented as a percentage of the control(\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eSmall Interfering RNA (siRNA) Transfection\u003c/h3\u003e\n\u003cp\u003eThe gene silence of Piezo1 or Vegfr2 in ECs was achieved via specific siRNA. The siRNAs against human Piezo1, Vegfr2, and negative control were designed and chemically synthesized (Tsingke Biotech, China). The designed siRNAs were as follows: Piezo1-targeting siRNA (Piezo1-si) sequences were sense (5'-3') CUCAAGUACUUCAUCAACU(dT)(dT), antisense (5'-3') AGUUGAUGAAGUACUUGAG(dT)(dT), Gene ID: 9780; and Vegfr2-targeting siRNA (Vegfr2-si) sequences were sense (5'-3') GGAAAUCUCUUGCAAGCUA(dT)(dT), antisense (5'-3') UAGCUUGCAAGAGAUUUCC(dT)(dT), Gene ID: 3791. For cell transfection, ECs were seeded on BioFlex 6-well culture plates coated with type IV collagen. The cells were serum-starved for 6 hours, and transfected with 40 nM siRNA (Piezo1-si, Vegfr2-si, or control siRNA) via Lipofectamine\u0026trade; 3000 reagent (L3000075, Invitrogen, Life Science) following the manufacturer\u0026rsquo;s instructions. The mRNA and protein expression was detected by RT-qPCR and Western blot at 48 h post transfection to confirm effect of knockdown.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eData Availability\u003c/h2\u003e \u003cp\u003eThe supporting data for this study are available from the corresponding authors upon reasonable request. The expanded methods are provided in the Supplementary Methods.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll data were presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Statistical analysis and graphic production were performed using GraphPad Prism 10 (GraphPad Software, USA). The Shapiro-Wilk test and Brown-Forsythe test were employed to assess the normality of data distribution and the homogeneity of variances, respectively. Comparisons between groups were conducted performed using unpaired two-tailed Student's t-test (for two groups) or one-way analysis of variance (ANOVA) followed by least significant difference test (for multiple groups), if the data conformed to normal distribution and equal variance; otherwise, the non-parametric Mann-Whitney U test or Kruskal-Wallis test with Dunn's post hoc correction was applied accordingly. Pearson correlation was applied to assessed the correlations between parameters. A P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePortal Hypertension Mediated GVB Disruption Promoted Intestinal and Systemic Inflammation in CCl\u003csub\u003e4\u003c/sub\u003e-induced Liver Cirrhosis\u003c/h2\u003e \u003cp\u003eLiver cirrhosis and related PH was induced by CCl4 administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). CCl\u003csub\u003e4\u003c/sub\u003e induced significant liver injury and fibrosis, with diffuse inflammatory cell infiltration and vacuolar fat changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), elevated serum ALT and AST levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), increased areas of collagen (Sirius red positive, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eE) and α-SMA expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). The portal venous pressure markedly increased in CCl\u003csub\u003e4\u003c/sub\u003e mice, and further elevated in the CCl\u003csub\u003e4\u003c/sub\u003e mice with PPVL (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). PH was associated with hepatic injury, manifesting as more severe inflammatory infiltration and elevation of serum ALT and AST in CCl\u003csub\u003e4\u003c/sub\u003e+PPVL mice compared with CCl\u003csub\u003e4\u003c/sub\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThere were obvious intestinal and systemic inflammation in CCl\u003csub\u003e4\u003c/sub\u003e-induced liver cirrhosis, and aggravate in the CCl\u003csub\u003e4\u003c/sub\u003e+PPVL mice. The levels of plasma endotoxin and circulating inflammatory cytokines, including TNF-α, IL-1β and IL-6, were increased in CCl\u003csub\u003e4\u003c/sub\u003e mice and CCl\u003csub\u003e4\u003c/sub\u003e+PPVL mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eG, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eH), which were positive correlated with the portal venous pressure (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Furthermore, the microvascular dilation, inflammatory infiltration and the concentrations of inflammatory TNF-α, IL-1β and IL-6 were also markedly increased in ileum and colon of CCl\u003csub\u003e4\u003c/sub\u003e mice and CCl\u003csub\u003e4\u003c/sub\u003e+PPVL mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eI-J; Figure S3). And the intestinal inflammation was accompanied by disruption of mucosal epithelial barrier, with reduced TER and increased FD4 permeability (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eK-L). All above indicated that PH may contribute to the local inflammatory injury and leaky gut, and meanwhile promote the circulating inflammation, in status of liver cirrhosis.\u003c/p\u003e \u003cp\u003eImportantly, it observed significantly GVB disruption in CCl\u003csub\u003e4\u003c/sub\u003e mice, and more serious GVB damage in CCl\u003csub\u003e4\u003c/sub\u003e+PPVL mice. As it shown, there was increased FD70 density detected in liver and spleen when administered through the intestine in CCl\u003csub\u003e4\u003c/sub\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eM, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eP-Q), which implied increased influx of enterogenous macromolecules into the bloodstream. The ileal and colonic PV1 expression, a marker of endothelial permeability, was also increased in CCl\u003csub\u003e4\u003c/sub\u003e mice which confirmed the GVB disruption in liver cirrhosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eN, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eR; Figure S4). On the other hand, the vascular leakage of circulating macromolecules into the gut was also increased in CCl\u003csub\u003e4\u003c/sub\u003e mice, with raised FD70 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eO, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eS) and evans blue (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eT) levels in the ileal and colonic mucosa when administered via tail-vein injection. Moreover, the GVB disruption was more remarkable in the CCl\u003csub\u003e4\u003c/sub\u003e+PPVL mice relative to the sham group. It further verified the inward and outward hyper-permeability of GVB in portal hypertension and liver cirrhosis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eImpaired GVB Initiates Gut Inflammation and Leakage, and Led to Systemic Injury in PPVL-induced Portal Hypertension\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePrehepatic PH was induced via partial portal vein ligation with 26G and 27G needles (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). PPVL led to significant increase in portal venous pressure (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), and presented with local inflammatory cell infiltration and blood stagnation in the hepatic sinusoid (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), but without elevation of serum ALT and AST levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Similarly, PPVL-induced PH appeared increased levels of plasma LPS and pro-inflammatory TNF-α, IL-1β and IL-6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-F), as well as inflammatory infiltration and increased concentrations of TNF-α, IL-1β and IL-6 in ileum and colon (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eG-H; Figure S5) with reduced TER and increased FD4 permeability (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eI-J). The GVB dysfunction was also observed in PH mice induced by PPVL, including increased endothelial PV1 expression in the ileum and colon (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eL, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eP; Figure S6), increased FD70 permeability from the gut to blood (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eK, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eN-O), and increased FD70 and evans blue leakage from blood to the gut (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eM, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eQ-R). It again pointed out that PH destroyed the GVB may initiate the gut inflammation and leakage, and cause systemic inflammation and injury.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eUpregulation of Piezo1 in the Intestinal Microvascular ECs Under Portal Hypertension of CCl\u003c/b\u003e \u003csub\u003e \u003cb\u003e4\u003c/b\u003e \u003c/sub\u003e \u003cb\u003eand PPVL Mice\u003c/b\u003e\u003c/p\u003e \u003cp\u003eExpression of mechanosensitive Piezo1 was up-regulated in the intestinal microvascular ECs at PH state. As it shown with immunofluorescence, Piezo1 intensity in CD31\u003csup\u003e+\u003c/sup\u003e ECs of the ileum and colon was enhanced in both the CCl\u003csub\u003e4\u003c/sub\u003e and PPVL induced PH mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Additionally, we further identified the CD45\u003csup\u003e\u0026minus;\u003c/sup\u003eCD90\u003csup\u003e\u0026minus;\u003c/sup\u003eCD31\u003csup\u003e+\u003c/sup\u003e vascular ECs in the ileum and colon via flow cytometry (Figure S7; Figure S8). The results revealed an increased average expression of Piezo1 in the intestinal vascular ECs from the CCl\u003csub\u003e4\u003c/sub\u003e and PPVL mice compared with the sham controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-I). It indicated that Piezo1 may mediate the pressure sensation of intestinal vascular ECs in PH and liver cirrhosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEndothelial Piezo1 Knockout Improved GVB Dysfunction and Leaky Gut Induced by Portal Hypertension\u003c/h2\u003e \u003cp\u003eSilence of endothelial Piezo1 effectively improved the GVB dysfunction, and relieved the intestinal and systemic inflammation in CCl\u003csub\u003e4\u003c/sub\u003e and PPVL induced PH mice. The PH-induced high expression of endothelial PV1 in the ileum and colon declined in the Piezo1\u003csup\u003eΔEC\u003c/sup\u003e mice compared to the Piezo1\u003csup\u003eWT\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eL, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eO; Figure S11). In Piezo1\u003csup\u003eΔEC\u003c/sup\u003e mice, PH-induced GVB hyper-permeability was obviously suppressed, which presented as decreased FD70 permeability from the gut to blood (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eM-N), and reduced FD70 and evans blue leakage from blood to the gut (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eK, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eP-Q). Piezo1 knockout relived the GVB function which was associated with declined circulating levels of LPS and pro-inflammatory cytokines TNF-α, IL-1β and IL-6 in CCl\u003csub\u003e4\u003c/sub\u003e and PPVL mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-E), and as well reduced hepatic inflammatory infiltration and injury (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B; Figure S9). Moreover, the restored GVB function also improved the intestinal inflammation, reduced mucosal TNF-α, IL-1β and IL-6 secretion (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eH-I; Figure S10), and recovered epithelial barrier and lessened gut leakage in the CCl\u003csub\u003e4\u003c/sub\u003e and PPVL mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eF-G).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePiezo1 Activation by Yoda1 and Hydrostatic Pressure Triggered the Increased Permeability of Intestinal Microvascular ECs\u003c/h2\u003e \u003cp\u003eYoda1 activated Piezo1 channel and triggered Ca\u003csup\u003e2+\u003c/sup\u003e influx in intestinal microvascular ECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-C). Piezo1 activation significantly increased the permeability of endothelial barrier, with decreased transendothelial cell resistance (TER) and increased passage of macromolecular FITC-BSA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-E), and also with increased PV1 levels and down-regulated expression of junctional proteins such as VE-cadherin, occludin, claudin-1, ZO-1 and JAM-A (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eF-L).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAdditionally, the hydrostatic pressure (2cm, 15cm, and 30cmH\u003csub\u003e2\u003c/sub\u003eO) induced Piezo1 activation was further tested (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). It indicated that elevated pressure stimulated Piezo1 expression and Ca\u003csup\u003e2+\u003c/sup\u003e influx in intestinal ECs, which could be obviously inhibited by Piezo1 silence with specific siRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-D). Also, there was marked reduce in TER and increase in FITC-BSA permeability in ECs as the pressure stimulus elevated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e6\u003c/span\u003eE-F). High hydrostatic pressure promoted the expression of PV1, and disrupted the adhesion and tight junctions among ECs, with weak expression of VE-cadherin, occludin, claudin-1, ZO-1 and JAM-A (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e6\u003c/span\u003eG-L; Figure S14). The pressure-mediated hyper-permeability and junctional damage of ECs could be blocked by Piezo1-siRNA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003ePiezo1 Promoted Endothelial Hyperpermeability and GVB Dysfunction via Activating the VEGFR2 Signaling\u003c/h2\u003e \u003cp\u003eThe differential genes were identified and exhibited via volcano plot based on the RNA sequencing data (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA), and the GSEA analysis illustrated the enrichment of the VEGF signaling pathway and the VEGFR regulation pathway in ECs treated with Yoda1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB-C; Figure S15). The RT-PCR assay further confirmed that Yoda1 and hydrostatic pressure significantly promoted the mRNA expression in ECs, which could be blocked by Piezo1-siRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eN). In addition, Piezo1 activation by Yoda1 (1\u0026micro;M and 5\u0026micro;M) and hydrostatic pressure (15cm and 30cmH\u003csub\u003e2\u003c/sub\u003eO) facilitated the endothelial expression of VEGFR2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE-H), particularly promoted the phosphorylated level of VEGFR2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI-M). And hydrostatic pressure (30cmH\u003csub\u003e2\u003c/sub\u003eO) induced high expression of VEGFR2 and p-VEGFR2 was obviously inhibited by Piezo1-siRNA. In vivo study also indicated a remarkable increase of VEGFR2 expression in ileal and colonic vascular ECs in CCl\u003csub\u003e4\u003c/sub\u003e and PPVL mice, while it was repressed in the Piezo1\u003csup\u003eΔEC\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eO; Figure S16). The mRNA and protein expression of VEGFR2, and the phosphorylation level, in the gut of CCl\u003csub\u003e4\u003c/sub\u003e and PPVL mice were evidently inhibited in the Piezo1 knockout state (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eP-T; Figure S16). All above suggested that endothelial Piezo1 may promote the GVB dysfunction via activating the VEGFR2 signaling.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eSilence of VEGFR2 Alleviated Piezo1-mediated Dysfunction and Hyper-permeability of Intestinal Microvascular ECs\u003c/h2\u003e \u003cp\u003eEC-model with silence of VEGFR2 was successfully established via specific siRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-E; Figure S17). It demonstrated that VEGFR2 knockdown ameliorated the dysfunction of endothelial barrier induced by Yoda1 (5\u0026micro;M) and hydrostatic pressure (30cmH\u003csub\u003e2\u003c/sub\u003eO), with increased TER and decreased FITC-BSA permeability (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eG-H). Moreover, VEGFR2-siRNA inhibited the PV1 expression in ECs upon Piezo1 activation by Yoda1 and hydrostatic pressure, and it in turn restored the cell junctions by promoting the expression of VE-cadherin, occludin, claudin-1, ZO-1 and JAM-A (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eI-N). These further clarified the role of VEGFR2 activation in Piezo1-induced hyper-permeability of intestinal microvascular ECs and GVB dysfunction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eGut dysfunction played an important role in the progression of chronic liver disease and cirrhosis, due to the closely anatomical and functional relations of the gut-liver axis(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). The degeneration of multiple intestinal barriers, including the biochemical, immunological and physical barrier, has been identified in liver injury(\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). It was highly concerned but several questions are still open, especially for the newly defined GVB which is last key guard from the intestine to liver. Here, we focused on the GVB disruption, to elucidate its role and mechanism in the development of liver cirrhosis.\u003c/p\u003e \u003cp\u003eGVB breakdown was remarkable in CCl\u003csub\u003e4\u003c/sub\u003e-induced cirrhosis mice, presented as upregulated endothelial PV1 expression, and increased permeability to macromolecular particles such as LPS, FD70 and Evans blue. The hyper-permeability of GVB was responsible for the dissemination of luminal bacteria in experimental cirrhosis(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Normally, the invaded pathogens and antigens escaped from the epithelial barrier are denied entry to the internal environment by GVB, and are finally clear by the mucosal immune system(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). This highlighted the importance of GVB integrity in preventing the aggravation of liver cirrhosis and systemic inflammatory damages.\u003c/p\u003e \u003cp\u003eAs we shown, the GVB manifested as bi-directional hyper-permeability in CCl\u003csub\u003e4\u003c/sub\u003e mice, namely, increased passage from the gut to blood (inward) and also increased leakage from blood to the gut (outward). On the one aspect, the increased in-flux permeability of GVB directly resulted in bacterial translocation to the portal vein, which may further give rise to liver injury with increased ALT and AST levels, increased hepatic inflammation and fibrosis, and also systemic inflammation with increased levels of serum IL-1β, IL-6 and TNF-α. On the other hand, the raised out-flux permeability of GVB with microvascular leakage may initiate local inflammation in the intestine and trigger or aggravate epithelial leakage. It was true that GVB damage was associated with increased inflammatory infiltration, and then decreased TER and elevated epithelial permeability to FD4 in ileum and colon. This may also be one potential explanation for the cause of intestinal damage and leaky gut in liver cirrhosis. The epithelial leakage, in turn, leads to more entries of luminal antigens forming a vicious cycle of systemic damage.\u003c/p\u003e \u003cp\u003ePortal hypertension was associated with GVB disruption in cirrhosis. Compared with the CCl\u003csub\u003e4\u003c/sub\u003e mice, the GVB damage was more evident in CCl\u003csub\u003e4\u003c/sub\u003e+PPVL mice, with more obvious intestinal and systemic inflammation and injury. Also, the extent of GVB damage was positive correlated with portal pressure in PPVL-induced prehepatic PH mice. As increasing portal pressure, the intestinal and systemic inflammation deteriorated in PH status, with raised plasma endotoxin and levels of local and serum cytokines such as IL-1β, IL-6 and TNF-α. These findings indicated the role of pressure-mediated GVB disruption in liver cirrhosis with PH. Similarly, it was reported that elevated microvessel pressure induced lung endothelial hyper-permeability and edema formation via disruption of adherens junction(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Intraluminal pressure remodeled the endothelial tissue, and regulated their barrier function via thinned junctions(\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). Our in vitro studies further revealed that elevated hydrostatic pressure destroyed the barrier function of intestinal microvascular ECs monolayer, with increased permeability to FITC-BSA and declined transendothelial resistance. Hydrostatic pressure facilitated endothelial barrier damage by increasing the expression of PV1, and breaking the endothelial adherens and tight junctions. Combined, PH-induced breakdown of endothelial integrity and increase of GVB permeability could be a potential explanation for the progress of liver cirrhosis and related systemic injury.\u003c/p\u003e \u003cp\u003eThe mechanism of endothelial sense to PH and subsequent GVB collapse was largely unknown. Notably, the mechanosensitive Piezo1 channel is distributed in ECs that directly sensing the mechanical stimuli, such as hydrostatic pressure, shear stress and substrate stiffness(\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). Piezo1 is crucial for the homeostasis and angiogenesis of various vascular endothelium(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e), including the intestinal microvessel ECs(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Endothelial Piezo1 stimulated angiogenesis to offer protection against intestinal ischemia-reperfusion injury(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Piezo1-mediated mechanotransduction contributes to endothelial inflammation activated by disturbed blood flow(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). Deletion of endothelial Piezo1 effectively prevented pressure-induced vascular hyper-permeability and lung edema(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). The expression of Piezo1 on intestinal vessel ECs was upregulated in CCl\u003csub\u003e4\u003c/sub\u003e- and PPVL-induced PH mice. And, specific knockout of Piezo1 in ECs improved the GVB function in PH mice, meanwhile inhibited the systemic and intestinal inflammation, and relieved the epithelial barrier injury. This further indicated the potential role of Piezo1 in PH-induced GVB impairment and related mucosal and circulating inflammation. It was further verified that Piezo1 activation by Yoda1 or hydrostatic pressure destroyed the barrier function of intestinal microvascular ECs monolayer, which could be alleviated by Piezo1 silence with specific siRNA. Also, activation of Piezo1 by ultrasonic stimulation promoted the hyper-permeability in human umbilical vein ECs (HUVECs), while the suppression of Piezo1 showed a decline in cell permeability(\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). Collectively, Piezo1 acted as a key sensor to PH and mediated the GVB impairment in liver cirrhosis, which could be a promising pharmacologic target to modulated GVB integrity.\u003c/p\u003e \u003cp\u003ePiezo1 activation in ECs by Yoda1 upregulated the VEGF and downstream signaling pathway and increased the expression of VEGFR2, deriving from the transcriptome results. Endothelial Piezo1 triggered VEGF/myc signaling by enhancing Ca\u003csup\u003e2+\u003c/sup\u003e influx could be responsible for driving the angiogenesis(\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). Piezo1 promoted angiogenesis in ileal ECs through the activation of the Ca\u003csup\u003e2+\u003c/sup\u003e/HIF-1α/VEGF pathway during intestinal ischemia-reperfusion injury(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). These data indicated the compact link of Piezo1 and VEGF signaling in endothelial function. We further confirmed that activation of Piezo1 by Yoda1 or hydrostatic pressure stimulated Ca\u003csup\u003e2+\u003c/sup\u003e influx, and promoted the expression and phosphorylation of VEGFR2 in ECs, which could be repressed by Piezo1-siRNA. Moreover, the high expression and activation of VEGFR2 in the ileum and colon of CCl\u003csub\u003e4\u003c/sub\u003e and PPVL mice were also inhibited in Piezo1\u003csup\u003eΔEC\u003c/sup\u003e mice. It also prompted that Piezo1/VEGFR2 collaboration may be involved in regulating the barrier function of ECs.\u003c/p\u003e \u003cp\u003eThe VEGFR2 kinase signaling is an important regulator of vascular permeability, however, the effect varies in different situations. For example, disrupt blood-retinal barrier function of diabetic retinopathy(\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e), promote vascular leakage and edema in myocardial infarction(\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e), strengthen retinal endothelial barrier via endothelial-pericyte interaction(\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e), and restore pulmonary microvascular barrier following ischemia reperfusion injury(\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e). Excessive VEGFR2 trafficking to the cell surface mediated vascular permeability, while inhibited this VEGFR2 recycling mitigated vascular leakage associated with inflammatory diseases(\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e). In intestinal microvascular ECs, as we shown, inhibition of VEGFR2 with siRNA knockdown protected Yoda1 and hydrostatic pressure induced barrier disruption, through inhibiting the expression of PV1, enhancing the adherens junctions (VE-cadherin) and tight junctions (occludin, claudin-1, ZO-1 and JAM-A). Piezo1 is required in force-induced Ca\u003csup\u003e2+\u003c/sup\u003e-dependent remodelling of cell-cell junctions and associated cytoskeleton, especially for adherens junctions(\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). Piezo1 sensing of high vascular pressures at the endothelial surface may promote disassembly of adherens junctions(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e). These further clarified the role of VEGFR2 activation in Piezo1-induced hyper-permeability of intestinal microvascular ECs and GVB dysfunction.\u003c/p\u003e \u003cp\u003eThis study provided a preliminary image that endothelial Piezo1 mediated GVB dysfunction in PH and promoted deterioration of liver cirrhosis. Nevertheless, several limitations should be acknowledged, which also offered the possible directions for future research. Firstly, our study focused on the role of Piezo1 in ECs, but did not preclude the involvement of other mechanosensitive channels or receptors(\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e) that may be responsible for pressure-induced endothelial hyperpermeability. Even so, the molecular pathway of Piezo1 in GVB impairment was currently limited, which should be multifaceted and requires further research to elucidate. Secondly, the actions of Piezo1 in modulating GVB function should be validated in PH patients with liver cirrhosis and related clinical manifestations of intestinal and systematic damage, for instance, through the study of gastrointestinal biopsy under endoscopy or surgery. And finally, the specific monoclonal antibody or fusion protein against Piezo1 is urgently needed, especially for the intestinal ECs-specific blocker with good in vivo safety and efficacy.\u003c/p\u003e \u003cp\u003eIn summary, we expounded that Piezo1 contributed to PH-induced damage of intestinal microvascular ECs and GVB dysfunction in cirrhosis via recruiting and activating the VEGFR2 signaling. Especially, the outward (from blood to the gut) and inward (from the gut to blood) hyper-permeability of GVB in PH status may initiate the intestinal inflammation, trigger or aggravate epithelial leakage, and in turn lead to more convenient bacterial translocation into the portal vein, facilitate liver and systemic damage, forming a vicious cycle. So that, Piezo1 mediated bi-directional hyper-permeability of the GVB should be a novel explanation and potential therapeutic target for preventing the exacerbation of liver cirrhosis with PH.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCCl\u003csub\u003e4\u003c/sub\u003e, carbon tetrachloride;\u003c/p\u003e\n\u003cp\u003ePPVL, partial portal vein ligation;\u003c/p\u003e\n\u003cp\u003ePH, portal hypertension;\u003c/p\u003e\n\u003cp\u003eALT, alanine aminotransferase;\u003c/p\u003e\n\u003cp\u003eAST, aspartate aminotransferase;\u003c/p\u003e\n\u003cp\u003eGVB, gut-vascular barrier;\u003c/p\u003e\n\u003cp\u003eECs, endothelial cells;\u003c/p\u003e\n\u003cp\u003eHIMECs, human intestinal microvascular endothelial cells;\u003c/p\u003e\n\u003cp\u003eLPS, lipopolysaccharide;\u003c/p\u003e\n\u003cp\u003eFD4, fluorescein isothiocyanate-labeled dextran 4kDa;\u003c/p\u003e\n\u003cp\u003eFD70, fluorescein isothiocyanate-labeled dextran 70kDa;\u003c/p\u003e\n\u003cp\u003eBSA, bovine serum albumin;\u003c/p\u003e\n\u003cp\u003eTER, trans-epithelial/endothelial cell resistance;\u003c/p\u003e\n\u003cp\u003ePV1, plasmalemma vesicle-associated protein 1;\u003c/p\u003e\n\u003cp\u003eVEGFR2, vascular endothelial growth factor receptor 2.\u003c/p\u003e\n\u003cp\u003eDEGs, differential expression genes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Dr. Liuying Chen (Division of Gastroenterology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology) for providing HIMECs and technical assistances.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Xiao Z:\u0026nbsp;\u003c/strong\u003emethodology, investigation, validation, data curation, writing - original draft; \u003cstrong\u003eHe J:\u0026nbsp;\u003c/strong\u003esoftware, formal analysis; \u003cstrong\u003eTian W: \u0026nbsp;\u003c/strong\u003einvestigation; \u003cstrong\u003eDu L:\u003c/strong\u003e investigation; \u003cstrong\u003eWang R:\u0026nbsp;\u003c/strong\u003evalidation; \u003cstrong\u003eBai T:\u0026nbsp;\u003c/strong\u003esoftware, formal analysis; \u003cstrong\u003eQian W:\u0026nbsp;\u003c/strong\u003emethodology, resources; \u003cstrong\u003eSong J:\u0026nbsp;\u003c/strong\u003econceptualization, supervision, writing - review \u0026amp; editing; \u003cstrong\u003eHou X:\u0026nbsp;\u003c/strong\u003eproject administration,funding acquisition, \u0026nbsp;writing - review \u0026amp; editing; \u003cstrong\u003eZhang L:\u003c/strong\u003e funding acquisition, conceptualization, methodology, investigation, data curation, writing - original draft.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003cstrong\u003etatement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Animal Care and Use Committee, Tongji Medical College, Huazhong University of Science and Technology, China\u0026nbsp;(Approval NO:3372).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they had full control over the data and complete access to the data, and no competing interest declared.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranscript profiling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw RNA-seq data was published in the Sequence Read Archive (SRA) database with Bioproject number PRJNA1415200.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original contributions presented in this study are included in this article and supplementary material, further inquiries can be directed to the corresponding authors.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eGrant support\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by grants from the National Natural Science Foundation of China (NSFC), China (No: 82470566, 81800463).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eVillanueva C, Tripathi D, Bosch J. 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J Clin Invest 2016;126:4527\u0026ndash;4536.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang L, Liu X, Gao L, Ji Y, Wang L, Zhang C, Dai L, et al. Activation of Piezo1 by ultrasonic stimulation and its effect on the permeability of human umbilical vein endothelial cells. Biomed Pharmacother 2020;131:110796.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLv W, Yang F, Ge Z, Xin L, Zhang L, Zhai Y, Liu X, et al. Aberrant overexpression of myosin 1b in glioblastoma promotes angiogenesis via VEGF-myc-myosin 1b-Piezo1 axis. J Biol Chem 2024;300:107807.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu S, Hou J, Tao Y, Xu Y, Liu J, Lin S, Tao L, et al. ELF3-regulated PES1 targets VEGFR2 to mediate angiogenesis and retinal inner barrier injury in diabetic retinopathy. 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The role of mechanosensitive ion channels in the gastrointestinal tract. Front Physiol 2022;13:904203.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFIFURE LEGENDS\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"experimental-and-molecular-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"emm","sideBox":"Learn more about [Experimental \u0026 Molecular Medicine](http://www.nature.com/emm/)","snPcode":"12276","submissionUrl":"https://mts-emm.nature.com/cgi-bin/main.plex","title":"Experimental \u0026 Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Piezo1, Endothelial cells, Gut-vascular barrier, Portal hypertension, Liver cirrhosis","lastPublishedDoi":"10.21203/rs.3.rs-9418908/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9418908/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eObjectives\u003c/b\u003e\u003c/p\u003e \u003cp\u003eGut-vascular barrier (GVB) disruption in cirrhotic portal hypertension (PH) exacerbates hepatic and systemic damage via the gut-liver-organ axis, but the underlying mechanism is unclear. The mechanosensitive channel Piezo1, known to regulate endothelial homeostasis, may mediate GVB dysfunction, requiring validation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e \u003cp\u003eCarbon tetrachloride (CCl\u003csub\u003e4\u003c/sub\u003e)-induced liver cirrhosis and partial portal vein ligation (PPVL)-induced pre-hepatic PH model were established in Piezo1\u003csup\u003eflox/flox\u003c/sup\u003e and endothelial cells (ECs)-specific Piezo1-deficient (Piezo1\u003csup\u003e△EC\u003c/sup\u003e) mice. Portal pressure, GVB permeability, intestinal and systemic inflammation, and Piezo1 expression in mucosal ECs were measured. Cultured intestinal microvascular ECs were treated with the Piezo1 agonist Yoda1 or hydrostatic pressure to investigate Piezo1-mediated endothelial barrier regulation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePH induced significant GVB breakdown in CCl\u003csub\u003e4\u003c/sub\u003e and PPVL mice, presented as increased permeability from the gut-to-blood and also leakage from blood-to-gut, accompanied by increased Piezo1 and VEGFR2 expression in ECs, along with systemic and intestinal inflammation and leaky epithelium. These effects were reversed in Piezo1\u003csup\u003e△EC\u003c/sup\u003e mice, and Piezo1 knockout in ECs reduced VEGFR2 expression which in turn inhibited intestinal leakage and inflammatory infiltration, and meanwhile improved systemic inflammation. Moreover, Yoda1 or hydrostatic pressure promoted VEGFR2 phosphorylation and ECs monolayer disruption, blocked by siRNA of \u003cem\u003ePiezo1\u003c/em\u003e or \u003cem\u003eVegfr2\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion\u003c/b\u003e\u003c/p\u003e \u003cp\u003eEndothelial Piezo1 contributed to GVB dysfunction in PH via VEGFR2 pathway. The outward and inward hyper-permeability of GVB in PH may initiate the intestinal inflammation and trigger epithelial leakage, and further cause systemic inflammation and injury. Therefore, Piezo1-mediated GVB damage may be a novel explanation and potential therapeutic target for deterioration of liver cirrhosis with PH.\u003c/p\u003e","manuscriptTitle":"Mechanosensing Piezo1 mediates gut-vascular barrier dysfunction in portal hypertension and promotes deterioration of liver cirrhosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-15 17:38:32","doi":"10.21203/rs.3.rs-9418908/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-05-11T06:04:08+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-05-07T04:30:17+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2026-05-07T04:28:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-20T23:48:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"Experimental \u0026 Molecular Medicine","date":"2026-04-20T12:43:25+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2026-04-14T23:26:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-14T18:48:59+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"experimental-and-molecular-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"emm","sideBox":"Learn more about [Experimental \u0026 Molecular Medicine](http://www.nature.com/emm/)","snPcode":"12276","submissionUrl":"https://mts-emm.nature.com/cgi-bin/main.plex","title":"Experimental \u0026 Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6eee9599-afed-42f2-a447-768a9b93ba70","owner":[],"postedDate":"May 15th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-05-11T06:04:08+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-05-07T04:30:17+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"2","date":"2026-05-07T04:28:30+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":67676448,"name":"Health sciences/Medical research/Experimental models of disease"},{"id":67676449,"name":"Health sciences/Diseases/Gastrointestinal diseases/Liver diseases/Portal hypertension"}],"tags":[],"updatedAt":"2026-05-15T17:38:32+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-15 17:38:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9418908","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9418908","identity":"rs-9418908","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}