VE-cadherin Levels Research Articles

Low-Dose X-Ray Increases Paracellular Permeability of Human Renal Glomerular Endothelial Cells.

Objective Glomerular endothelium functions as a filtration barrier of metabolites in the kidney. Although X-ray irradiation modulated the permeability of the vascular endothelium, the response of human renal glomerular endothelial cells (HRGECs) to low-dose X-ray irradiation has not been investigated. We evaluated the impacts of low-dose X-ray irradiation on HRGECs and revealed the underlying mechanism. Methods HRGECs were exposed to X-ray with doses of 0, 0.1, 0.5, 1.0, and 2.0 Gy. The proliferation, viability, and apoptosis of HRGECs were examined by MTT assay, trypan blue staining assay, and TUNEL staining, respectively. The paracellular permeability was assessed by paracellular permeability assay. The expression of VE-cadherin was investigated via immunofluorescence assay. Western blot and qRT-PCR detected the expression levels of VE-cadherin and CLDN5. Besides, the expression levels of pVE-cadherin (pY658), TGF-β, TGF-βRI, Src, p-Src, Smad2, p-Smad2, Smad3, p-Smad3, SNAIL, SLUG, and apoptosis-related proteins were tested by Western blot. Results The proliferation, viability, and apoptosis of HRGECs were not affected by low-dose (<2.0 Gy) X-ray irradiation. X-ray irradiation dose-dependently reduced the level of VE-cadherin, and VE-cadherin and CLDN5 levels were reduced with X-ray irradiation. The levels of pY658, p-Src, p-Smad2, and p-Smad3 were upregulated with the increase in X-ray dose. Besides, the paracellular permeability of HRGECs was increased by even low-dose (<2.0 Gy) X-ray irradiation. Therefore, low-dose X-ray irradiation reduced the cumulative content of VE-cadherin and increased the level of pY658 via activation of the TGF-β signaling pathway. Conclusion Even though low-dose X-ray exposure had no impact on proliferation, viability, and apoptosis of HRGECs, it increased the paracellular permeability by deterioration and downregulation of VE-cadherin through stimulating the TGF-β signaling pathway. This study built the framework for kidney response to low-dose irradiation exposure.

Increased Type I interferon signaling and brain endothelial barrier dysfunction in an experimental model of Alzheimer’s disease

Blood–brain barrier (BBB) dysfunction is emerging as a key pathogenic factor in the progression of Alzheimer’s disease (AD), where increased microvascular endothelial permeability has been proposed to play an important role. However, the molecular mechanisms leading to increased brain microvascular permeability in AD are not fully understood. We studied brain endothelial permeability in female APPswe/PS1∆E9 (APP/PS1) mice which constitute a transgenic mouse model of amyloid-beta (Aβ) amyloidosis and found that permeability increases with aging in the areas showing the greatest amyloid plaque deposition. We performed an unbiased bulk RNA-sequencing analysis of brain endothelial cells (BECs) in female APP/PS1 transgenic mice. We observed that upregulation of interferon signaling gene expression pathways in BECs was among the most prominent transcriptomic signatures in the brain endothelium. Immunofluorescence analysis of isolated BECs from female APP/PS1 mice demonstrated higher levels of the Type I interferon-stimulated gene IFIT2. Immunoblotting of APP/PS1 BECs showed downregulation of the adherens junction protein VE-cadherin. Stimulation of human brain endothelial cells with interferon-β decreased the levels of the adherens junction protein VE-cadherin as well as tight junction proteins Occludin and Claudin-5 and increased barrier leakiness. Depletion of the Type I interferon receptor in human brain endothelial cells prevented interferon-β-induced VE-cadherin downregulation and restored endothelial barrier integrity. Our study suggests that Type I interferon signaling contributes to brain endothelial dysfunction in AD.

Combination of polyglycerol sebacate coated with collagen for vascular engineering

Introduction: Here, we monitored the cytocompatibility of scaffolds consisting of poly (glycerol sebacate) (PGS) coated with collagen (Col) for endothelial cell activity after 72 hours. Methods: Human endothelial cells were allocated into Control, PGS, and PGS+Col groups. Scaffolds were characterized using FTIR and HNMR spectroscopy. Contact angel analysis and SEM were used to study wettability, surface morphology, and cell attachment. Cell survival was assessed using LDH leakage assay. Levels of Tie-1, Tie-2, VE-Cadherin, and VEGFR-2 were measured using western blotting and real-time PCR. Results: FTIR and HNMR analyses revealed the proper blending in PGS+Col group. SEM imaging exhibited a flat surface in the PGS group while thin Col fibers were detected in PGS+Col surface. The addition of Col to the PGS reduced the contract angle values from 97.3˚ to 81.1˚. Compared to PGS substrate alone, in PGS+Col group, cells appropriately attached to the surface. PGS and PGS+Col did not alter the leakage of LDH to the supernatant compared to control cells, showing the cytocopatiblity of PGS-based scaffolds. SOD and NO levels were increased significantly in PGS (p<0.05) and PGS+Col groups (p<0.001), respectively. We found that PGS+Col decreased Tie-1 content in endothelial cells whereas protein levels of Tie-2 and VE-Cadherin and expression of VEGFR-2 remained unchanged compared to PGS and control groups. Conclusion: Simultaneous application of Col and PGS can stimulate normal endothleial cell morphology without the alteration of tyrosine kinases receptors and cadherin.

Relationship between blood-brain permeability and antibodies against aquaporins in neuromyelitis optica spectrum disorders and multiple sclerosis patients.

To evaluate serum anti-aquaporin antibodies profile, to measure serum levels of cell-cell adhesion molecules as potential markers of blood-brain barrier (BBB) permeability, and to assess their relationship in neuromyelitis optica spectrum disorders (NMOsd) and multiple sclerosis (MS). Serum levels of cell-cell adhesion molecules could provide information about BBB disruption in demyelinating diseases of the central nervous system. Improved knowledge about differences in their profile in NMOsd and MS patients, as well as about their relationship with antibody serostatus, would facilitate early and accurate diagnosis. Sera from 20 NMOsd and 59 MS patients were collected and assessed for anti-aquaporin 4 antibodies (AQP4-IgG) and antibodies against myelin oligodendrocyte glycoprotein (MOG-Ab) using an indirect immunofluorescence test (IIFT). Anti-aquaporin 1 antibodies (AQP1-Ab), vascular endothelial growth factor (VEGF), and vascular endothelial cadherin (VE-Cadherin) levels were assessed using commercial enzyme-linked immunosorbent assay (ELISA) kits. For occludin (OCLN) and claudin-5 (CLDN5) serum levels, we employed home-made ELISAs elaborated in the Department of Neurochemistry and Neuropathology, Poznan University of Medical Sciences, Poland. AQP4-IgG appeared only in 6/20 NMOsd patients who were all originally AQP4-IgG seropositive. All MS and NMOsd patients were seronegative for MOG-Ab. Patients with MS had higher AQP1-Ab levels than those with NMOsd (median 782.32 vs. 203.16 pg/mL; p < 0.001). CLND5 levels were significantly higher in MS than in NMOsd patients (median 1.65 vs. 1.00 ng/mL; p = 0.004). No statistically significant differences between MS and NMOsd were found for OCLN, VEGF and VE-Cadherin serum levels. In MS, AQ1-Ab levels were significantly lower in MS patients treated with immunomodulatory drugs vs. the treatment-naive (median 712.46 pg/mL vs. 942.73 pg/mL, respectively). There was a positive correlation between CLDN5 and OCLN in both the MS and the NMOsd groups. There is a different BBB disruption profile in MS and NMOsd, reflected by significantly higher CLDN5 and AQP1-Ab levels in MS samples. AQP1-Ab can be considered as a promising indicator of BBB disruption possibly associated with astrocytopathy.

The Biomarker Like the Correlation between Vasculogenic Mimicry, Vascular Endothelial Cadherin, Sex-DeterminingRegion on Y-Box Transcription Factor 17, and Cyclin D1 in Oesophageal Squamous Cell Carcinoma.

Background This study aimed to explore the relationships between the sex-determining region on Y (SRY) box transcription factor 17 (SOX17), Cyclin D1, vascular endothelial cadherin (VE-cadherin), and vasculogenic mimicry (VM) in the occurrence and development of esophageal squamous cell carcinoma (ESCC). Methods The expressions of SOX17, Cyclin D1, and VE-cadherin, as well as VM, in tissues, were determined using immunohistochemistry. SOX17, Cyclin D1, and VE-cadherin mRNA in ESCC and their corresponding adjacent normal tissues were quantified using quantitative reverse transcription polymerase chain reaction analysis. Cell invasion, migration, and proliferation were determined after the silencing of VE-cadherin. SOX17, Cyclin D1, and VE-cadherin protein were quantified using Western blotting. Results The expression levels of SOX17, Cyclin D1, and VE-cadherin significantly correlated with the clinical characteristics of ESCC. After the VE-cadherin silencing, cell invasion, migration, and proliferation decreased, along with the Cyclin D1 levels, while the SOX17 levels increased. Conclusion SOX17, Cyclin D1, and VE-cadherin are involved in the development of ESCC.

Cobalamin status is negatively correlated with vascular endothelial-cadherin in vegetarian and vegan women with vitamin B12 deficiency

Optimal vitamin B12 status is important for vascular health. Vascular endothelial (VE) cadherin is an adherent junction protein involved in the maintenance of a functional endothelium. We hypothesized that vitamin B12 deficiency can negatively affect markers of vascular function, such as VE-cadherin. Within a human intervention study, we explored the possible association between cobalamin status (i.e., vitamin B12, holotranscobalamin, and homocysteine) and VE-cadherin (as marker of vascular health) in vegetarians/vegans (VEG) with B12 deficiency. The associations were evaluated at baseline and after 90-day supplementation with 2000 µg/wk of vitamin B12. On the whole, an inverse association between VE-cadherin and holotranscobalamin (P=.014) and a positive association between VE-cadherin and homocysteine (P=.041) was documented at baseline. VEG women showed higher levels of VE-cadherin compared with VEG men (P=.044), suggesting an increase in endothelial permeability. The intervention with vitamin B12 restored serum vitamin levels and improved the overall cobalamin status, whereas it did not affect VE-cadherin levels. The inverse association between holotranscobalamin and VE-cadherin was also maintained after intervention in women, corroborating the strong correlation between these 2 parameters. The results obtained seem to suggest a possible association between cobalamin status and VE-cadherin even if the intervention with B12 failed to positively affect VE-cadherin levels. Thus, further studies are needed to corroborate these findings and clarify the contribution of a vitamin B12 intervention on VE-cadherin levels in this target population. This trial was registered at ISRCTN registry (ISRCTN75099618).

Exercise training inhibits atherosclerosis progression and reduces VE-cadherin levels within atherosclerotic plaques in hypercholesterolemic mice

Vascular endothelial-cadherin (VE-cadherin), matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) have emerged as key-factors of atherogenesis. The aim of this study was to evaluate the effects of exercise training (ET) on those key-factors in relation to the progression of atherosclerotic lesions in hypercholesterolemic mice. Thirty male, apoE knockout (apoE−/−) mice were randomly assigned to the following equivalent groups: 1) CO-control: High-fat diet (HFD) administration for 12 weeks. 2) EX-exercise: HFD administration as in CO, and during the last 4 weeks (9th −12th week) ET on treadmill (5sessions/week, 60min/session). At the end of study, blood samples were obtained and all mice were sacrificed. Aortic roots were excised and analysed regarding the percentage of aortic stenosis, and the relative concentrations of collagen, elastin, VE-cadherin, MMP-8,-9 and TIMP-1,-2 within the atherosclerotic lesions. Aortic stenosis was significantly lower in the EX than the CO group (39.63 ± 7.22% vs 62.04 ± 8.55%; p < 0.001), along with considerable increase in fibrous cap thickness and of collagen and elastin contents within plaques (p < 0.05). Compared to controls, exercised-treated mice showed reduced intra-plaque relative concentrations of VE-cadherin (15.09 ± 1.89% vs 23.49 ± 3.01%, p < 0.001), MMP-8 (8.51 ± 2.24% vs 18.51 ± 4.08%, p < 0.001) and MMP-9 (12.1 ± 4.86% vs 18.88 ± 6.23%, p < 0.001). Inversely, the relative concentrations of TIMP-1 and TIMP-2 in the ET group were considerably higher by 62.5% and 31.2% than in the EX group (p < 0.05), respectively. Finally, body weight and lipids concentrations did not differ between groups at the end of the study (p > 0.05). ET treatment induced regression of established atherosclerotic lesions in apoE−/− mice and improved their stability. Those effects seemed to be mediated by favourable modification of VE-cadherin, MMPs and TIMPs.

Wenyang Huazhuo Tongluo formula alleviates pulmonary vascular injury and downregulates HIF-1α in bleomycin-induced systemic sclerosis mouse model

BackgroundVascular damage, autoimmune abnormalities, and fibrosis are the three pathological features of systemic sclerosis (SSc).However, pulmonary vascular damage is the main factor affecting the progression and prognosis of SSc. The main purpose of this study was to explore the molecular mechanism of Wenyang Huazhuo Tongluo Formula in alleviating pulmonary vascular injury in bleomycin-induced SSc mouse model.MethodsMasson staining and H&E staining were used to analyze the degree of pulmonary vascular fibrosis and the infiltration of leukocyte cells in lung tissue ofbleomycin-induced SSc mouse models treated with saline (BLM group), Wenyang Huazhuo Tongluo Formula (WYHZTL group) and HIF-1α inhibitor KC7F2 (KC7F2 group). Blood vessel exudation was determined by analyzing the cell number and albumin concentration in bronchoalveolar lavage fluid using a cell counter and ELISA assay, respectively. The degree of vascular injury was assessed by measuring the expression levels of vWF, E-selectin, ICAM-1, VCAM-1, VE-cadherin and claudin-5 in serum and pulmonary vascular endothelial cells using ELISA and immunofluorescence staining. Finally, the effect of Wenyang Huazhuo Tongluo Formula on the expression of HIF-1α was detected using immunofluorescence staining.ResultsWenyang Huazhuo Tongluo Formula and KC7F2 significantly inhibited bleomycin-induced pulmonary vascular fibrosis and the level of perivascular inflammatory cell infiltration. The number of cells and the concentration of albumin were significantly reduced in the bronchoalveolar lavage fluid of the WYHZTL group and KC7F2 group compared with the BLM group. In addition, treatment with Wenyang Huazhuo Tongluo Formula and KC7F2 significantly downregulated the expression levels of vWF, E-selectin, ICAM-1, VCAM-1 and HIF-1α, but upregulated the expression of VE-cadherin and claudin-5 in serum and pulmonary vascular endothelial cells, compared with treatment with saline.ConclusionsThis study reveals that Wenyang Huazhuo Tongluo Formula plays a new role in the treatment of SSc by alleviating pulmonary vascular damage. Furthermore, we found that Wenyang Huazhuo Tongluo Formula alleviates pulmonary vascular injury and inhibits HIF-1α expression.

Abstract 3637: Age-dependent loss of HAPLN1 erodes vascular integrity via upregulation of endothelial ICAM1 in melanoma

Abstract The 5-year survival rate for metastatic melanoma is only 23%, and patients over 55 years old have higher incidence of metastasis than younger patients independent of other prognostic factors. Our lab has shown that age-dependent changes in the tumor microenvironment drive this disparity. In particular, loss of a secreted extracellular matrix protein, HAPLN1, in the aged dermis confers increased metastasis via matrix breakdown and resulting cellular dysfunction. In this study we found that HAPLN1-mediated matrix changes impede the barrier function of blood vessels and upregulate vascular ligands which in turn increase the propensity for melanoma intravasation and metastasis via this route. VE-cadherin is a key endothelial cell junctional protein whose expression is directly correlated with vascular integrity. We showed via IHC that intradermal treatment of aged mice with recombinant HAPLN1 (rHAPLN1) is sufficient to significantly increase VE-cadherin levels in intratumoral blood vessels compared to untreated aged mice, equal to levels seen in young mice. Further, we isolated the contribution of the matrix by producing in vitro fibroblast derived matrices (FDMs) from the dermal fibroblasts of healthy young and aged donors. An impedance-based assay showed that resistance of human endothelial cell (HUVEC) monolayers is significantly decreased when cultured on aged matrix compared to young or aged+rHAPLN1 matrices. We found via qRT-PCR screen that HUVECs cultured on HAPLN1-low FDMs (aged, young+shHAPLN1) have upregulated ICAM1 compared those in HAPLN1-high environments (young, aged+rHAPLN1). ICAM1 is an endothelial cell receptor whose upregulation initiates a signaling cascade to downregulate VE-cadherin and increase vascular permeability. We confirmed the HAPLN1-dependent expression of ICAM1 in vivo via IHC of murine tumors. ICAM1 expression is known to be modulated in response to ECM stiffness. We have shown that matrices formed by aged fibroblasts are stiffer than those formed by young, and that this is due in part to the absence of HAPLN1. We created artificially stiffened FDMs via chemical cross-linking of otherwise pliable young FDMs to determine if stiffness alone was sufficient to upregulate ICAM1 on endothelial cells. We found via qRT-PCR that artificially stiffened young matrices were sufficient to increase endothelial ICAM1 levels on par with those in aged and young+shHAPLN1 conditions. This is critical, as endothelial ICAM1 is upregulated in melanoma and other cancers and is used as a ligand for cancer cells to move through the vasculature. In sum, we have found that age-dependent loss of HAPLN1 in the dermal ECM confers increased matrix stiffness which leads to the upregulation of endothelial ICAM1. This in turn increases vascular permeability to allow melanoma cells to more easily intravasate and metastasize via the hematogenous pathway in the aged system. Citation Format: Gloria E. Marino, Yash Chhabra, Amanpreet Kaur, Filipe Almeida, Ying Liu, Karin T. S. Eisinger-Mathason, Ashani T. Weeraratna. Age-dependent loss of HAPLN1 erodes vascular integrity via upregulation of endothelial ICAM1 in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3637.

Paeoniflorin improves myocardial injury via inhibition of Src/VE-cadherin pathway in septic rats

Objective: To study the protective effect and mechanism of paeoniflorin (pae) on myocardial injury in septic rats. Methods: Sprague-Dawley (SD) rats were randomly divided into 4 groups with 10 rats in each group. Rats were intraperitoneally injected with 1.4 ml normal saline and 1.4 ml 5% dimethyl sulfoxide (DMSO)solution independently in control group and DMSO group. Rats were intraperitoneally injected with 1.4 ml normal saline and 1.4 ml pae independently, then with 0.1 ml lipopolysaccharide (LPS) 1 hour later in sepsis group and pae group. Enzyme linked immunosorbent assay (ELISA) was used to detect serum cardiac troponin I (cTnI) levels and myocardial tissue tumor necrosis factor alpha (TNFα), interleukin(IL)-6, IL-1β, chemokine (C-X-C motif) ligand 1 (CXCL1), chemokine (C-X-C motif) ligand 2 (CXCL2), vascular cell adhesion molecule 1 (VCAM-1) levels. Evans blue (EB) method was used to detect the EB content of myocardial tissue. HE staining method was used to observe the pathological changes, real-time quantitative polymerase chain reaction (RT-qPCR) to detect mRNA expression levels of the above molecules, and Western-blot to detect vascular endothelium-cadherin (VE-cadherin), phosphorylated p38 mitogen-activated protein kinase (P-p38MAPK), phosphorylated Src protein (P-Src), Ras-Related C3 Botulinum Toxin Substrate 1 (Rac1) levels. Results: Compared with control group, cTnI level and the EB content in sepsis group increased significantly, and the myocardial inflammatory cell infiltration was obvious. The cTnI level and EB content in pae group were significantly reduced, and myocardial inflammatory cell infiltration was reduced [cTnI: (227.7±15.9)pg/ml vs. (312.9±17.9)pg/ml;EB: (13.2±2.3)μg/g vs. (23.8±2.9)μg/g; P<0.05]. Compared with control group, the levels of TNFα, IL-6, IL-1β, CXCL1, CXCL2, and VCAM-1 in sepsis group were increased. Compared with sepsis group, the above-mentioned molecular levels of pae group were significantly decreased [TNFα: (63.39±9.55)pg/ml vs. (126.54±19.17)pg/ml ;IL-6: (64.03±8.82)pg/ml vs. (85.60±9.52)pg/ml;IL-1β: (69.52±9.23)pg/ml vs. (130.45±15.10)pg/ml;CXCL1: (2 600.19±379.54)pg/ml vs. (4 903.89±533.42)pg/ml;CXCL2: (93.71±10.83)pg/ml vs. (127.24±13.92)pg/ml;VCAM-1: (112.22±13.49)pg/ml vs. (149.32±15.65)pg/ml, both P<0.05]. RT-qPCR results showed that the mRNA expressions of TNFα, IL-6, IL-1β, CXCL1, CXCL2 and VCAM-1 in the sepsis group were increased compared with the control group; Compared with sepsis group, the IL-6 mRNA (1.271±0.139 vs. 1.920±0.191, P<0.05), IL-1βmRNA (1.180±0.130 vs. 1.817±0.191, P<0.05), VCAM-1 mRNA (1.088±0.144 vs. 1.460±0.166, P<0.05) expression decreased significantly in the pae group. Compared with control group, the levels of P-p38MAPK and P-Src in sepsis group increased, and the level of VE-cadherin decreased. Compared with sepsis group, the levels of p38MAPK and P-p38MAPK in pae group were significantly decreased, and the level of VE-cadherin was increased (p38MAPK/β-actin: 1.125±0.078 vs. 1.520±0.164; P-p38MAPK protein: 1.639±0.133 vs. 2.112±0.222; both P<0.05). Conclusion: Paeoniflorin could improve the permeability of cardiac microvascular endothelium in sepsis rats and inhibit the secretion and expression of inflammation-related proteins and genes, which might be related to the inhibition of Src/VE-cadherin pathway by paeoniflorin.

Involvement of O-GlcNAcylation in vascular damage during septic shock

Septic shock is characterized by a systemic inflammatory response syndrome (SIRS) causing hypotension and systemic hypoperfusion leading to altered cellular metabolism. This pathology is accompanied by vascular leakage due to endothelial damage. O-GlcNAcylation is a post-translational modification involved in the stress response. Recently, stimulation of O-GlcNAcylation has been shown to improve cardiovascular function in several murine models of septic shock. An O-GlcNAcylome study identified VE-cadherin and ZO-1 as potential interesting target, which are two major proteins involved in vascular leakage. We hypothesize that stimulation of O-GlcNAcylation may be involved in the regulation of vascular damage during early septic shock. Septic shock was induced in rats ( n = 10–12) by cecal ligation and punction (CLP). At 8 h and 22 h post-surgery, CLP rats receive fluid therapy with or without OGA inhibitor, NButGT (10 mg/kg) to increase O-GlcNAcylation levels. Cardiovascular functions were assessed and aortic arteries and heart were harvested and analyzed at 24 h post-surgery. Expression levels of O-GlcNAcylation, VE-cadherin and ZO-1 were assessed by western blot. The localization of VE-cadherin and ZO-1 was analyzed in the aorta. Evaluation of O-GlcNAcylation on VE-cadherin and ZO-1 were performed by immunoprecipitation and wheat germ agglutinin (WGA) gel. The results of the involvement of NButGT treatment on CLP rats show a cardiovascular improvement through mean arterial pressure (SHAM: 101.2 ± 1.65; CLP: 87.8 ± 4.67; NButGT: 102.9 ± 3.42; n = 10–12; P < 0.05). VE-Cadherin expression in the heart seems to be decreased in CLP group and this expression was significantly reversed with the NButGT treatment (SHAM: 1.00 ± 0.05; CLP: 0.86 ± 0.11; NButGT: 1.27 ± 0.10; n = 6–12; P < 0.05). WGA gel has made it possible to confirm that VE-Cadherin is O-GlcNAcylated. This study will allow us to better understand the mechanisms involved in vascular damage following the improvement of cardiovascular functions by O-GlcNAcylation stimulation in our septic shock model.

Implications of Withaferin A for the metastatic potential and drug resistance in hepatocellular carcinoma cells via Nrf2-mediated EMT and ferroptosis

Hepatocellular carcinoma (HCC) constitutes a major global health threat due to the high incidence and mortality. Sorafenib is known as the first-line medication for advanced HCC; however, it only extends the limited benefit for HCC patients as the development of acquired resistance. Withaferin A exerts broad pharmaceutical applications in several cancers. However, its effects on HCC cell metastatic potential and sorafenib resistance remain elusive. Here, we corroborated that Withaferin A greatly restrained cell viability, invasion, vasculogenic mimicry (VM) formation, and VE-cadherin levels in HepG2 and SNU449 cells. Moreover, Withaferin A sensitized sorafenib (SR)-resistant HCC cells to sorafenib. In striking contrast to the parental cells, lower ferroptosis was observed in SR-resistant cells as the lower ROS, MDA, and higher intracellular GSH levels in SR-resistant cells. Of interest, Withaferin A enhanced ferroptosis in SR-resistant cells, which was reversed by ferroptosis antagonist liproxstation-1. Notably, Withaferin A elevated Keap1 expression to mitigate Nrf2 signaling activation-mediated epithelial to mesenchymal transition (EMT) and ferroptosis-related protein xCT expression. Importantly, blockage of the Keap1/Nrf2 signaling overturned Withaferin A-evoked ferroptosis and facilitated sorafenib resistance. In addition, knockdown of Keap1 antagonized the inhibitory efficacy of Withaferin A on HCC cell viability, invasion, and VM formation. Consequently, Withaferin A may attenuate the metastatic potential and sorafenib resistance by regulating Keap1/Nrf2-associated EMT and ferroptosis. Thus, Withaferin A may serve as a promising agent for HCC therapy, especially for advanced HCC.

Abstract 133: Coagulation FXI Regulates Endothelial Cell Barrier Function By Inducing VE-cadherin Proteolysis In An ADAM10-dependent Manner

Introduction: Proteolysis of vascular endothelial (VE)-cadherin is a biomarker for disruption of endothelial cell (EC) barrier function, with elevated levels of soluble VE-cadherin associated with atherosclerosis and sepsis. Factor XI (FXI), known for its role in the coagulation cascade, also plays a regulatory role in inflammation. While a relationship between FXI activity, VE-cadherin expression levels, and endothelial barrier function has been observed, the mechanisms by which this process is mediated are unknown. Hypothesis: Activated FXI (FXIa) regulates EC barrier function by inducing VE-cadherin proteolysis. Methods: Human umbilical vein endothelial cells were stimulated with FXIa (30 and 5 nM) for six hours. VE-cadherin proteolysis and expression levels were analyzed by Western blot and immunofluorescence. ECs permeability was quantified by measuring the flux of Evans blue-albumin across the ECs. Immunoprecipitation and Western blot analysis were used to study FXIa-PAI-1 or -very low density lipoprotein receptor (VLDLR) interactions. Results: FXIa (5 nM) generated a VE-cadherin C-terminal fragment and decreased VE-cadherin expression on ECs; this process was reversed by the serine protease inhibitor, PPACK. Thrombin, kallikrein, or FXIIa did not induce VE-cadherin proteolysis. FXIa increased ECs permeability, yet did not induce the expression of VCAM-1. In contrast, while VCAM-1 expression was upregulated by tumor necrosis factor alpha or vascular endothelial growth factor, neither of these induced VE-cadherin proteolysis. A pharmacological inhibitor of a disintegrin and metalloproteinase 10 (ADAM10) abrogated the effects of FXIa on ECs, including proteolysis of VE-cadherin. Similarly, the low density lipoprotein antagonist, receptor-associated protein (RAP), inhibited FXIa-induced VE-cadherin proteolysis by preventing FXIa complex formation with PAI-1 and VLDLR on ECs. Conclusion: These results suggest that FXIa disrupts VE-cadherin-mediated EC permeability by increasing ADAM10 activity through complex formation and internalization by PAI-1 and VLDLR. It remains to be seen whether this pathway represents a druggable target to protect EC barrier function in inflammatory diseases by inhibiting FXIa.

Placental IL‐1β impairs endothelial functions by reducing VE‐cadherin expression in PE

Preeclampsia (PE) is a pregnancy‐specific syndrome that affects 5~8% pregnancies and is a leading cause of maternal and neonatal morbidity and mortality, especially in low‐income and middle‐income countries. Widespread systemic inflammation and endothelial dysfunction are hallmarks of injury in PE, however, the molecular mechanisms linking inflammation and endothelial dysfunction remain largely unknown. We tested the hypothesis that placental IL‐1β impairs endothelial functions by inhibiting VE‐cadherin expression in PE. We found that NLRP3 inflammasome was overactivated in preeclamptic placenta, which induced more IL‐1β release. We also observed that IL‐1β significantly increased endothelial permeability of Human Umbilical Vein Endothelial Cells (HUVECs) in a dose‐response manner, and inhibition of IL‐1β receptor significantly attenuated the increase of endothelial permeability induced by IL‐1β. We further revealed that both mRNA and protein level of VE‐cadherin was reduced by IL‐1β in HUVECs. and overexpression of VE‐cadherin in endothelial cells reversed IL‐1β‐induced increase in endothelial permeability. Taken together, our preliminary data suggested that excessive placental IL‐1β significantly impaired endothelial functions by inhibiting VE‐cadherin expression.

Correlation of vasoactive and neuroendocrine mechanisms with the state of antioxidant defense in patients with arterial hypertension

Introduction. Antioxidants deficiency with excess of endoperoxides leads to damage of intracellular structures, aggravating the course of most diseases, including cardiovascular pathology. Given the important role of antioxidants in the regulation of physiological processes in cells, the aim of this research was to study the effect of the antioxidant status of blood serum on the expression of pro-inflammatory and vasoactive molecules by blood cells, as well as markers of the metabolic syndrome in the aspect of clarifying possible mechanisms of the pathogenesis of arterial hypertension (AH). Materials and methods. As part of the cohort study, 60 patients of both sexes with hypertension from 45 to 55 years and 15 practically healthy individuals were examined. During the study, we determined in the blood serum the concentrations of insulin, glucagon, apoA1, apoB100, nitric oxide (NO), angiotensin-II (AT-II), E-selectin, P-selectin, intercellular adhesion molecule ICAM1, vascular adhesion molecule VCAM1, VE-cadherin, epinephrine, norepinephrine, endothelin-1, vasopressin, brain natriuretic peptide (BNP), antioxidants (AOS), urokinase-type plasminogen (uPA), plasma endoperoxides (OXY), antiotensin II receptor type 1 (AT-IIR ), plasminogen inhibitor type 1 (PAI1), C-reactive protein (CRP). Results. In patients with AH an increase in the concentration of the studied adhesion molecules was revealed, with significant decrease in the level of VE-cadherin. An increase in the level of vasopressors and decrease NO production was also found. These changes were accompanied by a decrease in the level of apoA1, an increase of the levels of apoB100, insulin, BNP, uPA, PAI1, and CRP. At the same time, an increase in the level of OXY was noted, with a reduced concentration of AOS. It has been established that AOX promote decreasing of the adhesion molecules expression, as well as the production of studied vasopressors, including AT-II, endothelin-1, BNP, insulin, AROB, CRP and stimulates the level of VE-cadherin. Conclusions. AH proceed with activation of vasopressor mechanisms and sympathetic regulation, accompanied by an increase in the adhesive activity of blood cells and endothelium, as well as metabolic disorders and activation of peroxide oxidation of lipids. These changes are associated with AOS deficiency. A correlation between AOS deficiency, laboratory manifestations of a subclinical intravascular inflammatory process, increased aggregation of blood cells, dyslipidemia and dysmetabolic manifestations, as well as dysfunction of the vascular endothelium and myocardium has been established. At the same time, a high level of AOS, in comparison with its low level, is associated with a lower expression of adhesion molecules, a lower level of vasopressor molecules, especially AT-II, a decrease in the level of apoB100 and insulin, as well as a higher expression of VE-cadherin.

PD146176 affects human EA.hy926 endothelial cell function by differentially modulating oxylipin production of LOX, COX and CYP epoxygenase

Oxylipins are oxygenated derivatives of polyunsaturated fatty acids, generated by COX, LOX and CYP enzymes, that regulate various aspects of endothelial cell physiology. Although 15-LOX and its products are positively associated with atherosclerosis, the relevant mechanisms have not been explored. The current study examined the effects of PD146176 (PD), a putative 15-LOX inhibitor, on EA.hy926 endothelial cell functions in the growing and confluent states. The effects of PD on endothelial cell oxylipin production (profiled by LC/MS/MS), cell viability, proliferation, eNOS activity, ICAM-1 and VE-cadherin levels were assessed. The contribution of signaling pathways relevant to endothelial function (p38 MAPK, Akt, PPARα) were also investigated. PD treatment for 30 min did not block formation of individual 15-LOX oxylipins, but 20 μM PD stimulated the accumulation of total LOX and COX products, while reducing several individual CYP products generated by epoxygenase. At 20 μM, the accumulated total oxylipins were primarily LOX-derived (86%) followed by COX (12%) and CYP (2%). PD altered cell functions by upregulating p38 MAPK and PPARα and downregulating Akt in a dose-dependent fashion. These observations suggest a link between PD-induced changes in oxylipins and altered endothelial cell functions via specific signaling pathways. In conclusion, the results of this study imply that PD does not function as a 15-LOX inhibitor in EA.hy926 endothelial cells, and instead inhibits CYP epoxygenase. These findings suggest that the cellular function changes induced by PD may be contingent upon its ability to modulate total oxylipin production, particularly by the LOX and CYP families.

Cadmium exposure enhances VE-cadherin expression in endothelial cells via suppression of ROCK signaling.

Vascular endothelium is a target of cadmium (Cd), which is a global pollutant of the environment. However, the detailed effects and underlying mechanisms remain to be elucidated. In the present study, human umbilical vein endothelial cells (HUVECs) were treated with 0.1, 1, 5, 10, 50 µM cadmium chloride (CdCl2) for 12 h. It was found that vascular endothelial (VE)-cadherin mRNA and protein expression was upregulated by Cd in HUVECs in a dose-dependent manner. Higher levels of VE-cadherin were detected at cell-to-cell junctions in HUVECs treated with 10 µM CdCl2 compared with normal condition. The phosphorylation level of myosin-binding subunit of myosin phosphatase, a downstream substrate of Rho-associated protein kinase (ROCK), was reduced by 10 µM CdCl2, suggesting that Cd inhibited the Rho/ROCK pathway. Activation of ROCK by narciclasine reversed the Cd-induced increase of VE-cadherin expression. By contrast, ROCK pathway inhibitor Y27632 increased VE-cadherin expression in HUVECs. Following inhibition of the ROCK pathway, Cd did not significantly alter the level of VE-cadherin. Taken together, the results suggested that Cd exposure enhanced VE-cadherin expression in endothelial cells via suppression of ROCK signaling.

Pirfenidone exacerbates Th2-driven vasculopathy in a mouse model of systemic sclerosis-associated interstitial lung disease.

Systemic sclerosis (SSc) is an autoimmune disease characterised by severe vasculopathy and fibrosis of various organs including the lung. Targeted treatment options for SSc-associated interstitial lung disease (SSc-ILD) are scarce. We assessed the effects of pirfenidone in a mouse model of SSc-ILD. Pulmonary function, inflammation and collagen deposition in response to pirfenidone were assessed in Fra-2-overexpressing transgenic (Fra-2 TG) and bleomycin-treated mice. In Fra-2 TG mice, lung transcriptome was analysed after pirfenidone treatment. In vitro, pirfenidone effects on human eosinophil and endothelial cell function were analysed using flow cytometry-based assays and electric cell-substrate impedance measurements, respectively. Pirfenidone treatment attenuated pulmonary remodelling in the bleomycin model, but aggravated pulmonary inflammation, fibrosis and vascular remodelling in Fra-2 TG mice. Pirfenidone increased interleukin (IL)-4 levels and eosinophil numbers in lung tissue of Fra-2 TG mice without directly affecting eosinophil activation and migration in vitro. A pronounced immune response with high levels of cytokines/chemokines and disturbed endothelial integrity with low vascular endothelial (VE)-cadherin levels was observed in pirfenidone-treated Fra-2 TG mice. In contrast, eosinophil and VE-cadherin levels were unchanged in bleomycin-treated mice and not influenced by pirfenidone. In vitro, pirfenidone exacerbated the IL-4 induced reduction of endothelial barrier resistance, leading to higher leukocyte transmigration. This study shows that antifibrotic properties of pirfenidone may be overruled by unwanted interactions with pre-injured endothelium in a setting of high T-helper type 2 inflammation in a model of SSc-ILD. Careful ILD patient phenotyping may be required to exploit benefits of pirfenidone while avoiding therapy failure and additional lung damage in some patients.

Trimetazidine mitigates high glucose-induced retinal endothelial dysfunction by inhibiting PI3K/Akt/mTOR pathway-mediated autophagy

ABSTRACT Trimetazidine (TMZ), as a metabolic regulator, has been widely testified to exhibit positive therapeutic effects on various disease models, but its role in diabetic retinopathy has not been reported. Therefore, this study was designed with the purpose of exploring the effects of TMZ on high-glucose (HG)-induced retinal endothelial dysfunction and its underlying mechanism. To establish DR model in vitro, 30 mM glucose was applied to induce human retinal endothelial cells (HRECs). Cell proliferation, invasion, and migration were examined by means of Cell Counting Kit-8, transwell, and wound healing assays, respectively. The tubule formation experiment was used to test the tubulogenesis ability and fluorescein isothiocyanate (FITC)-albumin was utilized to measure the permeability of monolayer HRECs. In addition, immunofluorescence and Western blot were employed to detect protein expression. Compared with the HG-induced group, TMZ concentration dependently inhibited the proliferation, migration, and angiogenesis of HG-induced HRECs, decreased the permeability of monolayer HRECs, and increased the protein expression levels of Claudin-5 and VE-cadherin. In addition, TMZ intervention increased the expression of p-PI3K, p-AKT, and p-mTOR but decreased the expression of LC3I, LC3II, and Beclin 1, which were then partially reversed by P13 K inhibitor (LY294002). Moreover, the autophagy agonist rapamycin (RAPA) was also testified to reverse the inhibitory effects of TMZ on the proliferation, migration, and angiogenesis of HG-induced HRECs. In summary, TMZ inhibited excessive autophagy by activating PI3K/Akt/mTOR pathway, thereby improving retinal endothelial dysfunction induced by HG.

Dual VEGF/PDGF knockdown suppresses vasculogenic mimicry formation in choroidal melanoma cells via the Wnt5a/β-catenin/AKT signaling pathway

ObjectiveThis study aimed to explore the effects of knocking down both vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF) on vasculogenic mimicry (VM) formation in choroidal melanoma (CM) cells. MethodsCell counting Kit (CCK)−8, monoclonal formation, wound healing, transwell and flow cytometry assays were used to observe the cell effects in CM cell line, ocular choroidal melanoma-1 cells (OCM-1) with respect to proliferation, migration, invasion and apoptosis. Three-dimensional (3D) cultures were also used to characterize VM tube structural effects in OCM-1 cells and western blotting was used to characterize protein expression changes in VM-related markers. ResultsDual VEGF/PDGF knockdown suppressed cell proliferation, migration and invasion, but promoted cell apoptosis. It also reduced VM tube structures in OCM-1 cells. VM associated markers including, VE-cadherin, EphA2 and MT1-MMP were also down-regulated in OCM-1 cells. Similarly, Wnt5a, β-catenin and phosphorylated-AKT levels were also down-regulated. Western blotting and 3D cultures further demonstrated that combined Wnt5a silencing with dual VEGF/PDGF knockdown significantly decreased VE-cadherin and EphA2 levels and reduced VM tube structures in OCM-1 cells. ConclusionsDual VEGF/PDGF knockdown suppressed cell growth and metastasis in OCM-1 cells, and blocked the Wnt5a/β-catenin/AKT signaling pathway thereby inhibiting VM formation.