VE-cadherin Expression Research Articles

Novel miRNA-based drug CD5-2 reduces liver tumor growth in diethylnitrosamine-treated mice by normalizing tumor vasculature and altering immune infiltrate.

Liver cancers exhibit abnormal (leaky) vasculature, hypoxia and an immunosuppressive microenvironment. Normalization of tumor vasculature is an emerging approach to treat many cancers. Blockmir CD5-2 is a novel oligonucleotide-based inhibitor of the miR-27a interaction with VE-Cadherin, the endothelial-specific cadherin. The combination of a vasoactive medication with inhibition of immune checkpoints such as programmed cell death protein 1 (PD1) has been shown to be effective in treating liver cancer in humans. We aimed to study the effect of CD5-2 combined with checkpoint inhibition (using an antibody against PD1) on liver tumor growth, vasculature and immune infiltrate in the diethylnitrosamine (DEN)-induced liver tumor mouse model. We first analyzed human miR-27a and VE-Cadherin expression data from The Cancer Genome Atlas for hepatocellular carcinoma. CD5-2 and/or anti-PD1 antibody were given to the DEN-treated mice from age 7-months until harvest at age 9-months. Tumor and non-tumor liver tissues were analyzed using histology, immunohistochemistry, immunofluorescence and scanning electron microscopy. Human data showed high miR-27a and low VE-Cadherin were both significantly associated with poorer prognosis. Mice treated with CD5-2 plus anti-PD1 antibody had significantly smaller liver tumors (50% reduction) compared to mice treated with either agent alone, controls, or untreated mice. There was no difference in tumor number. Histologically, tumors in CD5-2-treated mice had less leaky vessels with higher VE-Cadherin expression and less tumor hypoxia compared to non-CD5-2-treated mice. Only tumors in the combination CD5-2 plus anti-PD1 antibody group exhibited a more favorable immune infiltrate (significantly higher CD3+ and CD8+ T cells and lower Ly6G+ neutrophils) compared to tumors from other groups. CD5-2 normalized tumor vasculature and reduced hypoxia in DEN-induced liver tumors. CD5-2 plus anti-PD1 antibody reduced liver tumor size possibly by altering the immune infiltrate to a more immunosupportive one.

Skeletal muscle-derived FSTL1 starting up angiogenesis by regulating endothelial junction via activating Src pathway can be upregulated by hydrogen sulfide.

Hydrogen sulfide (H2S) promotes microangiogenesis and revascularization after ischemia. Neovascularization starts with the destruction of intercellular junctions and is accompanied by various endothelial cell angiogenic behaviors. Follistatin-like 1 (FSTL1) is a cardiovascular-protective myokine that works against ischemic injury. The present study examined whether FSTL1 was involved in H2S-induced angiogenesis and explored the underlying molecular mechanism. We observed that H2S accelerated blood perfusion after ischemia in the mouse hindlimb ischemia model. Western blot analysis showed that H2S stabilized FSTL1 transcript and increased FSTL1 and Human antigen R (HuR) levels in skeletal muscle. RNA-interference HuR significantly inhibited the H2S-promoted increase in FSTL1 levels. Exogenous FSTL1 promoted the wound-healing migration of human umbilical vein endothelial cells (HUVECs) and increased monolayer endothelial barrier permeability. Immunostaining showed that FSTL1 increased interendothelial gap formation and decreased VE-Cadherin, Occludin, Connexin-43, and Claudin-5 expression. In addition, FSTL1 significantly increased the phosphorylation of Src and VEGFR2. However, the Src inhibitor, not the VEGFR2 inhibitor, could block FSTL1-induced effects in angiogenesis. In conclusion, we demonstrated that H2S could upregulate the expression of FSTL1 by increasing the HuR levels in skeletal muscle, and paracrine FSTL1 could initiate angiogenesis by opening intercellular junctions via the Src signaling pathway.NEW & NOTEWORTHY The myocyte-derived paracrine protein FSTL1 acts on vascular endothelial cells and initiates the process of angiogenesis by opening the intercellular junction via activating Src kinase. H2S can significantly upregulate FSTL1 protein levels in skeletal muscles by increasing HuR expression.

Vascular Immune Evasion of Mesenchymal Glioblastoma Is Mediated by Interaction and Regulation of VE-Cadherin on PD-L1.

The mesenchymal subtype of glioblastoma (mGBM), which is characterized by rigorous vasculature, resists anti-tumor immune therapy. Here, we investigated the mechanistic link between tumor vascularization and the evasion of immune surveillance. Clinical datasets with GBM transcripts showed that the expression of the mesenchymal markers YKL-40 (CHI3L1) and Vimentin is correlated with elevated expression of PD-L1 and poor disease survival. Interestingly, the expression of PD-L1 was predominantly found in vascular endothelial cells. Orthotopic transplantation of glioma cells GL261 over-expressing YKL-40 in mice showed increased angiogenesis and decreased CD8+ T cell infiltration, resulting in a reduction in mouse survival. The exposure of recombinant YKL-40 protein induced PD-L1 and VE-cadherin (VE-cad) expression in endothelial cells and drove VE-cad-mediated nuclear translocation of β-catenin/LEF, where LEF upregulated PD-L1 expression. YKL-40 stimulated the dissociation of VE-cad from PD-L1, rendering PD-L1 available to interact with PD-1 from CD8+-positive TALL-104 lymphocytes and inhibit TALL-104 cytotoxicity. YKL-40 promoted TALL-104 cell migration and adhesion to endothelial cells via CCR5-dependent chemotaxis but blocked its anti-vascular immunity. Knockdown of VE-cad or the PD-L1 gene ablated the effects of YKL-40 and reinvigorated TALL-104 cell immunity against vessels. In summary, our study demonstrates a novel vascular immune escape mechanism by which mGBM promotes tumor vascularization and malignant transformation.

Sodium Butyrate as Key Regulator of Mitochondrial Function and Barrier Integrity of Human Glomerular Endothelial Cells.

The gut microbiota has emerged as an important modulator of cardiovascular and renal homeostasis. The composition of gut microbiota in patients suffering from chronic kidney disease (CKD) is altered, where a lower number of bacteria producing short chain fatty acids (SCFAs) is observed. It is known that SCFAs, such as butyrate and acetate, have protective effects against cardiovascular diseases and CKD but their mechanisms of action remain largely unexplored. In the present study, we investigated the effect of butyrate and acetate on glomerular endothelial cells. Human glomerular microvascular endothelial cells (hgMVECs) were cultured and exposed to butyrate and acetate and their effects on cellular proliferation, mitochondrial mass and metabolism, as well as monolayer integrity were studied. While acetate did not show any effects on hgMVECs, our results revealed that butyrate reduces the proliferation of hgMVECs, strengthens the endothelial barrier through increased expression of VE-cadherin and Claudin-5 and promotes mitochondrial biogenesis. Moreover, butyrate reduces the increase in oxygen consumption induced by lipopolysaccharides (LPS), revealing a protective effect of butyrate against the detrimental effects of LPS. Taken together, our data show that butyrate is a key player in endothelial integrity and metabolic homeostasis.

Reactive astrocytes associated with prion disease impair the blood brain barrier

BackgroundImpairment of the blood-brain barrier (BBB) is considered to be a common feature among neurodegenerative diseases, including Alzheimer's, Parkinson's and prion diseases. In prion disease, increased BBB permeability was reported 40 years ago, yet the mechanisms behind the loss of BBB integrity have never been explored. Recently, we showed that reactive astrocytes associated with prion diseases are neurotoxic. The current work examines the potential link between astrocyte reactivity and BBB breakdown. ResultsIn prion-infected mice, the loss of BBB integrity and aberrant localization of aquaporin 4 (AQP4), a sign of retraction of astrocytic endfeet from blood vessels, were noticeable prior to disease onset. Gaps in cell-to-cell junctions along blood vessels, together with downregulation of Occludin, Claudin-5 and VE-cadherin, which constitute tight and adherens junctions, suggested that loss of BBB integrity is linked with degeneration of vascular endothelial cells. In contrast to cells isolated from non-infected adult mice, endothelial cells originating from prion-infected mice displayed disease-associated changes, including lower levels of Occludin, Claudin-5 and VE-cadherin expression, impaired tight and adherens junctions, and reduced trans-endothelial electrical resistance (TEER). Endothelial cells isolated from non-infected mice, when co-cultured with reactive astrocytes isolated from prion-infected animals or treated with media conditioned by the reactive astrocytes, developed the disease-associated phenotype observed in the endothelial cells from prion-infected mice. Reactive astrocytes were found to produce high levels of secreted IL-6, and treatment of endothelial monolayers originating from non-infected animals with recombinant IL-6 alone reduced their TEER. Remarkably, treatment with extracellular vesicles produced by normal astrocytes partially reversed the disease phenotype of endothelial cells isolated from prion-infected animals. ConclusionsTo our knowledge, the current work is the first to illustrate early BBB breakdown in prion disease and to document that reactive astrocytes associated with prion disease are detrimental to BBB integrity. Moreover, our findings suggest that the harmful effects are linked to proinflammatory factors secreted by reactive astrocytes.

Targeted long-term noninvasive treatment of choroidal neovascularization by biodegradable nanoparticles.

Choroidal neovascularization (CNV) is the main cause of vision loss in patients with wet age-related macular degeneration (AMD). Currently, treatment of these conditions requires repeated intravitreal injections, which may lead to complications such as infection and hemorrhage. So, we have developed a noninvasive method for treating CNV with nanoparticles, namely, Angiopoietin1-anti CD105-PLGA nanoparticles (AAP NPs), which targets the CNV to enhance drug accumulation at the site. These nanoparticles, with PLGA as a carrier, can slowly release encapsulated Angiopoietin 1 (Ang 1) and target the choroidal neovascularization marker CD105 to enhance drug accumulation, increases vascular endothelial cadherin (VE-cadherin) expression between vascular endothelial cells, effectively reduce neovascularization leakage and inhibit Angiopoietin 2(Ang 2) secretion by endothelial cells. In a rat model of laser-induced CNV, intravenous injection of AAP NPs exerted a good therapeutic effect in reducing CNV leakage and area. In short, these synthetic AAP NPs provide an effective alternative treatment for AMD and meet the urgent need for noninvasive treatment in neovascular ophthalmopathy. STATEMENT OF SIGNIFICANCE: This work describes the synthesis, injection-mediated delivery, in vitro and in vivo efficacy of targeted nanoparticles with encapsulated Ang1; via these nanoparticles, the drug can be targeted to choroidal neovascularization lesions for continuous treatment. The release of Ang1 can effectively reduce neovascularization leakage, maintain vascular stability, and inhibit Ang2 secretion and inflammation. This study provides a new approach for the treatment of wet age-related macular degeneration.

Ethanol Enhances Endothelial Rigidity by Targeting VE-Cadherin-Implications for Acute Aortic Dissection.

(1) Background: Acute aortic dissection (AAD) is caused by an endothelial entry tear followed by intimomedial delamination of the outer layers of the vessel wall. The established risk factors include hypertension and smoking. Another rising candidate risk factor is excessive alcohol consumption. This experimental study explores the effects of nicotine (Nic), angiotensin II (Ang II), and ethanol (EtOH) on human aortic endothelial cells (hAoEC). (2) Methods: HAoECs were exposed to Nic, Ang II, and EtOH at different dose levels. Cell migration was studied using the scratch assay and live-cell imaging. The metabolic viability and permeability capacity was investigated using the water-soluble tetrazolium (WST)-1 assay and an in vitro vascular permeability assay. Cell adherence was studied by utilizing the hanging drop assay. The transcriptional and protein level changes were analyzed by RT-qPCR, Western blotting and immunohistochemistry for major junctional complexing proteins. (3) Results: We observed reduced metabolic viability following Ang II and EtOH exposure vs. control. Further, cell adherence was enhanced by EtOH exposure prior to trituration and by all risk factors after trituration, which correlated with the increased gene and protein expression of VE-cadherin upon EtOH exposure. The cell migration capacity was reduced upon EtOH exposure vs. controls. (4) Conclusion: Marked functional changes were observed upon exposure to established and potential risk factors for AAD development in hAoECs. Our findings advocate for an enhanced mechanical rigidity in hAoECs in response to the three substances studied, which in turn might increase endothelial rigidity, suggesting a novel mechanism for developing an endothelial entry tear due to reduced deformability in response to increased shear and pulsatile stress.

The therapeutic value of adipose-derived pericyte transplantation after intracerebral hemorrhage in rats.

Intracerebral hemorrhage (ICH) is a devastating cerebrovascular associated with inflammation and BBB disruption. Pericytes plays a critical role in neurological diseases, while whether pericytes could be utilized to treat ICH remains to be elucidated. Here, we isolated CD146+CD34- pericytes from rat adipose tissues (ADPs). Fluorescence-activated cells maintained their cell morphology and differentiation potential and expressed pericytes markers (CD146, NG2, and PDGFRβ) but not endothelial markers (CD31, CD34, and CD45). ADPs transplantation improved the neuro-behavioral functions in ICH rats and resulted in decreased hematoma volume and neuron loss after ICH. Besides, ADPs graft restrained the infiltration of neutrophils and reactive microgliosis after ICH injury around the peri-hematoma area of rats, as evidenced by increased Iba1- and MPO immunoreactivity. The transplanted pericytes were covered on endothelial cells, and promoted angiogenesis and vascular basement membrane formation in the peri-hematoma area of ICH rats, as shown by double staining of PDGFRβ and CD31/CollagenIV. The decreased brain water content and Evans Blue leakage proved the protective role of ADPs graft on BBB permeability. Finally, transplanted ADPs increased the expression of VE-cadherin, ZO-1, and claudin-5, leading to stable endothelial cell-cell adhesion and tight junction. In conclusion, the transplantation of APDs improved neuronal after ICH, which involved different mechanisms including neuroinflammation regulation and BBB dysfunction recovery. Our results supported that ADPs might be the ideal cell type for ICH therapy and provided insights into the potential cell therapy for further ICH treatment.

A novel peptide P1-121aa encoded by STK24P1 regulates vasculogenic mimicry via ELF2 phosphorylation in glioblastoma

Glioblastoma (GBM) is the most common malignant tumor of the central nervous system. Vasculogenic mimicry (VM) is a hematological system composed of tumor cells that exert blood perfusion without relying on vascular endothelial cells. The current poor results of anti-vascular therapy for clinical GBM are associated with the presence of VM; therefore, it is important to investigate VM formation in GBM. Our results demonstrate that STK24P1 encodes P1-121aa with a kinase structural domain, and in vitro kinase assays demonstrated that P1-121aa mediates modification of ELF2 phosphorylation. ChIP and dual luciferase reporter gene assays demonstrated that the transcription factor ELF2 binds to VE-cadherin and the VEGFR2 promoter region, thereby promoting VM formation in glioma cells. P1-121aa, encoded by the pseudogene STK24P1, phosphorylates ELF2 at S107, increasing the stability of the ELF2 protein. ELF2 promotes VEGFR2 and VE-cadherin expression at the transcriptional level, which in turn promotes VM in GBM. This study demonstrates the important roles of STK24P1, P1-121aa, and ELF2 in regulating VM in GBM, which could provide potential targets for GBM treatment.

The ACE2 activator diminazene aceturate ameliorates colitis by repairing the gut-vascular barrier in mice.

Alleviating vascular barrier injury improves colitis. Angiotensin converting enzyme 2/angiotensin 1-7/Mas receptor (ACE2/Ang1-7/MasR) axis-related drugs have various biological properties, such as inhibition of inflammation and fibrosis, but their role in improving the gut-vascular barrier (GVB) has rarely been reported. This study aims to investigate the effects of diminazene aceturate (DIZE), an ACE2 activator, on vascular barrier damage in colitis. Mice were randomly divided into three groups: control, dextran sulfate sodium salt (DSS), and DIZE+DSS. Mice in the DSS group drank DSS for 8days starting on day 4. Mice in the DIZE+DSS group were pregavaged with DIZE for 3days and then drank DSS for 8days while continuing to be gavaged with DIZE for 4days. Mice were euthanized and samples were collected on the last day. Injury to colonic structure and colonic microvasculature was assessed by visual observation and appropriate staining. DSS-induced colonic and microvascular pathological damage in mice was substantially reversed by DIZE treatment. Molecular pathways were investigated by Western blot, quantitative real-time polymerase chain reaction (qRT-PCR), and enzyme linked immunosorbent assay (ELISA). DSS treatment upregulated angiotensin converting enzyme (ACE), angiotensin type 1 receptor (AT1R) protein, pro-inflammatory cytokines and inhibited tight junction-related protein expression. DIZE treatment activated ACE2/MasR protein expression and reversed epithelial barrier damage and inflammatory infiltration during DSS injury. In addition, DIZE treatment inhibited vascular endothelial growth factor A/vascular endothelial growth factor receptor 2/proto-oncogene tyrosine-protein kinase Src (VEGFA/VEGFR2/Src) pathway activation and restored vascular adhesion-linker protein vascular endothelial cadherin (VE-cadherin) expression during DSS injury. In conclusion, DIZE treatment ameliorated colitis, which was associated with balancing the two axes of the renin-angiotensin system (RAS) and repairing the GVB injury.

Bortezomib Increased Vascular Permeability by Decreasing Cell–Cell Junction Molecules in Human Pulmonary Microvascular Endothelial Cells

Bortezomib (BTZ), a chemotherapeutic drug used to treat multiple myeloma, induces life-threatening side effects, including severe pulmonary toxicity. However, the mechanisms underlying these effects remain unclear. The objectives of this study were to (1) investigate whether BTZ influences vascular permeability and (2) clarify the effect of BTZ on the expression of molecules associated with cell–cell junctions using human pulmonary microvascular endothelial cells in vitro. Clinically relevant concentrations of BTZ induced limited cytotoxicity and increased the permeability of human pulmonary microvascular endothelial cell monolayers. BTZ decreased the protein expression of claudin-5, occludin, and VE-cadherin but not that of ZO-1 and β-catenin. Additionally, BTZ decreased the mRNA expression of claudin-5, occludin, ZO-1, VE-cadherin, and β-catenin. Our results suggest that BTZ increases the vascular permeability of the pulmonary microvascular endothelium by downregulating cell–cell junction molecules, particularly claudin-5, occludin, and VE-cadherin.

Cerebellin-2 promotes endothelial-mesenchymal transition in hypoxic pulmonary hypertension rats by activating NF-κB/HIF-1α/Twist1 pathway

AimsEndothelial-mesenchymal transition (EndMT) is one of the critical factors leading to vascular remodeling in pulmonary hypertension (PH). Recent studies found that the expression of Cerebellin-2 (CBLN2) is significantly increased in the lung tissue of patients with PH, suggesting that CBLN2 may be closely related to the development of PH. This study aims to investigate the role and potential mechanism of CBLN2 in the hypoxia-induced EndMT of PH rats. Material and methodsHypoxia-induced PH rat model or EndMT cell model was constructed to investigate the role of CBLN2 in the process of endothelial mesenchymal transition during PH. The effects of CBLN2 siRNA, KC7F2 (HIF-1α inhibitor), and PDTC (NF-κB inhibitor) on hypoxia-induced EndMT were observed to evaluate the potential mechanism of CBLN2 in promoting EndMT. Key findingsThe right ventricular systolic pressure and pulmonary vascular remodeling index in hypoxia-treated rats were significantly increased. The transformation of endothelial cells (marked by CD31) to mesenchymal cells (marked by α-SMA) can be observed in the pulmonary vessels of PH rats, and the expression of CBLN2 in the intima was also significantly up-regulated. In the hypoxia-induced HPAECs, endothelial cell markers such as VE-cadherin and CD31 expression were significantly down-regulated, while mesenchymal-like cell markers such as α-SMA and vimentin were increased considerably, along with the increased expressions of CBLN2, p-p65, HIF-1α, and Twist1; CBLN2 siRNA, PDTC, and KC7F2 could inhibit those phenomena. SignificanceCBLN2 can promote EndMT by activating NF-κB/HIF-1α/Twist1 pathway. Therefore, CBLN2 may be a new therapeutic target for PH.

#4000 ROLE OF ENDOTHELIAL CELLS IN VASCULAR CALCIFICATION

Abstract Background and Aims Vascular calcification (VC) represents a common pathological feature of cardiovascular disease and VC caused by hyperphosphatemia is one of the hallmarks of chronic kidney disease (CKD). Therefore, elucidating the pathogenesis of VC may have significant clinical benefits for CKD patients. Vascular smooth muscle cells challenged with high-phosphate (Pi) actively participate to VC by trans-differentiating into simil-osteoblastic cells that acquire the capability to deposit hydroxyapatite crystals in the extracellular matrix of tunica media in arteries. The role of endothelial cells (ECs) in high-Pi calcification has been poorly investigated until now. Nonetheless, following particular stimuli ECs can lose their endothelial characteristics and acquire several different phenotypes transdifferentiating with a process known as endothelial-to-mesenchymal transition (EndMT). Given the central role of VC for CKD patients wellbeing we decided to investigate whether ECs may have a role in VC process. Method We set up an in vitro calcification model with human aortic ECs (HAECs) challenged with 2, 2.5, 3, 3.5 mM high-Pi for 7 days. Calcium deposition, cell viability and cell trans-differentiation were evaluated. Results In our model we observed deposition of calcium phosphate crystals induced by high-Pi that resulted significant from 3 up to 3.5 mM Pi (Ctr 0.43 ± 0.01; 3 mM Pi 6.72 ± 1.15; 3.5 mM Pi 17.31 ± 1.88; micromol Ca++/mg protein; P < .01; Fig 1A-B). We found that calcification is mediated by Pi-influx, as demonstrated by the inhibition of calcium deposition by Phosphonoformic acid (PFA), an inhibitor of Na/Pi cotransporter Pit-1 (3.5 mM Pi 15.52 ± 1.97; 1 mM PFA 0.6 ± 0.05; micromol Ca++/mg protein; P < .0001; Fig 1A, C). In addition, in our model high-Pi did not show any significant toxic effect at every concentration tested compared to control cells. In Pi-treated ECs we observed a clear change in cell-morphology from cobblestone to spindle shaped, a recognized sign of EndMT. Moreover, we observed a change in extracellular matrix composition more similar to osteoblastic matrix. In facts, ECs treated with Pi were Alcian Blue positive, a staining that binds glycosaminoglycans contained in bone matrix, with blue granules of calcium-phosphate in the cytoplasm of treated cells. Starting from day 5 up to day 7 the transition was confirmed by an up-regulation of SNAIL and N-cadherin mRNA expressions, one of the master genes and one the markers of EndMT process, respectively. A modification of endothelial phenotype was also supported by a progressive decrease in protein expression of Von Willebrand factor and VE-Cadherin observed at day 9 of calcification (−53.7% and −32.6% respectively). Moreover, from day 5 till day 7 we found some osteoblastic differentiation signs such as a progressive increase of RUNX2 and BMP2 mRNA expression and an increase of protective MGP mRNA levels. Conclusion Our data demonstrate that HAECs are able to calcify. High-Pi induces trans-differentiation in simil-osteoblastic cells and a progressive loss of endothelial characteristics through the EndMT process. More studies are needed to better elucidate the role for ECs in CKD VC process.

S1PR1 attenuates pulmonary fibrosis by inhibiting EndMT and improving endothelial barrier function

BackgroundIdiopathic pulmonary fibrosis (IPF) is a chronic fatal disease of unknown etiology. Its pathological manifestations include excessive proliferation and activation of fibroblasts and deposition of extracellular matrix. Endothelial cell-mesenchymal transformation (EndMT), a novel mechanism that generates fibroblast during IPF, is responsible for fibroblast-like phenotypic changes and activation of fibroblasts into hypersecretory cells. However, the exact mechanism behind EndMT-derived fibroblasts and activation is uncertain. Here, we investigated the role of sphingosine 1-phosphate receptor 1 (S1PR1) in EndMT-driven pulmonary fibrosis. MethodsWe treated C57BL/6 mice with bleomycin (BLM) in vivo and pulmonary microvascular endothelial cells with TGF-β1 in vitro. Western blot, flow cytometry, and immunofluorescence were used to detect the expression of S1PR1 in endothelial cells. To evaluate the effect of S1PR1 on EndMT and endothelial barrier and its role in lung fibrosis and related signaling pathways, S1PR1 agonist and antagonist were used in vitro and in vivo. ResultsEndothelial S1PR1 protein expression was downregulated in both in vitro and in vivo models of pulmonary fibrosis induced by TGF-β1 and BLM, respectively. Downregulation of S1PR1 resulted in EndMT, indicated by decreased expression of endothelial markers CD31 and VE-cadherin, increased expression of mesenchymal markers α-SMA and nuclear transcription factor Snail, and disruption of the endothelial barrier. Further mechanistic studies found that stimulation of S1PR1 inhibited TGF-β1-mediated activation of the Smad2/3 and RhoA/ROCK1 pathways. Moreover, stimulation of S1PR1 attenuated Smad2/3 and RhoA/ROCK1 pathway-mediated damage to endothelial barrier function. ConclusionsEndothelial S1PR1 provides protection against pulmonary fibrosis by inhibiting EndMT and attenuating endothelial barrier damage. Accordingly, S1PR1 may be a potential therapeutic target in progressive IPF.

Gap26 inhibited angiogenesis through the β-catenin-VE-cadherin-VEGFR2-Erk signaling pathway

PurposeTo investigate the effect of connexin 43 (Cx43) on corneal neovascularization and its regulation of VEGFR2 on vascular endothelial cells. MethodsIn vivo, we used mouse corneal suture model to induce corneal neovascularization and discovered the function of gap26 in corneal neovascularization. In vitro, the effect of gap26 on HUVEC was observed by cell proliferation, tube formation and scratch experiments. WB and PCR detected the changes in angiogenic protein and mRNA expression. Knockdown of key mRNA in neovascularization using siRNA confirmed that Cx43 regulates neovascularization through the β-catenin-VE-cadherin-VEGFR2-Erk signaling pathway. ResultsIn vivo, gap26 can reduce mouse corneal neovascularization. In vitro, we show that Cx43 expression is increased in the presence of VEGFA stimulation, and when we use gap26 to inhibit Cx43 can reduce vascular endothelial cell proliferation, tube formation and migration. We found that the expression of pVEGFR2 and pErk increased in response to VEGFA, while they decreased after using gap26. And the expression of β-catenin and VE-cadherin decreased in response to VEGFA, while they increased after using gap26. Furthermore, we found that Cx43 regulates angiogenesis through the β-catenin-VE-cadherin-VEGFR2-Erk pathway. ConclusionsGap26 can downregulate VEGFR2 phosphorylation by stabilizing the expression of β-catenin and VE-cadherin on the cell membrane, thereby inhibiting VEGFA-induced HUVECs proliferation, migration and tube formation and inhibiting corneal neovascularization.

Effects of collagen, chitosan and mixture on fibroblast responses and angiogenic activities in 2D and 3D in vitro models.

Despite accumulating evidences have demonstrated the potential of collagen and chitosan on tissue repair, it remains unclear on their combination effects. Here, we examined the regenerative effects of single collagen, chitosan and their mixture on fibroblasts and endothelial cells at cellular levels. The results showed that fibroblast responses, as indicated by high proliferative rate, increased spheroid diameter and migrated area existing from spheroid edge, and decreased wound area, were significantly promoted by either collagen or chitosan stimulation. Similarly, both collagen and chitosan resulted in increased endothelial cell proliferation and migration with accelerated tube-like network formation and upregulated VE-cadherin expression, although collagen strongly provided this effect. While the 1:1 mixture (100:100 μg/mL of chitosan to collagen) treatment caused a reduction in fibroblast viability, the lower ratio of chitosan (1:10 mixture; 10:100 μg/mL) did not produce any impact on both fibroblast and endothelial cell viabilities. The 1:10 mixture also significantly enhanced the additional effects on fibroblast responses and angiogenic activities as shown by higher endothelial growth, proliferation and migration with accelerated capillary-like network formation than those treated with the single substance. Further investigation of signaling proteins found that collagen significantly increased expressions of p-Fak, p-Akt and Cdk5 whereas chitosan upregulated p-Fak and Cdk5 expressions. Comparing to the single treatments, p-Fak, p-Akt and Cdk5 were higher expressed in the 1:10 mixture. These observations indicate that proper collagen-chitosan mixture provides the combination effects on fibroblast responses and angiogenic activities when a high concentration of collagen is used, possibly through Fak/Akt and Cdk5 signaling pathways. Therefore, this study helps to define the clinical use of collagen and chitosan as promising biomaterials for tissue repair.

Tumor-expressed B7-H3 promotes vasculogenic mimicry formation rather than angiogenesis in non-small cell lung cancer.

Vasculogenic mimicry (VM), an alternative microvascular circulation independent ofangiogenesis, is formed by aggressive cancer cells. Tumor-expressedB7-H3 has been reported to promote VM formation in hepatocellular carcinoma and modulate angiogenesis in breast cancer and colorectal cancer. However, its effects on VM generation and angiogenesis in non-small cell Lung cancer (NSCLC) remained to be elucidated. CRISPR/Cas9-mediated B7-H3 knockout (KO)was conducted in NSCLC A549 and H3255 cells. The expression of VM-related proteins, including vascular endothelial (VE)-cadherin and matrix metalloproteinase 14 (MMP14), and the secretion of vascular endothelial growth factor (VEGF) were measured by western blotting and chemiluminescence assay in both B7-H3 KO and mock-edited A549 and H3255 cells. To examine VM formation, a three-dimensional (3D)culture model was used for B7-H3 KO and mock A549 and H3255 cells. For in vivo analysis, xenograft mice models were established using B7-H3 KO and mock-edited A549 cells, and immunohistochemical (CD31) and histochemical (periodic acid-Schiff, PAS) double staining were performed to identify VM and endothelial vessels in tumor tissues. Finally, specific signaling inhibitors were used to analyze B7-H3-induced signaling pathway responsible for VE-cadherin and MMP14 expression and VM generation. Higher expression of B7-H3 was associated with a worse prognosis and moreadvanced T-category in NSCLC. CRISPR/Cas9-mediated B7-H3 KOin A549 and H3255 cells led to decreased expression of VE-cadherin and MMP14; however,the secretion of VEGFby the two cell lines remained unchanged. In the3D cell culture model, bothB7-H3 KO A549 and H3255cells showed a significant reduction in the formation of capillary-like tubular structures compared to mock-edited cells. In thein vivo xenograft model, mock-edited A549 cells formed excessive PAS+ CD31-VM channels, while B7-H3 KO restrained VM formation in the xenograft tumors. However, no significant differenceswere found in CD31+ endothelial vessels between xenografts formed by B7-H3 KO and mock-edited A549 cells. Finally, we analyzed the signaling pathway responsible for B7-H3-induced VM formation and found that selective inhibition of the phosphoinositide 3-kinase(PI3K)/protein kinase B(AKT) hyperactivation by LY294002 was associated with decreased expression of MMP14 and VE-cadherin, and in vitro VM formation by both A549 and H3255 cells. Tumor-expressed B7-H3 acts via PI3K/AKT signaling pathway to promote VM formation by NSCLC cells while bears no effects on angiogenesis in NSCLC.

Age specific differences in murine alveolar endothelial cells to pathological conditions

Abstract Neonatal pulmonary viral infections can have diverse outcomes ranging from bronchiolitis and pneumonia in RSV infection to more subdued response in SARS-CoV-2 infection. In this study, we investigated the responses of murine alveolar endothelial cells (MLEC) to neonate mice serum in presence of TLR agonists-treated macrophages, which can mimic SARS-Cov2 induced innate cell activation. We found that when poly I:C and ssRNA40 activated macrophages were cocultured with neonatal mice serum-treated MLECs, the expression of TNFα and IL-6 were significantly decreased. Moreover, macrophage co-culture leads to decreased ACE2 and VEGFR2 in adult MLECs, but macrophage co-culture increased ACE2 expression in neonatal MLECs under Poly I:C but not ssRNA40 stimulation. Addition of neonatal mice serum to MLECs increases the reactive oxygen species but downregulates multiple inflammatory signal pathways. Concurrently, the expression VE-cadherin and Shp2 was downregulated in neonatal mice serum exposed MLECs, which leads to compromised MLEC barrier integrity. Morphologically, neonatal mice serum, but not adult mice serum, triggered the lipid droplets formation and upregulated SR-BI and CD36 receptor expression in MLECs. Taken together, neonatal and adult MLECs differentially regulate cytokine-secretion from activated macrophages, and neonatal serum breaks the tight cell junction between MLECs by down regulating Shp2 and VE-Cadherin expression and induces the lipid droplets formation in MLECs through receptors SR-BI and CD36. The differences in neonatal and adult MLEC responses to pathologic conditions may reflect the age specific pulmonary manifestations to viral infections.

Schisandrin A ameliorates increased pulmonary capillary endothelial permeability accompanied with sepsis through inhibition of RhoA/ROCK1/MLC pathways.

Sepsis is a systemic inflammatory response, and vascular leakage associated with acute lung injury (ALI) is an important pathophysiological process during sepsis. Schisandrin A (SchA) is a bioactive lignan which has been reported to have the anti-inflammatory effects in many studies, while whether SchA can ameliorate ALI-related vascular leakage caused by sepsis is unknown. To evaluate the role and the underlying mechanism of SchA in increase of pulmonary vascular permeability induced by sepsis. The effect of SchA on pulmonary vascular permeability was examined in rat acute lung injury model. The effect of SchA on skin vascular permeability of mice was investigated through Miles assay. MTT assay was performed to detect the cell activity, and transwell assay was used to detect the effect of SchA on cell permeability. The effects of SchA on junction proteins and RhoA/ROCK1/MLC signaling pathway were manifested by immunofluorescence staining and western blot. The administration of SchA alleviated rat pulmonary endothelial dysfunction, relieved increased permeability in the mouse skin and HUVECs induced by lipopolysaccharide (LPS). Meanwhile, SchA inhibited the formation of stress fibers, reversed the decrease of expression of ZO-1 and VE-cadherin. Subsequent experiments confirmed that SchA inhibited RhoA/ROCK1/MLC canonical pathway in rat lungs and HUVECs induced by LPS. Moreover, overexpression of RhoA reversed the inhibitory effect of SchA in HUVECs, which suggested that SchA protected the pulmonary endothelial barrier by inhibiting RhoA/ROCK1/MLC pathway. In summary, our results indicate that SchA ameliorates the increase of pulmonary endothelial permeability induced by sepsis through inhibition of RhoA/ROCK1/MLC pathway, providing a potentially effective therapeutic strategy for sepsis.

Abstract 132: Mechanism By Which Coagulation Factor XI Regulates Endothelial Cell Permeability And Barrier Function In Vitro And In Vivo

Introduction: Proteolysis of vascular endothelial (VE)-cadherin is a biomarker for disruption of endothelial cell (EC) barrier function, with elevated levels of soluble VE (sVE)-cadherin associated with atherosclerosis and sepsis. Factor XI (FXI) plays a regulatory role in inflammation, and while a relationship between FXI activity and VE-cadherin expression levels has been observed, the mechanism by which this process is mediated is unknown. Hypothesis: Activated FXI (FXIa) induces VE-cadherin proteolysis. Methods: Human umbilical vein ECs were stimulated with FXIa (30 and 5 nM) for six hours. Western blot analysis was used to study VE-cadherin proteolysis, disintegrin and metalloproteinase 10 (ADAM10) expression, FXIa-PAI-1 or -very low density lipoprotein receptor (VLDLR) interactions, and cell signaling events. SVE-cadherin in baboons challenged with Staphylococcus aureus was detected by ELISA. Results: FXIa (5 nM) generated a VE-cadherin fragment and increased ECs permeability; the serine protease inhibitor, PPACK, and an inhibitor of ADAM10 reversed these processes. FXIa induced the expression of active ADAM10 on ECs. The receptor-associated protein (RAP) blocked FXIa-induced VE-cadherin proteolysis by preventing FXIa interaction with PAI-1 and VLDLR on ECs. FXIa-VLDLR interaction induced Dab1 and Src kinases phosphorylation. FXIa activated the VEGFR2-PLCgamma1-ERK1/2 signaling pathway. The ERK1/2 inhibitor, LY3214996, and the Src kinases inhibitor, PP2, inhibited VE-cadherin proteolysis by FXIa. PLCgamma1 phosphorylation was also prevented with PP2. The VEGFR2 kinase inhibitor, SU1498, prevented the cleavage of VE-cadherin by FXIa; in contrast, a VEGF blocking antibody had no effect on FXIa-induced VE-cadherin cleavage. Finally, the inhibition of FXI activation in a baboon model of sepsis significantly decreased the levels of sVE-cadherin. Conclusion: The complex formation of FXIa with PAI-1 and VLDLR promoted Src kinase phosphorylation. Src activation triggered the VEGFR2-PLCgamma1-ERK1/2 signaling pathway, which induced the trafficking of active ADAM10 to the EC surfaces inducing the cleavage of VE-cadherin. FXI may be a druggable target to protect EC barrier function in inflammatory diseases.