Endothelial Cell Integrins Research Articles

The Proangiogenic Effects of Melanoma-Derived Ectosomes Are Mediated by αvβ5 Integrin Rather than αvβ3 Integrin.

Ectosomes are carriers of proangiogenic factors during cancer progression. This study investigated whether the proangiogenic effect exerted by melanoma-derived ectosomes on recipient endothelial cells is mediated by ectosomal αvβ3 and αvβ5 integrins. Ectosomes were isolated from the conditioned culture media of four melanoma cell lines and melanocytes. Changes in gene and protein expression of αvβ3 and αvβ5 integrins, as well as VEGF and TNF-α were assessed in ectosome-treated endothelial cells. To confirm the functional involvement of ectosomal integrins in functional tests (Alamar Blue, wound healing and tube formation assays), ectosomes were also pretreated with anti-integrin antibodies and integrin-blocking peptides echistatin and cilengitide. Melanoma-derived ectosomes induced changes in the expression of αvβ3 and αvβ5 integrins in recipient endothelial cells, leading to increased viability, migratory properties, and tube formation potential. The extent of proangiogenic stimulation varied depending on the types of cells releasing ectosomes and the recipient cells. The use of anti-integrin antibodies and integrin-blocking peptides revealed a more significant role for the αvβ5 integrin/VEGF than the αvβ3 integrin/TNF-α pathway in the interactions between ectosomes and endothelial cells. The study demonstrated the functional role of ectosomal αvβ3 and αvβ5 integrins. It also provided a baseline understanding of ectosome-mediated αvβ3 integrin/TNF-α and αvβ5 integrin/VEGF signaling in angiogenesis.

Endothelial connexin-integrin crosstalk in vascular inflammation

Cardiovascular diseases including blood vessel disorders represent a major cause of death globally. The essential roles played by local and systemic vascular inflammation in the pathogenesis of cardiovascular diseases have been increasingly recognized. Vascular inflammation triggers the aberrant activation of endothelial cells, which leads to the functional and structural abnormalities in vascular vessels. In addition to humoral mediators such as pro-inflammatory cytokines and prostaglandins, the alteration of physical and mechanical microenvironment – including vascular stiffness and shear stress – modify the gene expression profiles and metabolic profiles of endothelial cells via mechano-transduction pathways, thereby contributing to the pathogenesis of vessel disorders. Notably, connexins and integrins crosstalk each other in response to the mechanical stress, and, thereby, play an important role in regulating the mechano-transduction of endothelial cells. Here, we provide an overview on how the inter-play between connexins and integrins in endothelial cells unfold during the mechano-transduction in vascular inflammation.

Computational Systems Biology Modeling of the Angiopoietin‐Tie Signaling Pathway and its Crosstalk with α5β1 Integrin in Endothelial Cells

The Angiopoietin (Ang)-Tie signaling pathway is a major signaling pathway regulating vascular growth (angiogenesis), stability, and permeability. Dysregulation in the pathway is known to lead to vascular dysfunction related to many diseases, including cardiovascular diseases, systemic inflammation, diabetic macular edema, and cancer. Integrin α5β1 has been demonstrated to play an important role in regulating Ang-Tie and its downstream signaling. The understanding of the molecular mechanisms of the α5β1/Ang-Tie crosstalk has been limited due to the complexity of the reaction network formed by the ligands, receptors, integrin interactions, and the molecular regulatory mechanisms. The present study uses a mechanistic computational model of the Ang-Tie signaling pathway and its crosstalk with α5β1 to demonstrate the ability of integrin α5β1 to modulate Tie2 activation by affecting the junctional localization of the co-receptor Tie1. The model provides a mechanistic explanation of how AXT107, an α5β1-modulating peptide, promotes the Tie2-activation function of Ang2 and predicts that simultaneously targeting α5β1 and VE-PTP can synergistically enhance the activation of Tie2 in endothelial cells in pathological states. The model is also used to make quantitative prediction of the effects of simultaneously targeting various aspects of the α5β1/Ang-Tie crosstalk, including the integrin, VE-PTP, and Ang2.

Optogenetics-based localization of talin to the plasma membrane promotes activation of β3 integrins

Interaction of talin with the cytoplasmic tails of integrin β triggers integrin activation, leading to an increase of integrin affinity/avidity for extracellular ligands. In talin KO mice, loss of talin interaction with platelet integrin αIIbβ3 causes a severe hemostatic defect, and loss of talin interaction with endothelial cell integrin αVβ3 affects angiogenesis. In normal cells, talin is autoinhibited and localized in the cytoplasm. Here, we used an optogenetic platform to assess whether recruitment of full-length talin to the plasma membrane was sufficient to induce integrin activation. A dimerization module (Arabidopsis cryptochrome 2 fused to the N terminus of talin; N-terminal of cryptochrome-interacting basic helix-loop-helix domain ended with a CAAX box protein [C: cysteine; A: aliphatic amino acid; X: any C-terminal amino acid]) responsive to 450 nm (blue) light was inserted into Chinese hamster ovary cells and endothelial cells also expressing αIIbβ3 or αVβ3, respectively. Thus, exposure of the cells to blue light caused a rapid and reversible recruitment of Arabidopsis cryptochrome 2–talin to the N-terminal of cryptochrome-interacting basic helix-loop-helix domain ended with a CAAX box protein [C: cysteine; A: aliphatic amino acid; X: any C-terminal amino acid]–decorated plasma membrane. This resulted in β3 integrin activation in both cell types, as well as increasing migration of the endothelial cells. However, membrane recruitment of talin was not sufficient for integrin activation, as membrane-associated Ras-related protein 1 (Rap1)–GTP was also required. Moreover, talin mutations that interfered with its direct binding to Rap1 abrogated β3 integrin activation. Altogether, these results define a role for the plasma membrane recruitment of talin in β3 integrin activation, and they suggest a nuanced sequence of events thereafter involving Rap1–GTP.

Single-Molecule Analysis Demonstrates Stress-Enhanced Binding between Staphylococcus aureus Surface Protein IsdB and Host Cell Integrins.

Binding of Staphylococcus aureus surface proteins to endothelial cell integrins plays essential roles in host cell adhesion and invasion, eventually leading to life-threatening diseases. The staphylococcal protein IsdB binds to β3-containing integrins through a mechanism that has never been thoroughly investigated. Here, we identify and characterize at the nanoscale a previously undescribed stress-dependent adhesion between IsdB and integrin αVβ3. The strength of single IsdB-αVβ3 interactions is moderate (∼100 pN) under low stress, but it increases dramatically under high stress (∼1000-2000 pN) to exceed the forces traditionally reported for the binding between integrins and Arg-Gly-Asp (RGD) sequences. We suggest a mechanism where high mechanical stress induces conformational changes in the integrin from a low-affinity, weak binding state to a high-affinity, strong binding state. This single-molecule study highlights that direct adhesin-integrin interactions represent potential targets to fight staphylococcal infections.

Integrin affinity modulation critically regulates atherogenic endothelial activation in vitro and in vivo

While vital to platelet and leukocyte adhesion, the role of integrin affinity modulation in adherent cells remains controversial. In endothelial cells, atheroprone hemodynamics and oxidized lipoproteins drive an increase in the high affinity conformation of α5β1 integrins in endothelial cells in vitro, and α5β1 integrin inhibitors reduce proinflammatory endothelial activation to these stimuli in vitro and in vivo. However, the importance of α5β1 integrin affinity modulation to endothelial phenotype remains unknown. We now show that endothelial cells (talin1 L325R) unable to induce high affinity integrins initially adhere and spread but show significant defects in nascent adhesion formation. In contrast, overall focal adhesion number, area, and composition in stably adherent cells are similar between talin1 wildtype and talin1 L325R endothelial cells. However, talin1 L325R endothelial cells fail to induce high affinity α5β1 integrins, fibronectin deposition, and proinflammatory responses to atheroprone hemodynamics and oxidized lipoproteins. Inducing the high affinity conformation of α5β1 integrins in talin1 L325R endothelial cells suggest that NF-κB activation and maximal fibronectin deposition require both integrin activation and other integrin-independent signaling. In endothelial-specific talin1 L325R mice, atheroprone hemodynamics fail to promote inflammation and macrophage recruitment, demonstrating a vital role for integrin activation in regulating endothelial phenotype.

RhoJ Regulates α5β1 Integrin Trafficking to Control Fibronectin Remodeling during Angiogenesis.

Rho guanosine triphosphatases (GTPases) are master regulators of cell shape and cell movement [1]. The archetypal family members RhoA, Rac1, and Cdc42 arose early in eukaryotic evolution and coordinate a diverse range of cell morphologies and migrations. Evolution of the vertebrates was paralleled by expansion of this family through gene duplication. Emergence of an adaptive immune system and morecomplex neural systems presented new roles for Rho GTPases, filled by new family members. Cdc42 underwent gene duplication to produce two related proteins-RhoQ and RhoJ [2]. RhoQ is active in neural dynamics; however, RhoJ is highly expressed in endothelial cells under control of the endothelial-specific promoter ERG [3, 4]. RhoJ is required for angiogenesis [5, 6] and has multiple rolesin this process [7, 8]. We recently demonstrated that RhoJ regulates the endosomal trafficking of podocalyxin during angiogenesis to control lumen formation [9]. Here, we use vesicle purification and proteomic analysis to identify the endothelial targets of RhoJ-mediated trafficking. We identify α5β1 integrin as a major RhoJ cargo and show that RhoJ regulates the intracellular trafficking of active α5β1 integrin in endothelial cells to repress fibronectin fibrillogenesis. Accordingly, mice lacking RhoJ show deregulated deposition of fibronectin around vessels during developmental angiogenesis. Intriguingly, we show that RhoJ acts in opposition to Cdc42 in this process through competition for a shared partner, PAK3. These studies identify a critical role for RhoJ in matrix remodeling during blood vessel formation and demonstrate a functional interrelationship between RhoJ and its evolutionary parent.

Angiopoietin-Tie Signaling Pathway in Endothelial Cells: A Computational Model.

SummaryThe angiopoietin-Tie signaling pathway is an important vascular signaling pathway involved in angiogenesis, vascular stability, and quiescence. Dysregulation in the pathway is linked to the impairments in vascular function associated with many diseases, including cancer, ocular diseases, systemic inflammation, and cardiovascular diseases. The present study uses a computational signaling pathway model validated against experimental data to quantitatively study various mechanistic aspects of the angiopoietin-Tie signaling pathway, including receptor activation, trafficking, turnover, and molecular mechanisms of its regulation. The model provides mechanistic insights into the controversial role of Ang2 and its regulators vascular endothelial protein tyrosine phosphatase (VE-PTP) and Tie1 and predicts synergistic effects of inhibition of VE-PTP, Tie1, and Tie2 cleavage on enhancing the vascular protective actions of Tie2.

Mechanostability of the Fibrinogen Bridge between Staphylococcal Surface Protein ClfA and Endothelial Cell Integrin αVβ3.

Binding of the Staphylococcus aureus surface protein clumping factor A (ClfA) to endothelial cell integrin αVβ3 plays a crucial role during sepsis, by causing endothelial cell apoptosis and loss of barrier integrity. ClfA uses the blood plasma protein fibrinogen (Fg) to bind to αVβ3 but how this is achieved at the molecular level is not known. Here we investigate the mechanical strength of the three-component ClfA-Fg-αVβ3 interaction on living bacteria, by means of single-molecule experiments. We find that the ClfA-Fg-αVβ3 ternary complex is extremely stable, being able to sustain forces (∼800 pN) that are much stronger than those of classical bonds between integrins and the Arg-Gly-Asp (RGD) tripeptide sequence (∼100 pN). Adhesion forces between single bacteria and αVβ3 are strongly inhibited by an anti-αVβ3 antibody, the RGD peptide, and the cyclic RGD peptide cilengitide, showing that formation of the complex involves RGD-dependent binding sites and can be efficiently inhibited by αVβ3 blockers. Collectively, our experiments favor a binding mechanism involving the extraordinary elasticity of Fg. In the absence of mechanical stress, RGD572-574 sequences in the Aα chains mediate weak binding to αVβ3, whereas under high mechanical stress exposure of cryptic Aα chain RGD95-97 sequences leads to extremely strong binding to the integrin. Our results identify an unexpected and previously undescribed force-dependent binding mechanism between ClfA and αVβ3 on endothelial cells, which could represent a potential target to fight staphylococcal bloodstream infections.

Abstract 1989: Breast cancer-derived exosomes modulate the endocytic pathway in brain endothelial cells

Abstract Breast cancer brain metastasis is associated with high mortality and has shown a rising trend in incidence during the recent years. Given the rapid progression of brain metastasis, elucidation of the early events that lead to brain metastasis will pave the way to identifying potential diagnostic and therapeutic targets for early intervention. To explore the early mechanisms of breast cancer brain metastasis, we focused on the role of breast cancer-derived exosomes in this process. While extensive studies have explored the role of tumor-derived exosomes in tumor progression, the current understanding of the contribution of tumor-derived exosomes to brain metastasis remains limited. Using a mouse model of brain metastasis, we showed that pre-treatment of mice with exosomes derived from the brain-seeking MDA-MB-231 breast cancer cell line (Br-Ex) increases brain metastasis growth. This facilitation of brain metastasis was not observed with exosomes derived from the parental MDA-MB-231 cells (P-Ex). It is widely acknowledged that during early stages of brain metastasis, tumor cells grow along the brain vasculature, the blood brain barrier (BBB). To understand the mechanisms of the exosome-derived facilitation of brain metastasis, we studied the interactions between exosomes and the BBB. Using state-of-the-art models of the BBB and high-resolution microscopy, we have identified, for the first time, the mechanisms by which Br-Ex modulate the endocytic pathway in brain endothelial cells to decrease exosome degradation. Interestingly, our mechanistic studies showed that these effects are induced through the transfer of exosomal miRNAs enriched in Br-EX. Moreover, we have shown that Br-Ex can exclusively change the expression of integrins in brain endothelial cells in a way that it could alter the microenvironment around the BBB in favor of metastatic tumor cells. These findings indicate that exosomes derived from a brain-seeking subpopulation of breast cancer cells can exclusively modify the physiological regulation of the BBB at multiple levels to accelerate metastatic growth in the brain microenvironment. Taken together, these studies increase our understanding of the early events that facilitate brain metastasis and provide multiple potential targets for diagnostic and therapeutic applications in brain metastasis. (This work was supported by the Breast Cancer Research Foundation and NIH R01CA185530.) Citation Format: Golnaz Morad, Christopher V. Carman, Marsha A. Moses. Breast cancer-derived exosomes modulate the endocytic pathway in brain endothelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1989.

Inhibition of α5β1integrin with the clinically validated small peptide, ATN‐161 stabilizes cerebral vasculature, reduces inflammation, and decreases blood‐brain barrier permeability after experimental ischemic stroke

Stroke is the second leading cause of death and the leading cause of long‐term disability worldwide. Blood‐brain barrier (BBB) dysfunction exacerbates reperfusion‐induced injury after recanalization in ischemic stroke. Endothelial cell integrin receptors, specifically the β1 subtype, play a direct role in this BBB dysfunction through regulation of barrier‐forming tight junction (TJ) proteins. We hypothesize that inhibition of a specific β1 integrin subtype, α5β1, will stabilize the BBB, and thereby reduce infarct volumes and improve functional recovery after experimental stroke.MethodMale C57BLJ6 mice underwent transient middle cerebral artery occlusion for 1 hour. Upon reperfusion, vehicle or the clinically validated α5β1 inhibitor, ATN‐161 (1mg/kg), was injected intraperitoneally and repeated 24 and 48 hours later. Infarct volume (TTC and MRI – DTI sequence) and edema volume (T2‐weighted MRI) was determined on PSD3. Functional recovery was determined by an 11‐point Neuroscore on PSD1‐PSD7. Physiological measurements, including pulse distention, heart rate and body temperature, were obtained before, during and after the initial dose of ATN‐161. Immunohistochemical analysis of α5β1, claudin‐5, CD45 (pan‐leukocyte), and IgG, expression was performed on PSD3. qPCR analysis on claudin‐5, collagen IV, MMP‐9, IL‐1β, and CXCL12 were performed on PSD3.ResultsATN‐161 administration reduced infarct volume, edema volume, and functional deficit. ATN‐161 also resulted in cerebral vascular stabilization by a decrease in MMP‐9 and increases in claudin‐5, and collagen IV. Furthermore, IL‐1β and CD45 was reducedupon ATN‐161 administration, suggesting a decrease in the post‐stroke inflammatory response. Together, the cerebrovascular stabilization and decrease in inflammation resulted in less IgG extraversion, suggesting a more intact BBB. Collectively, administration of ATN‐161 post‐stroke produced significant benefits, and thus could represent a novel stroke therapy worthy of further investigation.Support or Funding InformationThis work was supported by NIH R01NS065842.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract TP120: Inhibition of α5β1 Integrin With the Small Peptide ATN-161 Reduces Inflammation, Stabilizes the Blood-Brain Barrier, and is Neuroprotective After Experimental Ischemic Stroke

Endothelial cell integrin receptors, specifically the β 1 subtype, play a direct role in inflammation and blood-brain barrier (BBB) dysfunction, both implicated in poor ischemic stroke outcomes. We hypothesize that inhibition of a particular β1 integrin subtype, α5β1, (a pro-angiogenic fibronectin receptor acutely expressed after ischemic stroke) could be therapeutic in ischemic stroke via BBB stabilization. Methods: 3-month-old male, C57BL/J6 wildtype mice underwent transient middle cerebral artery occlusion for 1 hour. Mice were treated intraperitoneally with the small peptide α5β1 integrin inhibitor ATN-161 (1 mg/kg) upon reperfusion, post stroke day (PSD) 1 and PSD2. On PSD3, infarct volume (TTC and T2-weighted MRI) and vasogenic edema (MRI) were assessed. Neuroscore, an 11-point functional assessment, was performed on PSD1-14. Expression of MMP-9, claudin-5 and IL-1β levels were determined by qPCR. On PSD3, immunohistochemical analyses of α5 integrin, collagen IV, claudin-5, CD45, and DAPI expression were performed. In vitro oxygen-glucose deprivation (OGD) experiments on brain endothelial (bEND3) cells were also performed, evaluating ATN-161 administration (10 μM) effects on BEND3 layer permeability via FITC-dextran migration, and on immunocytochemistry of α5 integrin and claudin-5 expression. Results: Acute post-stroke administration of ATN-161 significantly decreased infarct volume and vasogenic edema, and improved functional recovery. Additionally, ATN-161 appeared to stabilize the BBB by increasing expression of the tight junction protein claudin-5, while decreasing expression of the extracellular matrix proteinase, MMP-9, inflammatory cytokine (IL-1β), and infiltrating leukocytes (CD45). Additionally, permeability was conserved with ATN-161 administration following OGD. Conclusion: Administration of the α5β1 inhibitor ATN-161 stabilizes the BBB barrier, and reduces inflammatory infiltration, while reducing infarct volume, vasogenic edema, and behavioral dysfunction following experimental stroke. Collectively, these results suggest inhibition of α5β1 via ATN-161 could represent a promising novel therapeutic approach for ischemic stroke.

Elucidation of Endothelial Cell Hemostatic Regulation with Integrin-Targeting Hydrogels.

Despite advances in the development of materials for cardiovascular devices, current strategies generally lack the thromboresistance of the native endothelium both in terms of efficacy and longevity. To harness this innate hemostatic regulation and improve long-term hemocompatibility, biohybrid devices are designed to promote endothelialization. Much of the research effort to date has focused on the use of extracellular matrix (ECM)-mimics and coatings to promote endothelial cell adhesion and migration with less attention given to the effect of the supported ECM binding events on hemostatic regulation. In this study, we developed integrin-targeted hydrogels to investigate the individual and combined effects of integrin binding events supported by many ECM-based coatings (α1β1, α2β1, α5β1, αvβ3). Targeted endothelial cell integrin interactions were first confirmed with antibody blocking studies and then correlated with gene expression of hemostatic regulators and a functional assay of platelet attachment and activation. Surfaces that targeted integrins α1β1 and α2β1 resulted in an endothelial cell layer that exhibited a thromboresistant phenotype with an associated reduction in platelet attachment and activation. It is anticipated that identification of specific integrins that promote endothelial cell adhesion as well as thromboresistance will enable the design of cardiovascular materials with improved long-term hemocompatibility.

Inhibition of Vascular Endothelial Cell Leak Following Escherichia coli Attachment in an Experimental Model of Sepsis.

The vascular endothelium is a major target of sepsis-induced events, and endothelial activation accounts for much of the pathology of sepsis. Urinary tract infections and pneumonia caused by Escherichia coli are among of the most common infections causing sepsis in both community and hospital settings. Currently, there are no approved drugs on the market to treat the underlying pathophysiology of sepsis. The aim of this study is to elucidate the molecular mechanism by which E. coli induces endothelial injury as a result of attachment. Laboratory research using a hemodynamic perfusion ex vivo model. Research Laboratories of Royal College of Surgeons in Ireland and Beaumont Hospital. Ex vivo human vascular endothelial cells. Addition of αVβ3 antagonist, cilengitide. Clinical strains of E. coli isolated from patients with sepsis bound to sheared human endothelial cells under static and hemodynamic shear conditions. Binding was dependent on E. coli cell membrane protein outer membrane protein A attaching directly to endothelial cell integrin αVβ3. Attachment resulted in disturbances in endothelial barrier integrity, as determined by loss of tight junction protein staining, permeability changes, and ultimately cell death by apoptosis. Using a low concentration of the αVβ3 antagonist cilengitide or using a strain deficient in outer membrane protein A resulted in a significant reduction in endothelial dysfunction following infection. Inhibition of E. coli binding to endothelial cell αVβ3 by cilengitide prevents endothelial dysfunction and may, therefore, present as a novel early therapeutic for the treatment of sepsis.

Computational modeling of synergistic interaction between αVβ3 integrin and VEGFR2 in endothelial cells: Implications for the mechanism of action of angiogenesis-modulating integrin-binding peptides

Cooperation between VEGFR2 and integrin αVβ3 is critical for neovascularization in wound healing, cardiovascular ischemic diseases, ocular diseases, and tumor angiogenesis. In the present study, we developed a rule-based computational model to investigate the potential mechanism by which the Src-induced integrin association with VEGFR2 enhances VEGFR2 activation. Simulations demonstrated that the main function of integrin is to reduce the degradation of VEGFR2 and hence stabilize the activation signal. In addition, receptor synthesis rate and recruitment from internal compartment were found to be sensitive determinants of the activation state of VEGFR2. The model was then applied to simulate the effect of integrin-binding peptides such as tumstatin and cilengitide on VEGFR2 signaling. Further, computational modeling proposed potential molecular mechanisms for the angiogenesis-modulating activity of other integrin-binding peptides. The model highlights the complexity of the crosstalk between αVβ3 integrin and VEGFR2 and the necessity of utilizing models to elucidate potential mechanisms in angiogenesis-modulating peptide therapy.

Abstract TMP28: Inhibition of Integrin α 5 β 1 With the Small Peptide Atn-161 Reduces Infarct Volume and Improves Functional Recovery Through Reduction of Blood-brain Barrier Permeability

Reperfusion-induced injury after recanalization is exacerbated by blood-brain barrier (BBB) dysfunction after ischemic stroke. Endothelial cell integrin receptors, specifically the β 1 subtype, play a direct role in BBB dysfunction through regulation of barrier-forming tight junction (TJ) proteins. We hypothesize that inhibition of an endothelial specific β 1 integrin, α 5 β 1 , after oxygen-glucose deprivation (OGD) in vitro and also after experimental stroke in vivo will stabilize the BBB through the TJ claudin-5, reducing infarct volumes and improving functional recovery. First, stroke was simulated in vitro by OGD or TNF- α treatment of brain endothelial cell monolayers. Treatment with the small peptide α 5 β 1 inhibitor ATN-161 (10 μM) resulted in decreased barrier permeability as measured by trans-endothelial cell electrical resistance and FITC-dextran permeability. Immunocytochemistry showed extracellular localization and upregulation of claudin-5 with ATN-161 treatment. Next, transient tandem middle cerebral artery occlusion was performed on 12-week-old male mice for 1 hour. On confocal and SEM imaging, post-stroke α 5 β 1 expression was upregulated primarily in the luminal vascular surface by post-stroke day (PSD) 2 rather than at the basement membrane (next to its extracellular matrix ligands). IP injection of ATN-161 (1 mg/kg) immediately after reperfusion and on PSD 1, and PSD2 caused a significant, but transient and acute (after the initial dose only) change in animal pulse distension, but no change in body temperature or heart rate. Infarct volumes, measured by TTC stain, were significantly reduced in ATN-161 treated animals. Furthermore, BBB integrity was improved by ATN-161 as determined by IgG staining and FITC-Dextran injection. Functional recovery, assessed by a 5-item Neuroscore, was also significantly improved. In conclusion, inhibition of α 5 β 1 by ATN-161 was well tolerated, reduced infarct volume, and improved behavioral recovery. Additional studies showed this result was likely due to reduced BBB permeability through the TJ claudin-5. Therefore, inhibition of α 5 β 1 with ATN-161 could represent a novel therapeutic approach for stroke worthy of further investigation.

Differential regulations of fibronectin and laminin in Smad2 activation in vascular endothelial cells in response to disturbed flow

BackgroundAtherosclerosis occurs in arterial curvatures and branches, where the flow is disturbed with low and oscillatory shear stress (OSS). The remodeling and alterations of extracellular matrices (ECMs) and their composition is the critical step in atherogenesis. In this study, we investigated the effects of different ECM proteins on the regulation of mechanotransduction in vascular endothelial cells (ECs) in response to OSS.MethodsThrough the experiments ranging from in vitro cell culture studies on effects of OSS on molecular signaling to in vivo examinations on clinical specimens from patients with coronary artery disease (CAD), we elucidated the roles of integrins and different ECMs, i.e., fibronectin (FN) and laminin (LM), in transforming growth factor (TGF)-β receptor (TβR)-mediated Smad2 activation and nuclear factor-κB (NF-κB) signaling in ECs in response to OSS and hence atherogenesis.ResultsOSS at 0.5±12 dynes/cm2 induces sustained increases in the association of types I and II TβRs with β1 and β3 integrins in ECs grown on FN, but it only transient increases in ECs grown on LM. OSS induces a sustained activation of Smad2 in ECs on FN, but only a transient activation of Smad2 in ECs on LM. OSS-activation of Smad2 in ECs on FN regulates downstream NF-κB signaling and pro-inflammatory gene expression through the activation of β1 integrin and its association with TβRs. In contrast, OSS induces transient activations of β1 and β3 integrins in ECs on LM, which associate with type I TβR to regulate Smad2 phosphorylation, resulting in transient induction of NF-κB and pro-inflammatory gene expression. In vivo investigations on diseased human coronary arteries from CAD patients revealed that Smad2 is highly activated in ECs of atherosclerotic lesions, which is accompanied by the concomitant increase of FN rather than LM in the EC layer and neointimal region of atherosclerotic lesions.ConclusionsOur findings provide new insights into the mechanisms of how OSS regulates Smad2 signaling and pro-inflammatory genes through the complex signaling networks of integrins, TβRs, and ECMs, thus illustrating the molecular basis of regional pro-inflammatory activation within disturbed flow regions in the arterial tree.

Endothelial \u03b16\u03b24 integrin protects during experimental autoimmune encephalomyelitis-induced neuroinflammation by maintaining vascular integrity and tight junction protein expression

BackgroundExtracellular matrix (ECM) proteins play critical functions regulating vascular formation and function. Laminin is a major component of the vascular basal lamina, and transgenic mice deficient in astrocyte or pericyte laminin show defective blood-brain barrier (BBB) integrity, indicating an important instructive role for laminin in cerebral blood vessels. As previous work shows that in the normal brain, vascular expression of the laminin receptor α6β4 integrin is predominantly restricted to arterioles, but induced on all vessels during neuroinflammation, it is important to define the role of this integrin in the maintenance of BBB integrity.Methodsα6β4 integrin expression was analyzed using dual immunofluorescence (dual-IF) of brain sections taken from the mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). To investigate the role of endothelial α6β4 integrin, transgenic mice lacking β4 integrin in endothelial cells (β4-EC-KO) and wild-type (WT) littermates were subject to EAE, and clinical score and various neuropathological parameters were examined by immunofluorescence. In addition, β4 integrin null brain endothelial cells (BECs) were examined in culture for expression of tight junction proteins using immunocytochemistry and flow cytometry.ResultsCerebrovascular expression of β4 integrin was markedly upregulated during EAE progression, such that by the acute stage of EAE (day 21), the vast majority of blood vessels expressed β4 integrin. In the EAE model, while the β4-EC-KO mice showed the same time of disease onset as the WT littermates, they developed significantly worse clinical disease over time, resulting in increased clinical score at the peak of disease and maintained elevated thereafter. Consistent with this, the β4-EC-KO mice showed enhanced levels of leukocyte infiltration and BBB breakdown and also displayed increased loss of the endothelial tight junction proteins claudin-5 and ZO-1. Under pro-inflammatory conditions, primary cultures of β4KO BECs also showed increased loss of claudin-5 and ZO-1 expression.ConclusionsTaken together, our data suggest that α6β4 integrin upregulation is an inducible protective mechanism that stabilizes the BBB during neuroinflammatory conditions.

Endothelium-derived extracellular vesicles promote splenic monocyte mobilization in myocardial infarction.

Transcriptionally activated monocytes are recruited to the heart after acute myocardial infarction (AMI). After AMI in mice and humans, the number of extracellular vesicles (EVs) increased acutely. In humans, EV number correlated closely with the extent of myocardial injury. We hypothesized that EVs mediate splenic monocyte mobilization and program transcription following AMI. Some plasma EVs bear endothelial cell (EC) integrins, and both proinflammatory stimulation of ECs and AMI significantly increased VCAM-1–positive EV release. Injected EC-EVs localized to the spleen and interacted with, and mobilized, splenic monocytes in otherwise naive, healthy animals. Analysis of human plasma EV-associated miRNA showed 12 markedly enriched miRNAs after AMI; functional enrichment analyses identified 1,869 putative mRNA targets, which regulate relevant cellular functions (e.g., proliferation and cell movement). Furthermore, gene ontology termed positive chemotaxis as the most enriched pathway for the miRNA-mRNA targets. Among the identified EV miRNAs, EC-associated miRNA-126-3p and -5p were highly regulated after AMI. miRNA-126-3p and -5p regulate cell adhesion– and chemotaxis-associated genes, including the negative regulator of cell motility, plexin-B2. EC-EV exposure significantly downregulated plexin-B2 mRNA in monocytes and upregulated motility integrin ITGB2. These findings identify EVs as a possible novel signaling pathway by linking ischemic myocardium with monocyte mobilization and transcriptional activation following AMI.

Hypoxia upregulates integrin gene expression in microvascular endothelial cells and promotes their migration and capillary-like tube formation.

Tissue hypoxia affects gene expression through the hypoxia-inducible transcription factors, HIF-1 and HIF-2, in both physiological and pathological angiogenesis. Angiogenesis is a complex response of endothelial cells integrating cell proliferation, migration, tube formation, and their interaction with the extracellular matrix through integrin receptors. In this report, we studied the effect of hypoxia on the angiogenic functions of human microvascular endothelial cells (HMEC-1) as well as on expression of the angiogenic integrins αν β3 , αν β5 , and α5 β1 . Exposure of HMEC-1 to hypoxia (1% O2 ) or to DMOG, a prolyl-4-hydroxylase inhibitor, caused significant reduction to their proliferation rate, whereas their migration ability toward laminin-1 or collagen IV and capillary-like tube formation were significantly enhanced. In addition, αv , β1 , β3 , and β5 integrins expression was increased under hypoxia in HMEC-1, while α5 integrin was not affected. Both HIF-1 and HIF-2 protein expression and transcriptional activity were induced under hypoxia in HMEC-1. The knockdown of either HIF-1α or HIF-2α inhibited integrin β3 hypoxic stimulation, suggesting a HIF-dependent induction of β3 integrin in HMEC-1. Taken together, our results indicate that hypoxia transcriptionally up-regulates angiogenic integrins in microvascular endothelial cells along with promoting migration and tube formation of HMEC-1.