CD93 Signaling via Rho Proteins Drives Cytoskeletal Remodeling in Spreading Endothelial Cells

During angiogenesis, cell adhesion molecules expressed on the endothelial cell surface promote the growth and survival of newly forming vessels. Hence, elucidation of the signaling pathways activated by cell-to-matrix adhesion may assist in the discovery of new targets to be used in antiangiogenic therapy. In proliferating endothelial cells, the single-pass transmembrane glycoprotein CD93 has recently emerged as an important endothelial cell adhesion molecule regulating vascular maturation. In this study, we unveil a signaling pathway triggered by CD93 that regulates actin cytoskeletal dynamics responsible of endothelial cell adhesion. We show that the Src-dependent phosphorylation of CD93 and the adaptor protein Cbl leads to the recruitment of Crk, which works as a downstream integrator in the CD93-mediated signaling. Moreover, confocal microscopy analysis of FRET-based biosensors shows that CD93 drives the coordinated activation of Rac1 and RhoA at the cell edge of spreading cells, thus promoting the establishment of cell polarity and adhesion required for cell motility.

Tyrosine phosphorylation of S1PR1 leads to chaperone BiP-mediated import to the endoplasmic reticulum

Cell surface G protein–coupled receptors (GPCRs), upon agonist binding, undergo serine–threonine phosphorylation, leading to either receptor recycling or degradation. Here, we show a new fate of GPCRs, exemplified by ER retention of sphingosine-1-phosphate receptor 1 (S1PR1). We show that S1P phosphorylates S1PR1 on tyrosine residue Y143, which is associated with recruitment of activated BiP from the ER into the cytosol. BiP then interacts with endocytosed Y143-S1PR1 and delivers it into the ER. In contrast to WT-S1PR1, which is recycled and stabilizes the endothelial barrier, phosphomimicking S1PR1 (Y143D-S1PR1) is retained by BiP in the ER and increases cytosolic Ca2+ and disrupts barrier function. Intriguingly, a proinflammatory, but non-GPCR agonist, TNF-α, also triggered barrier-disruptive signaling by promoting S1PR1 phosphorylation on Y143 and its import into ER via BiP. BiP depletion restored Y143D-S1PR1 expression on the endothelial cell surface and rescued canonical receptor functions. Findings identify Y143-phosphorylated S1PR1 as a potential target for prevention of endothelial barrier breakdown under inflammatory conditions.

Leukocyte adhesion is governed by endolysosomal two pore channel 2 (TPC2)

In response to pro-inflammatory challenges including pathogenic attack and tissue damage, the endothelial cell surface is rearranged to present leukocyte-engaging cell surface receptors. The initial contact needed for leukocyte tethering and rolling is mediated via adhesion demand-driven exocytosis of Weibel-Palade bodies (WPB) that contain the leukocyte receptor P-selectin together with the stabilizing co-factor CD63. We found that diminished expression of the endolysosomal non-selective cation channel TPC2 or inhibition of TPC2-mediated Ca2+-release via trans-Ned 19 led to reduced endolysosomal Ca2+ efflux, and blocked transfer of CD63 from late endosomes/lysosomes (LEL) to WPB, and a concomitant loss of P-selectin on the endothelial cell surface. Accordingly, P-selectin-mediated leukocyte recruitment to trans-Ned 19-treated HUVEC under flow was significantly reduced without disturbing VWF exocytosis. Our findings establish the endolysosome-related TPC2 Ca2+ channel as a key element in the maintenance of proper endothelial functions and a potential pharmacological target in the control of inflammatory leukocyte recruitment.

Annexin A2 in Fibrinolysis, Inflammation and Fibrosis

As a cell surface tissue plasminogen activator (tPA)-plasminogen receptor, the annexin A2 (A2) complex facilitates plasmin generation on the endothelial cell surface, and is an established regulator of hemostasis. Whereas A2 is overexpressed in hemorrhagic disease such as acute promyelocytic leukemia, its underexpression or impairment may result in thrombosis, as in antiphospholipid syndrome, venous thromboembolism, or atherosclerosis. Within immune response cells, A2 orchestrates membrane repair, vesicle fusion, and cytoskeletal organization, thus playing a critical role in inflammatory response and tissue injury. Dysregulation of A2 is evident in multiple human disorders, and may contribute to the pathogenesis of various inflammatory disorders. The fibrinolytic system, moreover, is central to wound healing through its ability to remodel the provisional matrix and promote angiogenesis. A2 dysfunction may also promote tissue fibrogenesis and end-organ fibrosis.

Proteoglycans contribute to the functional integrity of the glomerular endothelial cell surface layer and are regulated in diabetic kidney disease

All capillary endothelia, including those of the glomeruli, have a luminal cell surface layer (ESL) consisting of glycoproteins, glycolipids, proteoglycans (PGs) and glycosaminoglycans. Previous results have demonstrated that an intact ESL is necessary for a normal filtration barrier and damage to the ESL coupled to proteinuria is seen for example in diabetic kidney disease (DKD). We used the principles of ion exchange chromatography in vivo to elute the highly negatively charged components of the ESL with a 1 M NaCl solution in rats. Ultrastructural morphology and renal function were analyzed and 17 PGs and hyaluronan were identified in the ESL. The high salt solution reduced the glomerular ESL thickness, led to albuminuria and reduced GFR. To assess the relevance of ESL in renal disease the expression of PGs in glomeruli from DKD patients in a next generation sequencing cohort was investigated. We found that seven of the homologues of the PGs identified in the ESL from rats were differently regulated in patients with DKD compared to healthy subjects. The results show that proteoglycans and glycosaminoglycans are essential components of the ESL, maintaining the permselective properties of the glomerular barrier and thus preventing proteinuria.

Shear-Regulated Extracellular Microenvironments and Endothelial Cell Surface Integrin Receptors Intertwine in Atherosclerosis

Mechanical forces imposed by blood flow shear stress directly modulate endothelial gene expression and functional phenotype. The production of extracellular matrix proteins and corresponding cell-surface integrin receptors in arterial endothelial cells is intricately regulated by blood flow patterns. Laminar blood flow promotes mature and atheroresistant endothelial phenotype, while disturbed flow induces dysfunctional and atheroprone endothelial responses. Here, we discuss how hemodynamic changes orchestrate the remodeling of extracellular microenvironments and the expression profile of the integrin receptors in endothelial cells leading to oxidative stress and inflammation. Targeting the interaction between matrix proteins and their corresponding integrins is a potential therapeutic approach for atherosclerosis.

Effect of Heparanase and Heparan Sulfate Chains in Hemostasis

Heparanase, the only mammalian enzyme known to degrade heparan sulfate chains, affects the hemostatic system through several mechanisms. Along with the degrading effect, heparanase engenders release of syndecan-1 from the cell surface and directly enhances the activity of the blood coagulation initiator, tissue factor, in the coagulation system. Upregulation of tissue factor and release of tissue factor pathway inhibitor from the cell surface contribute to the prothrombotic effect. Tissue factor pathway inhibitor and the strongest physiological anticoagulant antithrombin are attached to the endothelial cell surface by heparan sulfate. Hence, degradation of heparan sulfate induces further release of these two natural anticoagulants from endothelial cells. Elevated heparanase procoagulant activity and heparan sulfate chain levels in plasma, demonstrated in cancer, pregnancy, oral contraceptive use, and aging, could suggest a potential mechanism for increased risk of thrombosis in these clinical settings. In contrast to the blood circulation, accumulation of heparan sulfate chains in transudate and exudate pleural effusions induces a local anticoagulant milieu. The anticoagulant effect of heparan sulfate chains in other closed spaces such as peritoneal or subdural cavities should be further investigated.

Fibrinolytic Serine Proteases, Therapeutic Serpins and Inflammation: Fire Dancers and Firestorms

The making and breaking of clots orchestrated by the thrombotic and thrombolytic serine protease cascades are critical determinants of morbidity and mortality during infection and with vascular or tissue injury. Both the clot forming (thrombotic) and the clot dissolving (thrombolytic or fibrinolytic) cascades are composed of a highly sensitive and complex relationship of sequentially activated serine proteases and their regulatory inhibitors in the circulating blood. The proteases and inhibitors interact continuously throughout all branches of the cardiovascular system in the human body, representing one of the most abundant groups of proteins in the blood. There is an intricate interaction of the coagulation cascades with endothelial cell surface receptors lining the vascular tree, circulating immune cells, platelets and connective tissue encasing the arterial layers. Beyond their role in control of bleeding and clotting, the thrombotic and thrombolytic cascades initiate immune cell responses, representing a front line, “off-the-shelf” system for inducing inflammatory responses. These hemostatic pathways are one of the first response systems after injury with the fibrinolytic cascade being one of the earliest to evolve in primordial immune responses. An equally important contributor and parallel ancient component of these thrombotic and thrombolytic serine protease cascades are theserineproteaseinhibitors, termedserpins. Serpins are metastable suicide inhibitors with ubiquitous roles in coagulation and fibrinolysis as well as multiple central regulatory pathways throughout the body. Serpins are now known to also modulate the immune response, either via control of thrombotic and thrombolytic cascades or via direct effects on cellular phenotypes, among many other functions. Here we review the co-evolution of the thrombolytic cascade and the immune response in disease and in treatment. We will focus on the relevance of these recent advances in the context of the ongoing COVID-19 pandemic. SARS-CoV-2 is a “respiratory” coronavirus that causes extensive cardiovascular pathogenesis, with microthrombi throughout the vascular tree, resulting in severe and potentially fatal coagulopathies.