Reticular adhesions are assembled at flat clathrin lattices and opposed by active integrin α5ß1.

Reticular adhesions (RAs) consist of integrin αvß5 and harbor flat clathrin lattices (FCLs), long-lasting structures with similar molecular composition as clathrin-mediated endocytosis (CME) carriers. Why FCLs and RAs colocalize is not known. Here, we show that RAs are assembled at FCLs in a process controlled by fibronectin (FN) and its receptor, integrin α5ß1. We observed that cells on FN-rich matrices displayed fewer FCLs and RAs. CME machinery inhibition abolished RAs and live-cell imaging showed that RA establishment requires FCL coassembly. The inhibitory activity of FN was mediated by the activation of integrin α5ß1 at Tensin1-positive fibrillar adhesions. Conventionally, endocytosis disassembles cellular adhesions by internalizing their components. Our results present a novel paradigm in the relationship between these two processes by showing that endocytic proteins can actively function in the assembly of cell adhesions. Furthermore, we show this novel adhesion assembly mechanism is coupled to cell migration via unique crosstalk between cell-matrix adhesions.

Lysophosphatidylcholine in phospholipase A2-modified LDL triggers secretion of angiopoietin 2.

BACKGROUND AND AIMS: Secretory phospholipase A2 (PLA2) hydrolyzes LDL phospholipids generating modified LDL particles (PLA2-LDL) with increased atherogenic properties. Exocytosis of Weibel-Palade bodies (WPB) releases angiopoietin 2 (Ang2) and externalizes P-selectin, which both play important roles in vascular inflammation. Here, we investigated the effects of PLA2-LDL on exocytosis of WPBs. METHODS: Human coronary artery endothelial cells (HCAECs) were stimulated with PLA2- LDL, and its uptake and effect on Ang2 release, leukocyte adhesion, and intracellular calcium levels were measured. The effects of PLA2-LDL on Ang2 release and WPB exocytosis were measured in and ex vivo in mice. RESULTS: Exposure of HCAECs to PLA2-LDL triggered Ang2 secretion and promoted leukocyte-HCAEC interaction. Lysophosphatidylcholine was identified as a critical component of PLA2-LDL regulating the WPB exocytosis, which was mediated by cell-surface proteoglycans, phospholipase C, intracellular calcium, and cytoskeletal remodeling. PLA2-LDL also induced murine endothelial WPB exocytosis in blood vessels in and ex vivo, as evidenced by secretion of Ang2 in vivo, P-selectin translocation to plasma membrane in intact endothelial cells in thoracic artery and tracheal vessels, and reduced Ang2 staining in tracheal endothelial cells. Finally, in contrast to normal human coronary arteries, in which Ang2 was present only in the endothelial layer, at sites of advanced atherosclerotic lesions, Ang2 was detected also in the intima, media, and adventitia. CONCLUSIONS: Our studies reveal PLA2-LDL as a potent agonist of endothelial WPB exocytosis, resulting in increased secretion of Ang2 and translocation of P-selectin. The results provide mechanistic insight into PLA2-LDL-dependent promotion of vascular inflammation and atherosclerosis.

Targeting ß1-integrin inhibits vascular leakage in endotoxemia.

Loss of endothelial integrity promotes capillary leakage in numerous diseases, including sepsis, but there are no effective therapies for preserving endothelial barrier function. Angiopoietin-2 (ANGPT2) is a context-dependent regulator of vascular leakage that signals via both endothelial TEK receptor tyrosine kinase (TIE2) and integrins. Here, we show that antibodies against ß1-integrin decrease LPS-induced vascular leakage in murine endotoxemia, as either a preventative or an intervention therapy. ß1-integrin inhibiting antibodies bound to the vascular endothelium in vivo improved the integrity of endothelial cell-cell junctions and protected mice from endotoxemia-associated cardiac failure, without affecting endothelial inflammation, serum proinflammatory cytokine levels, or TIE receptor signaling. Moreover, conditional deletion of a single allele of endothelial ß1-integrin protected mice from LPS-induced vascular leakage. In endothelial monolayers, the inflammatory agents thrombin, lipopolysaccharide (LPS), and IL-1ß decreased junctional vascular endothelial (VE)-cadherin and induced actin stress fibers via ß1- and α5-integrins and ANGPT2. Additionally, ß1-integrin inhibiting antibodies prevented inflammation-induced endothelial cell contractility and monolayer permeability. Mechanistically, the inflammatory agents stimulated ANGPT2-dependent translocation of α5ß1-integrin into tensin-1-positive fibrillar adhesions, which destabilized the endothelial monolayer. Thus, ß1-integrin promotes endothelial barrier disruption during inflammation, and targeting ß1-integrin signaling could serve as a novel means of blocking pathological vascular leak.

Endothelial destabilization by angiopoietin-2 via integrin ß1 activation.

Angiopoietins regulate vascular homeostasis via the endothelial Tie receptor tyrosine kinases. Angiopoietin-1 (Ang1) supports endothelial stabilization via Tie2 activation. Angiopoietin-2 (Ang2) functions as a context-dependent Tie2 agonist/antagonist promoting pathological angiogenesis, vascular permeability and inflammation. Elucidating Ang2-dependent mechanisms of vascular destablization is critical for rational design of angiopoietin antagonists that have demonstrated therapeutic efficacy in cancer trials. Here, we report that Ang2, but not Ang1, activates ß1-integrin, leading to endothelial destablization. Autocrine Ang2 signalling upon Tie2 silencing, or in Ang2 transgenic mice, promotes ß1-integrin-positive elongated matrix adhesions and actin stress fibres, regulating vascular endothelial-cadherin-containing cell-cell junctions. The Tie2-silenced monolayer integrity is rescued by ß1-integrin, phosphoinositide-3 kinase or Rho kinase inhibition, and by re-expression of a membrane-bound Tie2 ectodomain. Furthermore, Tie2 silencing increases, whereas Ang2 blocking inhibits transendothelial tumour cell migration in vitro. These results establish Ang2-mediated ß1-integrin activation as a promoter of endothelial destablization, explaining the controversial vascular functions of Ang1 and Ang2.