Caveolae are an essential component of the pathway for endothelial cell signaling associated with abrupt reduction of shear stress

Abstract

Abrupt cessation of flow representing the acute loss of shear stress (simulated ischemia) to flow-adapted pulmonary microvascular endothelial cells (PMVEC) leads to reactive oxygen species (ROS) generation that signals for EC proliferation. We evaluated the role of caveolin-1 on this cellular response with mouse PMVEC that were preconditioned for 72 h to laminar flow at 5 dyn/cm(2) followed by stop of flow ("ischemia"). Preconditioning resulted in a 2.7-fold increase in cellular expression of K(ATP) (K(IR) 6.2) channels but no change in expression level of caveolin-1, gp91(phox), or MAP kinases. The initial response to ischemia in wild type cells was cell membrane depolarization that was abolished by gene targeting of K(IR) 6.2. The subsequent response was increased ROS production associated with activation of NADPH oxidase (NOX2) and then phosphorylation of MAP kinases (Erk, JNK). After 24 h of ischemia in wild type cells, the cell proliferation index increased 2.5 fold and the % of cells in S+G(2)/M phases increased 6-fold. This signaling cascade (cell membrane depolarization, ROS production, MAP kinase activation and cell proliferation) was abrogated in caveolin-1 null PMVEC or by treatment of wild type cells with filipin. These studies indicate that caveolin-1 functions as a shear sensor in flow-adapted EC resulting in ROS-mediated cell signaling and endothelial cell proliferation following the abrupt reduction in flow.

Peroxiredoxin 6 Phosphorylation and Subsequent Phospholipase A2 Activity Are Required for Agonist-mediated Activation of NADPH Oxidase in Mouse Pulmonary Microvascular Endothelium and Alveolar Macrophages

“…Isolation of Cells and Experimental Design-Microvascular endothelial cells (PMVEC) were isolated from lungs of wildtype, Prdx6 null, and NOX2 null mice as reported previously (12,13). Briefly, minced lungs were treated with collagenase (3 mg/ml), the digest was forced through an 18-gauge needle and centrifuged, binding buffer (6.5 mM sodium phosphate, pH 7.4) was added to the pellet, and the cell suspension was incubated with anti-PECAM antibody followed by incubation with prewashed Dynabeads (Dynal, Oslo, Norway) coated with sheep anti-rat IgG (beads coated with anti-mouse IgG were not available).…”

PECAM-1 and caveolae form the mechanosensing complex necessary for NOX2 activation and angiogenic signaling with stopped flow in pulmonary endothelium

“…42) and on reports of PECAM-1 activation (tyrosine phosphorylation) upon flow (9), we investigated whether PECAM-1, by virtue of its junctional location and cytoskeletal linkage, might serve as a mechanosensor for the loss of blood flow. Our earlier studies on mechanotransduction showed that lack of caveolin-1 (and thus caveolae) prevented the pulmonary endothelial ROS production with cessation of flow (33,45). We thus investigated the role of PECAM-1 in mechanosensing and the association (if any) of PECAM-1 and caveolin-1 (and thus caveolae) in the pulmonary endothelium.…”

The serpentine path to a novel mechanism-based inhibitor of acute inflammatory lung injury

“…After extensive further study, we found that the source of ROS with ischemia was the endothelium, and the mechanism was related to altered mechanotransduction, namely the cessation of the normal shear forces applied to the pulmonary endothelial cells by the pulmonary blood flow (3,71,75). Additional studies showed that shear in pulmonary endothelium is sensed by caveolae and is somehow related to endothelial platelet-endothelial adhesion molecule (PECAM) (61,63 pulmonary endothelial ATP-sensitive K ϩ channels in the open configuration, contributing to the endothelial cell transmembrane potential; cessation of flow leads to ATP-sensitive K ϩ channel closure, endothelial cell membrane depolarization, and activation of NOX2 with subsequent O 2 ·Ϫ generation (4,14,16) (Fig. 3).…”