No abstract

We studied the formation of capillary tubes by endothelial cells which were sandwiched between two fibrin gels under serum-free conditions. After formation of the overlying fibrin gel, the endothelial cell monolayer rearranged into an extensive net of capillary tubes. Tube formation was apparent at 5 h and was fully developed by 24 h. The capillary tubes were vacuolated, and both intracellular and intercellular lumina were present. Maximal tube formation was observed with fibrin II (which lacks both fibrinopeptide A and B), minimal tube formation with fibrin I (which lacks only fibrinopeptide A), and complete absence of tube formation with fibrin 325 (which lacks the NH2- terminal beta 15-42 sequence, in addition to fibrinopeptides A and B). The inability of fibrin 325 to stimulate capillary tube formation supports the idea that beta 15-42 plays an important role in this process, and its importance was confirmed by the finding that exogenous soluble beta 15-42 inhibited fibrin II-induced capillary tube formation. This effect was specific for fibrin, since beta 15-42 did not inhibit tube formation by endothelial cells sandwiched between collagen gels. The interaction of the apical surface of the endothelial cell with the overlying fibrin II gel, as opposed to the underlying fibrin gel upon which the cells were seeded, was necessary for capillary tube formation. These studies suggest that the beta 15-42 sequence of fibrin interacts with a component of the apical cell surface and that this interaction plays a fundamental role in the induction of endothelial capillary tube formation.

We studied the interaction of [125I]fibronectin with human umbilical vein endothelial cells. Endothelial cell monolayers cross-linked [125I]fibronectin which had been preadsorbed to gelatin-coated dishes. The cross-linking of the substrate-immobilized [125I]fibronectin was mediated by cell-associated tissue transglutaminase and occurred more rapidly during the first 30 min after endothelial cell seeding but also continued for several hours after the cells were fully spread. The processing of the [125I]fibronectin was associated with the basolateral surface of the endothelial cell, as demonstrated by the finding that cross-linking did not occur when [125I]fibronectin was presented to the apical surface of confluent monolayers. Transglutaminase activity was not necessary for attachment and spreading of HUVEC on a fibronectin/gelatin matrix. The presence of a nonpeptidyl transglutaminase inactivator rendered the cells more susceptible to detachment by trypsin and destabilized the association of fibronectin with the subendothelial extracellular matrix. Thus, endothelial cells process fibronectin into cross-linked multimers due to the expression of tissue transglutaminase at the basal surface of the cell. This process may serve to stabilize the extracellular matrix and to firmly anchor the cells to the basement membrane.

Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a key regulator of oxidative stress and cellular repair and can be activated through inhibition of its cytoplasmic repressor, Kelch-like ECH-associated protein 1 (Keap1). Several small molecule disrupters of the Nrf2-Keap1 complex have recently been tested and/or approved for human therapeutic use but lack either potency or selectivity. The main goal of our work was to develop a potent, selective activator of NRF2 as protection against oxidative stress. In human bronchial epithelial cells, our Nrf2 activator, 3-(pyridin-3-ylsulfonyl)-5-(trifluoromethyl)-2-chromen-2-one (PSTC), induced Nrf2 nuclear translocation, Nrf2-regulated gene expression, and downstream signaling events, including induction of NAD(P)H:quinone oxidoreductase 1 (NQO1) enzyme activity and heme oxygenase-1 protein expression, in an Nrf2-dependent manner. As a marker of subsequent functional activity, PSTC restored oxidant (-butyl hydroperoxide)-induced glutathione depletion. The compound's engagement of the Nrf2 signaling pathway translated to an in vivo setting, with induction of Nrf2-regulated gene expression and NQO1 enzyme activity, as well as restoration of oxidant (ozone)-induced glutathione depletion, occurring in the lungs of PSTC-treated rodents. Under disease conditions, PSTC engaged its target, inducing the expression of Nrf2-regulated genes in human bronchial epithelial cells derived from patients with chronic obstructive pulmonary disease, as well as in the lungs of cigarette smoke-exposed mice. Subsequent to the latter, a dose-dependent inhibition of cigarette smoke-induced pulmonary inflammation was observed. Finally, in contrast with bardoxolone methyl and sulforaphane, PSTC did not inhibit interleukin-1-induced nuclear factor-B translocation or insulin-induced S6 phosphorylation in human cells, emphasizing the on-target activity of this compound. In summary, we characterize a potent, selective Nrf2 activator that offers protection against pulmonary oxidative stress in several cellular and in vivo models.