Protective role of Cav-1 in pneumolysin-induced endothelial barrier dysfunction

Pneumolysin (PLY) is a bacterial pore forming toxin and primary virulence factor of Streptococcus pneumonia, a major cause of pneumonia. PLY binds cholesterol-rich domains of the endothelial cell (EC) plasma membrane resulting in pore assembly and increased intracellular (IC) Ca2+ levels that compromise endothelial barrier integrity. Caveolae are specialized plasmalemma microdomains of ECs enriched in cholesterol. We hypothesized that the abundance of cholesterol-rich domains in EC plasma membranes confers cellular susceptibility to PLY. Contrary to this hypothesis, we found increased PLY-induced IC Ca2+ following membrane cholesterol depletion. Caveolin-1 (Cav-1) is an essential structural protein of caveolae and its regulation by cholesterol levels suggested a possible role in EC barrier function. Indeed, Cav-1 and its scaffolding domain peptide protected the endothelial barrier from PLY-induced disruption. In loss of function experiments, Cav-1 was knocked-out using CRISPR-Cas9 or silenced in human lung microvascular ECs. Loss of Cav-1 significantly enhanced the ability of PLY to disrupt endothelial barrier integrity. Rescue experiments with re-expression of Cav-1 or its scaffolding domain peptide protected the EC barrier against PLY-induced barrier disruption. Dynamin-2 (DNM2) is known to regulate caveolar membrane endocytosis. Inhibition of endocytosis, with dynamin inhibitors or siDNM2 amplified PLY induced EC barrier dysfunction. These results suggest that Cav-1 protects the endothelial barrier against PLY by promoting endocytosis of damaged membrane, thus reducing calcium entry and PLY-dependent signaling

Maternal hyperleptinemia is associated with male offspring’s altered vascular function and structure in mice

Children of mothers with gestational diabetes have greater risk of developing hypertension but little is known about the mechanisms by which this occurs. The objective of this study was to test the hypothesis that high maternal concentrations of leptin during pregnancy, which are present in mothers with gestational diabetes and/or obesity, alter blood pressure, vascular structure and vascular function in offspring. Wildtype (WT) offspring of hyperleptinemic, normoglycemic, Lepr db/+ dams were compared to genotype matched offspring of WT-control dams. Vascular function was assessed in male offspring at 6, and at 31 weeks of age after half the offspring had been fed a high fat, high sucrose diet (HFD) for 6 weeks. Blood pressure was increased by HFD but not affected by maternal hyperleptinemia. On a standard diet, offspring of hyperleptinemic dams had outwardly remodeled mesenteric arteries and an enhanced vasodilatory response to insulin. In offspring of WT but not Leprdb/+ dams, HFD induced vessel hypertrophy and enhanced vasodilatory responses to acetylcholine, while HFD reduced insulin responsiveness in offspring of hyperleptinemic dams. Offspring of hyperleptinemic dams had stiffer arteries regardless of diet. Therefore, while maternal hyperleptinemia was largely beneficial to offspring vascular health under astandard diet, it had detrimental effects in offspring fed HFD. These results suggest that circulating maternal leptin concentrations may interact with other factors in the pre- and post-natal environments to contribute to altered vascular function in offspring of diabetic pregnancie

Collagen IV contributes to nitric oxide-induced angiogenesis of lung endothelial cells

Nitric oxide (NO) mediates endothelial angiogenesis via inducing the expression of integrin αvβ3. During angiogenesis, endothelial cells adhere to and migrate into the extracellular matrix through integrins. Collagen IV binds to integrin αvβ3, leading to integrin activation, which affects a number of signaling processes in endothelial cells. In the present study, we evaluated the role of collagen IV in NO-induced angiogenesis. We found that NO donor 2,2′-(hydroxynitrosohydrazino)bis-ethanamine (NOC-18) causes increases in collagen IV mRNA and protein in lung endothelial cells and collagen IV release into the medium. Addition of collagen IV into the coating of endothelial culture increases endothelial monolayer wound repair, proliferation, and tube formation. Inhibition of collagen IV synthesis using gene silencing attenuates NOC-18-induced increases in monolayer wound repair, cell proliferation, and tube formation as well as in the phosphorylation of focal adhesion kinase (FAK). Integrin blocking antibody LM609 prevents NOC-18-induced increase in endothelial monolayer wound repair. Inhibition of protein kinase G (PKG) using the specific PKG inhibitor KT5823 or PKG small interfering RNA prevents NOC-18-induced increases in collagen IV protein and mRNA and endothelial angiogenesis. Together, these results indicate that NO promotes collagen IV synthesis via a PKG signaling pathway and that the increase in collagen IV synthesis contributes to NO-induced angiogenesis of lung endothelial cells through integrin-FAK signaling. Manipulation of collagen IV could be a novel approach for the prevention and treatment of diseases such as alveolar capillary dysplasia, severe pulmonary arterial hypertension, and tumor invasion

Heme oxygenase-1 induction contributes to renoprotection by G-CSF during rhabdomyolysis-associated acute kidney injury

Granulocyte colony-stimulating factor (G-CSF) is renoprotective during acute kidney injury (AKI) induced by ischemia and cisplatin nephrotoxicity; however, the underlying mechanism is not entirely clear. Rhabdomyolysis is another important clinical cause of AKI, due to the release of nephrotoxins (e.g., heme) from disrupted muscles. The current study has determined the effects of G-CSF on rhabdomyolysis-associated AKI using in vivo and in vitro models. In C57BL/6 mice, intramuscular injection of glycerol induced AKI, which was partially prevented by G-CSF pretreatment. Consistently, glycerol-induced renal tissue damage was ameliorated by G-CSF. In addition, animal survival following the glycerol injection was improved from ∼30 to ∼70% by G-CSF. In cultured renal tubular cells, hemin-induced apoptosis was also suppressed by G-CSF. Interestingly, G-CSF induced heme oxygenase-1 (HO-1, a critical enzyme for heme/hemin degradation and detoxification) in both cultured tubular cells and mouse kidneys. Blockade of HO-1 with protoporphyrin IX zinc(II) (ZnPP) could largely diminish the protective effects of G-CSF. Together, these results demonstrated the renoprotective effects of G-CSF in rhabdomyolysis-associated AKI. Notably, G-CSF may directly protect against tubular cell injury under the disease condition by inducing HO-1

KNK437 exaggerates hyperoxia-induced endothelial apoptosis and the increases in caspase-3 activity and nuclear AIF protein level.

<p>PAECs were exposed to normoxia and hyperoxia in the absence and presence of KNK437 (50–100 μM) for 48 h after which TUNEL staining, caspase-3 activity and nuclear AIF protein level were determined. (A) Representative images of TUNEL staining of PAECs exposed to hyperoxia and/or KNK437 for 48 h. (B) Bar graph depicting the changes in the numbers of TUNEL-positive cells. (C) Changes in caspase-3 activity. (D) Representative immunoblots of AIF. (C) Bar graph depicting the changes in nuclear AIF protein levels. Results are expressed as mean ± SE; n = 4. *<i>P</i><0.05 vs. normoxia; **<i>P</i><0.05 vs. normoxia+vehicle; #<i>P</i><0.05 vs. hyperoxia+vehicle.</p

Hsp70/AIF interaction and the effects of NAC and KNK437 on the Hsp70/AIF interaction in hyperoxic PAECs.

<p>PAECs were exposed to normoxia (nor) and hyperoxia (hyper) in the absence and presence of NAC (5 mM) or KNK437 (50–100 μM) for 48 h after which co-immunoprecipitations of Hsp70 and AIF in the cytosolic fraction of cell lysates were performed. (A and C) Representative immunoblots of Hsp70 and AIF. (B and D) Bar graph depicting the changes in Hsp70/AIF ratio. Results are expressed as mean ± SE; n = 4. *<i>P</i><0.05 vs. normoxia; #<i>P</i><0.05 vs. hyperoxia+vehicle.</p