Person:

Kalwa, Hermann

Lung ischaemia–reperfusion-induced oedema (LIRE) is a life-threatening condition that causes pulmonary oedema induced by endothelial dysfunction. Here we show that lungs from mice lacking nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox2(^{y/−})) or the classical transient receptor potential channel 6 TRPC6(^{−/-}) are protected from LIR-induced oedema (LIRE). Generation of chimeric mice by bone marrow cell transplantation and endothelial-specific Nox2 deletion showed that endothelial Nox2, but not leukocytic Nox2 or TRPC6, are responsible for LIRE. Lung endothelial cells from Nox2- or TRPC6-deficient mice showed attenuated ischaemia-induced Ca(^{2+}) influx, cellular shape changes and impaired barrier function. Production of reactive oxygen species was completely abolished in Nox2(^{y/−}) cells. A novel mechanistic model comprising endothelial Nox2-derived production of superoxide, activation of phospholipase C-γ, inhibition of diacylglycerol (DAG) kinase, DAG-mediated activation of TRPC6 and ensuing LIRE is supported by pharmacological and molecular evidence. This mechanism highlights novel pharmacological targets for the treatment of LIRE.

Nitric oxide (NO) and hydrogen peroxide (H(_2)O(_2)) play key roles in physiological and pathological responses in cardiac myocytes. The mechanisms whereby H(_2)O(_2)–modulated phosphorylation pathways regulate the endothelial isoform of nitric oxide synthase (eNOS) in these cells are incompletely understood. We show here that H(_2)O(_2) treatment of adult mouse cardiac myocytes leads to increases in intracellular Ca(^{2+}) ([Ca(^{2+})](_i)), and document that activity of the L-type Ca(^{2+}) channel is necessary for the H(_2)O(_2)-promoted increase in sarcomere shortening and of [Ca(^{2+})](_i). Using the chemical NO sensor Cu(_2)(FL2E), we discovered that the H(_2)O(_2)-promoted increase in cardiac myocyte NO synthesis requires activation of the L-type Ca(^{2+}) channel, as well as phosphorylation of the AMP-activated protein kinase (AMPK), and mitogen-activated protein kinase kinase 1/2 (MEK1/2). Moreover, H(_2)O(_2)-stimulated phosphorylations of eNOS, AMPK, MEK1/2, and ERK1/2 all depend on both an increase in [Ca(^{2+})](_i) as well as the activation of protein kinase C (PKC). We also found that H(_2)O(_2)-promoted cardiac myocyte eNOS translocation from peripheral membranes to internal sites is abrogated by the L-type Ca(^{2+}) channel blocker nifedipine. We have previously shown that kinase Akt is also involved in H(_2)O(_2)-promoted eNOS phosphorylation. Here we present evidence documenting that H(_2)O(_2)-promoted Akt phosphorylation is dependent on activation of the L-type Ca(^{2+})channel, but is independent of PKC. These studies establish key roles for Ca(^{2+})- and PKC-dependent signaling pathways in the modulation of cardiac myocyte eNOS activation by H(_2)O(_2).

Caveolin-1 is a scaffolding/regulatory protein that interacts with diverse signaling molecules. Caveolin-1null mice have marked metabolic abnormalities, yet the underlying molecular mechanisms are incompletely understood. We found the redox stress plasma biomarker plasma 8-isoprostane was elevated in caveolin-1null mice, and discovered that siRNA-mediated caveolin-1 knockdown in endothelial cells promoted significant increases in intracellular H2O2. Mitochondrial ROS production was increased in endothelial cells after caveolin-1 knockdown; 2-deoxy-D-glucose attenuated this increase, implicating caveolin-1 in control of glycolytic pathways. We performed unbiased metabolomic characterizations of endothelial cell lysates following caveolin-1 knockdown, and discovered strikingly increased levels (up to 30-fold) of cellular dipeptides, consistent with autophagy activation. Metabolomic analyses revealed that caveolin-1 knockdown led to a decrease in glycolytic intermediates, accompanied by an increase in fatty acids, suggesting a metabolic switch. Taken together, these results establish that caveolin-1 plays a central role in regulation of oxidative stress, metabolic switching, and autophagy in the endothelium, and may represent a critical target in cardiovascular diseases.