Abstract
AbstractSeveral studies demonstrate that hemolysis and free heme in circulation cause endothelial barrier dysfunction and are associated with severe pathological conditions such as acute respiratory distress syndrome, acute chest syndrome, and sepsis. However, the precise molecular mechanisms involved in the pathology of heme-induced barrier disruption remain to be elucidated. In this study, we investigated the role of free heme in the endothelial barrier integrity and mechanisms of heme-mediated intracellular signaling of human lung microvascular endothelial cells (HLMVECs). Heme, in a dose-dependent manner, induced a rapid drop in the endothelial barrier integrity of HLMVECs. An investigation into barrier proteins revealed that heme primarily affected the tight junction proteins zona occludens-1, claudin-1, and claudin-5, which were significantly reduced after heme exposure. The p38MAPK/HSP27 pathway, involved in the regulation of endothelial cytoskeleton remodeling, was also significantly altered after heme treatment, both in HLMVECs and mice. By using a knockout (KO) mouse for MKK3, a key regulator of the p38MAPK pathway, we showed that this KO effectively decreased heme-induced endothelial barrier dysfunction. Taken together, our results indicate that targeting the p38MAPK pathway may represent a crucial treatment strategy in alleviating hemolytic diseases.
Highlights
Elevated levels of circulating free heme have been shown to correlate with increased risk of several hemolytic disorders and are associated with endothelial barrier dysfunction
In this study, we used human lung microvascular endothelial cells (HLMVECs) to assess barrier dysfunction because they are most susceptible to injury with severe implications
We demonstrate a strong association between the p38MAPK pathway and heme-induced barrier dysfunction by using a knockout (KO) mouse model of MKK3, a key regulator of the p38 pathway
Summary
Elevated levels of circulating free heme have been shown to correlate with increased risk of several hemolytic disorders and are associated with endothelial barrier dysfunction. ZO-1 has been shown to bind to F-actin and has been linked to regulation of the actomyosin cytoskeleton.[9] An investigation into the other junction proteins by western blotting revealed that ZO-1, CLDN1, and CLDN-5 were significantly decreased at all time points tested, with a slight drop in VE-cadherin seen only at 30 minutes after heme treatment (Figure 1F-J).
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