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  • Cell-free hemoglobin-mediated human lung microvascular endothelial barrier dysfunction is not mediated by cell death.

Cell-free hemoglobin-mediated human lung microvascular endothelial barrier dysfunction is not mediated by cell death.

Biochemical and biophysical research communications (2021-04-14)
Toria Tomasek, Lorraine B Ware, Julie A Bastarache, Jamie E Meegan
ABSTRACT

Circulating cell-free hemoglobin (CFH) contributes to endothelial injury in several inflammatory and hemolytic conditions. We and others have shown that CFH causes increased endothelial permeability, but the precise mechanisms of CFH-mediated endothelial barrier dysfunction are not fully understood. Based on our previous study in a mouse model of sepsis demonstrating that CFH increased apoptosis in the lung, we hypothesized that CFH causes endothelial barrier dysfunction through this cell death mechanism. We first confirmed that CFH causes human lung microvascular barrier dysfunction in vitro that can be prevented by the hemoglobin scavenger, haptoglobin. While CFH caused a small but significant decrease in cell viability measured by the membrane impermeable DNA dye Draq7 in human lung microvascular endothelial cells, CFH did not increase apoptosis as measured by TUNEL staining or Western blot for cleaved caspase-3. Moreover, inhibitors of apoptosis (Z-VAD-FMK), necrosis (IM-54), necroptosis (necrostatin-1), ferroptosis (ferrostatin-1), or autophagy (3-methyladenine) did not prevent CFH-mediated endothelial barrier dysfunction. We conclude that although CFH may cause a modest decrease in cell viability over time, cell death does not contribute to CFH-mediated lung microvascular endothelial barrier dysfunction.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Necrostatin-1, ≥98% (HPLC)
Sigma-Aldrich
Necrosis Inhibitor, IM-54, The Necrosis Inhibitor, IM-54, also referenced under CAS 861891-50-1, selectively blocks oxidative stress-induced necrotic cell death. This small molecule/inhibitor is primarily used for Cancer applications.
Sigma-Aldrich
Z-VAD-FMK, solid
Sigma-Aldrich
3-Methyladenine, autophagy inhibitor
Sigma-Aldrich
Ferrostatin-1, ≥95% (HPLC)
Roche
In Situ Cell Death Detection Kit, Fluorescein, sufficient for ≤50 tests, suitable for detection