Abstract Body (Do not enter title and authors here): Background: Increasing evidence support a vasoprotective role of the major endothelial NADPH oxidase isoform 4 (NOX4). NOX4 is notably induced by hypoxia.
Research Question: What is the functional role of NOX4 under hypoxic conditions in the vessel wall?
Aim: We aimed to elucidate NOX4’s significance in endothelial and vascular function under hypoxia.
Methods: Primary cultures of human endothelial cells were exposed to hypoxia of 1% O₂. Internal mammary arteries were obtained from patients undergoing coronary artery bypass grafting surgery. Surgical specimens of the artery wall proximal to occlusion were obtained from patients with peripheral arterial disease. Human NOX4 was downregulated by lentiviral shNOX4. Primary microvascular lung endothelial cells were isolated from wild-type and Nox4^-/- mice. Vascular function was assessed under hypoxia in murine mesenteric arteries and aortas in a Mulvany myograph. Laminar flow was applied by cone-and-plate viscometer or ibidi pump system. NO was measured by Griess reaction, H₂O₂ by Amplex Red Assay and gene expression by real-time PCR and Western blot.
Results: Hypoxia significantly elevated NOX4 expression and activity in endothelial cells. Inhibition of prolyl hydroxylase domain (PHD) enzymes, which stabilize hypoxia-inducible factors (HIFs), increased NOX4 expression even under normoxic conditions. In human hypoxia-prone vessels, NOX4 expression strongly correlated with genes relevant for vascular function, such as prostaglandin I2 synthase (PTGIS). Endothelial function assessments under hypoxia revealed elevated contraction and endothelial dysfunction in both wild-type and Nox4^-/- mice with Nox4^-/- mice exhibiting the most severe alterations in endothelium-dependent vasodilation. Laminar shear stress attenuated the hypoxic response in endothelial cells, reducing HIF1a and NOX4 expression while enhancing eNOS expression. NO synthase inhibition under combined hypoxia and laminar flow conditions upregulated NOX4. Furthermore, NOX4 deletion affected PECAM1 expression and endothelial cell adhesion. Inhibition of NOX4 under combined hypoxia and laminar flow increased the number of endothelial cells not aligned in the direction of flow.
Conclusions: Hypoxia-induced NOX4 ensures vasodilation in both conductive and resistant arteries. Laminar blood flow restores eNOS expression and mitigates the hypoxic response on NOX4. NOX4 deletion affects PECAM1 expression, reduces endothelial cell adhesion and alignment.