A Novel Functional Variant of nNOS Mediates Anoxic Injury in Brain Microvascular Endothelial Cells via Mitochondrial Superoxide Generation

Abstract

Deletions of endothelial (eNOS) and neuronal (nNOS) nitric oxide synthase isoforms in mice have been shown to elicit opposite effects on ischemic brain injury following experimental stroke. Recently, we have demonstrated the expression of nNOS in human, rat, and mouse primary brain microvascular endothelial cells (MECs). The constitutively active endothelial nNOS (enNOS) was found to be functionally distinct from the nNOS expressed in neurons and the eNOS but sensitive to inhibition by pharmacological nNOS inhibitors. Under physiological conditions, enNOS was found to produce superoxide instead of nitric oxide (NO), unlike all known NOS isoforms. The objective of the present study was to examine the pathophysiological role of enNOS in endothelial injury. We hypothesized that enNOS mediates the anoxic injury underlying the blood‐brain barrier (BBB) dysfunction and cell death by instigating mitochondrial oxidative stress. We utilized primary human brain MECs (passage# 7–9) and employed inhibitors of nNOS (N‐ω‐Propyl‐L‐arginine or NPA and ARL‐17477; 1 μmol/L each) and eNOS (L‐N5‐(1‐Iminoethyl)ornithine or NIO, 1 μmol/L). Electron spin resonance spectrometry measurements with mitochondria‐targeted mito‐TEMPO‐H (50 μmol/L) showed that cells treated with enNOS inhibitor (ARL‐17477, 165±16 a.u, n=7; p < 0.05) reduced the mitochondrial superoxide in compared to the untreated MECs (108±10 a.u, n=7). Oxygen consumption rate (OCR; pmol/minute/pg protein) measured in MECs by Seahorse Analyzer showed increase in maximal respiration and decreased non‐mitochondrial OCR with NPA treatment (355 ± 60 and 7 ± 4, respectively; n=11; p < 0.05) compared to untreated cells (189 ± 49 and 19 ± 4, respectively; n=11). Transendothelial electrical resistance (TEER) measurements were made to quantitate the BBB integrity in confluent monolayers of hMECs grown in transwells and subjected to oxygen‐glucose deprivation (OGD; 6 h). Inhibition of enNOS with ARL‐17477 (50 ± 3 vs. 39 ± 2 untreated, n=6–8; p < 0.05) and NPA (46 ± 2 vs. 36 ± 2 untreated, n=7–9; p < 0.05) increased TEER (Ω.cm2) in MECs. In addition, ARL‐17477 and NPA (119 ± 15 and 116 ± 9, n=9 each; p<0.05) sustained recovery of TEER (% change) at 30 hours following OGD compared to the eNOS inhibited (89 ± 5, n=9; p=NS) and untreated cells (75 ± 8, n=9). Western blot studies have shown that treatment with enNOS inhibitors increases the levels of the tight junction protein, Zona Occludens‐1, in MECs compared to untreated cells. In contrast, viability measurements using CCK‐8 assay (% viable cells) observed that MECs treated with each NOS inhibitor (NPA: 42 ± 3; ARL‐17477: 52 ± 3; NIO: 70 ± 5; n = 16, p < 0.05) and exposed to OGD increased cell survival compared to untreated cells (26 ± 2; n = 16). Thus, enNOS mediates anoxic‐injury leading to the impairment of BBB integrity as well as cell death.Support or Funding InformationAHA Predoctoral (VNS: 16PRE31450006) and postdoctoral (IR: 15POST23040005) Fellowship Grant, AHA Scientist Development Grant (PVK: 14SDG20490359), and NIH grants (DWB: HL093554 and PVK: NS094834).