Glutamate induces oxidative stress and apoptosis in cerebral vascular endothelial cells: contributions of HO-1 and HO-2 to cytoprotection

Abstract

In cerebral circulation, epileptic seizures associated with excessive release of the excitatory neurotransmitter glutamate cause endothelial injury. Heme oxygenase (HO), which metabolizes heme to a vasodilator, carbon monoxide (CO), and antioxidants, biliverdin/bilirubin, is highly expressed in cerebral microvessels as a constitutive isoform, HO-2, whereas the inducible form, HO-1, is not detectable. Using cerebral vascular endothelial cells from newborn pigs and HO-2-knockout mice, we addressed the hypotheses that 1) glutamate induces oxidative stress-related endothelial death by apoptosis, and 2) HO-1 and HO-2 are protective against glutamate cytotoxicity. In cerebral endothelial cells, glutamate (0.1-2.0 mM) increased formation of reactive oxygen species, including superoxide radicals, and induced major keystone events of apoptosis, such as NF-kappaB nuclear translocation, caspase-3 activation, DNA fragmentation, and cell detachment. Glutamate-induced apoptosis was greatly exacerbated in HO-2 gene-deleted murine cerebrovascular endothelial cells and in porcine cells with pharmacologically inhibited HO-2 activity. Glutamate toxicity was prevented by superoxide dismutase, suggesting apoptotic changes are oxidative stress related. When HO-1 was pharmacologically upregulated by cobalt protoporphyrin, apoptotic effects of glutamate in cerebral endothelial cells were completely prevented. Glutamate-induced reactive oxygen species production and apoptosis were blocked by a CO-releasing compound, CORM-A1 (50 microM), and by bilirubin (1 microM), consistent with the antioxidant and cytoprotective roles of the end products of HO activity. We conclude that both HO-1 and HO-2 have anti-apoptotic effects against oxidative stress-related glutamate toxicity in cerebral vascular endothelium. Although HO-1, when induced, provides powerful protection, HO-2 is an essential endogenous anti-apoptotic factor against glutamate toxicity in the cerebral vascular endothelium.

Cerebromicrovascular endothelial cells are resistant to l-glutamate

“…Indeed, the morphology of cultures shown in that study (33) do not show much resemblance to the characteristic features of CMVEC cultures (Fig. 1, A and B) but rather look like the cells in our CMVPC cultures (Fig.…”

“…As a novel approach to the controversy on CMVEC excitotoxicity, we studied the effect of L-glut on CMVPC cultures, since the study of Parfenova et al (33) used 20% FBSsupplemented culture medium and prolonged culturing, which in our experience promote CMVPC proliferation (21). Indeed, the morphology of cultures shown in that study (33) do not show much resemblance to the characteristic features of CMVEC cultures (Fig.…”

“…In piglet CMVECs, the presence of NMDA receptors was suggested by [ 3 H]L-glut binding assays (34); however, these binding sites may not be ionotropic receptors but rather the well-known L-glut transporters of CMVECs. We restricted our efforts to seek L-glut receptor expression in CMVECs to the study of NMDA receptor subunit NR1 expression, since most of the positive studies demonstrating L-glut excitotoxicity emphasized the role of NMDA receptors in the mechanism of CMVEC injury (1,(33)(34)(35)(36)45). NR1 subunits are ubiquitous components of any functional NMDA receptors (6); thus the lack of NR1 immunopositivity in our CMVEC and CMVPC cultures is consistent with the insensitivity and resistance of these cells to the high doses of L-glut/ NMDA shown in the present study.…”

“…L-Glutamine was praised as a growth supplement in rat CMVEC cultures by the same authors in a full paper that did not mention L-glut toxicity (11). More recently, L-glut (1-3 mM, 30 min-3 h) has been suggested to induce apoptotic cell death in murine and piglet CMVECs caused by oxidative stress, and the protective role of inducible hemoxygenase-2 activity was emphasized (33,45). However, Andras et al (1) found no decrease in CMVEC viability with the same dose of L-glut (1 mM, 24 h) in rat CMVECs, and we confirmed this finding in both rat and piglet CMVEC cultures in the present study.…”

“…CMVECs have been reported to possess various L-glut receptor subunits or L-glut binding sites in rat, piglet, or human CMVECs (1,5,27,34,36,45). High concentrations of L-glut (1-3 mM), at least in vitro, are claimed to result in N-methyl-D-aspartate (NMDA) receptor activation with subsequent elevations in intracellular Ca 2ϩ levels ([Ca 2ϩ ] i ) and subsequent production of reactive oxygen species leading to cell dysfunction/death of CMVECs (33,35,36,45).However, the attractive CMVEC excitotoxicity hypothesis is challenged by several lines of evidence. First, many studies have failed to find any functional glutamate receptors in ovine, bovine, rat, and human cerebral microvessels or cerebral arteries (2, 31, 44).…”