Chi Dae Kim

Vascular NAD(P)H Oxidase Triggers Delayed Cerebral Vasospasm After Subarachnoid Hemorrhage in Rats

Background and Purpose– To clarify the role of vascular NAD(P)H oxidase in the pathogenesis of cerebral vasospasm after subarachnoid hemorrhage (SAH), both the activity and/or activation mechanisms of NAD(P)H oxidase in the cerebral vasculature and the effect of oxidase inhibition on SAH-induced cerebral vasospasm were assessed. Methods— The changes in the luminal perimeter of the middle cerebral artery were measured histologically after SAH was induced according to a 2-hemorrhage model in rats. The NAD(P)H oxidase activity in the cerebral vasculature was measured with a lucigenin assay at different time intervals from 12 hours to 14 days after injection of autologous blood into cisterna magna. The membrane translocation of p47phox and the protein expression of membrane subunits (gp91phox and p22phox) of NAD(P)H oxidase were analyzed using Western blot analysis. Results— The luminal perimeter of the middle cerebral artery started to decrease on day 1 and peaked on day 5 after a second injection of blood, and these changes were significantly ameliorated by treatment with an NAD(P)H oxidase inhibitor, diphenyleneiodonium. At 24 hours after the second injection of blood, both vascular production of superoxide anion and NAD(P)H oxidase activity were markedly increased with enhanced membrane translocation of p47phox, but by 48 hours the enzyme activity had regained normal values. However, no significant changes in the expression of gp91phox and p22phox were observed throughout the experiments. Conclusions— These findings suggest that the activation of NAD(P)H oxidase through enhanced assembly of the oxidase components in the early stages of SAH might contribute to the delayed cerebral vasospasm in SAH rats.

Neuroprotection by cilostazol, a phosphodiesterase type 3 inhibitor, against apoptotic white matter changes in rat after chronic cerebral hypoperfusion.

In the present study, we elucidated effect of cilostazol to prevent the occurrence of vacuolation and rarefaction of the white matter in association with apoptosis induced by bilateral occlusion of common carotid arteries in the male Wistar rats. Rats orally received vehicle (DMSO) or 60 mg kg(-1) day(-1) (orally) cilostazol for 3, 7, 14 or 30 days. In the vehicle group, increased vacuolation and rarefactions in the white matter were accompanied by extensive activation of both microglial and astroglial cells with suppression of oligodendrocytes in association with increased TNF-alpha production, caspase-3 immunoreactivity and TUNEL-positive cells in the white matter including optic tract. Post-treatment with cilostazol (60 mg kg(-1) day(-1)) strongly suppressed not only elevated activation of astroglia and microglia but also diminished oligodendrocytes following chronic cerebral hypoperfusion. In conclusion, cilostazol (60 mg kg(-1) day(-1), orally) significantly reduced the apoptotic cell death in association with decreased TNF-alpha production and caspase-3-positive cells in the white matter of rat brains subjected to bilateral occlusion of common carotid arteries, consequently ameliorating vacuoles and rarefaction changes in the white matter.

NAD(P)H oxidase participates in the signaling events in high glucose-induced proliferation of vascular smooth muscle cells.

Reactive oxygen species (ROS) have been implicated in the pathogenesis of vascular dysfunction in diabetes mellitus, and NAD(P)H oxidase is known as the most important source of ROS in the vasculatures. To determine whether NAD(P)H oxidase is a major participant in the critical intermediary signaling events in high glucose (HG, 25 mM)-induced proliferation of vascular smooth muscle cells (VSMC), we investigated in explanted aortic VSMC from rats the role of NAD(P)H oxidase on the HG-related cellular proliferation and superoxide production. VSMC under HG condition had increased proliferative capacity that was inhibited by tiron (1 mM), a cell membrane permeable superoxide scavenger, but not by SOD, which is not permeable to cell membrane. The nitroblue tetrazolium staining in the HG-exposed VSMC was more prominent than that of VSMC under normal glucose (5.5 mM) condition, which was significantly inhibited by DPI (10 microM), an NAD(P)H oxidase inhibitor, but not by inhibitors for other oxidases such as NADH dehydrogenase, xanthine oxidase, and nitric oxide synthase. In the VSMC under HG condition, the enhanced NAD(P)H oxidase activity with increased membrane translocation of Rac1 was observed, but the protein expression of p22phox and gp91phox was not increased. These data suggest that HG-induced changes in VSMC proliferation are related to the intracellular production of superoxide through enhanced activity of NAD(P)H oxidase.

Impairment of autoregulatory vasodilation by NAD(P)H oxidase-dependent superoxide generation during acute stage of subarachnoid hemorrhage in rat pial artery.

This study assessed the mechanism(s) by which the autoregulatory vasodilation of rat pial artery in response to acute hypotension during the acute phase of subarachnoid hemorrhage (SAH) was markedly blunted. Increased superoxide production from the cerebral vessels in response to NAD(P)H at 24 hours after SAH +NG-nitro-L-arginine methyl ester (L-NAME) (10 mg/kg) was inhibited by intracisternal administration of a tyrosine kinase inhibitor genistein (10 μmol/L) and Rac inhibitor Clostridium difficile toxin B (1 ng/mL) and a flavoenzyme inhibitor diphenyleneiodonium (10 μmol/L). The expression of gp91phox was enhanced by SAH + L-NAME from 12 to 24 hours, which was inhibited by genistein and toxin B, but not the p22phox. Increased membrane translocation of Rac after SAH + L-NAME was attenuated by both genistein and toxin B, whereas increased tyrosine kinase activity was blocked by genistein, but not by toxin B. The blunted autoregulatory vasodilation to acute hypotension was effectively recovered by genistein and C. difficile toxin B as well as by diphenyleneiodonium. In conclusion, SAH during acute stage causes an increase in NAD(P)H oxidase—dependent superoxide formation in cerebral vessels, which is due to activation of tyrosine phosphorylation-dependent increased expression of gp91phox mRNA and translocation of Rac protein, thereby resulting in a significant reduction of autoregulatory vasodilation.

Effect of ω-Conotoxin GVIA and ω-Agatoxin IVA on the Capsaicin-Sensitive Calcitonin Gene-Related Peptide Release and Autoregulatory Vasodilation in Rat Pial Arteries

This study assesses the effect of neuronal voltage-sensitive Ca2+ channel blockers, ω-conotoxin GVIA (CTX), and ω-agatoxin IVA (AgTX) on the vasodilation and release of calcitonin gene-related peptide (CGRP), both of which were induced by either application of capsaicin or acute stepwise hypotension. Changes in pial arterial diameter were determined directly through a closed cranial window. The vasodilation of pial artery induced by either CGRP (0.1 μmol/L) or capsaicin (0.3 μmol/L) was significantly inhibited by CGRP8–37 (0.1 μmol/L) (P < 0.05 and P < 0.05, respectively). The autoregulatory vasodilation to acute stepwise hypotension was severely attenuated by pretreatment with either CTX or AgTX. When the hypotension was kept for 2, 4, and 10 minutes, the releasable CGRP-like immunoreactivity (CGRP-LI) level (vehicle, 13.4 ± 1.5 fmol/mm2/30 min) by 10 μmol/L capsaicin from the isolated pial arteries was significantly reduced in the 4- and 10-minute hypotension groups (11.3 ± 1.2 fmol/mm2/30 min, P < 0.05, and 11.1 ± 1.5 fmol/mm2/30 min, P < 0.05, respectively), but not in 2-min group. Moreover, the CGRP-LI level released by 10 μmol/L capsaicin (13.7 ± 0.9 fmol/mm2/30 min) also was significantly depressed by pretreatment with 1 μmol/L CTX to 10.4 ± 1.0 fmol/mm2/30 min (P < 0.01) and with 0.1 μmol/L AgTX to 8.7 (1.7 fmol/mm2/30 min (P < 0.001), as well as by pretreatment with 10 μmol/L capsaicin (6.0 ± 1.6 fmol/mm2/30 min, P < 0.001). These results suggest that the neuronal N- and P-type voltage-sensitive Ca2+ channels are implicated in the release of CGRP from capsaicin-sensitive perivascular sensory nerves in response to acute hypotension, and that the released CGRP may contribute to the autoregulatory vasodilation in the cerebral microcirculation.

Suppression of PU.1-linked TLR4 expression by cilostazol with decrease of cytokine production in macrophages from patients with rheumatoid arthritis

The present study assessed the effects of cilostazol on LPS‐stimulated TLR4 signal pathways in synovial macrophages from patients with rheumatoid arthritis (RA). These effects were confirmed in collagen‐induced arthritis (CIA) in mice.

Prevention of Impairment of Cerebral Blood Flow Autoregulation during Acute Stage of Subarachnoid Hemorrhage by Gene Transfer of Cu/Zn SOD-1 to Cerebral Vessels

The present study determined whether gene transfer of human copper/zinc superoxide dismutase-1 (Cu/Zn SOD-1) prevented the autoregulatory impairment of CBF induced by subarachnoid hemorrhage (SAH). After application of recombinant adenovirus (100 μL of 1 × 1010 pfu/mL, intracisternally) encoding human Cu/Zn SOD-1 3 days before experiments, Cu/Zn SOD-1 activity significantly increased in association with increase in Cu/Zn SOD-1 mRNA and protein expression in the cerebral vasculature of both sham-operated and SAH rats as determined by reverse transcriptase-polymerase chain reaction, Western blotting, and immunohistochemistry, and SAH-induced increase in superoxide anion was markedly reduced in accordance with increased nitric oxide production. In line with these findings, rats that received human Cu/Zn SOD-1 therapy showed the prominent restoration of blunted vasodilation of the pial artery in response to calcitonin gene-related peptide and levcromakalim, and the recovery of impaired autoregulatory vasodilation in response to acute hypotension, thereby leading to significant restoration of CBF autoregulation. These results provide a rational basis for application of Cu/Zn SOD-1 gene therapy for protection of the impairment of autoregulatory CBF during the acute stage of SAH.

Decreased CGRP level with increased sensitivity to CGRP in the pial arteries of spontaneously hypertensive rats

It was aimed to investigate the importance of calcitonin gene-related peptide (CGRP) in maintenance of normal cerebral microcirculation. We examined both the functional (in vivo) and biochemical effects (in vitro) of CGRP on the pial arteries of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). When mock cerebrospinal fluid containing capsaicin (3 x 10(-7) M) was suffused over the cortical surface, the diameter of pial arteries of SHR was transiently increased and rapidly returned to the baseline level, while the capsaicin-induced increase in pial arterial diameters of WKY was large and sustained for a longer duration (> 10 min). Capsaicin-induced vasodilation was significantly attenuated by pretreatment with CGRP8-37, a CGRP1, receptor antagonist, in both WKY and SHR. On the other hand, cortical suffusion with CGRP (10(-9) approximately 10(-6) M) exerted a larger enhancement in the vasodilation of pial artery of SHR than WKY. The CGRP-induced vasodilation was significantly antagonized by CGRP8-37 in both WKY and SHR. The released level of CGRP-like immunoreactivity (CGRP-LI) from the pial artery was significantly lower in SHR (12.3 +/- 1.2 fmol/mm2/hr) than that in WKY (24.5 +/- 3.9 fmol/mm2/hr). CGRP (10(-6) M)-induced stimulation of cyclic AMP formation was rather larger in the pial arteries from SHR (50.2 +/- 5.8 fmol/mm2/30 min, p < 0.05) than those from WKY (34.5 +/- 3.8 fmol/mm2/30 min). These data suggest that, in the pial arteries of SHR, the transient vasodilation to capsaicin and enhanced vasodilation to CGRP are related to the decreased CGRP level in the cerebral microvascular beds, consequently leading to increased sensitivity of the CGRP receptors to CGRP.

Involvement of NADH/NADPH oxidase-derived superoxide in experimental vasospasm induced by periarterial blood in rat femoral artery.

To elucidate the mechanism(s) involved in periarterial blood-mediated vasospasm in the rat femoral artery, vascular production of superoxide and related expression of intercellular adhesion molecule-1 (ICAM-1) were assessed with subsequent perivascular mobilization of granulocytes and macrophages. Arterial vasospasm characterized by increased wall thickness and decreased lumen size was observed on the side exposed to blood at 7 to 12 days, and these vascular changes were significantly ameliorated by pretreatment with NADH/NADPH oxidase inhibitor, diphenyleneiodonium (200 microM, locally). Increased mobilization of granulocytes was paralleled with the expression of ICAM-1 in the vessels at 24 hours after periarterial application of blood to the femoral artery, and then both declined. Subsequently, infiltration of macrophage progressively increased at all layers throughout 7 to 12 days. In in vitro study, a large amount of superoxide that was inhibitable by diphenyleneiodonium (20 and 100 microM) was produced at 3 hours upon application of 10% autologous blood to the aortic segments. Furthermore, ICAM-1 expression by autologous blood was well correlated with generation of superoxide anion in the aortic segment (r=0.975, P<0.05). Taken together, it is suggested that NADH/NADPH oxidase-derived superoxide is implicated in periarterial blood-induced vasospasm via increased expression of ICAM-1 with subsequent mobilization of granulocyte/macrophage.