Activation of cyclic GMP-dependent protein kinase blocks alcohol-mediated cell death and calcium disruption in cerebellar granule neurons.

Alcohol during brain development leads to the widespread neuronal death observed in fetal alcohol spectrum disorders (FASD). In comparison, the mature brain is less vulnerable to alcohol. Studies into maturation-acquired alcohol resistance uncovered a protective mechanism that reduces alcohol-induced neuronal death through nitric oxide-cGMP-cyclic GMP-dependent protein kinase (NO-cGMP-cGK) signaling. However, the downstream processes underlying this neuroprotection remain unclear. Alcohol can disrupt levels of intracellular calcium ([Ca2+]i) in vulnerable neuronal populations to trigger cell death in both in vivo and in vitro models of FASD. Since cGK has been demonstrated to regulate and inhibit intracellular Ca2+ release, we examined the hypothesis that cGK confers alcohol resistance by preventing [Ca2+]i disruptions. Alcohol resistance, determined by neuronal survival after 24 h of alcohol exposure, was examined in primary cerebellar granule neuron (CGN) cultures derived from 5 to 7 day-old neonatal mice with an activator, 8-Br-cGMP, and/or an inhibitor, Rp-8-pCPT-cGMPS, of cGK signaling. Intracellular Ca2+ responses to alcohol were measured by ratiometric Ca2+ imaging in Fura-2-loaded CGN cultures after 8-Br-cGMP treatment. Our results indicate that activating cGK with 8-Br-cGMP before alcohol administration provided neuroprotection, which the cGK inhibitor, Rp-8-pCPT-cGMPS, blocked. Alcohol exposure elevated [Ca2+]i, whereas 8-Br-cGMP pretreatment reduced both the level of the alcohol-induced rise in [Ca2+]i as well as the number of cells that responded to alcohol by increasing [Ca2+]i. These findings associate alcohol resistance, mediated by cGK signaling, to reduction of the persistent and toxic increase in [Ca2+]i from alcohol exposure.

Intracellular calcium plays a critical role in the alcohol-mediated death of cerebellar granule neurons.

Alcohol is a potent neuroteratogen that can trigger neuronal death in the developing brain. However, the mechanism underlying this alcohol-induced neuronal death is not fully understood. Utilizing primary cultures of cerebellar granule neurons (CGN), we tested the hypothesis that the alcohol-induced increase in intracellular calcium [Ca(2+)](i) causes the death of CGN. Alcohol induced a dose-dependent (200-800 mg/dL) neuronal death within 24 h. Ratiometric Ca(2+) imaging with Fura-2 revealed that alcohol causes a rapid (1-2 min), dose-dependent increase in [Ca(2+)](i), which persisted for the duration of the experiment (5 or 7 min). The alcohol-induced increase in [Ca(2+)](i) was observed in Ca(2+) -free media, suggesting intracellular Ca(2+) release. Pre-treatment of CGN cultures with an inhibitor (2-APB) of the inositol-triphosphate receptor (IP(3) R), which regulates Ca(2+) release from the endoplasmic reticulum (ER), blocked both the alcohol-induced rise in [Ca(2+)](i) and the neuronal death caused by alcohol. Similarly, pre-treatment with BAPTA/AM, a Ca(2+) -chelator, also inhibited the alcohol-induced surge in [Ca(2+) ](i) and prevented neuronal death. In conclusion, alcohol disrupts [Ca(2+)](i) homeostasis in CGN by releasing Ca(2+) from intracellular stores, resulting in a sustained increase in [Ca(2+)](i). This sustained increase in [Ca(2+)](i) may be a key determinant in the mechanism underlying alcohol-induced neuronal death.

Increased expression of Nox1 in neointimal smooth muscle cells promotes activation of matrix metalloproteinase-9.

OBJECTIVE: Vascular injury causes neointimal hypertrophy, which is characterized by redox-mediated matrix degradation and smooth muscle cell (SMC) migration and proliferation. We hypothesized that, as compared to the adjacent medial SMCs, neointimal SMCs produce increased superoxide via NADPH oxidase, which induces redox-sensitive intracellular signaling to activate matrix metalloproteinase-9 (MMP-9). METHODS AND RESULTS: Two weeks after balloon injury, rat aorta developed a prominent neointima, containing increased expression of NADPH oxidase and reactive oxygen species (ROS) as compared to the medial layer. Next, SMCs were isolated from either the neointima or the media and studied in culture. Neointimal-derived SMCs exhibited increased Nox1 expression and ROS levels as compared to medial SMCs. Neointimal SMCs had higher cell growth rates than medial SMCs. ROS-dependent ERK1/2 phosphorylation was greater in neointimal SMCs. MMP-9 activity, as detected by gel zymography, was greater in neointimal SMCs under resting and stimulated conditions and was prevented by expression of an antisense to Nox1 or treatment with an ERK1/2 inhibitor. CONCLUSIONS: Following vascular injury, the increased expression of Nox1 in SMCs within the neointima initiates redox-dependent phosphorylation of ERK1/2 and subsequent MMP-9 activation.

Nox1 transactivation of epidermal growth factor receptor promotes N-cadherin shedding and smooth muscle cell migration.

AIMS: In atherosclerosis and restenosis, vascular smooth muscle cells (SMCs) migrate into the subendothelial space and proliferate, contributing to neointimal formation. The goal of this study was to define the signalling pathway by which Nox1 NAPDH oxidase mediates SMC migration. METHODS AND RESULTS: SMCs were cultured from thoracic aorta from Nox1(-/y) (Nox1 knockout, KO) and wild-type (WT) mice. In response to thrombin, WT but not Nox1 KO SMCs generated increased levels of reactive oxygen species (ROS). Deficiency of Nox1 prevented thrombin-induced phosphorylation of Src and the subsequent transactivation of the epidermal growth factor receptor (EGFR) at multiple tyrosine residues. Next, activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and matrix metalloproteinase-9 (MMP-9) by thrombin was inhibited by the EGFR inhibitor AG1478 and in Nox1 KO SMCs. Thrombin-induced shedding of N-cadherin from the plasma membrane was dependent on the presence of Nox1 and was blocked by AG1478 and an inhibitor of metalloproteinases. Migration of SMCs to thrombin was impaired in the Nox1 KO SMCs and was restored by expression of Nox1. Finally, treatment of WT SMCs with AG1478 abrogated Nox1-dependent SMC migration. CONCLUSIONS: The Nox1 NADPH oxidase signals through EGFR to activate MMP-9 and promote the shedding of N-cadherin, thereby contributing to SMC migration.

Activation of NADPH oxidase 1 increases intracellular calcium and migration of smooth muscle cells.

Redox-dependent migration and proliferation of vascular smooth muscle cells (SMCs) are central events in the development of vascular proliferative diseases; however, the underlying intracellular signaling mechanisms are not fully understood. We tested the hypothesis that activation of Nox1 NADPH oxidase modulates intracellular calcium ([Ca(2+)](i)) levels. Using cultured SMCs from wild-type and Nox1 null mice, we confirmed that thrombin-dependent generation of reactive oxygen species requires Nox1. Thrombin rapidly increased [Ca(2+)](i), as measured by fura-2 fluorescence ratio imaging, in wild-type but not Nox1 null SMCs. The increase in [Ca(2+)](i) in wild-type SMCs was inhibited by antisense to Nox1 and restored by expression of Nox1 in Nox1 null SMCs. Investigation into potential mechanisms by which Nox1 modulates [Ca(2+)](i) showed that thrombin-induced inositol triphosphate generation and thapsigargin-induced intracellular calcium mobilization were similar in wild-type and Nox1 null SMCs. To examine the effects of Nox1 on Ca(2+) entry, cells were either bathed in Ca(2+)-free medium or exposed to dihydropyridines to block L-type Ca(2+) channel activity. Treatment with nifedipine or removal of extracellular Ca(2+) reduced the thrombin-mediated increase of [Ca(2+)](i) in wild-type SMCs, whereas the response in Nox1 null SMCs was unchanged. Sodium vanadate, an inhibitor of protein tyrosine phosphatases, restored the thrombin-induced increase of [Ca(2+)](i) in Nox1 null SMCs. Migration of SMCs was impaired with deficiency of Nox1 and restored with expression of Nox1 or the addition of sodium vanadate. In summary, we conclude that Nox1 NADPH oxidase modulates Ca(2+) mobilization in SMCs, in part through regulation of Ca(2+) influx, to thereby promote cell migration.

Glutathione peroxidase-deficient smooth muscle cells cause paracrine activation of normal smooth muscle cells via cyclophilin A.

BACKGROUND/AIMS: Reduced activity of the antioxidant glutathione peroxidase-1 (GPx1) correlates with increased risk of cardiovascular events in patients with coronary artery disease. However, it remains unclear whether this imbalance in antioxidant capacity directly contributes to activation of vascular cells. In response to oxidative stress, smooth muscle cells (SMCs) secrete the pro-inflammatory immunomodulator cyclophilin A (CyPA). We hypothesized that reduction in vascular cell GPx1 activity causes secretion of CyPA and paracrine-mediated activation of NF-κB and proliferation of SMCs. METHODS/RESULTS: Using a murine model of GPx1 deficiency (GPx1(+/-)), we found elevated levels of hydrogen peroxide levels and increased secretion of CyPA in both arterial segments and cultured SMCs as compared to wild type (WT). Conditioned media from GPx1(+/-) SMCs caused increased NF-κB activation of quiescent WT SMCs, and this was inhibited by the antioxidant N-acetyl-l-cysteine or by cyclosporine A (CsA). In co-culture experiments, SMCs derived from GPx1(+/-) aorta caused increased proliferation of WT SMCs, which was also inhibited by CsA. CONCLUSIONS: Reduction in vascular cell GPx1 activity and the associated increase in oxidative stress cause CyPA-mediated paracrine activation of SMCs. These findings identify a novel mechanism by which an imbalance in antioxidant capacity may contribute to vascular disease.

Inhibition of apoptotic signaling and neointimal hyperplasia by tempol and nitric oxide synthase following vascular injury.

OBJECTIVES: We hypothesized that redox-mediated apoptosis of medial smooth muscle cells (SMC) during the acute phase of vascular injury contributes to the pathophysiology of vascular disease. METHODS: Apoptosis of medial SMC (1-14 days following balloon injury) was identified in rat carotid arteries by in situ DNA labeling. NADPH-derived superoxide and expression of Bcl-xL, Bax, caspase-3 and caspase-9 were assessed. The antioxidant tempol was administered in drinking water throughout the experimental period, and local adenoviral-mediated gene transfer of eNOS was performed prior to vascular injury. RESULTS: Balloon injury increased NADPH-dependent superoxide production, medial SMC apoptosis, Bax-positive medial SMC index, Bax/Bcl-xL ratio, and caspase-3 and caspase-9 expression in the injured arteries. Treatment with tempol or eNOS gene transfer decreased superoxide levels and medial SMC apoptosis, with a concomitant increase in medial SMC density. Inhibition of superoxide was associated with a decreased Bax/Bcl-xL ratio, and caspase-3 and -9 expression. Tempol treatment and eNOS gene therapy significantly reduced neointima formation. CONCLUSION: Vascular generation of reactive oxygen species participates in Bax activation and medial SMC apoptosis. These effects likely contribute to the shedding of cell-cell adhesion molecules and promote medial SMC migration and proliferation responsible for neointimal hyperplasia.

Tempol therapy attenuates medial smooth muscle cell apoptosis and neointima formation after balloon catheter injury in carotid artery of diabetic rats.

Accumulating data support the hypothesis that reactive oxygen species (ROS) play a critical role in the vascular complications observed in diabetes. However, the mechanisms of ROS-mediated vascular complications in diabetes are not clear. We tested the hypothesis that ROS-mediated increase in proapoptotic factor Bax expression leads to medial smooth muscle cell (SMC) apoptosis that is associated with neointima formation. We used a fructose-rich diet for 4 wk to model Type 2 diabetes in rats. SOD mimetic membrane-permeable 4-hydroxy-2,2,6,6,-tetramethylpiperidine-1-oxyl (Tempol, 1 mM) was administered in drinking water to scavenge superoxide starting 1 day before surgery and continued during the duration of the experiment. Vascular injury resulted in a significant increase in medial SMC apoptosis that was associated with neointima formation. The number of medial SMC positive for Bax immunostaining significantly increased in injured arteries compared with uninjured arteries. Superoxide scavenging by Tempol treatment inhibited both the Bax-positive index as well as the apoptotic index of medial SMC in response to vascular injury. Tempol treatment inhibited apoptotic loss of medial SMC, thus increasing their density in the injured arteries. These alterations in the media were associated with a marked decrease in neointima formation in injured arteries. We conclude that Bax expression may play an important role in vascular SMC apoptosis and, finally, that this regulatory mechanism is redox sensitive.

Inhibition of matrix metalloproteinase activity by TIMP-1 gene transfer effectively treats ischemic cardiomyopathy.

BACKGROUND: Enhanced activity of matrix metalloproteinases (MMPs) has been associated with extracellular matrix degradation and ischemic heart failure in animal models and human patients. This study evaluated the effects of MMP inhibition by gene transfer of TIMP-1 in a rat model of ischemic cardiomyopathy. METHODS AND RESULTS: Rats underwent ligation of the left anterior descending coronary artery with direct intramyocardial injection of replication-deficient adenovirus encoding TIMP-1 (n=8) or null virus as control vector (n=8), and animals were analyzed after 6 weeks. Both systolic and diastolic cardiac function was significantly preserved in the TIMP-1 group compared with control animals (maximum left ventricular [LV] pressure: TIMP-1 70+/-10 versus control 56+/-12 mmHg, P<0.05; maximum dP/dt 2697+/-842 versus 1622+/-527 mmHg/sec, P<0.01; minimum dP/dt -2900+/-917 versus -1195+/-593, P<0.001). Ventricular geometry was significantly preserved in the TIMP-1 group (LV diameter 13.0+/-0.7 versus control 14.4+/-0.4 mm, P<0.001; border-zone wall thickness 1.59+/-0.11 versus control 1.28+/-0.19 mm, P<0.05), and this was associated with a reduction in myocardial fibrosis (2.36+/-0.87 versus control 3.89+/-1.79 microg hydroxyproline/mg tissue, P<0.05). MMP activity was reduced in the TIMP-1 animals (1.5+/-0.9 versus control 43.1+/-14.9 ng of MMP-1 activity, P<0.05). CONCLUSIONS: TIMP-1 gene transfer inhibits MMP activity and preserves cardiac function and geometry in ischemic cardiomyopathy. The reduction in myocardial fibrosis may be primarily responsible for the improved diastolic function in treated animals. TIMP-1 overexpression is a promising therapeutic target for continued investigation.