Ramesh C. Bhalla

eNOS Gene Transfer Inhibits Smooth Muscle Cell Migration and MMP-2 and MMP-9 Activity

Vascular smooth muscle cell (SMC) migration is a critical step in the development of neointima after angioplasty. Matrix metalloproteinases (MMPs) degrade the basement membrane and the extracellular matrix, facilitating SMC migration. Transfer of the endothelial nitric oxide synthase (eNOS) gene to the injury site inhibits neointima formation. Neither the signaling pathways leading to NO-mediated inhibition of SMC migration and proliferation nor the alterations in these pathways have been characterized. We hypothesize that NO inhibits SMC migration in part by regulating MMP activity. To test this hypothesis, we transfected cultured rat aortic SMCs with replication-deficient adenovirus containing bovine eNOS gene and analyzed the conditioned medium for MMP activity. We observed that eNOS gene transfer significantly (P<0.05) inhibited SMC migration and significantly (P<0.05) decreased MMP-2 and MMP-9 activities in the conditioned medium. Similarly, addition of the NO donor DETA NONOate and 8-bromo-cGMP to the culture medium significantly decreased MMP-2 and MMP-9 activities in the conditioned medium collected 24 hours after treatment. Furthermore, Western blot analysis of the conditioned medium collected from eNOS gene-transfected SMCs showed a significant increase in tissue inhibitor of metalloproteinases-2 (TIMP-2) levels. Our data suggest that NO decreases MMP-2 and MMP-9 activities and increases TIMP-2 secretion, and this shifts the balance of MMP activity, which may favor the inhibition of cell migration because of inhibition of extracellular matrix degradation.

Altered calcium sequestration by subcellular fractions of vascular smooth muscle from spontaneously hypertensive rats

Abstract The calcium sequestration characteristics of microsomal and mitochondrial fractions prepared from aortae of spontaneously hypertensive (SHR), Kyoto Wistar normotensive (NWR), and normotensive Sprague-Dawley (NSDR) rats were studied. Calcium uptake by the microsomal vesicles of SHR was significantly lower as compared to NWR and NSDR. However, mitochondria isolated from SHR aortae did not differ from NWR in their calcium uptake characteristics. Microsomal vesicles of NSDR aortae accumulated consistently more calcium than NWR. These findings illustrate the significance of relevant controls for SHR. Calcium-dependent ATPase activity in the microsomal fraction was significantly increased in SHR as compared to NWR. This may indicate a compensatory phenomenon on the part of the smooth muscle cell to increase Ca 2+ uptake by the microsomal vesicles. These results suggest that a decreased ability to sequester calcium by the microsomal fraction of vascular smooth muscle of SHR may result in altered calcium distribution in the muscle cell.

Mechanical stimulation increases intracellular calcium concentration in nodose sensory neurons.

The cellular mechanisms involved in activation of mechanosensitive visceral sensory nerves are poorly understood. The major goal of this study was to determine the effect of mechanical stimulation on intracellular calcium concentration ([Ca2+]i) using nodose sensory neurons grown in culture. Primary cultures of nodose sensory neurons were prepared by enzymatic dispersion from nodose ganglia of 4-8 week old Sprague-Dawley rats. Whole cell [Ca2+]i was measured by a microscopic digital image analysis system in fura-2 loaded single neurons. Brief mechanical stimulation of individual nodose sensory neurons was achieved by deformation of the cell surface with a glass micropipette. In 31 of 50 neurons (62%), mechanical stimulation increased [Ca2+]i from 125 +/- 8 to 763 +/- 89 nM measured approximately 10 s after stimulation. [Ca2+]i then declined gradually, returning to near basal levels over a period of minutes. [Ca2+]i failed to increase after mechanical stimulation in the remaining 19 neurons. The mechanically-induced rise in [Ca2+]i was essentially abolished after the neurons were incubated for 5-10 min in zero Ca2+ buffer (n = 7) or after addition of gadolinium (10 microM), a blocker of stretch-activated ion channels (n = 5). The effect of gadolinium was reversed after removal of gadolinium. The results indicate that: (1) mechanical stretch increases [Ca2+]i in a subpopulation of nodose sensory neurons in culture, and (2) the stretch-induced increase in [Ca2+]i is dependent on influx of Ca2+ from extracellular fluid and is reversibly blocked by gadolinium. The findings suggest that opening of stretch-activated ion channels in response to mechanical deformation leads to an increase in Ca2+ concentration in visceral sensory neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Vascular Effects of Metformin Possible Mechanisms for its Antihypertensive Action in the Spontaneously Hypertensive Rat

Metformin, an antidiabetic agent, potentiates insulin action and reduces insulin resistance. We examined the antihypertensive effects and vascular effects of metformin in spontaneously hypertensive rats (SHR). Wistar-Kyoto normotensive (WKY) and SHR were injected with metformin (100 mg/kg) or saline subcutaneously twice daily for 4 weeks. Blood pressure was recorded by a tail-cuff plethesmographic method. Metformin treatment significantly attenuated (P < .05) the increase in blood pressure in metformin treated SHR versus untreated control SHR. At the end of the experimental period of 4 weeks, metformin-treated SHR had a mean blood pressure that was 34 mm lower than that of untreated SHR. Metformin treatment had no significant effect on blood pressure in WKY rats. Treatment of SHR aortic smooth muscle (SM) cells with metformin (2 micrograms/mL) for 24 h significantly decreased (P < .05) arginine vasopressin- and thrombin- stimulated increase in [Ca2+]i. However, metformin treatment did not have a significant effect on the basal [Ca+]i. Incubation of SHR aortic SM cells with OH-L-arginine (25 to 100 mumol/L) for 24 h increased nitrite production in a dose dependent manner. Metformin (5 micrograms/mL) treatment of SM cells increased nitrite production at all concentrations of OH-L-arginine; however, differences were significant (P < .05) only at 25 and 50 mumol/L OH-L-arginine. These results suggest that metformin may be decreasing arterial pressure in the SHR, at least in part, by attenuating the agonist-stimulated [Ca2+]i response in SHR vascular smooth muscle cells.

Estrogen receptor-α gene transfer into bovine aortic endothelial cells induces eNOS gene expression and inhibits cell migration

OBJECTIVES It has been suggested that estrogen may improve endothelial cell function to delay the onset of atherosclerosis in pre-menopausal females, though its mechanism of action is not fully understood. We examined the hypothesis that human estrogen receptor-alpha (ER alpha) gene transfection improves the endothelial cell function. METHODS A replication deficient adenoviral vector was used to transfect the ER alpha gene into bovine aortic endothelial cells (BAEC) and a GFP gene containing vector was used as control. Expression of the eNOS gene was determined by Northern blot analysis and enzyme activity assay; cell migration was assayed using a Transwell apparatus; and tyrosine phosphorylation of FAK was estimated by Western blot analysis. RESULTS ER alpha gene transfection of endothelial cells produced a 2-3-fold increase in eNOS mRNA and protein levels as well as a significant increase (P < 0.05) in NOS activity as measured by citrulline assay and nitrite accumulation in the media in response to bradykinin stimulation. Treatment of cells with estrogen blocking agent ICI 182780 inhibited eNOS induction in response to ER alpha transfection. ER alpha gene transfection significantly inhibited (P < 0.05) bFGF-induced chemotactic migration of endothelial cells but increased cell attachment to fibronectin, laminin, and type I and IV collagens. ER alpha gene transfer also inhibited bFGF-stimulated tyrosine phosphorylation of FAK. CONCLUSION Our results suggest that the atheroprotective effects of estrogen may in part be mediated by ER alpha-induced upregulation of eNOS gene expression and maintenance of endothelial cell function and integrity.

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.

Ontogenetic development of isoproterenol subsensitivity of myocardial adenylate cyclase and β-adrenergic receptors in spontaneously hypertensive rats

[3H]Dihydroalprenolol binding and adenylate cyclase activity in the myocardial membranes of Kyoto Wistar normotensive rats and spontaneously hypertensive rats were compared at various stages of postnatal development ranging from 2 to 36 weeks. Basal as well as agonist-stimulated myocardial adenylate cyclase activity was consistently decreased in spontaneously hypertensive rats as compared to normotensive rats as early as 2 weeks of age with significant differences (P < 0.05) observed after 6 weeks of age. When results were expressed as percent stimulation over the basal activity, only isoproterenol plus GTP-stimulated enzyme activity was reduced by 25--30% in spontaneously hypertensive rats, suggesting a specific loss of stimulation by isoproterenol in hypertensive animals. The number of [3H]dihydroalprenolol binding sites of KD for dihydroalprenolol binding were comparable between spontaneously hypertensive and normotensive rats at 3, 6 and 12 weeks of age. The competition of isoproterenol with [3H]dihydroalprenolol for the specific binding sites showed that the affinity of isoproterenol binding was decreased 3--4-fold in spontaneously hypertensive compared with normotensive rats. With postnatal development in age, basal as well as agonist-stimulated activities decreased progressively in both spontaneously hypertensive and normotensive rats. Similarly, the number of [3H]dihydroalprenolol binding sites decreased with the development in age, whereas affinity of dihydroalprenolol binding increased up to 12 weeks of age. These results therefore suggest that adenylate cyclase activity and the number of beta-adrenergic receptors in rat heart, decrease with age and that in hypertension, specific decrease in isoproterenol stimulation of cyclase appears at all stages of development.

Protein Kinase Translocation as an Early Event in the Hormonal Control of Uterine Contraction

β-Adrenergic stimulation with isoproterenol inhibits contractility, increases cyclic adenosine monophosphate (AMP) concentration, decreases the concentration of unsaturated cyclic AMP receptor sites, and increases cyclic AMP-independent kinase in the uterus of ovariectomized rats. The total soluble kinase activity is reduced. The protein kinase activity lost from the cytosol was translocated to the microsomal fraction mostly in a cyclic AMP-independent form, suggesting a particulate substrate for the activated enzyme.

NOS gene transfer inhibits expression of cell cycle regulatory molecules in vascular smooth muscle cells

The mechanisms of nitric oxide (NO)-mediated inhibition of vascular smooth muscle (VSM) cell proliferation are still obscure. Cyclins A and E in association with cyclin-dependent kinase 2 (cdk2) serve as positive regulators for mammalian cell cycle progression through the G1/S checkpoint of the cell cycle and subsequent cell proliferation. Therefore, we have tested the effect of adenovirus-mediated transfection of the endothelial nitric oxide synthase (eNOS) gene into guinea pig coronary VSM cells on platelet-derived growth factor (BB homodimer) (PDGF-BB)-stimulated cell proliferation and the expression of cell cycle regulatory molecules. Transfection of the eNOS gene (eNOS) into VSM cells significantly inhibited (P < 0.05) [3H]thymidine incorporation into the DNA in response to PDGF-BB stimulation compared with lacZ-transfected control cells. The eNOS transfer significantly inhibited (P < 0.05) PDGF-BB-induced proliferating cell nuclear antigen (PCNA) and cyclin A expression in VSM cells compared with cells transfected with the control vector. The time course of cyclin E expression in response to PDGF-BB stimulation was delayed in eNOS-transfected cells. Levels of cyclin-dependent kinase inhibitors p21 and p27 were not significantly affected by eNOS transfer. eNOS transfer did not decrease PDGF-beta receptor number, affinity, and autophosphorylation measured by radioreceptor assay and Western analysis. These results suggest that inhibition of PDGF-stimulated expression of cyclin A, cyclin E, and PCNA is the target of NO action. These findings could explain, at least in part, NO-mediated inhibition of VSM cell proliferation.The mechanisms of nitric oxide (NO)-mediated inhibition of vascular smooth muscle (VSM) cell proliferation are still obscure. Cyclins A and E in association with cyclin-dependent kinase 2 (cdk2) serve as positive regulators for mammalian cell cycle progression through the G1/S checkpoint of the cell cycle and subsequent cell proliferation. Therefore, we have tested the effect of adenovirus-mediated transfection of the endothelial nitric oxide synthase (eNOS) gene into guinea pig coronary VSM cells on platelet-derived growth factor (BB homodimer) (PDGF-BB)-stimulated cell proliferation and the expression of cell cycle regulatory molecules. Transfection of the eNOS gene ( eNOS) into VSM cells significantly inhibited ( P < 0.05) [3H]thymidine incorporation into the DNA in response to PDGF-BB stimulation compared with lacZ-transfected control cells. The eNOS transfer significantly inhibited ( P < 0.05) PDGF-BB-induced proliferating cell nuclear antigen (PCNA) and cyclin A expression in VSM cells compared with cells transfected with the control vector. The time course of cyclin E expression in response to PDGF-BB stimulation was delayed in eNOS-transfected cells. Levels of cyclin-dependent kinase inhibitors p21 and p27 were not significantly affected by eNOStransfer. eNOS transfer did not decrease PDGF-β receptor number, affinity, and autophosphorylation measured by radioreceptor assay and Western analysis. These results suggest that inhibition of PDGF-stimulated expression of cyclin A, cyclin E, and PCNA is the target of NO action. These findings could explain, at least in part, NO-mediated inhibition of VSM cell proliferation.

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 &mgr;g 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.