Caffeic acid inhibits vascular smooth muscle cell proliferation induced by angiotensin II in stroke-prone spontaneously hypertensive rats.

Epidemiological studies have linked the consumption of phenolic acids with reduced risk of cardiovascular diseases. In the present study, we sought to investigate whether caffeic acid, a phenolic acid which is abundant in normal diet, can antagonize angiotensin II (Ang II)-induced vascular smooth muscle cell (VSMC) proliferation in stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto (WKY) rats, and if so, to elucidate the underlying cell signaling mechanisms. We exposed VSMCs to Ang II and caffeic acid and found that caffeic acid significantly inhibited intracellular superoxide anion generation (decreased from 127 +/- 6.3% to 100.3 +/- 6.6% of the control cells) and the cell proliferation induced by Ang II. Furthermore, caffeic acid significantly abolished the tyrosine phosphorylation of JAK2 (decreased from 7.4 +/- 0.6-fold to 2.4 +/- 0.6-fold at 2 min) and STAT1 (decreased from 1.8 +/- 0.2-fold to 0.5 +/- 0.1-fold at 2 min) and the phosphorylation of ERK1/2 (decreased from 99.2 +/- 10.2-fold to 49.8 +/- 10.9-fold at 2 min) that were induced by Ang II. These effects of caffeic acid were consistent with the inhibition of the proliferation of VSMCs by DPI, an NADPH oxidase inhibitor, and by AG-490, a JAK2 inhibitor. In conclusion, our findings suggest that caffeic acid attenuates the proliferative reaction of VSMCs to Ang II stimulation in both SHRSP and WKY rats by inhibiting the generation of reactive oxygen species and then partially blocking the JAK/STAT signaling cascade and the Ras/Raf-1/ERK1/2 cascade.     

Hyperglycemia inhibits vascular smooth muscle cell apoptosis through a protein kinase C-dependent pathway

We hypothesized that the pathogenesis of diabetic vasculopathy involves the abnormal regulation of vascular smooth muscle cell (VSMC) apoptosis. In nondiabetic mice, a reduction in carotid artery blood flow resulted in a significant loss of medial VSMCs via apoptosis (normal flow 84+/-1 viable VSMCs, reduced flow 70+/-5 viable VSMCs; n=12, P:<0.01). In contrast, flow-induced VSMC apoptosis was markedly attenuated in streptozotocin-induced diabetic mice (normal flow 85+/-2 viable VSMC, reduced flow 82+/-4 viable VSMC; n=13, NS). In accord with our in vivo findings, the exposure of cultured rat and human VSMCs to high glucose (17.5 mmol/L) significantly attenuated the induction of apoptosis in response to serum withdrawal (rat VSMCs in normal [5.5 mmol/L] glucose 28+/-1%, high D-glucose 19+/-2%; P:<0.0001). High glucose also inhibited apoptosis induced by Fas ligand (100 ng/mL) (normal 23+/-2%, high D-glucose 13+/-2%; P:<0.006). Supplementation with the nonmetabolized enantiomer L-glucose had no effect. We confirmed reports that high glucose activates protein kinase C (PKC) and demonstrated that PKC blockade with long-term phorbol ester treatment or calphostin C prevented the antiapoptotic effect (P:<0. 001). Moreover, the upregulation of either PKCalpha or PKCbetaII expression was sufficient to inhibit serum withdrawal-induced apoptosis (control 25+/-2%, PKCalpha 11+/-2%, PKCbetaII 8+/-2%; P:<0. 0001), whereas the upregulation of PKCdelta had no significant effect. Taken together, these findings demonstrate that hyperglycemia inhibits VSMC apoptosis via a PKC-dependent pathway.     

Inhibitory effects of PPAR-γ on endothelin-1-induced inflammatory pathways in vascular smooth muscle cells from normotensive and hypertensive rats

The present study evaluated the effects of endothelin (ET)-1 and the peroxisome proliferator activated receptor γ (PPAR-γ) agonist, rosiglitazone, on inflammatory markers in vascular smooth muscle cells (VSMCs) from normotensive (WKY) and hypertensive (SHRSP) rats. Rat VSMC-derived mesenteric arteries from WKY and SHRSP were treated with ET-1 (100 mmol/L) and rosiglitazone (1μmol/L) or ET type A (ET_A) or type B (ET_B) receptor antagonists. Nuclear factor kappa-B (NFκB) binding activity was assessed by electrophoretic mobility shift assay and phospho-inhibitory κB (IκB); vascular cell adhesion molecule (VCAM)-1, intercellular adhesion molecule (ICAM)-1, and cyclooxygenase (COX)-2 expression was determined using Western blotting. ET-1 significantly increased NFκB binding, and VCAM-1, ICAM, and COX-2 expression to a greater degree in SHRSP than in WKY VSMC. These changes were associated with increased phosphorylation of IκB, thus resulting in decreased NFκB inhibition. Co-incubation with PPAR-γ activator rosiglitazone, or ET_A or ET_B receptor antagonism prevented ET-1-stimulated vascular proinflammatory effects in both WKY and SHRSP VSMC. Proinflammatory effects of ET-1 in VSMCs are mediated via both ET_A and ET_B receptor subtypes. These effects may be abrogated by the PPAR-γ activator rosiglitazone. PPAR-γ activators may thus prevent deleterious ET-1-dependent proinflammatory vascular effects in VSMC in hypertension.     

Differential effects of antihypertensive agents on proliferation of vascular smooth muscle cells from spontaneously hypertensive rats.

We have previously shown that Ca-antagonists and alpha-blockers substantially inhibit the cellular proliferation of cultured rat vascular smooth muscle cells (VSMC). This study explored whether these inhibitory effects on cellular proliferation differ between cultured VSMC from spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). SHR VSMC proliferated much faster than WKY VSMC in 10% FCS. Cellular proliferation, determined by both cell number count and 3H-thymidine incorporation, was significantly blunted in the presence of either nifedipine (Nif) or bunazosin (Bun). The magnitude of these inhibitory effects was more pronounced for SHR cells than WKY cells (% reduction of 3H-thymidine uptake with Nif: 62.1 +/- 7.8% for SHR vs 75.3 +/- 10.2% for WKY, n = 6, p less than 0.05, and with Bun: 70.2 +/- 7.8% for SHR vs 82.1 +/- 9.9% for WKY, n = 6, p less than 0.05). In contrast, the intracellular water volume was unaffected by these antihypertensive agents based on equilibrium distribution of 3-O-methyl-D-glucose14C. It is concluded that SHR VSMC grow much faster than WKY VSMC and that this abnormality is innate to the SHR cells. It is also concluded that both Ca-antagonists and alpha-blockers exerted a substantial inhibitory effect on cellular proliferation of the cultured VSMC of either SHR or WKY. Furthermore, the greater inhibition of proliferation in the SHR VSMC suggests that Ca mediated- and/or alpha-receptor mediated processes of cellular proliferation of SHR could differ from that of WKY and that these abnormalities may contribute to the hyperproliferative changes of VSMC in this model.     

Negative regulation of RhoA/Rho kinase by angiotensin II type 2 receptor in vascular smooth muscle cells: role in angiotensin II-induced vasodilation in stroke-prone spontaneously hypertensive rats

OBJECTIVE: To test whether angiotensin II (Ang II) through the Ang II type 2 receptor (AT2R), downregulates RhoA/Rho kinase, which plays a role in AT1 receptor (AT1R)-mediated function. METHODS: In vitro studies were performed in A10 vascular smooth muscle cells (VSMC) and in vivo studies in mesenteric arteries from Wistar-Kyoto (WKY) and stroke-prone spontaneously hypertensive (SHRSP) rats. VSMC were stimulated with Ang II (10 mol/l), CGP42112A (10 mol/l, a selective AT2R agonist) +/- valsartan (10 mol/l, an AT1R antagonist), or the Rho kinase inhibitor fasudil (10 mol/l). AT1R and AT2R expression and myosin light chain (MLC) phosphorylation were determined by immunoblotting. RhoA activity was assessed by measuring membrane translocation. Functional significance between AT2R, RhoA/Rho kinase and vasodilation was assessed in arteries from valsartan-treated (30 mg/kg per day, 14 days) WKY and SHRSP rats. Vasodilatory responses to Ang II (10-10 mol/l) were performed in norepinephrine pre-contracted vessels +/- valsartan(10 mol/l), PD123319 (10 mol/l, an AT2R antagonist) or fasudil (10 mol/l). RESULTS: A10 VSMC expressed AT1R and AT2R. In valsartan-treated cells, Ang II-induced RhoA translocation was reduced versus controls (42 +/- 6%, P < 0.05). Similar responses were obtained with CGP42112A (45 +/- 6%, P < 0.05). This was associated with decreased MLC activation. Fasudil abrogated Ang II- and CGP42112A-mediated effects. Ang II evoked a significant vasodilatory response only in valsartan-treated SHRSP (max dilation 40 +/- 7%). PD123319 blocked these effects. Fasudil increased AngII-induced relaxation in SHRSP vessels. AT2R expression was increased by valsartan (two- to three-fold) in SHRSP arteries. RhoA translocation was increased two-fold in untreated SHRSP (P < 0.05) and was reduced by valsartan (P < 0.05). These changes were associated with decreased MLC phosphorylation. CONCLUSIONS: Ang II/AT2R negatively regulates vascular RhoA/Rho kinase/MLC phosphorylation. These processes may play a role in Ang II-mediated vasodilation in conditions associated with vascular AT2R upregulation, such as in SHRSP chronically treated with AT1R blockers, which may contribute to blood pressure lowering by these antihypertensive agents.     

Coculture of vascular endothelial cells and smooth muscle cells from spontaneously hypertensive rats

The effect of endothelium-released vasoactive factors on vascular smooth muscle cell (VSMC) proliferation was studied in a coculture system. Isolated aortic endothelial cells and smooth muscle cells from 4-week-old spontaneously hypertensive rats (SHR) and age-matched Wistar-Kyoto (WKY) rats were cocultured. After coculture, the VSMC proliferation rate was examined by 3H-thymidine incorporation assay and the levels of the vasoactive factors in medium were determined by enzyme immunoassay (EIA). The results indicate that the proliferation rate of VSMCs in SHR was significantly higher than in WKY rats when VSMCs were cultured alone. When SHR vascular endothelial cells (VECs) were cocultured with VSMCs, the proliferation rate of SHR VSMCs was enhanced; however, there was no growth promoting effect in WKY VSMCs. When WKY VECs were cocultured with VSMCs, no VSMC proliferation effect was observed. When VSMCs were cultured alone, the endothelin-1 (ET-1) secretion in SHR was significantly higher than in WKY rats. When VECs and VSMCs were cocultured, the ET-1 concentration increased in both SHR VEC and WKY VEC coculture groups in a similar manner; but the SHR VECs tended to release more thromboxaneA2 (TXA2) and less PGI2 than WKY VECs. These results suggest that some kind of interaction between SHR VSMCs and SHR VECs is responsible for the high proliferation of SHR VSMCs but not the effects of SHR VECs per se.     

RhoA activation in vascular smooth muscle cells from stroke-prone spontaneously hypertensive rats.

RhoA is commonly activated in the aorta in various hypertensive models, indicating that RhoA seems to be a molecular switch in hypertension. The molecular mechanisms for RhoA activation in stroke-prone spontaneously hypertensive rats (SHRSP) were here investigated using cultured aortic smooth muscle cells (VSMC). The level of the active form of RhoA was higher in VSMC from SHRSP than in those from Wistar-Kyoto rats (WKY). The phosphorylation level of myosin phosphatase target subunit 1 (MYPT1) at the inhibitory site was also significantly higher in SHRSP, and the phosphorylation levels in both VSMCs were strongly inhibited to a similar extent by treatment with Y-27632, a Rho-kinase inhibitor. The expression levels of RhoA/Rho-kinase related molecules, namely RhoA, Rho-kinase, MYPT1, CPI-17 (inhibitory phosphoprotein for myosin phosphatase) and myosin light chain kinase, were not different between SHRSP and WKY. Valsartan, an angiotensin II (Ang II)- type 1 receptor antagonist, selectively and significantly reduced the RhoA activation in VSMC from SHRSP. The expression levels of the Rho GDP-dissociation inhibitor (RhoGDI) and leukemia-associated Rho-specific guanine nucleotide exchange factor (RhoGEF) did not differ between SHRSP and WKY. In cyclic nucleotide signaling, cyclic GMP (cGMP)-dependent protein kinase Ialpha (cGKIalpha) was significantly downregulated in SHRSP cells, although there were no changes in the expression levels of guanylate cyclase beta and cyclic AMP (cAMP)-dependent protein kinase or the intracellular contents of cGMP and cAMP between the two rat models. These results suggest that the possible mechanisms underlying RhoA activation in VSMC from SHRSP are autocrine/paracrine regulation by Ang II and/or cGKIalpha downregulation.     

Effects of chronic nitric oxide synthase inhibition on cerebral arterioles in Wistar-Kyoto rats

OBJECTIVE: Cerebral arterioles in stroke-prone spontaneously hypertensive rats (SHRSP), but not in Sprague-Dawley rats with hypertension induced by nitric oxide (NO) synthase inhibition, undergo inward remodeling. The goal of this study was to determine whether development of vascular inward remodeling may depend on genetic factors. DESIGN: We examined effects of NO synthase inhibition on the structure of cerebral arterioles in Wistar-Kyoto rats (WKY), a rat strain genetically distinct from Sprague-Dawley. METHODS: Pressure (servonull), diameter (cranial window) and cross-sectional area of the vessel wall (CSA, histologically) were measured in maximally dilated (EDTA) cerebral arterioles in WKY, untreated (n = 8) or treated for 3 months with the NO synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME) (10 mg/kg per day, n = 10) in the drinking water, and in untreated SHRSP (n = 7). RESULTS: Treatment with L-NAME in WKY increased mean cerebral arteriolar pressure (69 +/- 7 versus 47 +/- 7 mmHg, P < 0.05) and pulse pressure (30 +/- 3 versus 17 +/- 1 mmHg, P < 0.05) to levels significantly lower than in SHRSP (98 +/- 5 and 35 +/- 1 mmHg respectively, P < 0.05). CSA was significantly greater in L-NAME-treated WKY and SHRSP than in untreated WKY (1692 +/- 50 and 1525 +/- 98 microm respectively, versus 1224 +/- 85, P < 0.05). External diameter was significantly less in L-NAME-treated WKY than in untreated WKY (119 +/- 5 versus 135 +/- 4 microm, P < 0.05) but significantly greater than in SHRSP (98 +/- 1 microm, P < 0.05). CONCLUSION: Cerebral arterioles undergo hypertrophy and remodeling in WKY with L-NAME-induced hypertension. These findings suggest that genetic factors present in WKY and SHRSP may play a role in the development of vascular inward remodeling during chronic hypertension in rats.     

Coculture of Vascular Endothelial Cells and Smooth Muscle Cells from Spontaneously Hypertensive Rats

The effect of endothelium‐released vasoactive factors on vascular smooth muscle cell (VSMC) proliferation was studied in a coculture system. Isolated aortic endothelial cells and smooth muscle cells from 4‐week‐old spontaneously hypertensive rats (SHR) and age‐matched Wistar–Kyoto (WKY) rats were cocultured. After coculture, the VSMC proliferation rate was examined by ³H‐thymidine incorporation assay and the levels of the vasoactive factors in medium were determined by enzyme immunoassay (EIA). The results indicate that the proliferation rate of VSMCs in SHR was significantly higher than in WKY rats when VSMCs were cultured alone. When SHR vascular endothelial cells (VECs) were cocultured with VSMCs, the proliferation rate of SHR VSMCs was enhanced; however, there was no growth promoting effect in WKY VSMCs. When WKY VECs were cocultured with VSMCs, no VSMC proliferation effect was observed. When VSMCs were cultured alone, the endothelin‐1 (ET‐1) secretion in SHR was significantly higher than in WKY rats. When VECs and VSMCs were cocultured, the ET‐1 concentration increased in both SHR VEC and WKY VEC coculture groups in a similar manner; but the SHR VECs tended to release more thromboxaneA₂ (TXA₂) and less PGI₂ than WKY VECs. These results suggest that some kind of interaction between SHR VSMCs and SHR VECs is responsible for the high proliferation of SHR VSMCs but not the effects of SHR VECs per se.     

Genetic and gender influences on sensitivity to focal cerebral ischemia in the stroke-prone spontaneously hypertensive rat

We have investigated genetic transmission of increased sensitivity to focal cerebral ischemia and the influence of gender in the stroke-prone spontaneously hypertensive rat (SHRSP). Halothane-anesthetized, 3- to 5-month-old male and female Wistar-Kyoto rats (WKY), SHRSP, and the first filial generation rats (F1 crosses 1 and 2) underwent distal (2 mm) permanent middle cerebral artery occlusion (MCAO) by electrocoagulation. Infarct volume was measured by using hematoxylin-eosin-stained sections and image analysis 24 hours after ischemia and expressed as a percentage of the volume of the ipsilateral hemisphere. Infarct volume in males and females grouped together were significantly larger in SHRSP, F1 cross 1 (SHRSP father), and F1 cross 2 (WKY father), at 36.6+/-2.3% (mean+/-SEM, P<0.001, n=15), 25.4+/-2.4% (P<0.01, n=14), and 33. 9+/-1.6% (P<0.001, n=18), respectively, compared with WKY (14+/-2%, n=17). Male F1 cross 1 (18.9+/-2.4%, n=6) developed significantly smaller infarcts than male F1 cross 2 (32.8+/-2%, n=8, P<0.005). Females, which underwent ischemia during metestrus, developed larger infarcts than respective males. A group of females in which the cycle was not controlled for developed significantly smaller infarcts than females in metestrus. Thus, the increased sensitivity to MCAO in SHRSP is retained in both F1 cross 1 and cross 2 hybrids, suggesting a dominant or codominant trait; response to cerebral ischemia appears to be affected by gender and stage in the estrous cycle. In addition, the male progenitor of the cross (ie, SHRSP versus WKY) influences stroke sensitivity in male F1 cohorts.     

Cellular adhesion to elements of the extracellular matrix in the hypertensive rat modulates the effect of angiotensin II]

This study was to assess the role of different components of the extracellular matrix (ECM) on the mobilization of Cai++ induced by angiotensin II in vascular smooth muscle cells (VSMC) from hypertensive (SHR) and normotensive (WKY) rats. The effect of AII (10-6 M) on Cai++ release was studied in VSMC isolated from the aorta of 5-week-old WKY and SHR using fluorescent imaging microscopy (fura-2). Cai++ mobilization was characterized by amplitude, slope of Cai++ increase and total amount of Cai++. Cells were cultured on glass coverslips (control) or coated with either collagen I, collagen IV, vitronectin, fibronectin and extracellular matrix (ECM) and studied at confluence between passage 3 and 9. A significant increase of Cai++ released by AII has been observed with cells from WKY cultured on collagen I (meam +/- SEM, amplitude: 192 +/- 12% of control values, slope: 194 +/- 13%, total amount Cai++: 173 +/- 12%, n = 270, p < or = 0.0001 for each, unpaired t-test). Conversely, response with SHR was not significatively modified. Cai++ mobilization was not significatively modified after culture of VSMC from SHR and WKY on collagen IV. A significative decrease of the slope (WKY: 66 +/- 6%, p < or = 0.0001; SHR: 83 +/- 5%, p < or = 0.03) and of the amount of Cai++ (WKY: 74 +/- 7%, p < or = 0.01; SHR: 74 +/- 5%, p < or = 0.01) has been observed after culture of VSMC from the 2 strains on vitronectin. A decrease in amplitude (53 +/- 3%, p < or = 0.0001), slope (38 +/- 4%, p < or = 0.0001) and Cai++ release (69 +/- 5%, p < or = 0.004, n = 106) has been observed in VSMC from SHR seeded on fibronectin. Conversely, in VSMC from WKY, Cai++ mobilisation has not been modified compared with control cells. Culture of VSMC from SHR on ECM induced a significative decrease of amplitude (49 +/- 2%), slope (54 +/- 4%) and Cai++ release (53 +/- 3%, p < or = 0.0001 for each, n = 122), while in WKY, ECM induced a significative stimulation of these parameters (amplitude: 157 +/- 11%, slope: 149 +/- 13% and Cai++ release: 130 +/- 9%, p < or = 0.0001 for each, n = 247). These results show that the Cai++ mobilization induced by AII is modified by the adhesion of cells to different ECM components. This suggests a modulation of the A II-associated signalling events via the focal adhesion points. Furthermore, a difference in this modulation is observed between SHR and WKY when cells are seeded on collagen I, fibronectin or ECM. These modulations of Cai++ mobilization could play a role in the regulation of growth and differentiation of cells during the development of hypertension.     

Endothelium-dependent responses of cerebral blood vessels during chronic hypertension

Acetylcholine produces less dilatation of pial arterioles in stroke-prone spontaneously hypertensive rats (SHRSP) than in normotensive (WKY) rats. Responses of cerebral vessels to acetylcholine and bradykinin appear to involve different mechanisms. Our first goal was to determine whether responses of pial arterioles to bradykinin are impaired in SHRSP. Diameter of pial arterioles (20-60 microns) was measured using intravital microscopy in WKY rats and SHRSP (9-12 months old). Superfusion of bradykinin (3 x 10(-7) M) dilated pial arterioles by 35 +/- 6% (mean +/- SEM) in WKY rats, but only 21 +/- 3% in SHRSP (p less than 0.05 versus WKY rats). Both nitric oxide (5 x 10(-7) M) and nitroglycerin (10(-5) M) produced similar vasodilatation in WKY rats and SHRSP. Our second goal was to determine whether alteration of postreceptor mechanisms contributes to impairment of endothelium-dependent cerebral vasodilatation in SHRSP. Calcium ionophore A23187 (10(-5) M) produced more vasodilatation in WKY rats than in SHRSP (32 +/- 8% versus 9 +/- 4%, p less than 0.05). Responses to A23187 (10(-5) M) were inhibited by indomethacin (46 +/- 13% versus 15 +/- 5%, p less than 0.05) in WKY rats, whereas responses to A23187 (10(-6) M) were potentiated modestly by indomethacin (-3 +/- 2% versus 4 +/- 2%, p less than 0.05) in SHRSP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic association of hypertension and vascular changes in stroke-prone spontaneously hypertensive rats

Isolated tail arteries from stroke-prone spontaneously hypertensive rats (SHRSP) exhibit oscillatory contractile activity in response to norepinephrine, whereas those from normotensive Wistar-Kyoto rats (WKY) do not. To determine whether the norepinephrine-induced oscillations are related to high blood pressure or to separable genetic differences between strains, the response to norepinephrine was studied in adult SHRSP, WKY, and progeny of genetic crosses of SHRSP and WKY (F1, F2, F1 X SHRSP, F1 X WKY). Helical tail artery strips were mounted in a tissue bath for isometric force recording. Rats were classified as responders if oscillatory activity in the presence of 1.8 X 10(-7) M norepinephrine exceeded 250 mg/10 min (milligrams of force amplitude during a 10-minute interval). The blood pressures (mm Hg +/- SEM; tail cuff method) and percentage of rats exhibiting norepinephrine-induced oscillations were as follows: WKY: 109 +/- 3, 0%; F1: 129 +/- 4, 0%; F2: 150 +/- 4, 38%; F1 X WKY: 137 +/- 3, 9%; F1 X SHRSP: 188 +/- 7, 71%; SHRSP: 207 +/- 7, 100%. The distribution of the frequency of animals with oscillatory activity among the progenies was consistent with the hypothesis that a single gene locus determines the observed difference in oscillatory activity between the WKY and SHRSP strains. The allele from the SHRSP that determines the activity phenotype is recessive to the allele contributed by the normotensive WKY strain. In the segregating F2 progeny, the blood pressure of the responders was higher than that of the nonresponders (161 +/- 6 vs 144 +/- 4 mm Hg; p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

C-Peptide induces vascular smooth muscle cell proliferation: involvement of SRC-kinase, phosphatidylinositol 3-kinase, and extracellular signal-regulated kinase 1/2

Increased levels of C-peptide, a cleavage product of proinsulin, circulate in patients with insulin resistance and early type 2 diabetes mellitus. Recent data suggest a potential causal role of C-peptide in atherogenesis by promoting monocyte and T-lymphocyte recruitment into the vessel wall. The present study examined the effect of C-peptide on vascular smooth muscle cells (VSMCs) proliferation and evaluated intracellular signaling pathways involved. In early arteriosclerotic lesions of diabetic subjects, C-peptide colocalized with VSMCs in the media. In vitro, stimulation of human or rat VSMCs with C-peptide induced cell proliferation in a concentration-dependent manner with a maximal 2.6+/-0.8-fold induction at 10 nmol/L human C-peptide (P<0.05 compared with unstimulated cells; n=9) and a 1.8+/-0.2-fold induction at 0.5 nmol/L rat C-peptide (P<0.05 compared with unstimulated cells; n=7), respectively, as shown by [H3]-thymidin incorporation. The proliferative effect of C-peptide on VSMCs was inhibited by Src short interference RNA transfection, PP2, an inhibitor of Src-kinase, LY294002, an inhibitor of PI-3 kinase, and the ERK1/2 inhibitor PD98059. Moreover, C-peptide induced phosphorylation of Src, as well as activation of PI-3 kinase and ERK1/2, suggesting that these signaling molecules are involved in C-peptide-induced VSMC proliferation. Finally, C-peptide induced cyclin D1 expression as well as phosphorylation of Rb in VSMCs. Our results demonstrate that C-peptide induces VSMC proliferation through activation of Src- and PI-3 kinase as well as ERK1/2. These data suggest a novel mechanism how C-peptide may contribute to plaque development and restenosis formation in patients with insulin resistance and early type 2 diabetes mellitus.     

Atherosclerotic abdominal aortic aneurysm and the interaction between autologous human plaque-derived vascular smooth muscle cells, type 1 NKT, and helper T cells

Immune cell infiltration, vascular smooth muscle cell (VSMC) proliferation, and apoptosis are pathological hallmarks of atherosclerosis. The multifocal, chronic, and inflammatory nature of this disease of the cardiovascular system complicates targeted cellular therapy and emphasizes the need to understand the role and interaction of immune cells with VSMCs. We characterized the immune cell subsets present in human atherosclerotic tissue derived from atherosclerotic abdominal aortic aneurysm (AAA) and expanded them to study their interaction with autologous plaque-derived VSMCs in vitro. We show here that apart from T lymphocytes, plaque infiltrates consist of lots of NK cells and significant proportions of NKT cells that express T cell receptor (TCR) alphabeta, CD4, and the NK markers CD56 and CD161. The infiltrates are predominantly IFN-gamma-producing Type 1 lymphoid cells. When cocultured, the T and NKT cells adhere to VSMCs. CD4+ T cells enhance VSMC proliferation. VSMCs in turn enhance CD4+CD161+ NKT but not CD4+ or CD8+ T cell proliferation. CD4+CD161+ NKT cells inhibit VSMC proliferation by inducing apoptosis. Our results suggest that the interactions of Type 1 CD4+ T and CD4+CD161+ NKT cells with VSMCs may regulate VSMC proliferation and death respectively in atherosclerosis and the balance of these interactions could determine plaque stability.     

Responses of cerebral arterioles to adenosine 5'-diphosphate, serotonin, and the thromboxane analogue U-46619 during chronic hypertension

The goal of this study was to determine whether responses of cerebral arterioles to products released by platelets are impaired in stroke-prone spontaneously hypertensive rats (SHRSP). The diameter of pial arterioles was measured during suffusion with adenosine 5'-diphosphate (ADP), serotonin, and the thromboxane analogue U-46619, using intravital microscopy in normotensive Wistar-Kyoto rats (WKY) and SHRSP (7-10 months old). Responses of cerebral arterioles to ADP and serotonin were profoundly impaired in SHRSP. ADP (10(-5) M) increased pial arteriolar diameter 17 +/- 3% (mean +/- SE) in WKY and only 4 +/- 1% in SHRSP. Serotonin (10(-5) M) increased pial arteriolar diameter 15 +/- 2% in WKY and, in contrast, reduced the diameter 13 +/- 1% in SHRSP. Nitroglycerin produced a similar dilatation of cerebral arterioles in WKY and SHRSP, suggesting that impairment of dilatation in SHRSP in response to ADP and serotonin was not related to nonspecific impairment of vasodilatation in SHRSP. The thromboxane analogue U-46619 produced a similar constriction of arterioles in WKY and SHRSP. We also examined the possibility that impaired dilator responses of cerebral arterioles in SHRSP in response to ADP and serotonin may be related to production of a cyclooxygenase vasoconstrictor substance. Indomethacin (10 mg/kg i.v.) partially restored dilator responses to ADP and serotonin in SHRSP, without altering responses in WKY. Thus, we speculate that vasoactive substances released by platelets may release a prostanoid constrictor substance from cerebral vessels of SHRSP and thereby predispose SHRSP to cerebral ischemia and, perhaps, stroke.     

Role of endothelium in the response to endothelin in hypertension

The relation between endothelin and acetylcholine (ACh) was examined and compared in aortas from Wistar-Kyoto (WKY) rats and from stroke-prone spontaneously hypertensive rats (SHRSP). The relaxation produced by ACh in an endothelin-induced contraction was less in aortas from WKY rats than in those from SHRSP. In aortas from WKY rats but not in those from SHRSP, the contraction produced by endothelin was augmented when the intact aortic rings were treated with methylene blue (10(-5) M). This augmentation was also found in preparations of the WKY rat aortic rings in which the endothelium had been removed. The augmentation was not present in SHRSP aortic rings that had been similarly denuded. Treatment with indomethacin (5 x 10(-6) M) had no effect on endothelin-induced contraction in either WKY rat or SHRSP aortic rings. Our findings indicate that endothelin and ACh have in common the ability to release endothelium-derived relaxing factor (EDRF) in WKY rat aortic rings. The reduced endothelium-dependent relaxation in response to ACh in the WKY rat probably reflects the fact that endothelin had already released the EDRF in rings from this strain of rats. The release of EDRF by endothelin is less in SHRSP than it is in WKY rats. Because of this failure of endothelin to release EDRF in SHRSP, endothelin may contribute to the increase in total peripheral resistance in this form of hypertension.     

Superoxide anion production is increased in a model of genetic hypertension: role of the endothelium.

The hypothesis that the decreased nitric oxide (NO) availability observed in spontaneously hypertensive stroke-prone rats (SHRSP) is due to excess superoxide (O2-) was examined. O2- generation, measured by lucigenin chemiluminescence, was studied in 12- to 16-week male and female Wistar-Kyoto rats (WKY) and SHRSP. In addition, expression of the gene encoding endothelial NO synthase, the enzyme involved in NO generation, was investigated. O2- generation was increased in male and female SHRSP (4.11+/-0.24 and 3. 84+/-0.28 nmol O2-. min-1. mg-1 respectively) compared with their WKY counterparts and was significantly higher in male than female WKY (1.22+/-0.08 in males and 0.8+/-0.08 nmol O2-. min-1. mg-1 respectively) (SHRSP versus WKY P<0.0001, 95% CI -3.39, -2.51; male versus female WKY P=0.0029, 95% CI -0.67, -0.17). Removal of the endothelium by rubbing or addition of NO synthase inhibitors attenuated O2- generation in SHRSP but not WKY. In males, removal of the endothelium reduced O2- generation from 3.86+/-0.12 to 1.35+/-0. 08 nmol. min-1. mg-1 (P<0.0001, 95% CI 2.29, 2.81), whereas addition of L-NAME caused a reduction from 4.13+/-0.17 to 1.32+/-0.16 nmol. min-1. mg-1 (P<0.0001, 95% CI 2.36, 2.83). Similar reductions were observed in females. L-arginine had no significant effect, but tetrahydrobiopterin significantly decreased O2- generation in SHRSP from 4.04+/-0.11 to 2.36+/-0.40 nmol. min-1. mg-1 (P=0.0026, 95% CI 0.89, 2.44). Endothelial NO synthase mRNA expression was significantly greater in SHRSP than in WKY and in WKY males than in WKY females. These results show that O2- generation is increased in SHRSP and that the tissue and enzymatic sources of this excess O2- appear to be the endothelium and eNOS, respectively. The increase in O2- generation could explain the decreased availability of basal NO observed in this model of genetic hypertension.     

Regenerative electrical activity and arterial contraction in hypertensive rats

Isolate tail arteries from spontaneously hypertensive rats-stroke prone strain (SHRSP) display oscillatory contractile responses to norepinephrine. These oscillations are not observed in tail arteries from normotensive Wistar-Kyoto rats (WKY). The mechanism underlying these oscillatory contractions was investigated by simultaneous measurement of isometric force development and membrane potential (Em) from tail artery strips in vitro. After equilibration in physiological salt solution containing 1.6 mM calcium (37 degrees C), resting Em was not different between WKY (-52 +/- 1.1 mV) and SHRSP (-52 +/- 0.4 mV). Norepinephrine (3 x 10(-7) M) produced a similar degree of depolarization in tissues from the two strains (WKY = (-42.5 +/- 0.9, SHRSP = -41 +/- 0.8). However, while Em recordings from WKY arteries were quiescent, those from SHRSP displayed bursts of electrical spiking activity that were temporally associated with the rising phase of oscillations in contractile force. The frequency and duration of these bursts of action potentials increased with the concentration of norepinephrine. Action potentials were not observed in calcium-free solution or in presence of nifedipine (3 x 10(-7) M). Releasing the passive stretch on the tissues caused a decrease in the rate of spiking. These studies demonstrate catecholamine-induced regenerative electrical activity in tail arteries from SHRSP that is dependent on extracellular calcium. This activity is unique to tail arteries from this strain.     

Effect of felodipine on blood pressure and vascular reactivity in stroke-prone spontaneously hypertensive rats

Isolated tail arteries from stroke-prone spontaneously hypertensive rats (SHRSP), but not from normotensive Wistar-Kyoto rats (WKY), exhibit oscillatory contractions in response to norepinephrine. Previous studies indicate that the mechanism for these oscillations involves altered membrane calcium and/or potassium handling, and that this vascular change is a genetic defect associated with hypertension in SHRSP. The purpose of this experiment was to determine whether treatment of SHRSP with the calcium entry blocker felodipine would alter oscillatory activity. Adult SHRSP and WKY rats were treated orally with felodipine for 8 weeks. Felodipine treatment produced a significant decrease in blood pressure in SHRSP (control SHRSP: 240 +/- 7 mmHg, n = 6; felodipine-treated SHRSP: 164 +/- 8 mmHg, n = 5, P less than 0.05; tail-cuff method). Helically-cut tail artery strips from all rats were mounted in tissue baths for isometric force recording and exposed to norepinephrine (6 x 10(-9) to 6 x 10(-6) mol/l) for 20 min at each concentration. Oscillatory activity was defined as the sum of the magnitudes of all phasic contractions occurring during the final 10 min of norepinephrine incubation. Oscillatory activity was markedly reduced in tail arteries from felodipine-treated SHRSP when compared with control SHRSP. Felodipine also inhibited oscillatory activity when added directly to the tissue bath. It seems, therefore, that felodipine may lower blood pressure in SHRSP, at least in part, by correcting the genetic defect responsible for oscillatory activity.