Role of extracellular matrix in angiotensin II signalling in aortic smooth muscle cells: relationship with arterial hypertension

Angiotensin II (Ang II) is involved in hypertension-related arterial wall hypertrophy [1]. Regulation of AT II transduction pathway in vascular smooth muscle cells (VSMC) may involve cytoskeleton and extracellular matrix (ECM) [2]. We assessed the role of components of ECM on Cai2+ increase induced by Ang II in Wistar-Kyoto (WKY) and Spontaneously Hypertensive Rats (SHR) aortic VSMC. The effect of Ang II (1 mumol) on Ca2+ mobilization was studied in cultured VSMC isolated from the aorta of 6-wk old WKY (MAP (m +/- SE) = 98 +/- 4 mmHg) and SHR (136 +/- 5 mmHg; p < 0.05), using fluorescent imaging microscopy (Fura-2 AM). Cai2+ release from internal stores and Ca2+ influx were assessed in the absence and upon reintroduction of external Ca2+ respectively. Cells were cultured on uncoated glass coverslips (control) or coated with either collagen I (10 micrograms/mL), collagen IV (7 micrograms/mL), vitronectin (0.1 microgram/mL), fibronectin (3 micrograms/mL) and extracellular matrix extract (matrigel, 1/10) and studied at confluence. Paxillin was located in cells by indirect immunofluorescence micrography. Results are expressed in % of Control. Statistical significance (p < 0.05) was assessed with Student's t-test for unpaired data. The effects on Ang II-induced Ca2+ mobilization of growing cells on ECM are in Table. Paxillin in Control cells appeared as dots at the cell boundaries. Density increased in cells grown on collagen I with a diffuse distribution in the WKY cells. On matrigel, paxillin was located in a belt-like fashion at the periphery of the cell. These effects were not linked to differences in cell cycle (flux cytometry).     

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.     

Transforming growth factor-beta1 modulates angiotensin II-induced calcium release in vascular smooth muscle cells from spontaneously hypertensive rats

OBJECTIVES: To investigate the role of transforming growth factor-beta1 (TGF-beta1) on Ca2+-dependent mechanisms elicited by angiotensin II in aortic vascular smooth muscle cells (VSMC) of Wistar- Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). METHODS: Cai2+ release induced by angiotensin II (1 micromol/ l) was studied in cultured VSMC isolated from the aortas of 6-week-old WKY rats and SHR. Intracellular Ca2+ (Cai2+) was assessed in Fura-2 loaded cells using fluorescent imaging microscopy. Angiotensin II receptors were analysed by binding studies. RESULTS: Pretreatment of VSMC for 24 h with TGF-beta1 significantly increased angiotensin II-induced Cai2+ mobilization from internal stores in SHR, while Ca2+ influx was not altered. This effect involves tyrosine kinase and is not due to an increase in angiotensin II binding sites, or a change in the affinity of the receptors. By contrast, TGF-beta1 did not modify the response of VSMC from WKY rats to angiotensin II. CONCLUSIONS: These results help our understanding of the interactions between the pathways activated by TGF-beta1 and the G protein-coupled receptor signalling pathway, and their role in genetic hypertension.     

Normalization of active urinary kallikrein excretion by young spontaneously hypertensive rats by chronic inhibition of conversion enzyme

This study was designed to investigate the decreased urinary kallikrein excretion (Ukall.V) in Okamoto-Aoki spontaneously hypertensive rats (SHR) and the effect of long-term converting enzyme inhibition. From ages 4 to 7 weeks, Ukall.V was determined (amidolytic assay: nanokatals/wk) in 4 groups of 6 male rats housed into individual metabolic cages and fed a normal sodium diet: SHR and normotensive Wistar-Kyoto rats (WKY); SHR-C and WKY-C which were given captopril: 30 mg/kg BW every 12 hours by gavage. Ukall.V was each time lower in SHR than in age-matched WKY, even at 4 wks of age (54.6 +/- 9.1 vs 108.5 +/- 16.1 nkat/wk; p less than .01) when systolic blood pressure (s.BP) was already higher. In SHR-C, s.BP was identical or slightly lower to that in WKY. Ukall.V was still lower at wk 4 when captopril was first administered (60.9 +/- 8.4 nkat/wk; p less than .01), but identical to that in WKY at each subsequent age (105.7 +/- 25.9 vs 114.1 +/- 5.6 nkat/wk at wk 5; 219.8 +/- 44.5 vs 253.4 +/- 22.4 nkat/wk at wk 7). Excretion of active kallikrein was highly correlated to s.BP in WKY (r = .87), SHR (r = 0.91) and SHR-C (r = 0.95). The slope of the regression line relating Ukall.V with s.BP was significantly less in SHR than WKY (1.33 +/- 0.35 vs 3.36 +/- 0.84 nkat/wk/mmHg; p less than .01); the slope in SHR-C (3.35 +/- 0.77 nkat/wk/mmHg) was significantly steeper than in SHR (p less than .01) and identical to that in WKY.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of mitogen-activated protein/extracellular signal-regulated kinase improves endothelial function and attenuates Ang II-induced contractility of mesenteric resistance arteries from spontaneously hypertensive rats

OBJECTIVES: Extracellular signal-regulated kinases (ERK1/2) modulate vascular smooth muscle cell (VSMC) growth and contractility, important factors in blood pressure regulation. In the present in vivo study, we investigated whether short-term inhibition of ERK1/2-dependent signaling pathways influences vascular function and blood pressure (BP) in spontaneously hypertensive rats (SHR). METHODS: SHR and Wistar-Kyoto (WKY) rats were injected subcutaneously with either PD98059, selective MEK1/2 inhibitor (20 mg/kg), or vehicle. BP was measured by telemetry. Rats were killed 24 h after injection and small mesenteric arteries mounted as pressurized systems for morphometric analysis and assessment of endothelial function and angiotensin II (Ang II)-induced contractility. ERK1/2 phosphorylation was measured by Western blots, using protein extracts from mesenteric arteries, aorta, heart and kidneys. RESULTS: BP was higher (P < 0.01) in SHR than in WKY rats. PD98059 did not influence BP in either group. Endothelial-dependent relaxation (acetylcholine-induced), which was impaired in SHR, was improved by PD98059 (P < 0.05). Ang II increased contraction, with greater responses in SHR (Emax = 25 +/- 4%) than WKY (Emax = 9 +/- 3%) (P < 0.01). PD98059 reduced Ang II-induced contraction in SHR (Emax = 5.8 +/- 0.4%) and WKY (Emax = 4 +/- 0.4%). Vascular structure was unaltered by PD98059. Vascular and renal ERK1/2 phosphorylation, which was higher in SHR than WKY, was decreased by PD98059 in SHR. CONCLUSION: Short-term treatment with PD98059 improves endothelial function and vascular contractility without influencing BP in SHR. These findings provide evidence that vascular ERK1/2 activity is upregulated and that MEK1/2-sensitive signaling pathways play an important role in the regulation of vascular function in SHR. Acute inhibition of MEK1/2 does not alter blood pressure despite improved endothelial function and reduced arterial reactivity to Ang II.     

Different cell cycle regulation of vascular smooth muscle in genetic hypertension

Vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) proliferate faster than those from Wistar-Kyoto rats (WKY). Therefore regulation of cell cycle progression was examined in VSMC from both strains. Analysis of G1 progression was performed in VSMC synchronized by serum starvation. Double staining for propidium iodide and bromodeoxyuridine revealed that G1 progression was faster in SHR as compared with WKY. Indeed, 59+/-6% of VSMC from SHR but only 14+/-10% of those from WKY had left G1 phase after 24 hours of mitogenic stimulation. Moreover, 15+/-2% of SHR cells had already completed the cycle at this time point. Western blot analysis demonstrated that the level of cyclin D, cyclin E, and cyclin A was higher in SHR cells progressing through G1 phase, whereas expression of cyclin-dependent kinase 2 as well as the cyclin-dependent kinase inhibitors p21 and p27 were similar in the two groups. Consistent with a higher level of cyclins, the activity of cyclin-dependent kinase 2 was more pronounced in SHR cells. Analysis of G2 progression was performed in VSMC synchronized by treatment with aphidicolin and revealed an additional difference in cell cycle regulation between SHR and WKY. Indeed, the level of cell division cycle kinase 2 was higher in cells from SHR, whereas that of its catalytic partner cyclin B was similar. Consistent with this pattern of expression, the activity of cell division cycle kinase 2 was more pronounced in VSMC from SHR as compared with WKY. Thus, these data demonstrate that the different proliferation of VSMC from SHR and WKY is related to a different progression in G1 phase as the result of the expression of cyclin D, cyclin A, and cyclin E as well as a different progression in G2 phase caused by expression of cell division cycle kinase 2.     

Alterations of calcium channels in vascular smooth muscle cells from spontaneously hypertensive rats.

We examined the possible alterations in calcium handling through the calcium channels of spontaneously hypertensive rats (SHR) using 45Ca2+ uptake measurements in cultured aortic cells. Primary cultures of vascular smooth muscle cells (VSMC) were obtained by enzymatic dissociation of the thoracic aortas from 8-week-old SHR and age-matched Wistar-Kyoto rats (WKY). The functions of voltage sensitive calcium channels (VSCC) and receptor operated calcium channels (ROCC) were estimated from the activated 45Ca2+ uptake in VSMC with high K+ depolarization and arginine vasopressin (AVP), respectively. Compared to basal conditions, depolarization with 55 mM KCl increased 45Ca2+ uptake at 20 min by 94 +/- 17 (SE) % in SHR and 38 +/- 6% in WKY. The activated 45Ca2+ uptake was significantly greater in SHR than in WKY (p < 0.01). There was no significant difference in 45Ca2+ uptake at 20 min in the presence of 5 x 10(-8)M AVP between SHR and WKY. These results suggest that calcium uptake, at least through VSCC, is increased in VSMC of SHR. This enhanced activity may be implicated in the hypertensive mechanisms in this model of hypertension.     

Urinary kallikrein activity in conscious spontaneously-hypertensive rats: development as a function of age

Urinary excretion of active kallikrein was determined every day (amidolytic assay) in 6 male Okamoto-Aoki spontaneously hypertensive rats (SHR) and in 6 male normotensive Wistar-Kyoto rats (WKY) from ages 3 to 6 weeks and from 12 to 16 weeks. The rats were placed into individual metabolic cages and allowed free access to food having normal sodium content and to tap water. Urinary kallikrein excretion (UKall V, nKat/24 h) was lower in 3-week-old SHR (7.8 +/- 1.4 nKat/24 h) than in WKY (15.5 +/- 2.3 nKat/24 h, p less than 0.01) at a moment when systolic blood pressure (BP) in SHR (85.5 +/- 4.0 mmHg) was already higher than in WKY (76.3 +/- 4.6 mmHg, p less than 0.01). The slope of the increase in kallikrein excretion from week 3 to week 6 was not different for SHR and WKY (y = 6.39 x - 12.09, r = 0.95 vs y = 7.49 x - 9.40, r = 0.93). In contrast, from week 12 to week 15, this slope was slightly negative for SHR (y = 1.08 x + 59.38, r = 0.66) and became significantly different (p less than 0.05) from the slope in WKY which remained positive (y = 5.09 x + 7.05, r = 0.48). The relation between kallikrein excretion and systolic BP was an exponential curve for both SHR and WKY. But the curve of SHR (y = 1.22.e0.03x, r = 0.91) was significantly different (p less than 0.01) from the curve of WKY (y = 1.08.e0.03x, r = 0.95). For each identical systolic BP, UKall V was always lower in SHR than in WKY.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thapsigargin-insensitive calcium pools in vascular smooth muscle cells.

Since sarcoplasmic Ca2+-ATPase may play an important role for the regulation of cytosolic free calcium concentration ([Ca2+]i) and may be altered in primary hypertension, the effects of thapsigargin and bradykinin on intracellular calcium pools in cultured vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats of the Münster strain (SHR) and normotensive Wistar-Kyoto (WKY) rats were investigated. VSMC were cultured on glass cover slips and [Ca2+]i was measured using the fluorescent dye fura2. To exclude transplasmamembrane calcium influx all experiments were performed in a calcium free medium. Thapsigargin, a selective inhibitor of the sarcoplasmic Ca2+-ATPase, and bradykinin, that is known to induce inositol trisphosphate release, dose dependently caused an increase of [Ca2+]i by emptying intracellular Ca2+ stores. The peak increase of [Ca2+]i after addition of saturation doses of thapsigargin (1 micromol/L) was not significantly different in the two strains (SHR: 69 +/- 11 nmol/L, n=24; WKY: 58 +/- 12 nmol/L, n=20; mean +/- SEM). When 10 micromol/L bradykinin was added after depletion of the thapsigargin-sensitive pools, still a release of [Ca2+]i could be observed. The bradykinin-induced [Ca2+]i increase was similar in the absence and presence of thapsigargin in VSMC from SHR (62 +/- 12 nmol/L, n=20; vs 52 +/- 18 nmol/L, n=22). In contrast, in the VSMC from WKY a significant reduction of the bradykinin induced [Ca2+]i-increase could be observed after the depletion of the thapsigargin sensitive calcium pools (70 +/- 8 nmol/L, n=21, vs. 33 +/- 7, n=20; p<0.002). It is concluded that bradykinin releases calcium from a pool that is not refilled by the common, thapsigargin-sensitive Ca2+-ATPase. In contrast to VSMC from normotensive WKY, in VSMC from spontaneously hypertensive rats thapsigargin and bradykinin sensitive pools may be regulated separately.     

Role of oxidative stress in the sex differences in blood pressure in spontaneously hypertensive rats

OBJECTIVE: The hypothesis was tested that differences in oxidative stress play a role in the sex differences in the development and maintenance of hypertension in spontaneously hypertensive rats (SHR). DESIGN AND METHODS: Male and female SHR [and Wistar-Kyoto (WKY) rats in the long-term study] (n = 6-12 per group) received tempol (30 mg/kg per day) or tap water for 6 weeks from 9 to 15 weeks of age or from birth until 15 weeks of age. Blood pressure [mean arterial pressure (MAP)] and kidney tissue F2-isoprostane (IsoP) were measured at 15 weeks of age. RESULTS: In SHR given tempol for 6 weeks, blood pressure and IsoP were reduced in males, but not in females. In SHR given tempol from birth, MAP was higher in SHR than WKY rats (SHR males, 181 +/- 2 mmHg; SHR females, 172 +/- 3 mmHg; WKY males, 100 +/- 2 mmHg; WKY females, 101 +/- 2 mmHg, P < 0.01), and tempol reduced MAP by 14% (156 +/- 3) and 26% (127 +/- 4) in male and female SHR, respectively, but had no effect on WKY rats. IsoP was higher in SHR than WKY rats and higher in male SHR than female SHR (SHR males, 5.18 +/- 0.23 ng/mg; SHR females, 3.71 +/- 0.19 ng/mg, P < 0.01; WKY males, 1.72 +/- 0.45 ng/mg; WKY females, 2.21 +/- 0.08 ng/mg, P < 0.05, compared with SHR). Tempol reduced IsoP in SHR to levels found in WKY rats, but had no effect on IsoP in WKY rats. CONCLUSIONS: Development of hypertension in SHR is mediated in part by oxidative stress independent of sex. Also, tempol is effective in reducing blood pressure in females only when given prior to the onset of hypertension.     

Sympathetic nerves and adrenal medulla: contributions to cardiovascular-conditioned emotional responses in spontaneously hypertensive rats

This report investigates the contributions of the sympathetic nerves and adrenal medulla to resting mean arterial pressure (MAP) and to emotionally conditioned MAP and heart rate (HR) responses in unrestrained spontaneously hypertensive rats (SHR) and Wistar-Kyoto normotensive control rats (WKY). Resting MAP (in mm Hg), which was higher in SHR (WKY = 120 +/- 4; SHR = 163 +/- 4; p less than 0.01), did not differ in the two strains following chemosympathectomy (WKY = 105 +/- 2; SHR = 101 +/- 2; n.s.). Adrenal medullectomy did not affect resting MAP in WKY (125 +/- 6; n.s.) but lowered it in SHR (146 +/- 5; p less than 0.05), relative to controls (see above). The conditioned pressor response (in mm Hg) in controls consisted of two peaks (I, II) in both strains, but was exaggerated in SHR (I = WKY, 13 +/- 1; SHR, 25 +/- 2; p less than 0.01; II = WKY 10 +/- 2; SHR 20 +/- 2; p less than 0.01). Chemosympathectomy suppressed (relative to controls) the first peak, but not the second, in both strains (WKY: I = 4 +/- 1, p less than 0.01; II = 12 +/- 2, n.s.; SHR: I = 6 +/- 1, p less than 0.01; II = 15 +/- 2, n.s.). Adrenal medullectomy alone had little effect on the pressor response, but when combined with chemosympathectomy both peaks were largely eliminated (WKY: I = 2 +/- I; II = 5 +/- 1; SHR: I = 1 +/- 0; II = 2 +/- 0). These data indicate that: 1) hypertension in conscious, freely behaving SHR is largely sustained by the sympathetic vasomotor nerves but that the adrenal medulla contributes to the magnitude of the elevation; 2) the early component of the exaggerated pressor response during aversive stimulation is mediated by sympathetic vasomotor excitation; and 3) the later component of the exaggerated pressor response reflects coactivation of the sympathetic vasomotor nerves and the adrenal medulla.     

Increased expression of vascular endothelial growth factor and capillary density in hearts of spontaneously hypertensive rats

OBJECTIVE: The role of VEGF in vascular remodeling of target organs exposed to chronic hypertension is poorly understood. The authors compared capillary density (CD), capillary-to-fiber ratio (C/F), and VEGF mRNA expression in the hearts (left ventricle [LV]), and skeletal muscles (soleus and anterior tibialis [AT]) of 18-week-old male spontaneously hypertensive rats (SHR) and age-matched male Wistar-Kyoto (WKY) and Sprague-Dawley (SD) rats. METHODS: CD or C/F in LV, soleus, and AT of SHR, WKY, and SD rats was determined by analysis of randomly acquired digital images of cryosections stained with FITC-conjugated GS-I lectin. VEGF mRNA expressions in the tissues were determined by Northern blot. RESULTS: VEGF mRNA expressions in LV of SHR were 3.84- and 5.05-fold higher, compared to SD and WKY rats, respectively (n = 6; p < .01). There were no significant differences in VEGF mRNA expression in soleus or AT among SHR, WKY, and SD rats (p > .05). CD in LV of SHR (4975 +/- 167) was significantly higher than WKY or SD rats, 4151 +/- 169 and 3807 +/- 187 mm(-2), respectively (p < .05). In LV of SHR, C/F increased (35%) more significantly than CD (increased 20%), compared to WKY rats. CD, or C/F in soleus or AT of SHR was similar to that observed in WKY or 8D rats. CONCLUSIONS: VEGF expression, CD, and C/F in the heart (LV) of SHR are significantly increased, compared to WKY and SD rats. The data are consistent with the possibility that VEGF may contribute to capillary growth as a compensatory response to hypertension.     

Nitric oxide synthase induction by ouabain in vascular smooth muscle cells from normotensive and hypertensive rats

OBJECTIVE: To investigate the effect of ouabain on inducible nitric oxide synthase (iNOS) activity and expression in cytokine-stimulated vascular smooth muscle cells (VSMC) from normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). METHODS: VSMC were treated for 24 h and afterwards, nitric oxide (NO) release was determined by the production of nitrite, a stable metabolite of NO. Activity of iNOS was measured by the conversion of [3H]-L-arginine to [3H]-L-citrulline and iNOS protein expression by Western blotting. RESULTS: Ouabain (0.01-1 mmol/l) further enhanced interleukin-1beta (II-1beta)-induced nitrite production by WKY and SHR VSMC, although a more pronounced effect was observed in SHR cells (maximum response 52.1 +/- 5.2 and 71.2 +/- 6.4% of 11-1beta effect in WKY and SHR cells, respectively). Such response on NO release was mimicked by the calcium ionophore A 23187 (0.01-1 micromol/l) and abolished by the voltage-operated calcium channels (VOCC) nifedipine (0.1 micromol/l). Expression of iNOS showed that ouabain increased the synthesis of the enzyme in WKY and SHR VSMC stimulated with II-1beta, and this effect was higher in SHR cells. The increased iNOS expression was significantly reduced by nifedipine. CONCLUSIONS: Ouabain stimulation of iNOS expression and activity in II-1beta-stimulated VSMCs from WKY rats and SHR seems to be related to increased intracellular calcium influx through VOCC. The more pronounced effect observed in SHR VSMC could be explained by an altered calcium entry in the hypertensive strain.     

Effects of dihydropyridines on tension and calcium-45 influx in isolated mesenteric resistance vessels from spontaneously hypertensive and normotensive rats

Contractile tension responses to norepinephrine and depolarizing potassium (80 mM K+), as well as calcium-45 influx stimulated by these agents, were studied in isolated mesenteric resistance vessels (each 100 microM internal diameter) from spontaneously hypertensive rats (SHRs) and from normotensive Wistar Kyoto rats (WKYs). Inhibitory effects of 2 dihydropyridine Ca++ antagonists, PN 200-110 (isradipine) and nisoldipine, on these parameters were also determined. Contractile responses to 80 mM K+ were inhibited by both Ca++ antagonists with the same potency and efficacy in SHR compared with WKY vessels (PN 200-110 IC50 = 2.8 +/- 1.3 X 10(-8) M in SHRs and 2.5 +/- 1.5 X 10(-8) M in WKYs; nisoldipine IC50 = 1.1 +/- 0.4 X 10(-8) M in SHRs and 1.2 +/- 0.9 X 10(-8) M in WKYs). However, contractile responses to norepinephrine (10(-4) M) were inhibited less potently by nisoldipine in SHR vessels (IC50 = 2.2 +/- 0.3 X 10(-9) M) compared with WKY vessels (IC50 = 1.6 +/- 0.6 X 10(-10) M). Similarly, PN 200-110 tended to be less (but not significantly less) potent in SHR vessels (IC50 = 3.3 +/- 1.8 X 10(-8) M) than in WKY vessels (IC50 = 3.4 +/- 0.9 X 10(-9) M); its efficacy was significantly depressed in the SHR vessels (by approximately 20%). When norepinephrine-stimulated calcium-45 influx was determined in the presence of these Ca++ antagonists, a similar profile emerged with respect to a comparison of SHR and WKY vessels. These results support a previously hypothesized alteration in receptor-activated Ca++ influx pathways in SHR mesenteric resistance vessels.     

Cardioreparative effects of lisinopril in rats with genetic hypertension and left ventricular hypertrophy.

BACKGROUND. In genetic and acquired hypertension, a structural remodeling of the nonmyocyte compartment of the myocardium, including the accumulation of fibrillar collagen within the interstitium and adventitia of intramyocardial coronary arteries and a medial thickening of these vessels, represents a determinant of pathological hypertrophy that leads to ventricular dysfunction. METHODS AND RESULTS. To evaluate the benefit of angiotensin converting enzyme inhibition in reversing this interstitial and vascular remodeling in the rat with genetic spontaneous hypertension (SHR) and established left ventricular hypertrophy (LVH), we treated 14-week-old male SHR with oral lisinopril (average dose, 15 mg/kg/day) for 12 weeks. Myocardial stiffness and coronary vascular reserve to adenosine (800 micrograms/min) were examined in the isolated heart; myocardial collagen and intramural coronary artery architecture were analyzed morphometrically. In lisinopril-treated SHR compared with 14-week-old baseline or 26-week-old untreated SHR and age- and sex-matched Wistar-Kyoto (WKY) controls, we found 1) a regression in LVH and normalization of blood pressure, 2) a complete regression of interstitial fibrosis, represented by a decrease of interstitial collagen volume fraction from 7.0 +/- 1.3% to 3.2 +/- 0.3% (p less than 0.025; WKY, 2.8 +/- 0.5%), 3) normalization of myocardial stiffness constant from 19.5 +/- 0.9 to 13.7 +/- 1.3 (p less than 0.025; WKY, 13.8 +/- 2.2), 4) a reversal of intramural coronary artery remodeling, including a decrease in the ratio of perivascular fibrosis to vessel lumen size from 1.4 +/- 0.2 to 0.4 +/- 0.1 (p less than 0.025; WKY, 0.6 +/- 0.1) and medial thickening from 12.3 +/- 0.6 to 7.4 +/- 0.5 microns (p less than 0.005; WKY, 7.4 +/- 0.4 microns), and 4) a restoration of coronary vasodilator response to adenosine from 12.3 +/- 0.9 to 26.0 +/- 1.4 ml/min/g (p less than 0.005; WKY, 21.8 +/- 2.2 ml/min/g). Thus, in SHR with LVH and adverse structural remodeling of the cardiac interstitium, lisinopril reversed fibrous tissue accumulation and medial thickening of intramyocardial coronary arteries and restored myocardial stiffness and coronary vascular reserve to normal. CONCLUSIONS. These cardioreparative properties of angiotensin converting enzyme inhibition may be valuable in reversing left ventricular dysfunction in hypertensive heart disease.     

Prolonged QT interval is associated with blood pressure rather than left ventricular mass in spontaneously hypertensive rats

QT interval is prolonged in hypertensive individuals, although the factors responsible for this increase are not completely understood. We questioned whether enhanced left ventricular mass (LVM) or increased systemic blood pressure represents the principal factor determining QT prolongation in the period of development of hypertension and left ventricular hypertrophy (LVH) in spontaneously hypertensive rats (SHR). In 12-and 20-week-old SHR (SHR12 and SHR20) and age-matched normotensive Wistar-Kyoto rats (WKY12 and WKY20), arterial systolic blood pressure (sBP) was measured using tail-cuff technique. Orthogonal Frank ECG was registered in anaesthetized animals in vivo, and bipolar ECG was measured in spontaneously beating isolated hearts in vitro. Progressive increase of sBP and LVM resulted in significant QT prolongation in SHR20 as compared to WKY12, WKY20, and also to SHR12 in vivo (WKY12: 82 +/- 9 ms, WKY20: 81 +/- 9 ms, SHR12: 88 +/- 15 and SHR20: 100 +/- 10, respectively; p < 0.05) but not in isolated hearts (WKY20: 196 +/- 39 ms and SHR20: 220 +/- 55, respectively; NS). In whole animals, QT duration was positively related to sBP (r = 0.6842; p < 0.001) but not to LVM (r = 0.1632, NS) in SHR20. The results suggest that QT prolongation in SHR developing hypertension and LVH depends on blood pressure rather than increase in LVM. In this period, myocardial hypertrophy is probably the predisposition for QT prolongation, but the significant change manifests only in the presence of elevated systemic factors.     

Vascular oxidative stress precedes high blood pressure in spontaneously hypertensive rats

This study examines whether longitudinal antioxidant treatment initiated in prehypertensive spontaneously hypertensive rats (SHR) can attenuate vascular oxidant stress and prevent blood pressure elevation during development. Male SHR and age-matched Wistar-Kyoto rats (WKY) were treated from 6 to 11 weeks of age with Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidinoxyl) (1 mmol/l in drinking water), a membrane-permeable superoxide dismutase mimetic. Mean systolic blood pressures (SBPs) were measured by tail-cuff Agonist-induced and basal O2- production was measured in thoracic aortas of 6- and 11-week-old SHR and WKY by lucigenin-derived chemiluminescence and oxidative fluorescent microscopy, respectively. SBP of 6-week-old SHR (131 +/- 5 mmHg) and WKY (130 +/- 4 mmHg) were not different; however, 11-week-old SHR SBP (171 +/- 4 mmHg) was significantly greater (p = .0001) than 11-week-old WKY SBP (143 +/- 5 mmHg). Tempol treatment completely, but reversibly, prevented this age-related rise in SHR SBP (SHR + Tempol: 137 +/- 4 mmHg; p < .0001 versus untreated SHR). Agonist-induced vascular O2- was increased in 6- (p = .03) and 11-week-old SHR (p < .0001) and 11-week-old WKY (p = .03) but not in 6-week-old WKY. Long-term Tempol treatment significantly lowered O2- production in both strains. Basal O2- measurements in both 6- and 11-week-old SHR were qualitatively increased compared with age-matched WKY; this increase in SHR was inhibited with in vitro Tempol treatment. These data show that antioxidant treatment to reduce oxidative stress prevents the age-related development of high blood pressure in an animal model of genetic hypertension.     

Alterations in catecholamine release in the central nervous system of spontaneously hypertensive rats.

The aim of the present study was to investigate alterations in catecholamine release in the central nervous system of spontaneously hypertensive rats. Slices of hypothalamus, medulla oblongata and striatum were prepared from spontaneously hypertensive rats (SHR: 9-10 weeks old) and age-matched Wistar Kyoto rats (WKY). The slices were incubated with (3H)norepinephrine (NE) or (3H)dopamine (DA), superfused with Krebs-solution in vitro, and the release of the catecholamines was compared between the two strains. The basal release of hypothalamic (3H)NE did not differ between SHR and WKY slices. However, stimulation (1 Hz)-evoked (3H)NE release was significantly greater in SHR than in WKY (percent fractional release of total tissue NE: WKY 0.494 +/- 0.019%, n = 6, SHR 0.730 +/- 0.053%, n = 6, p less than 0.05). The stimulation-evoked (3H)NE release from the medulla oblongata did not differ significantly between SHR and WKY slices. Finally stimulation-evoked release of striatal (3H)DA was significantly depressed in SHR (percent fractional release of total tissue DA: WKY 2.048 +/- 0.24%, n = 6, SHR 1.460 +/- 0.068%, n = 6, p less than 0.05). These results indicate that the release of hypothalamic NE and striatal DA are altered in SHR. It is suggested that enhanced hypothalamic noradrenergic activity and reduced striatal dopaminergic activity can increase sympathetic outflow to the periphery, which may play a role in the pathogenesis of this form of hypertension.     

Myocardial collagen and mechanics after preventing hypertrophy in hypertensive rats

To determine if a remodeling of the collagen matrix would occur in the absence of hypertrophy and cell necrosis and if such a remodeling could alter active and passive stiffness of the intact myocardium, five rats with genetic hypertension (SHR) were treated (SHRT) with hydralazine for 32 weeks, beginning at four weeks of age, and compared to six age- and sex-matched SHR and seven Wistar-Kyoto genetic control rats (WKY). Left ventricular (LV) weight of SHRT was 17% lower (P less than .001) than that of SHR and 19% higher (P less than .01) than that of WKY. Collagen volume fraction of SHR (13.7 +/- 3.2%) and SHRT (9.9 +/- 1.8%) were greater (P less than .01) than WKY (5.0 +/- 1.9%). Diastolic and systolic stress-strain relations were determined in the isolated heart. A comparison of these relations revealed: 1) a 24% increase in passive stiffness for SHR and SHRT; and 2) a reduced zero-strain intercept (41% to 54%) and slope (36% to 48%) of the developed stress-strain relation for the SHRT. Thus, in SHR, collagen remodeling occurred in the absence of hypertrophy which suggests that the muscular and collagenous compartments of the myocardium are under separate controls. The excess accumulation of collagen in SHR and SHRT leads to abnormal passive stiffness, and the prevention of hypertrophy with hydralazine reduces active stiffness.     

Hypertensive myocardial hypertrophy and rhythm disorders: 2 possible origins

It has been suggested that complex ventricular arrhythmias commonly occur in hypertensive patients with left ventricular hypertrophy. We have previously demonstrated that coronary artery ligation in anesthetized spontaneously hypertensive rats (SHR) and their normotensive controls (WKY) resulted in a significantly increased incidence and duration of ventricular fibrillation in SHR compared with WKY. The object of the present study was to characterize the structural and electrophysiological abnormalities in hypertrophied hearts, associated with the occurrence of arrhythmias. We used a double tissue bath in which a ventricular strip was exposed simultaneously to normal and to altered conditions (low pH, hypoxia and high potassium). Electrical activity recorded using standard micro-electrode techniques showed the occurrence of arrhythmias in all preparations and the development of major alterations in conduction (a conduction block appeared at 11 +/- 1 mn in SHR vs 16 +/- 1 mn in WKY, p less than 0.05), and maximal upstroke velocity (Vmax values before and 3 mn after the beginning of ischemia were 229 +/- 12 to 46 +/- 7 v/s for the SHR and 227 +/- 10 to 106 +/- 12 v/s for the WKY; p less than 0.001). These changes were associated in hypertrophied ventricles with a marked sub-endocardial collagen fibrosis as estimated by the use of automated image analysis (subendocardial collagen density = 4.39 +/- 0.34 p. 100 in SHR vs 1.66 +/- 0.15 p. 100 in WKY; p less than 0.001). Action potential duration measured using conventional glass micro-electrodes in a single chamber tissue bath revealed a highly significant difference (p less than 0.001) in APD 90 p. 100 of papillary muscles between SHR (114.7 +/- 2.8 ms) and WKY (76.9 +/- 1.7 ms). The addition of tetra-ethylammonium to block potassium channels induced triggered activity arising from early afterdepolarizations only in muscles hypertrophied SHR hearts.(ABSTRACT TRUNCATED AT 250 WORDS)