Elevated production of 20-HETE in the cerebral vasculature contributes to severity of ischemic stroke and oxidative stress in spontaneously hypertensive rats

Hypertension is a major risk factor for stroke, but the factors that contribute to the increased incidence and severity of ischemic stroke in hypertension remain to be determined. 20-hydroxyeicosatetraenoic acid (20-HETE) has been reported to be a potent constrictor of cerebral arteries, and inhibitors of 20-HETE formation reduce infarct size following cerebral ischemia. The present study examined whether elevated production of 20-HETE in the cerebral vasculature could contribute to the larger infarct size previously reported after transient middle cerebral artery occlusion (MCAO) in hypertensive strains of rat [spontaneously hypertensive rat (SHR) and spontaneously hypertensive stroke-prone rat (SHRSP)]. The synthesis of 20-HETE in the cerebral vasculature of SHRSP measured by liquid chromatography-tandem mass spectrometry was about twice that seen in Wistar-Kyoto (WKY) rats. This was associated with the elevated expression of cytochrome P-450 (CYP)4A protein and CYP4A1 and CYP4A8 mRNA. Infarct volume after transient MCAO was greater in SHRSP (36+/-4% of hemisphere volume) than in SHR (19+/-5%) or WKY rats (5+/-2%). This was associated with a significantly greater reduction in regional cerebral blood flow (rCBF) in SHR and SHRSP than in WKY rats during the ischemic period (78% vs. 62%). In WKY rats, rCBF returned to 75% of control following reperfusion. In contrast, SHR and SHRSP exhibited a large (166+/-18% of baseline) and sustained (1 h) postischemic hyperperfusion. Acute blockade of the synthesis of 20-HETE with N-hydroxy-N'-(4-butyl-2-methylphenyl)-formamidine (HET0016; 1 mg/kg) reduced infarct size by 59% in SHR and 87% in SHRSP. HET0016 had no effect on the fall in rCBF during MCAO but eliminated the hyperemic response. HET0016 also attenuated vascular O2*- formation and restored endothelium-dependent dilation in cerebral arteries of SHRSP. These results indicate the production of 20-HETE is elevated in the cerebral vasculature of SHRSP and contributes to oxidative stress, endothelial dysfunction, and the enhanced sensitivity to ischemic stroke in this hypertensive model.     

Muscle RING Finger-1 Promotes a Maladaptive Phenotype in Chronic Hypoxia-Induced Right Ventricular Remodeling

Exposure to chronic hypoxia (CH) induces elevated pulmonary artery pressure/resistance, leading to an eventual maladaptive right ventricular hypertrophy (RVH). Muscle RING finger-1 (MuRF1) is a muscle-specific ubiquitin ligase that mediates myocyte atrophy and has been shown to play a role in left ventricular hypertrophy and altered cardiac bioenergetics in pressure overloaded hearts. However, little is known about the contribution of MuRF1 impacting RVH in the setting of CH. Therefore, we hypothesized that MuRF1 deletion would enhance RVH compared to their wild-type littermates, while cardiac-specific overexpression would reduce hypertrophy following CH-induced pulmonary hypertension. We assessed right ventricular systolic pressure (RVSP), right ventricle to left ventricle plus septal weight ratio (RV/LV+S) and hematocrit (Hct) following a 3-wk isobaric CH exposure. Additionally, we conducted dual-isotope SPECT/CT imaging with cardiac function agent ²⁰¹Tl-chloride and cell death agent ^99mTc-annexin V. Predictably, CH induced pulmonary hypertension, measured by increased RVSP, RV/LV+S and Hct in WT mice compared to normoxic WT mice. Normoxic WT and MuRF1-null mice exhibited no significant differences in RVSP, RV/LV+S or Hct. CH-induced increases in RVSP were also similar between WT and MuRF1-null mice; however, RV/LV+S and Hct were significantly elevated in CH-exposed MuRF1-null mice compared to WT. In cardiac-specific MuRF1 overexpressing mice, RV/LV+S increased significantly due to CH exposure, even greater than in WT mice. This remodeling appeared eccentric, maladaptive and led to reduced systemic perfusion. In conclusion, these results are consistent with an atrophic role for MuRF1 regulating the magnitude of right ventricular hypertrophy following CH-induction of pulmonary hypertension.     

Decreased content in left atrium and increased plasma concentration of atrial natriuretic polypeptide in spontaneously hypertensive rats (SHR) and SHR stroke-prone

The atrial contents and concentrations, and the plasma concentrations of atrial natriuretic polypeptide (ANP) in spontaneously hypertensive rats (SHR) and SHR stroke-prone (SHRSP) were measured and compared with those of age-matched Wistar Kyoto rats (WKY) using a specific radioimmunoassay (RIA) for alpha-rat ANP (alpha-rANP). The contents of alpha-rANP-LI in the atria of SHR (19.0 +/- 0.9 micrograms, mean +/- SEM) and SHRSP (19.3 +/- 0.6 micrograms) were significantly lower than that of WKY (22.8 +/- 1.4 micrograms) (p less than 0.05). The atrial concentration of alpha-rANP-LI was also significantly lower in SHR (248.2 +/- 11.3 ng/mg, p less than 0.05) and tended to be lower in SHRSP (272.2 +/- 12.4 ng/mg) than that of WKY (300.0 +/- 14.2 ng/mg). Furthermore, the concentrations in the left auricles of SHR and SHRSP were significantly lower than that of WKY (p less than 0.01 and p less than 0.05, respectively). In contrast, no significant difference was observed in the alpha-rANP-LI concentrations in the right auricles of WKY, SHR and SHRSP. Gel filtration studies coupled with RIA showed that gel filtration profiles of the extracts from the right and left auricles of WKY, SHR and SHRSP were essentially identical. The plasma alpha-rANP-LI levels in SHR (260 +/- 34 pg/ml) and SHRSP (319 +/- 19 pg/ml) were significantly higher than that in WKY (170 +/- 17 pg/ml) (p less than 0.05 and p less than 0.01, respectively). These results suggest that the secretion of ANP from the heart is increased in SHR and SHRSP compared with WKY.     

自发性高血压大鼠血管α1肾上腺素受体亚型的改变

本工作在离体与整体条件下比较易卒中型自发性高血压(SHRSP)大鼠与WKY大鼠血管中α_1受体的两种亚型。在离体灌流的主动脉、肾动脉与肠系膜动脉,50μmol/L氯甲基可乐定(CEC)预温育30min可使α_1受体激动时引起的最大收缩张力在SHRSP与WKY大鼠分别降为对照时的31.4±8.3％与35.2±2.9％,68.4±8.2％与80.1±7.3％,68.4±6.3％与55.4±7.0％,两者间均无显著性差别。但10μmol/L硝苯吡啶对α_1受体收缩效应的阻断作用则在SHRSP大鼠大大超过WKY大鼠,最大收缩张力分别降为对照时的3.1±1.5％与56.5±4.8％(P<0.01),9.0±4.1％与23.6±3.5％(P<0.05),5.9±2.5％与28.0±0.8％(P<0.01)。整体动物实验也显示硝苯吡啶的降血压作用及对苯肾上腺素升血压效应的阻断作用在SHRSP大鼠都较WKY大鼠显著增强。离体主动脉a_1受体激动时的快速相与持续相收缩均主要由α_(1B)亚型激动引起,硝苯吡啶对快速相收缩的阻断作用在SHRSP与WKY大鼠无显著性差别,但对持续相收缩的阻断作用则在SHRSP大鼠显著强于WKY大鼠。上述结果提示SHRSP大鼠血管α_1受体两种亚型的分布没有显著改变,但α_(1B)受体激动时继发性细胞外Ca~(2+)进入的途径由非双氢吡啶敏感性钙通道转变为双氢吡啶敏感性钙通道。     

NO supports right ventricular flow dominance and whole body O(2) utilization in midgestation fetal lambs

It is unknown if nitric oxide (NO) modulates the relative levels of left (LV) and right (RV) ventricular output, fetal O2 consumption, or blood flow distribution between the body and placenta at midgestation. To address these questions, six fetal lambs were instrumented at 89-96 days gestation (term 147 days), and blood flows were measured with radioactive microspheres 3-4 days later at baseline and after inhibition of NO synthesis with 10 mg/kg (L-NNA10) and 25 mg/kg (L-NNA25) N(omega)-nitro-L-arginine. LV output fell by 74 +/- 15 ml. min(-1). kg(-1) at L-NNA10 (P < 0.005), whereas RV output decreased by 90 +/- 18 ml. min(-1). kg(-1) at L-NNA10 (P < 0.02) and by a further 80 +/- 22 ml. min(-1). kg(-1) at L-NNA25 (P < 0.05). As a result, RV output exceeded LV output at baseline (P = 0.03) and L-NNA10 (P < 0.02) but not at L-NNA25. Fetal body blood flow fell by 95 +/- 25 ml. min(-1). kg(-1) at L-NNA10 (P < 0.01), but because placental blood flow decreased by 70 +/- 22 ml. min(-1). kg(-1) at L-NNA10 (P < 0.01) and a further 71 +/- 21 ml. min(-1). kg(-1) at L-NNA25 (P < 0.01), the fetal body-to-placental blood flow ratio was near unity at baseline and L-NNA10 but rose to 1.5 +/- 0.3 at L-NNA25 (P < 0.05). In association with these flow changes, fetal O2 consumption declined by 1.4 +/- 0.3 ml. min(-1). kg(-1) at L-NNA10 (P < 0.05) and by a further 1.5 +/- 0.6 ml. min(-1). kg(-1) at L-NNA25 (P < 0.02). These findings suggest that, in midgestation fetal lambs, NO supports an RV flow dominance, whole body O2 utilization, and the maintenance of a near-equal fetoplacental blood flow distribution.     

Acetylcholine-induced endothelium-derived contracting factor in hypoxic pulmonary hypertensive rats.

We determined the role of an endothelium-derived contracting factor in the impaired relaxation response to ACh of conduit pulmonary arteries (PAs) isolated from rats with hypoxic pulmonary hypertension (PH). A PGH2/thromboxane A2 (TxA2)-receptor antagonist (ONO-3708) partially restored the impairment of ACh-induced relaxation, whereas TxA2 synthase inhibitors (OKY-046 and CV-4151) did not affect the impaired relaxation in phenylephrine-precontracted hypertensive PAs. Endothelium-denuded hypertensive PA rings showed no difference in the response to ACh between preparations with and without ONO-3708. In both endothelium-denuded control and hypertensive PAs, exogenous PGH2 induced contractions, and the magnitude of the contractions was greater in the control than in hypoxic PH preparations. An endothelin A-receptor antagonist (BQ-485), an endothelin B-receptor antagonist (BQ-788), and a superoxide anion scavenger (superoxide dismutase) did not restore the impaired response to ACh in hypertensive PAs. These findings suggest that PGH2 produced from the conduit PAs of rats with chronic hypoxic PH may be the endothelium-derived contracting factor responsible for the impairment of ACh-mediated vasorelaxation.     

VEGF-induced relaxation of pulmonary arteries is mediated by endothelial cytochrome P-450 hydroxylase

The cytochrome P-450 metabolite 20-HETE induces calcium-, endothelial-, and nitric oxide (NO)-dependent relaxation of bovine pulmonary arteries (PA). VEGF is an NO-dependent dilator of systemic arteries and plays a key role in maintaining the integrity of the pulmonary vasculature. We tested the effect of VEGF on PA diameter and tone and the contribution of cytochrome P-450 family 4 (CYP4) to vasoactive effects of VEGF. Bovine PA rings (1 mm in diameter) relaxed with VEGF (0.1-10 nM) in an endothelial- and eNOS-dependent manner. This response was blunted by pretreatment with the CYP4 inhibitor dibromododecynyl methyl sulfonamide (DDMS) as well as a mechanistically different CYP4 inhibitor N-hydroxy-N'-(4-butyl-2-methylphenyl)formamidine. PAs also increased in diameter by 6-12% in the presence of VEGF (10 nM), and this increase was attenuated by DDMS. In contrast to that shown in PAs, 20-HETE constricted bovine renal arteries and did not increase intracellular Ca(2+) in renal artery endothelial cells as observed in bovine pulmonary artery endothelial cells (BPAECs). VEGF-evoked increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) in BPAECs were blunted by treatment with DDMS. Both VEGF (10 nM) and 20-HETE (1-5 microM) stimulated NO release from cultured BPAECs, and once again VEGF-induced increases were attenuated by pretreating the cells with DDMS. We conclude that CYP4/20-HETE contributes to VEGF-stimulated NO release and vasodilation in bovine PAs. Given the unique expression of 20-HETE-forming CYP4 in BPAECs vs. systemic arterial endothelial cells, CYP4 may be an important mediator of endothelial-dependent vasoreactivity in PAs.     

Dynamic microRNA expression during the transition from right ventricular hypertrophy to failure

MicroRNAs (miRs) are small, noncoding RNAs that are emerging as crucial regulators of cardiac remodeling in left ventricular hypertrophy (LVH) and failure (LVF). However, there are no data on their role in right ventricular hypertrophy (RVH) and failure (RVF). This is a critical question given that the RV is uniquely at risk in patients with congenital right-sided obstructive lesions and in those with systemic RVs. We have developed a murine model of RVH and RVF using pulmonary artery constriction (PAC). miR microarray analysis of RV from PAC vs. control demonstrates altered miR expression with gene targets associated with cardiomyocyte survival and growth during hypertrophy (miR 199a-3p) and reactivation of the fetal gene program during heart failure (miR-208b). The transition from hypertrophy to heart failure is characterized by apoptosis and fibrosis (miRs-34, 21, 1). Most are similar to LVH/LVF. However, there are several key differences between RV and LV: four miRs (34a, 28, 148a, and 93) were upregulated in RVH/RVF that are downregulated or unchanged in LVH/LVF. Furthermore, there is a corresponding downregulation of their putative target genes involving cell survival, proliferation, metabolism, extracellular matrix turnover, and impaired proteosomal function. The current study demonstrates, for the first time, alterations in miRs during the process of RV remodeling and the gene regulatory pathways leading to RVH and RVF. Many of these alterations are similar to those in the afterload-stressed LV. miRs differentially regulated between the RV and LV may contribute to the RVs increased susceptibility to heart failure.     

The Soluble Guanylate Cyclase Stimulator Riociguat Ameliorates Pulmonary Hypertension Induced by Hypoxia and SU5416 in Rats

Background

The nitric oxide (NO)–soluble guanylate cyclase (sGC)–cyclic guanosine monophosphate (cGMP) signal-transduction pathway is impaired in many cardiovascular diseases, including pulmonary arterial hypertension (PAH). Riociguat (BAY 63–2521) is a stimulator of sGC that works both in synergy with and independently of NO to increase levels of cGMP. The aims of this study were to investigate the role of NO–sGC–cGMP signaling in a model of severe PAH and to evaluate the effects of sGC stimulation by riociguat and PDE5 inhibition by sildenafil on pulmonary hemodynamics and vascular remodeling in severe experimental PAH.

Methods and Results

Severe angioproliferative PAH was induced in rats by combined exposure to the vascular endothelial growth factor receptor antagonist SU5416 and hypoxia (SUHx). Twenty-one days thereafter rats were randomized to receive either riociguat (10 mg/kg/day), sildenafil (50 mg/kg/day) or vehicle by oral gavage, for 14 days until the day of the terminal hemodynamic measurements. Administration of riociguat or sildenafil significantly decreased right ventricular systolic pressure (RVSP). Riociguat significantly decreased RV hypertrophy (RVH) (0.55±0.02, p<0.05), increased cardiac output (60.8±.8 mL/minute, p<0.05) and decreased total pulmonary resistance (4.03±0.3 mmHg min⁻¹ ml⁻¹ 100 g BW, p<0.05), compared with sildenafil and vehicle. Both compounds significantly decreased the RV collagen content and improved RV function, but the effects of riociguat on tricuspid annular plane systolic excursion and RV myocardial performance were significantly better than those of sildenafil (p<0.05). The proportion of occluded arteries was significantly lower in animals receiving riociguat than in those receiving vehicle (p<0.05); furthermore, the neointima/media ratio was significantly lower in those receiving riociguat than in those receiving sildenafil or vehicle (p<0.05).

Conclusion

Riociguat and sildenafil significantly reduced RVSP and RVH, and improved RV function compared with vehicle. Riociguat had a greater effect on hemodynamics and RVH than sildenafil.     

Pulmonary vascular effects of sildenafil on the development of chronic pulmonary hypertension in the ovine fetus

We investigated the pulmonary vascular effects of prophylactic use of sildenafil, a specific phosphodiesterase-5 inhibitor, in late-gestation fetal lambs with chronic pulmonary hypertension. Fetal lambs were operated on at 129 +/- 1 days gestation (term = 147 days). Ductus arteriosus (DA) was compressed for 8 days to cause chronic pulmonary hypertension. Fetuses were treated with sildenafil (24 mg/day) or saline. Pulmonary vascular responses to increase in shear stress and in fetal PaO2 were studied at, respectively, day 4 and 6. Percent wall thickness of small pulmonary arteries (%WT) and the right ventricle-to-left ventricle plus septum ratio (RVH) were measured after completion of the study. In the control group, DA compression increased PA pressure (48 +/- 5 to 72 +/- 8 mmHg, P < 0.01) and pulmonary vascular resistance (PVR) (0.62 +/- 0.08 to 1.15 +/- 0.11 mmHg x ml(-1) x min(-1), P < 0.05). Similar increase in PAP was observed in the sildenafil group, but PVR did not change significantly (0.54 +/- 0.06 to 0.64 +/- 0.09 mmHg x ml(-1) x min(-1)). Acute DA compression, after brief decompression, elevated PVR 25% in controls and decreased PVR 35% in the sildenafil group. Increased fetal PaO2 did not change PVR in controls but decreased PVR 60% in the sildenafil group. %WT and RVH were not different between groups. Prophylactic sildenafil treatment prevents the rise in pulmonary vascular tone and altered vasoreactivity caused by DA compression in fetal lambs. These results support the hypothesis that elevated PDE5 activity is involved in the consequences of chronic pulmonary hypertension in the perinatal lung.     

Nitric oxide contributes to right coronary vasodilation during systemic hypoxia

As arterial partial pressure of O(2) (Pa(O(2))) is reduced during systemic hypoxia, right ventricular (RV) work and myocardial O(2) consumption (MVo(2)) increase. Mechanisms responsible for maintaining RV O(2) demand/supply balance during hypoxia have not been delineated. To address this problem, right coronary (RC) blood flow and RV O(2) extraction were measured in nine conscious, instrumented dogs exposed to normobaric hypoxia. Catheters were implanted in the right ventricle for measuring pressure, in the ascending aorta for measuring arterial pressure and for sampling arterial blood, and in an RC vein. A flow transducer was placed around the RC artery. After recovery from surgery, dogs were exposed to hypoxia in a chamber ventilated with N(2), and blood samples and hemodynamic data were collected as chamber O(2) was reduced progressively to approximately 8%. After control measurements were made, the chamber was opened and the dog was allowed to recover. N(omega)-nitro-L-arginine (L-NNA) was then administered (35 mg/kg, via RV catheter) to inhibit nitric oxide (NO) production, and the hypoxia protocol was repeated. RC blood flow increased during hypoxia due to coronary vasodilation, because RC conductance increased from 0.65 +/- 0.05 to 1.32 +/- 0.12 ml x min(-1) x 100 g(-1) x L-NNA blunted the hypoxia-induced increase in RC conductance. RV O(2) extraction remained constant at 64 +/- 4% as Pa(O(2)) was decreased, but after L-NNA, extraction increased to 70 +/- 3% during normoxia and then to 78 +/- 3% during hypoxia. RV MVo(2) increased during hypoxia, but after L-NNA, MVo(2) was lower at any respective Pa(O(2)). The relationship between heart rate times RV systolic pressure (rate-pressure product) and RV MVo(2) was not altered by l-NNA. To account for L-NNA-mediated decreases in RV MVo(2), O(2) demand/supply variables were plotted as functions of MVo(2). Slope of the conductance-MVo(2) relationship was depressed by L-NNA (P = 0.03), whereas the slope of the extraction-MVo(2) relationship increased (P = 0.003). In summary, increases in RV MVo(2) during hypoxia are met normally by increasing RC blood flow. When NO synthesis is blocked, the large RV O(2) extraction reserve is mobilized to maintain RV O(2) demand/supply balance. We conclude that NO contributes to RC vasodilation during systemic hypoxia.     

Sex-specific differences in cerebral arterial myogenic tone in hypertensive and normotensive rats

Cerebral blood flow (CBF) is maintained constant despite changes in systemic blood pressure (BP) through multiple mechanisms of autoregulation such as vascular myogenic reactivity. Our aim was to determine myogenic characteristics of cannulated middle cerebral arteries (MCA) in male and female stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto rats (WKY) at 12 wk of age under pressurised no-flow conditions. MCA pressure-diameter relationships (20-200 mmHg) were constructed in active (with calcium) and passive (without calcium) conditions, and myogenic and mechanical properties were determined. Myogenic reactivity in WKY (P < 0.05) and SHRSP (P < 0.05) males was impaired compared with their female counterparts. Comparison of SHRSP with WKY in males revealed similar myogenic reactivity, but in females SHRSP exhibited augmented myogenic reactivity (P < 0.05). In both sexes, myogenic tone yielded at lower pressure in SHRSP compared with WKY vessels (120-140 vs. 140-180 mmHg). Stress-strain relationships and elastic moduli in WKY rats showed that vessels were stiffer in females than in males. Conversely, in SHRSP, male vessels were stiffer than female vessels. Comparison of strains in males indicated that stiffness was increased in SHRSP compared with WKY vessels, whereas the converse was observed in females. These findings demonstrate that MCA myogenic and distensibility characteristics exhibit significant sex- and strain-dependent differences. Inappropriate myogenic adaptation and augmented vascular stiffness, particularly in male SHRSP, are potential limiting factors in blood flow autoregulation and may increase the predisposition for stroke-related cerebrovascular events.     

Alterations in atrial and plasma atrial natriuretic factor (ANF) content during development of hypoxia-induced pulmonary hypertension in the rat

Distension of the atrial wall has been proposed as a signal for the increased release of atrial natriuretic factor (ANF) from atrial myocytes in response to perceived volume overload. To determine whether pressure changes resulting from hypertension in the pulmonary circulation may stimulate release of ANF, rats were exposed to chronic hypobaric hypoxia for 3 or 21 days and the ANF concentration in the atria and plasma were determined by specific radioimmunoassay. Exposure to chronic hypoxia resulted in significant increases in hematocrit at both 3 (p less than 0.025) and 21 days (p less than 0.005) and in the development of right ventricular hypertrophy (RVH) expressed as the ratio of the weight of the right ventricle to the weight of the left ventricle and septum (RV/LV+S) at both 3 (RV/LV+S = 0.278 +/- 0.005) and 21 days (RV/LV+S = 0.536 +/- 0.021). After 21 days, left atrial (LA) ANF content was significantly increased in hypoxic rats compared to controls (508 +/- 70 ng/mg tissue vs 302 +/- 37 ng/mg), while right atrial (RA) ANF content was significantly reduced (440 +/- 45 vs 601 +/- 58 ng/mg). At this time, plasma ANF concentration was significantly elevated compared to controls (238 +/- 107 pg/ml vs 101 +/- 10 pg/ml). These results suggest that the development of pulmonary hypertension following chronic hypobaric exposure induces altered atrial ANF content and increased plasma ANF concentration as a result of altered distension of the atrial wall.     

Voltage-gated K(+)-channel activity in ovine pulmonary vasculature is developmentally regulated

To examine mechanisms underlying developmental changes in pulmonary vascular tone, we tested the hypotheses that 1) maturation-related changes in the ability of the pulmonary vasculature to respond to hypoxia are intrinsic to the pulmonary artery (PA) smooth muscle cells (SMCs); 2) voltage-gated K(+) (K(v))-channel activity increases with maturation; and 3) O(2)-sensitive Kv2.1 channel expression and message increase with maturation. To confirm that maturational differences are intrinsic to PASMCs, we used fluorescence microscopy to study the effect of acute hypoxia on cytosolic Ca(2+) concentration ([Ca(2+)](i)) in SMCs isolated from adult and fetal PAs. Although PASMCs from both fetal and adult circulations were able to sense an acute decrease in O(2) tension, acute hypoxia induced a more rapid and greater change in [Ca(2+)](i) in magnitude in PASMCs from adult compared with fetal PAs. To determine developmental changes in K(v)-channel activity, the effects of the K(+)-channel antagonist 4-aminopyridine (4-AP) were studied on fetal and adult PASMC [Ca(2+)](i). 4-AP (1 mM) caused PASMC [Ca(2+)](i) to increase by 94 +/- 22% in the fetus and 303 +/- 46% in the adult. K(v)-channel expression and mRNA levels in distal pulmonary arteries from fetal, neonatal, and adult sheep were determined through the use of immunoblotting and semiquantitative RT-PCR. Both Kv2.1-channel protein and mRNA expression in distal pulmonary vasculature increased with maturation. We conclude that there are maturation-dependent changes in PASMC O(2) sensing that may render the adult PASMCs more responsive to acute hypoxia.     

Mineralocorticoids upregulate arterial contraction to epidermal growth factor

The present studies test the hypothesis that contraction to EGF is dependent on mineralocorticoids and/or an elevation in systolic blood pressure (SBP). Endothelium-denuded thoracic aortas from sham normotensive, N(omega)-nitro-L-arginine (L-NNA) hypertensive, Wistar-Kyoto (WKY), and spontaneously hypertensive rats (SHR) were used in isolated tissue-bath experiments. Maximal contraction to epidermal growth factor [EGF; percentage of phenylephrine (PE; 10 umol/l)-induced contraction] was greater in strips from L-NNA (32 +/- 5%) and SHR (53 +/- 8%) rats compared with sham and WKY rats (17 +/- 1 and 12 +/- 4%, respectively). Wistar-Furth rats became only mildly hypertensive when given DOCA salt (134 +/- 6 mmHg) compared with Wistar rats (176 +/- 9 mmHg), but aortas from both strains had a similarly enhanced contraction to EGF (approximately 9 times the maximal contraction of sham aorta). Furthermore, in vitro incubation of aortas from Wistar and Wistar-Furth rats with aldosterone (10 nmol/l) increased EGF-receptor mRNA expression by >50%. These data indicate that arterial contraction to EGF may occur independent of hypertension and be stimulated by mineralocorticoids.     

慢性缺氧对大鼠肺动胍内皮依赖性舒张反应及CGMP含量的影响

This experiment was designed to investigate whether chronic hypoxia affect rat pulmonary artery (PA) endothelium-dependent relaxation and the content of cGMP in PA. Both ACh and ATP could induce endothelium-dependent relaxation of PA, not prevented by indomethacin, but completely abolished by methylene blue. These results indicated that vasodilatation of PA induced by both ACh and ATP is mediated by EDRF (endothelium-derived relaxing factor). Chronic hypoxia significantly depressed PA endothelium-dependent relaxation. The percent relaxation of IPPA and EPPA by 10(-6) mol/L ACh was 61.3% and 59.2% of those in control, and the percent relaxation of IPPA and EPPA by 1.8 x 10(-5) mol/L ATP was 64.9% and 55.3% respectively of the control. Chronic hypoxia also depressed SNP-induced endothelium-independent relaxation. Chronic hypoxia significantly decreased the content of cGMP in PA. The basic level of cGMP was 51.9 +/- 5.7 (n = 14) in hypoxia group and 84.9 +/- 9.7 (n = 14) pmol/g wet wt. in control group (P less than 0.01). After treatment of PA with ACh (10(-7) mol/L), the content of cGMP was 91.4 +/- 7.3 (n = 5) pmol/g wet wt. in hypoxic group and 240.8 +/- 30.6 (n = 5) pmol/g wet wt. in control group (P less than 0.01). Our data suggest that chronic hypoxia might depress rat pulmonary artery endothelium-dependent relaxation through the inhibition of soluble guanylate cyclase in vascular smooth muscle cells.     

Progressive development of pulmonary hypertension leading to right ventricular hypertrophy assessed by echocardiography in rats.

The present study aimed to evaluate the development of pulmonary hypertension by serial echocardiography, including measurements of pulmonary artery (PA) flow velocities, and correlate echocardiographic indices with pathological findings in rats administered monocrotaline (MCT). MCT (60 mg/kg body weight) or physiologic saline was administered to a total of 9 male Wistar rats at the age of 4 weeks (MCT group: n = 4, control group: n = 5, respectively). Echocardiography was performed serially until the age of 8 weeks. The ratio of right ventricular (RV) outflow tract dimensions to aortic dimensions increased progressively in the MCT group and became significantly greater than that of the control group after the age of 6 weeks. Peak PA velocity (Peak V) in the MCT group was significantly less than that of the control group at the ages of 7 and 8 weeks. The ratio of acceleration time to ejection time (AT/ET) in PA flow waveforms declined progressively and was significantly less than that of the control group after the age of 6 weeks. The ratio of RV weight to body weight (RVW/BW) in the MCT group was significantly greater than that of the control group. Both AT/ET ratio and Peak V were significantly inversely correlated with RVW/BW ratio. Furthermore, these echocardiographic findings were also significantly inversely correlated with the mean cross-sectional RV myocyte area. In conclusion, the progressive development of pulmonary hypertension leading to RV hypertrophy can be evaluated appropriately by echocardiography including PA flow Doppler indices in rats.     

Alterations in heme oxygenase/carbon monoxide system in pulmonary arteries in hypertension

Enhancement of the heme oxygenase/carbon monoxide (HO/CO) system has been shown to lower blood pressure (BP) in young (8 weeks), but not in adult (20 weeks) spontaneously hypertensive (SHR) rats. The reasons for this selective effect still remain puzzling. We investigated the effects of hemin on the HO/CO system of the pulmonary artery (PA) in SHR and Wistar-Kyoto (WKY) rats at different ages and evaluated the hemin-dependent changes in sGC and cGMP pathways. Hemin administration resulted in an evident reduction of BP (from 148.6 +/- 3.2 to 125.8 +/- 2.6 mmHg, P < 0.01) in young, but not in prehypertensive (4 weeks) or adult SHR or WKY rats at all ages. Coadministration of the HO inhibitor, chromium mesoporphyrin, with hemin, cancelled the BP-lowering effect of hemin. Remarkably, lower expression levels of HO-1, HO-2, and sGC paralleled with reduced HO activity and cGMP content were observed in PA from 8-week SHR rats, but not from adult SHR or WKY rats of all ages. Interestingly, hemin treatment restored these deficiencies, although the expression level of non-inducible HO-2 protein remained unchanged. We conclude that in young and prehypertensive SHR rats, an impaired HO/CO-sGC/cGMP system in the PA might be indicative of the pathogenesis and development of hypertension. In contrast, the HO/CO system in the PA of adult SHR rats was upregulated as a compensatory reaction to elevated BP and desensitization of the downstream targets of the sGC/cGMP pathway occurred.     

Chronic hypoxia induces right heart failure in caveolin-1-/- mice

Caveolin-1 (Cav-1)-/- mice develop mild pulmonary hypertension as they age. In this study, we sought to determine the effect of chronic hypoxia, an established model of pulmonary hypertension, on young Cav-1-/- mice with no measurable signs of pulmonary hypertension. Exposure of Cav-1-/- mice to chronic hypoxia resulted in an initial rise in right ventricular (RV) systolic pressure (RVSP) similar to wild-type (WT) mice. By three weeks RVSP decreased in the Cav-1-/- mice, whereas it was maintained in WT mice. The drop in RVSP in Cav-1-/- mice was accompanied by decreased cardiac output, increased RV hypertrophy, RV interstitial fibrosis, decreased RV sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a mRNA and decreased RV function compared with WT mice. Importantly, minimal differences were noted in pulmonary vascular remodeling between WT and Cav-1-/- mice, and left ventricular function was normal in hypoxic Cav-1-/- mice. Mechanistically, increased endothelial nitric oxide synthase uncoupling and increased tyrosine nitration of protein kinase G were detected in the RV of Cav-1-/- mice. These hemodynamic, histological, and molecular changes were prevented in Cav-1-/- mice expressing an endothelial-specific Cav-1 transgene or by nitric oxide synthase inhibition. These data suggest that, in Cav-1-/- mice, increased oxidative/nitrosative stress due to endothelial nitric oxide synthase uncoupling modifies the response of the RV to pressure overload, accelerating the deterioration of RV function.     

钙调神经磷酸酶信号通路参与前列腺素F2α诱导的心肌肥厚

利用野百合碱（monocrotaline,MCT）诱导大鼠右心室肥厚模型和培养乳鼠心肌细胞,研究前列腺素F2α（prostaglandin F2α,PGF2α）在心肌肥厚中的作用及钙调神经磷酸酶（calcineurin,CaN）信号通路征其中的作用。在雄性Sprague-Dawley大鼠中,用MCT（60mg／kg）单次i．p．诱导右心室肥厚,同时用塞来旨布（20mg／kg）预防／治疗给药2周。用病理检测、电镜观察等方法观察心肌肥厚时组织病理改变;EIA试剂盒检测心肌组织PGF2α含量;RT-PCR检测心房钠尿肽（atrial natriuretic peptide,ANP）和CaNmRNA的表达;用蛋白免疫印迹法检测CaN及其下游因了NFAT3和GATA4蛋门质的表达。以心肌细胞直径、蛋白含量和ANP mRNA表达的变化为0．1μmol／L PGF2α诱导心肌细胞肥大的指标。以CaN mRNA表达作为该信号通路的主要指标,并观察CaN抑制剂环孢素A对PGF2α所致心肌细胞肥人和CaN mRNA表达的影响。结果显示：MCT注射2周（M2W组）,右心室肥厚指数（RVHI）、右心室／体重比及肺重／体重比分别增加了47％、53％和118％;注射后4周（M4W组）增加了64％、94％及156％。电镜观察发现右心室组织损伤。同时,右心室组织PGF2α含量在M2W和M4W组分别增加了44％和51％,与RVHI、ANP和CaN的mRNA表达,及CaN／NFAT3／GATA4的蛋白质表达均呈正相关。环氧酶抑制剂塞来昔布预防和治疗给药均明显改善MCT诱导的组织病理学改变。在高体细胞培养中,PGF2α（0．1μmol／L）明显使心肌细胞增大,蛋白质含量增加,ANP和CaN mRNA表达增强：同时,CaN抑制剂环孢素A明显抑制PGF2α诱导的心肌细胞肥大和CaN mRNA表达。上述结果提示：心肌组织局部PGF2α参与了MCT诱导的心肌肥厚过程,CaN信号通路是其细胞内信号转导通路之一。