Cell Tracking Suggests Pathophysiological and Therapeutic Role of Bone Marrow Cells in Sugen5416/Hypoxia Rat Model of Pulmonary Arterial Hypertension

BackgroundThe mechanism of vascular remodelling in pulmonary arterial hypertension (PAH) remains unclear. Hence, defining the origin of cells constituting intractable vascular lesions in PAH is expected to facilitate therapeutic progress. Herein, we aimed to evaluate the origin of intractable vascular lesions in PAH rodent models via bone marrow (BM) and orthotopic lung transplantation (LT).MethodsTo trace BM-derived cells, we prepared chimeric rats transplanted with BM cells from green fluorescent protein (GFP) transgenic rats. Male rats were transplanted with lungs obtained from female rats and vice versa. Pulmonary hypertension was induced in the transplanted rats via Sugen5416 treatment and subsequent chronic hypoxia (Su/Hx).ResultsIn the chimeric Su/Hx models, GFP-positive cells were observed in the pulmonary vascular area. Moreover, the right ventricular systolic pressure was significantly lower compared with wild-type Su/Hx rats without BM transplantation (P = 0.009). PAH suppression was also observed in rats that received allograft transplanted BM transplantation. In male rats that received LT and Su/Hx, BM-derived cells carrying the Y chromosome were also detected in neointimal occlusive lesions of the transplanted lungs received from female rats.ConclusionsBM-derived cells participate in pulmonary vascular remodelling in the Su/Hx rat model, whereas BM transplantation may contribute to suppression of development of PAH.     

Inhaled Rho kinase inhibitors are potent and selective vasodilators in rat pulmonary hypertension

We have found in chronically hypoxic rats that acute intravenous administration of the Rho kinase inhibitor Y-27632 nearly normalizes the pulmonary hypertension (PH) but has no pulmonary vascular selectivity. In this study, we tested if oral or inhaled Y-27632 would be an effective and selective pulmonary vasodilator in hypoxic PH. Although acute oral Y-27632 caused a marked and sustained decrease in mean pulmonary arterial pressure (MPAP), it also decreased mean systemic arterial pressure (MSAP). In contrast, 5 minutes of inhaled Y-27632 decreased MPAP without reducing MSAP. The hypotensive effect of inhaled Y-27632 on hypoxic PH was greater than that of inhaled nitric oxide, and the effect lasted for at least 5 hours. Inhaled fasudil, another Rho kinase inhibitor, caused selective MPAP reductions in monocrotaline-induced PH and in spontaneous PH in fawn-hooded rats, as well as in chronically hypoxic rats. These results suggested that inhaled Y-27632 was more effective than inhaled nitric oxide as a selective pulmonary vasodilator in hypoxic PH, and that Rho kinase-mediated vasoconstriction was also involved in the other models of PH. Inhaled Rho kinase inhibitors might be useful for acute vasodilator testing in patients with PH, and future work should evaluate their efficacy in the long-term treatment of PH.     

The effects of antiangiogenic compound SU5416 in a rat model of pulmonary arterial hypertension

Several lines of evidence indicate that vascular endothelial growth factor (VEGF) plays a prosurvival and antiapoptotic role in endothelial cells. SU5416 is the first VEGF receptor 2 inhibitor to enter clinical development for cancer therapy. A phase I/II study of SU5416 has been completed, and the results show that SU5416 is well tolerated in patients with terminal cancers. It has been shown that VEGF receptor blockade using SU5416 combined with chronic hypoxia results in severe angioproliferative pulmonary hypertension (PAH) with neointimal changes in adult rats. Although classic animal models of pulmonary hypertension (that is, the monocrotaline and hypoxic models) do not form obstructive intimal lesions in the peripheral pulmonary arteries, the SU5416 model has shown pulmonary arterial changes resembling plexiform lesions. Therefore, the SU5416 model of PAH has been used for some time, and it has thus contributed to a better understanding of the pulmonary hypertensive process. However, the mechanism by which SU5416 combined with chronic hypoxia can result in PAH with plexiform-like lesions in adult rats is complex and still remains to be fully elucidated. The most likely explanation is that there is increased apoptosis of endothelial cells in response to the loss of the survival signaling, creating conditions favoring the emergence of apoptosis-resistant cells with increased growth potential, that is, the endothelial cell hyperproliferation that might characterize the plexiform lesions of human PAH. The aim of the present review is to provide information useful for understanding a potent inhibitor of VEGF receptor tyrosine kinase, SU5416, and to better understand its use for generating animal models of PAH.     

Development and characterization of an animal model of severe pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a serious pathological phenomenon with poor prognosis, which is associated with morphological as well as hemodynamic alteration of the pulmonary circulation. To establish an animal model mimicking severe human PAH, we combined 2 well-described procedures, i.e. exposure to hypobaric chronic hypoxia and administration of monocrotaline hydrochloride in rats. Compared to a single procedure, the combined procedure induced more severe right ventricle hypertrophy and an increase in right ventricle systolic pressure. Histological examination on the combined procedure model revealed a severe medial hypertrophy as well as occlusive vascular changes of the intra-acinar pulmonary arteries with endothelial lesions. It is noteworthy that severe alterations including concentric neointimal thickening, abnormal endothelial proliferation, plexiform lesions and vascular occlusion with fibrin thrombi were observed in the combined pulmonary hypertension model when exposed to a long period of hypoxia. The present data indicate that a combined treatment of monocrotaline injection and hypobaric chronic hypoxia exposure produces more severe hemodynamic changes and histological alterations. Since human PAH diagnosed in clinical practice is often severe, this combined treatment animal model could be useful to identify relevant therapeutic targets acting on both hemodynamic and structural alterations of the pulmonary circulation.     

p27Kip1在大鼠动脉粥样硬化斑块中的表达及法舒地尔的干预作用

目的 观察p27Kip1在大鼠动脉粥样硬化斑块中的表达及盐酸法舒地尔的干预作用.方法 将30只健康雄性Wistar大鼠随机分为三组:正常对照组、动脉粥样硬化组和法舒地尔组.正常对照组大鼠行假球囊损伤手术后给予正常饮食,动脉粥样硬化组和法舒地尔组大鼠给予维生素D3右下肢肌肉注射后用球囊行动脉拉伤,在基础饲料中加入胆固醇、胆酸钠、丙基硫氧嘧啶、维生素D3粉剂、猪油等饲养,法舒地尔组同时给予法舒地尔腹腔注射.喂养9周后抽血并处死,自主动脉弓下约1 cm处留取动脉组织行HE染色,用免疫组织化学法检测主动脉壁中Rho激酶和p27Kip1蛋白的表达.结果 HE染色显示,动脉粥样硬化组均形成了典型的粥样斑块;免疫组织化学检测发现,Rho激酶在正常的血管壁即有表达,在动脉粥样硬化组的表达明显高于正常对照组和法舒地尔组(P<0.01),在法舒地尔组的表达明显高于正常对照组(P<0.05).p27Kip1蛋白在正常血管壁表达较多,在动脉粥样硬化组的表达明显低于正常对照组和法舒地尔组(P<0.01),在法舒地尔组的表达明显低于正常对照组(P<0.05).结论 动脉粥样硬化病变时,粥样硬化灶内Rho激酶表达明显增加,p27Kip1蛋白的表达明显下调;法舒地尔明显抑制粥样硬化灶内的血管平滑肌细胞增殖,抑制Rho激酶的表达上调及p27Kip1蛋白的表达下调.     

Inhibition of Rho-kinase attenuates hypoxia-induced angiogenesis in the pulmonary circulation

Pulmonary hypertension (PH) is a common complication of chronic hypoxic lung diseases, which increase morbidity and mortality. Hypoxic PH has previously been attributed to structural changes in the pulmonary vasculature including narrowing of the vascular lumen and loss of vessels, which produce a fixed increase in resistance. Using quantitative stereology, we now show that chronic hypoxia caused PH and remodeling of the blood vessel walls in rats but that this remodeling did not lead to structural narrowing of the vascular lumen. Sustained inhibition of the RhoA/Rho-kinase pathway throughout the period of hypoxic exposure attenuated PH and prevented remodeling in intra-acinar vessels without enlarging the structurally determined lumen diameter. In chronically hypoxic lungs, acute Rho kinase inhibition markedly decreased PVR but did not alter the alveolar to arterial oxygen gap. In addition to increased vascular resistance, chronic hypoxia induced Rho kinase-dependent capillary angiogenesis. Thus, hypoxic PH was not caused by fixed structural changes in the vasculature but by sustained vasoconstriction, which was largely Rho kinase dependent. Importantly, this vasoconstriction had no role in ventilation-perfusion matching and optimization of gas exchange. Rho kinase also mediated hypoxia-induced capillary angiogenesis, a previously unrecognized but potentially important adaptive response.     

Vascular endothelial growth factor in diabetes induced early retinal abnormalities

Increased vascular permeability and blood flow alterations are characteristic features of diabetic retinal microangiopathy. The present study investigated vascular endothelial growth factor (VEGF) and its interactions with endothelin (ET) 1 and 3, endothelial, and inducible nitric oxide synthase (eNOS, iNOS) in mediating diabetes induced retinal vascular dysfunction. Male Sprague Dawley rats with streptozotocin (STZ) induced diabetes, with or without VEGF receptor signal inhibitor SU5416 treatment (high or low dose) were investigated after 4 weeks of follow-up. Colour Doppler ultrasound of the ophthalmic/central retinal artery, retinal tissue analysis with competitive RT-PCR and microvascular permeability were studied. Diabetes caused increased microvascular permeability along with increased VEGF mRNA expression. Increased vascular permeability was prevented by SU5416 treatment. Diabetic animals showed higher resistivity index (RI), indicative of vasoconstriction with increased ET-1 and ET-3 mRNA expression, whereas eNOS and iNOS mRNA expressions were un-affected. SU5416 treatment corrected increased RI via increased iNOS in spite of increased ET-1, ET-3 and VEGF mRNA expression. Cell culture (HUVEC) studies indicate that in part, an SU5416 induced iNOS upregulation may be mediated though a MAP kinase signalling pathway. The present data suggest VEGF is important in mediating both vasoconstriction and permeability in the retina in early diabetes.     

Effects of Oral Rho Kinase Inhibitor Fasudil on Detrusor Overactivity after Bladder Outlet Obstruction in Rats

Objectives: Bladder outlet obstruction (BOO)‐related detrusor hypertrophy is associated with upregulation of Rho‐kinase (ROCK) activity in an experimental animal model, and has been implicated in BOO‐induced bladder dysfunction. The aim of this study was to test whether chronic oral administration of an oral ROCK inhibitor, fasudil (HA1077, 5‐isoquinolinesulfonyl homopiperazine), could prevent the development of both detrusor hypertrophy and detrusor overactivity in rat model. Methods: Thirty five‐week‐old male Sprague‐Dawley rats were divided into three groups (n = 10 per group): control (sham surgical) with no treatment (group 1); 6‐week obstructed rats (group 2); and 6‐week obstructed rats treated for 6 weeks with fasudil (group 3). Results: The BOO group showed increased detrusor overactivity. Treatment with fasudil partly but significantly ameliorated the development of detrusor overactivity. The expression of RhoA protein in detrusor muscle was significantly greater in the BOO group than in the control group and subsequently decreased with fasudil treatment in the BOO‐induced rat. Conclusion: These findings suggest that fasudil, a specific inhibitor of Rho‐kinase, ameliorates BOO‐induced detrusor overactivity in a rat model. Thus, ROCK inhibitor might be used as a novel agent to treat overactive bladder symptoms.     

法舒地尔治疗先天性心脏病相关性重度肺动脉高压短期疗效观察

目的 探讨法舒地尔治疗先天性心脏病相关性重度肺动脉高压短期应用的有效性及安全性。方法 入选2011年1月2012年8月我科住院的先天性心脏病相关性重度肺动脉高压患者,法舒地尔60 mg静脉滴注,每天2次,连续用药14 d后观察6分钟步行距离、血流动力学参数、超声心动图指标以及血浆N末端脑钠尿肽前体(NTpro BNP)变化情况。结果 共纳入21(男4,女17)例患者,年龄16~54(32±14)岁;用药14 d后6分钟步行距离由(445±49)m显著增加至(471±40)m,差异有统计学意义;且WHO肺动脉高压心功能分级显著改善(P<0.05)。血浆NT-pro BNP亦明显下降,从(788±623)pg/ml降低至(464±393)pg/ml(P<0.05)。右房平均压从(6.1±2.3)mm Hg降低至(5.2±1.8)mm Hg(P<0.05);右向左分流量从(23±16)%明显减少至(18±16)%(P<0.05);而肺动脉平均压、肺血管阻力、体肺循环血流量比值、体肺循环阻力比值、股动脉氧饱和度、心脏指数等指标虽无显著统计学差异,但较用药前均有所改善,仅体循环压力及阻力均无统计学差异。结论 法舒地尔短期内应用可显著提高先天性心脏病相关重度肺动脉高压患者的运动耐量、WHO肺动脉高压心功能分级,有效改善肺动脉高压的血流动力学指标,而且安全性与耐受性良好。     

Attenuation of pulmonary hypertension secondary to left ventricular dysfunction in the rat by Rho‐kinase inhibitor fasudil

Pulmonary hypertension (PH) in left ventricular dysfunction is attributable not only to backward failure of the left ventricle, but also to increased pulmonary vascular resistance (PVR) in some patients. Recently, Rho‐kinase has been known as a potent growth stimulator and mediator of vasoconstriction, and Rho‐kinase inhibitors could ameliorate PVR, little is known about the role of Rho‐kinase in left ventricular dysfunction‐induced PH. We utilized the ascending aortic‐banded rat and assessed the effect of Rho‐kinase inhibitor fasudil on the development of PH secondary to left ventricular dysfunction. Subsequently, in rats subjected to aortic banding for 6 weeks, there were increases in mean pulmonary arterial pressure, pulmonary arteriolar medial thickness, active RhoA, Rho‐kinase II, Rho‐kinase activity, endothelial nitric oxide synthase (eNOS) and endothelin‐1(ET‐1) concomitant with decreased levels in NO and cGMP in the lung. Treatment with fasudil at a dose of 30 mg/kg/day from days 1 to 28 or from days 29 to 42 decreased the mean pulmonary arterial pressure by 57% and 56%, right ventricular hypertrophy by 31% and 30%, pulmonary arteriolar medial thickness by 50% and 50%, and pulmonary expression of Rho‐kinase II by 41% and 28%, respectively, as well as augmented pulmonary expression of eNOS by 16% and 31% and NO by 50% and 76%, respectively, when compared with the vehicle controls. In conclusion, these results suggest that inhibition of Rho‐kinase may provide therapeutic potential for preventing and attenuating the development of PH in left ventricular dysfunction. Further translational study in human is needed to substantiate the findings. Pediatr Pulmonol. 2011; 46:45–59. © 2010 Wiley‐Liss, Inc.     

Sildenafil prevents and reverses transverse-tubule remodeling and Ca(2+) handling dysfunction in right ventricle failure induced by pulmonary artery hypertension

Right ventricular (RV) failure (RVF) is the main cause of death in patients with pulmonary artery hypertension (PAH). Sildenafil, a phosphodiesterase type 5 inhibitor, was approved recently for treatment of PAH patients. However, the mechanisms underlying RV contractile malfunction and the benefits of sildenafil on RV function are not well understood. We aimed to investigate the following: (1) the ultrastructural and excitation-contraction coupling alterations underlying PAH-induced RVF; (2) whether the ultrastructural changes are reversible; and (3) the mechanisms underlying the therapeutic benefits of sildenafil in PAH-RVF. We used a single injection of monocrotaline in Wistar rats to induce pulmonary vascular proliferation, which led to PAH and RVF. RV myocytes displayed severe transverse (T)-tubule loss and disorganization, as well as blunted and dys-synchronous sarcoplasmic reticulum Ca(2+) release. Sildenafil prevented and reversed the monocrotaline-induced PAH and LV filling impairment. Early intervention with sildenafil prevented RV hypertrophy and the development of RVF, T-tubule remodeling, and Ca(2+) handling dysfunction. Although late treatment with sildenafil did not reverse RV hypertrophy in animals with established RVF, RV systolic function was improved. Furthermore, late intervention partially reversed both the impairment of myocyte T-tubule integrity and Ca(2+) handling protein and sarcoplasmic reticulum Ca(2+) release function in monocrotaline-treated rats. In conclusion, PAH-induced increase in RV afterload causes severe T-tubule remodeling and Ca(2+) handling dysfunction in RV myocytes, leading to RV contractile failure. Sildenafil prevents and partially reverses ultrastructural, molecular, and functional remodeling of failing RV myocytes. Reversal of pathological T-tubule remodeling, although incomplete, is achievable without the regression of RV hypertrophy.     

Pulmonary vascular reactivity in the spontaneously hypertensive rat.

We examined the pulmonary vascular reactivity of normotensive rats (NR) and spontaneously hypertensive rats (SHR) to acute and chronic pressor stimuli. In rats kept at low altitude (1,520 m), SHR had a slight degree of right ventricular hypertrophy, but there was no difference between SHR and NR in either right ventricular systolic pressure or pulmonary artery wall thickness. When compared to blood-perfused lungs from low altitude NR, lungs from low altitude SHR were normoresponsive to acute airway hypoxia, hyporesponsive to intra-arterial angiotensin II, and hyperresponsive to intra-arterial prostaglandin F2alpha. After exposing rats to simulated high altitude (4--6 weeks at 4,270 m) to induce hypoxic pulmonary hypertension, SHR had a higher right ventricular systolic pressure, a greater degree of right ventricular hypertrophy, and more pulmonary artery medial thickening than did NR. The results indicate that although the pulmonary vasculature of SHR does not become hypertensive spontaneously, it might have an increased tendency to develop hypertension when exposed to an appropriate stimulus, i.e., chronic airway hypoxia.     

Rho/Rho激酶在压力负荷心力衰竭大鼠心肌组织的表达

目的　探讨升主动脉缩窄压力超负荷心力衰竭 (心衰 )大鼠心肌组织Rho/Rho激酶的表达及Rho激酶抑制剂法舒地尔 (fasudil)对心衰的影响。方法　结扎大鼠升主动脉 ,同时制备假手术模型。 2 0周后成功建立慢性心衰模型。随机分为三组 ,每组 10只大鼠 :(1)假手术组 :生理盐水 0 1ml,腹腔注射 ,每日 2次 ;(2 )心衰组 :生理盐水 0 1ml,腹腔注射 ,每日 2次 ;(3)fasudil组 :fasudil 5mg/kg ,腹腔注射 ,每日 2次 ,疗程 4周。治疗前后检测各组大鼠血流动力学指标 ,疗程结束后处死大鼠 ,检测左室肥厚指数、心肌组织RhoA、Rho激酶mRNA表达及Ca2 + 浓度即 [Ca2 + ]i 的变化。结果心衰组与假手术组相比 ,左室舒张末压明显增高 ,而左室收缩压和左室压力变化最大上升和下降速率明显减低 ,P <0 0 1;左室肥厚指数明显增加 ,P <0 0 1;心肌组织RhoA、Rho激酶mRNA表达显著增高 ,P <0 0 1;心肌组织内 [Ca2 + ]i 显著增高 ,P <0 0 1。fasudil组与心衰组相比 ,左室舒张末压明显减低 ,左室收缩压和左室压力变化最大上升和下降速率明显增高 ,P <0 0 1;左室肥厚指数下降 ,P <0 0 1;心肌组织RhoA、Rho激酶mRNA表达明显下降 ,P <0 0 1;心肌细胞内 [Ca2 + ]i 变化无统计学意义 ,P >0 0 5。结论　心衰大鼠心肌组织RhoA、Rho激     

血管紧张素转化酶2激活预防肺动脉高压的实验研究

目的:探讨血管紧张素转化酶2(ACE2)激活,对肺叶切除联合野百合碱注射,诱导重度肺动脉高压(PAH)的预防作用。方法:左肺切除+野百合碱注射建立大鼠重度PAH模型。雄性SD大鼠50只,随机分为5组,A组为空白对照组、B组为Resorcinolnaphthalein(Res)对照组,C组为PAH组,D组为Res预防组,E组为Res+A-779干预组。测量平均肺动脉压力(mPAP)、右心室肥厚指数(RVHI);弹力纤维染色分析肺血管病变;ELISA、Western Blot分析肾素血管紧张素系统各主要成分的水平。结果:处理3 w后,C组大鼠mPAP、RVHI明显升高,出现了严重的肺动脉中膜肥厚、内膜增生(与A组比较,P<0.05),D组上述4项指标明显减轻(与C组比较,P<0.05);E组则比D组病变严重(P<0.05)。ACE、Ang-II及AT1R水平,D组明显低于C组(P<0.05),而ACE2、Ang-(1-7)及Mas水平D组明显高于C组(P<0.05)。结论:Res激活ACE2可以抑制重度肺动脉高压的形成,调节肾素血管紧张素系统的平衡,可能是其作用的主要机制之一。     

Effects of cigarette smoking on pulmonary vasoreactivity]

The effects of cigarette smoking on hemodynamics and hypoxic pulmonary vasoconstriction were studied in intact rats, in isolated rat lungs and in humans. The results show that smoking increases the vascular tone of pulmonary arterioles and venules, and increases their reactivity to alveolar hypoxia; leukotrienes, prostaglandins and sympathetic nerve mediate the smoking--induced pulmonary vasoconstriction; and the augmentation of pulmonary vasoreactivity to hypoxia is mainly mediated by leukotrienes and sympathetic nerve, possibly by prostaglandins as well in humans.     

Do statins reduce blood pressure?: a meta-analysis of randomized, controlled trials

The RhoA/Rho kinase (ROCK) pathway is a new mechanism of remodeling and vasoconstriction. Few data are available regarding ROCK activation when angiotensin I-converting enzyme is high and blood pressure is normal. We hypothesized that ROCK is activated in the vascular wall in normotensive rats with genetically high angiotensin I-converting enzyme levels, and it causes increased vascular expression of genes promoting vascular remodeling and also oxidative stress. Aortic ROCK activation, mRNA and protein levels (of monocyte chemoattractant protein-1, transforming growth factor [TGF]-beta(1), and plasminogen activator inhibitor-1 [PAI-1]), NADPH oxidase activity, and O(2)(*-) production were measured in normotensive rats with genetically high (Brown Norway [BN]) and low (Lewis) angiotensin-I-converting enzyme levels and in BN rats treated with the ROCK antagonist fasudil (100 mg/kg per day) for 7 days. ROCK activation was 12-fold higher in BN versus Lewis rats (P<0.05) and was reduced with fasudil by 100% (P<0.05). Aortic TGF-beta1, PAI-1, and monocyte chemoattractant protein-1 mRNA levels were higher in BN versus Lewis rats by 300%, 180%, and 1000%, respectively (P<0.05). Aortic TGF-beta1, PAI-1, and monocyte chemoattractant protein-1 protein levels were higher in BN versus Lewis rats (P<0,05). Fasudil reduced TGF-beta1 and PAI-1 mRNA and TGF-beta1, PAI-1, and monocyte chemoattractant protein-1 protein aortic levels to those observed in Lewis rats. Aortic reduced nicotinamide-adenine dinucleotide phosphate oxidase activity and (*)O(2)(-) production were increased by 88% and 300%, respectively, in BN rats (P<0.05) and normalized by fasudil. In conclusion, ROCK is significantly activated in the aortic wall in normotensive rats with genetically high angiotensin-I-converting enzyme and angiotensin II, and it causes activation of genes that promote vascular remodeling and also increases vascular oxidative stress.     

Effect of potassium channel openers on hypoxic pulmonary vasoconstriction]

The ATP-sensitive potassium channel (K+ATP) has been suggested as an important mechanism for the reactivity of vascular smooth muscle. We investigated the effects of K+ channel openers (lemakalim, pinacidil) on hypoxic pulmonary vasoconstriction (HPV) and angiotensin II (Ag II) induced vasoconstriction in isolated rat lungs (Sprague-Dawley rats: 300-450 g). Ventilation with hypoxic gas (2% O2, 5% CO2) was performed for 6 min after the injection of Ag II (0.1 microgram). Isolated lungs were perfused under constant flow (0.04 ml/g/min) using 20 ml of blood from donor rat. The perfusion pressure was used as the pulmonary artery pressure. Lemakalim or pinacidil was pre-administered through the reservoir. Pretreatment with pinacidil (10(-4) M) or lemakalim (10(-5) M) inhibited the pressor response to hypoxia, but did not inhibit the response to angiotensin II. Although the effect of lemakalim on HPV was reversed by administration of glibenclamide (10(-5) M) or tolbutamide (10(-3) M), the effect of pinacidil on HPV was not influenced by either drug. These results suggest that 1) K+ channel openers (lemakalim and pinacidil) inhibit the pressor response to hypoxia, and 2) lemakalim seems to act through K+ATP, whereas pinacidil may have other mechanisms of inhibition of vascular smooth muscle contraction. K+ATP may play an important role in the regulation of pulmonary vascular reactivity to hypoxia.     

Absence of T cells confers increased pulmonary arterial hypertension and vascular remodeling

RATIONALE: Severe pulmonary arterial hypertension (SPH) is a frequently lethal condition characterized by pulmonary vascular remodeling and right heart strain or failure. SPH is also often associated with autoimmune and collagen vascular disorders. OBJECTIVES: To study the effects of T cells on the development of experimental SPH. METHODS: Athymic nude rats lacking T cells were treated with a single subcutaneous injection of vascular endothelial growth factor (VEGF) receptor blocker SU5416 (20 mg/kg) to induce pulmonary vascular endothelial cell apoptosis. Immunohistochemical analysis and IL-4 levels of the lung tissue were performed. Cell death and proliferation were assessed by Western blot and immunohistochemistry. MEASUREMENTS AND MAIN RESULTS: In contrast to SU5416-treated euthymic rats that develop SPH only in combination with chronic hypoxia, athymic nude rats developed SPH and vascular remodeling (similar to clinical SPH) at normoxic conditions as demonstrated by measurements of pulmonary artery pressure and right ventricle hypertrophy. Pulmonary arterioles became occluded with proliferating endothelial cells and were surrounded by mast cells, B cells, and macrophages. IL-4, proliferating cell nuclear antigen, and collagen type I levels were markedly increased in SU5416-treated athymic rat lungs. Antibody deposition was noted along the vascular endothelium in rats with SPH. Finally, protection from SPH was conferred by immune challenge with spleen cells from euthymic nude rats. CONCLUSIONS: These studies demonstrate the importance of a complete, intact immune system in protecting against pulmonary angioproliferation in this new model of SPH as well as the importance of intact VEGF receptor signaling for lung endothelial cell homeostasis.     

Intermedin modulates hypoxic pulmonary vascular remodeling by inhibiting pulmonary artery smooth muscle cell proliferation

BackgroundHypoxic pulmonary arterial hypertension (PAH) is a disabling disease with limited treatment options. Hypoxic pulmonary vascular remodeling is a major cause of hypoxic PAH. Pharmacological agents that can inhibit the remodeling process may have great therapeutic value.ObjectiveTo examine the effect of intermedin (IMD), a new calcitonin gene-related peptide family of peptide, on hypoxic pulmonary vascular remodeling.MethodsRats were exposed to normoxia or hypoxia (∼10% O₂), or exposed to hypoxia and treated with IMD, administered by an implanted mini-osmotic pump (6.5 μg/rat/day), for 4 weeks. The effects of IMD infusion on the development of hypoxic PAH and right ventricle (RV) hypertrophy, on pulmonary vascular remodeling, on pulmonary artery smooth muscle cell (PASMC) proliferation and apoptosis, and on the activations of l-arginine nitric oxide (NO) pathway and endoplasmic reticulum stress apoptotic pathway were examined.ResultsRats exposed to hypoxia developed PAH and RV hypertrophy. IMD treatment alleviated PAH and prevented RV hypertrophy. IMD inhibited hypoxic pulmonary vascular remodeling as indicated by reduced wall thickness and increased lumen diameter of pulmonary arterioles, and decreased muscularization of distal pulmonary vasculature in hypoxia-exposed rats. IMD treatment inhibited PASMC proliferation and promoted PASMC apoptosis. IMD treatment increased tissue level of constitutive NO synthase activity and tissue NO content in lungs, and enhanced l-arginine uptake into pulmonary vascular tissues. IMD treatment increased cellular levels of glucose-regulated protein (GRP) 78 and GRP94, two major markers of endoplasmic reticulum (ER) stress, and increased caspase-12 expression, the ER stress-specific caspase, in lungs and cultured PASMCs.ConclusionsThese results demonstrate that IMD treatment attenuates hypoxic pulmonary vascular remodeling, and thereby hypoxic PAH mainly by inhibiting PASMC proliferation. Promotion of PASMC apoptosis may also contribute to the inhibitory effect of IMD. Activations l-arginine–NO pathway and of ER stress-specific apoptosis pathway could be the mechanisms mediating the anti-proliferative and pro-apoptotic effects of IMD.     

Thiazide-like diuretics attenuate agonist-induced vasoconstriction by calcium desensitization linked to Rho kinase

Lowering blood pressure using thiazide-like diuretics, including chlorthalidone and hydrochlorothiazide, has been proven to be effective in clinical studies. However, the mechanisms by which thiazide-like diuretics lower blood pressure are still poorly understood. To evaluate whether thiazide-like diuretics cause calcium desensitization in smooth muscle cells, we measured their effects on agonist-induced increase of blood pressure in Wistar rats in vivo and on agonist-induced vasoconstriction of aortic rings, DNA synthesis, and protein synthesis, RhoA, Rho kinase, and intracellular calcium in vascular smooth muscle cells in vitro. Thiazide-like diuretics significantly attenuated angiotensin II-induced or norepinephrine-induced increase of systolic blood pressure in rats. Thiazide-like diuretics inhibited agonist-induced vasoconstriction of aortic rings in a concentration-dependent manner in the presence and absence of endothelium. The inhibitory effects of thiazide-like diuretics were similar to that of the specific Rho kinase inhibitor Y27632. RT-PCR and immunoblotting showed that RhoA and Rho kinase were significantly reduced in vascular smooth muscle cells after administration of thiazide-like diuretics. In contrast, thiazide-like diuretics did not affect protein tyrosine phosphatase-2 (SHP-2) expression. Agonist-induced changes of intracellular calcium were not affected by thiazide-like diuretics. The study indicates that thiazide-like diuretics inhibit agonist-induced vasoconstriction by calcium desensitization in smooth muscle cells linked to the Rho-Rho kinase pathway.