【作者】 李晓晖；

【导师】 李元建； Nicholas W.Morrell；

【作者基本信息】 中南大学 ， 药理学， 2011， 博士

【摘要】 肺动脉高压(Pulmonary Arterial Hypertension, PAH)是以肺动脉压力增高为特征的一类疾病,其发病机制至今仍不明了。已知血管重构是肺动脉高压的重要病理特征,其主要病理改变是血管平滑肌层增厚造成管腔狭窄、闭塞。肺血管重构可引起肺动脉压力升高,肺血管阻力增大而加重右心室后负荷,严重时导致右室肥厚、心力衰竭甚至死亡。延缓肺血管重构,改善心肺功能是药物治疗肺动脉高压,提高患者生存率的关键靶点。骨形成蛋白(Bone Morphogenetic Proteins, BMPs)信号通路与肺动脉高压的发病密切相关。骨形成蛋白Ⅱ型受体(BMPR-Ⅱ)基因突变是导致遗传性肺动脉高压的主要发病因素,也与特发性肺动脉高压患者有关。BMP信号通路失调被认为是肺动脉高压的重要发病原因。但是BMPR-Ⅱ基因敲除小鼠并不一定发生肺动脉高压,提示可能另有BMP信号通路下游效应蛋白在发病过程中起关键作用,或者存在第二重打击而触发PAH的发病过程。Id蛋白是BMP信号通路的主要下游蛋白,在组织发育,细胞分化,细胞周期调控等方面扮演十分重要的角色。本论文第一章将探讨BMPR-Ⅱ突变对Id蛋白的影响及其在调节肺动脉平滑肌细胞增殖功能中的作用。药物治疗是目前临床治疗肺动脉高压的主要手段,常用药物包括前列环素类药物,磷酸二酯酶5 (PDE5)抑制药,内皮素(ET)受体抑制药以及钙离子通道拮抗药(CCB)等几大类,主要通过抑制血管收缩,改善血管舒张功能,延缓血管重构和肌化等而发挥作用,但其疗效并不十分满意。本文在确证BMP信号通路在调节PASMCs增殖功能中起重要作用的基础上,将从在体和离体两个水平进一步研究BMP信号通路在肺动脉高压药物治疗中的作用并探讨其作为药物治疗靶点的研究前景。第一章Id蛋白介导BMP对肺动脉平滑肌细胞功能的调节第一节BMPR-Ⅱ介导BMP诱导的Id蛋白表达研究背景肺动脉高压发病机制至今仍不明了。血管重构被认为是肺动脉高压的重要病理特征,肺小血管肌化可引起肺血管阻力增加,肺动脉压力升高并最终导致失代偿性心衰甚至死亡。肺动脉平滑肌细胞(Pulmonary arterial smooth muscle cells, PASMCs)的过度增殖是引起血管重构的关键因素。越来越多的研究以肺血管重构机制为关注点,临床治疗也多以改善血管重构,延缓肺动脉高压病程,提高患者生存率为目的。骨形成蛋白(Bone morphogenetic proteins, BMPs)是转化生长因子(Transforming Growth Factor, TGF)超家族成员之一。BMP可结合位于细胞膜上的丝氨酸／络氨酸受体复合物(由BMPR-Ⅰ和BMPR-Ⅱ组成),进而磷酸化激活胞内Smad信号分子,Smads入核启动下游基因表达。研究发现,骨形成蛋白Ⅱ型受体(BMPR—Ⅱ)突变是导致遗传性肺动脉高压的主要发病因素,也发现于25%左右的特发性肺动脉高压患者。在低氧诱导和野百合碱注射诱导的肺动脉高压动物模型也发现BMPR-Ⅱ表达下调,BMP信号通路功能受损。因此,BMP信号通路被认为是PAH发生发展的重要调节通路。但是,BMPR-Ⅱ基因敲除小鼠并不一定发生肺动脉高压,提示BMPR-Ⅱ基因突变可能仅是肺动脉高压发病的遗传学基础或易感因素,另有BMP信号通路下游效应蛋白在发病过程中起关键作用,或者存在第二重打击而触发肺动脉高压的发病过程。Id蛋白是BMP信号通路主要的下游蛋白,包括Id1-Id4四种亚型,其表达与功能各有不同。Idl和Id3被认为主要与血管生理有关。我们最近研究发现,BMPR-Ⅱ基因缺失杂合型小鼠肺组织Idl蛋白表达水平低于野生型小鼠,BMP抑制肺动脉平滑肌细胞增殖可能与Idl有关,但是Id蛋白在BMP信号通路中的作用,BMPR-Ⅱ突变对Id蛋白的影响尚不明了。本研究主要以人肺动脉平滑肌细胞为研究对象,探讨BMPR-Ⅱ在介导BMP诱导四种Id蛋白亚型的表达与功能中的作用。另外基于BMPR-Ⅱ+/-小鼠与野生型小鼠在常氧和低氧状态下肺血管Id蛋白表达的差异,以及BMP信号通路激活十分迅速,Id蛋白上游Smad蛋白磷酸化在BMP刺激后半小时左右即可发生并达到高峰,我们也观察了BMP对Id蛋白调节的时间效应。方法(1)BMPR-Ⅱ+/-小鼠与野生型小鼠肺组织提取及免疫组织化学分析WKY雄性小鼠,分设4组(n=5)：野生型组(WT).BMPR-Ⅱ基因敲除组(BMPR-Ⅱ+/-).低氧野生型组(Hypoxia+WT).低氧BMPR-Ⅱ基因敲除组(Hypoxia+BMPR-Ⅱ+/-)。肺组织一部分行石蜡包埋,以备免疫组织化学检测,一部分采用磁珠分选法分离培养肺动脉平滑肌细胞。(2)人肺动脉平滑肌细胞培养,SiRNA干扰及RNA,蛋白检测人肺动脉平滑肌细胞来源于肺移植病人或肺切除术患者,之前已作BMPR-Ⅱ基因型分析。常规以含10%胎牛血清(FBS)的DMEM培基培养。不同因素处理后,提取细胞总RNA及蛋白进行相关检测。SiRNA直接购于或合成于公司。结果1.BMP可诱导人肺动脉平滑肌细胞四种Id蛋白的表达；2.SiRNA沉默BMPR-Ⅱ表达取消BMP诱导的Id蛋白表达；3.含BMPR-Ⅱ突变基因的人肺动脉平滑肌细胞Idl和Id3表达明显低于BMPR-Ⅱ基因野生型正常细胞；4.Id3蛋白主要表达于小鼠肺血管平滑肌层；常氧和低氧状态下,BMPR-Ⅱ+/-小鼠Id3表达都明显低于野生型小鼠；5.BMP诱导Idl和Id3表达具有短期与长期两次效应；结论BMPR-Ⅱ基因缺失抑制了人肺动脉平滑肌细胞Id蛋白的表达,BMPR-Ⅱ介导BMP诱导的Id蛋白表达；BMP诱导Idl和Id3蛋白表达具有早期和长期效应。第二节Id蛋白介导BMP对PASMCs增殖和细胞周期相关蛋白的调节研究背景Id蛋白是一类转录因子,属于茎-环-茎(Helix-Loop-Helix, HLH)大类。Id蛋白广泛表达于人体各个组织,在胚胎发育,血管新生,细胞分化,细胞周期调控等多种事件中扮演重要角色。Id基因还被认为是促癌基因,是决定细胞命运的关键因子。研究发现,Id蛋白在增殖细胞普遍高水平表达,在非增殖细胞、沉默细胞表达量很低甚至缺失,提示Id蛋白参与细胞增殖功能的调节。研究表明,Id蛋白调控细胞增殖主要与其调节细胞周期有关,调节点位于G1期入S期的时相阶段。细胞周期依赖性激酶(cyclin dependent kinase, CDK)的抑制蛋白如p21和p27等是Id蛋白的主要调控对象。肺动脉平滑肌细胞增殖也受BMP信号通路的调控,但是具体机制不明,Id蛋白是否介导这一调节,其作用是否与调控细胞周期有关等问题都有待解决。本研究着眼于肺动脉平滑肌细胞增殖功能的调控,探讨BMP/Id信号通路在调节PASMCs增殖和细胞周期相关蛋白表达的作用。方法人PASMCs常规培养,观察BMP对细胞增殖的影响。同时观察BMP对细胞周期相关调节蛋白如p21, p27, pRb的作用。之后以SiRNA干扰沉默Idl和Id3蛋白表达,探讨Id蛋白在调节细胞周期、PASMCs增殖中的作用。结果1.细胞计数显示,BMP4抑制野生型肺动脉平滑肌细胞的增殖；2. BMP4对BMPR-Ⅱ基因突变细胞增殖无明显影响；3.Westernblot实验显示,BMP4对PASMCs中P21蛋白表达的调节在野生型细胞和BMPR-II基因突变细胞存在差异；4.SiRNA干扰有效抑制Id1和Id3表达；5.Id1和Id3介导了BMP4对细胞周期相关蛋白P21和pRb蛋白的作用；结论Id1和Id3蛋白对细胞周期相关蛋白的调控可能介导了BMP4对肺动脉平滑肌细胞增殖的抑制作用。第二章以BMP信号通路为靶点的药物治疗机制研究第一节曲前列素治疗肺动脉高压与调节BMP信号通路有关研究背景肺动脉高压患者死亡率高,生存率低,至今仍无特效治疗方案。除了晚期进行肺移植手术之外,药物治疗仍然是绝大部分肺动脉高压患者的主要治疗手段。常用药物包括前列环素类药物,磷酸二酯酶5(Phosphodiesterase 5, PDE5)抑制药,内皮素(Endothelin, ET)受体抑制药以及钙离子通道拮抗药(Calcium channel block, CCB)等几大类,主要发挥抑制血管收缩、改善血管舒张功能、延缓血管重构和肌化等药理作用。前列环素类药物如曲前列素能在体内激活腺苷酸环化酶(Adenyl cyclase, AC),促进环磷酸腺苷(Cyclic Adenosine monophosphate, cAMP)的生成,达到舒张血管的作用。研究发现,前列环素类药物还具有改善肺血管重构的作用,但是其作用机制尚不明确。TGF信号通路在肺动脉高压发病过程中的作用至今仍有争议,一般认为其与BMIP信号通路作用相反,是维持血管稳态平衡的两个平衡端。本研究在以野百合碱诱导的肺动脉高压大鼠模型,给予曲前列素治疗,观察其对肺动脉压力、右心室功能、肺小血管肌化及重构的影响。基于第一章研究发现BMP/Id信号通路在调节PASMCs增殖中的作用,本节也将进一步探讨曲前列素对BMP/Id信号通路及TGF信号通路的影响及其意义。方法(1)野百合碱诱导肺动脉高压大鼠模型及药物治疗：雄性SD品系野生型大鼠,分为三个组别：对照组,野百合碱+治疗空白对照组,野百合碱+曲前列素治疗组。给予单次皮下细注射野百合碱(monocrotanine, MCT 60mg/kg)诱导肺动脉高压模型。野百合碱注射三周后,背部皮下植入渗透性微泵注射曲前列素治疗2周。药物浓度0.15mg/ml,注射速度为45ng/kg/min。对照组给予等量等张生理盐水。微泵植入时以芬太尼(300ug/kg)与曼陀罗碱(300ug/kg)麻醉大鼠,术后30分钟动物复苏。(2)动物手术及组织提取：实验终点时以戊巴比妥麻醉大鼠,行气管切开呼吸机维持呼吸。左颈动脉插管监测系统血压,右心导管由右颈静脉插入,监测右心功能、肺血管阻力等指标。动物放血后,左肺存于10%福尔马林以备组织学实验,右肺速冻于液氮以备RNA、蛋白提取,心脏分离出右心室、左心室加室间隔称重计算RV/(LV+S)比值。结果1.野百合碱诱导的大鼠肺动脉压力、右室收缩压和肺血管阻力明显增高,血管重构明显,肺小血管肌化比例高,右心室显著肥厚。曲前列素治疗降低肺血管阻力、右室收缩压,改善血管重构,肺小血管肌化及右心室肥厚。2.免疫组织化学分析显示,野百合碱诱导的大鼠肺组织Id1表达下调,增殖细胞增多。曲前列素治疗升高了Id1的表达,增殖细胞数目明显减少。3. Westernblot和Real-time PCR实验显示,野百合碱诱导的大鼠肺组织BMPR-II蛋白表达明显减少,Smad1/5磷酸化水平降低,Id1mRNA水平显著下调。曲前列素不能恢复BMPR-II的表达,但是升高了Smad1/5磷酸化水平及Id1mRNA水平的表达。4. Westernblot和Real-time PCR实验显示,野百合碱诱导的大鼠肺组织Smad3磷酸化水平升高,PAI-1mRNA水平上调。曲前列素治疗降低了Smad3磷酸化水平,抑制了PAI-1mRNA水平的表达。结论曲前列素治疗肺动脉高压,改善肺血管重构可能与其抑制肺动脉平滑肌细胞TGFβ信号通路,激活BMP信号通路有关。BMP信号通路可能是PPH治疗的重要靶点。第二节西地那非抑制PASMCs增殖及其对BMP信号通路的调节研究背景BMP信号通路的调节是PAH研究关注的热点,但至今所知甚少。新近发现cGMP依赖性蛋白激酶I(cGMP dependent protein kinase I,cGKI)是调节BMP信号通路的重要蛋白,cGKI可直接结合于BMPR-II,促进受体配体结合,之后还可以与Smad结合,促进其磷酸化并一同入核调节下游基因表达。cGKI在体内主要由环磷酸鸟苷(cyclic guanylic acid, cGMP)激活。NO激活鸟苷酸环化酶(guanylate cyclase, GC)可生成cGMP,其降解则主要通过磷酸二酯酶水解代谢。磷酸二酯酶抑制药如西地那非(Sildeanfil)已广泛用于肺动脉高压治疗,其药理机制在于抑制cGMP的降解而升高其体内浓度,达到舒张血管、增加血流的目的。基于新近发现cGKI是调节BMP信号通路的重要蛋白,可直接结合并调节BMP信号通路的各个阶段。本节拟研究西地那非对PASMCs细胞增殖的作用,探讨其作用是否与升高cGMP生成,激活cGKI,进而调节BMP信号通路有关。目的在于揭示BMP信号通路在PAH药物治疗中的作用,前瞻性探讨BMP信号通路作为新的PAH药物治疗靶点的可行性。方法(1)观察西地那非对人肺动脉平滑肌细胞增殖的影响：人肺动脉平滑肌细胞计数实验分为5组(n=3)：无血清饥饿对照组：0.1%FBS的DMEM培基；常规培养对照组：10%FBS的DMEM培基；BMP4处理组：lOng/ml浓度的BMP4；西地那非处理组：1μM浓度西地那非；BMP4合用西地那非处理组：10ng/ml浓度的BMP4合用1μM浓度西地那非。细胞按20000/well密度接种于24孔培养板,分别在处理后第1,4,6天计数。培基隔天更换。(2)观察西地那非对人肺动脉平滑肌细胞BMP信号通路的作用：西地那非(1-31μM)单独使用或与BMP4合用孵育人肺动脉平滑肌细胞,采用Real-time PCR和Westernblot等技术检测细胞BMP信号通路中各蛋白的表达水平。(3)观察cGMP/cGKI在介导西地那非抑制肺动脉平滑肌细胞增殖、调节BMP信号通路中作用：以cGKI抑制剂Rp-8-Br-PET-cGMPs和SiRNA分别抑制、干扰cGKI的表达和功能,观察cGKI被抑制前后西地那非对PASMCs增殖及BMP信号通路的影响。结果1.西地那非抑制PASMCs增殖,合用BMP4可进一步增强BMP4对PASMCs增殖的抑制作用；2.合用西地那非增强BMP4激活的BMP信号下游通路；3.合用cGMP内源性拟似物8-br-cGMP增强BMP4激活的BMP信号通路下游通路；4.使用SiRNA干扰沉默cGKI表达后,BMP4合用西地那非增强PASMCs中BMP信号通路的作用减弱；5.使用cGKI抑制物Rp-8-Br-PET-cGMPs后,BMP4合用西地那非增强PASMCs中BMP信号通路的作用减弱；结论西地那非抑制人肺动脉平滑肌细胞增殖,增强BMP4诱导的BMP信号通路与激活cGKI有关。cGKI可能是调节BMP信号的重要靶点。更多还原

【Abstract】 Pulmonary arterial hypertension (PAH) is characterized by elevated pulmonary arterial pressure. Vascular remodeling is the common response in various classes of PAH and proliferation of pulmonary arterial smooth muscle cells (PASMCs) contributes to vascular remodeling mostly. Persistent high pulmonary arterial pressure leads to increased vascular resistence, right ventricle hypertrophy, heart failure and even death. There is a growing interest in identifying the signaling mechanisms underlying the development of vascular remodeling and the transition to heart failure.Bone morphogenetic protein (BMP) signaling plays a critical role in PAH. BMP receptor II (BMPR-II) is responsible to majority of herediatal PAH and some cases of idopathic PAH. Dysfunction of BMP signaling has been thought as an important reason of PAH. Id proteins are major downstream factors of BMP signaling and play a key role during several biological processes such as development, cell differention and cell cycle control. We will first determine the Id proteins’function in regulating VSMC proliferation underlying BMPR-II mutation.Drugs are the first choice in PAH therapy. Commonly used drugs, including phosphodiesterase 5 inhibitors, endothelin receptor inhibitors and calcium channel antagonist and other broad categories, mainly play a role through inhibition of vascular contraction, improvement of vasodilation, inhibition of vascular remodeling and so on. But the efficacy is not satisfactory. Searching for special therapeutic target is still the important mission.Based on our discovery that BMP/Id signaling is crucial in regulating VSMC proliferation, we will continue to investigate the effect of Treprostinil and Sildenafil on BMP/Id signaling and discuss the potential that treat BMP/Id signaling as a new therapeutic target from in vivo and in vitro levels.Chapter I Id proteins are involved in regulation of pulmonary arterial SMC proliferation by BMPsPart One BMPs induce Id proteins expression via BMPR-IIBACKGROUNDPathological mechanism of PAH is still unknown. Vascular remodeling is thought a very important character of PAH. Persistent high pulmonary arterial pressure leads to increased vascular resistence, right ventricle hypertrophy, heart failure and even death. There is a growing interest in identifying the signaling mechanisms underlying the development of vascular remodeling. BMPs belong to TGF family. BMP signaling has been recognized to play the critical role during development of PAH. The BMPs activate signaling by binding to serine/threonine protein kinases receptor complex (type I and type II) on the cell membrane. First, BMPs bind to type II receptor, which also activate type II receptor by phosphorylation. This binding triggers a receptor complex formation and phosphorylation of type I receptor, which further phosphorylate regulatory Smad proteins (usually Smad1,5 and 8). Activated R-Smads then can traslacate into the nucleus together with co-mediator Smad4 and regulates downstream genes transcription. BMP receptor II (BMPR-II) is responsible to majority of herediatal PAH and about 25% cases of idopathic PAH. Dysfunction of BMP signaling has also been found in experimental PAH. But the fact that BMPR-II+/- mice does not spontaneously develop PAH reveals that other second hits or further dysfunction of BMP signaling is necessary to accelerate initiation and progress of PAH.Id proteins are transcription factors and belong to helix-loop-helix class. Id proteisn are widely expressed and play a key role in embryo development, angiogenesis, cell differentiation and cell cycle refulation. There are four Id proteisn, Idl,2,3 and Id4. Each Id protein has individual expression pattern and function. Id4 expression is much less than other three Ids. Id2 is closely related to immunity while Idl and Id3 are mainly involved in vessel physiology. It is reported that Id proteins regulate cell proliferation through cell cycle control. The regulation site is at the stage from G1 phase to S phase. Cyclin dependent kinase (CDK) inhibitors such as p21 and p27 are major targets of Id proteins.Id proteins are major downstream proteins of BMP signaling. Recently we find Idprotein expression is downregulated in BMPR-Ⅱ+/-mouse than in wildtype mouse, and it is involved in regulation of proliferation of PASMCs by BMPs. The present study investigates the effect on Id proteins expression and function underlying BMPR-II mutant, reveals the role of Id proteins in regulation of proliferation of PASMCs. In conclusion, our research reveals that Id proteins are critical downstream effectors of BMP signaling in PASMCs proliferation regulation.METHODS(1) Animal experiment on BMPR-II+/- mice and wildtype mice:Sex (male)-matched C57BL6 mice were divided into 4 groups (n=5):wild type group (WT), BMPR-Ⅱknock out group(BMPR-Ⅱ+/-), Wild type under hypoxia group (hypoxia+WT), and BMPR-Ⅱknock out under hypoxia group (hypoxia+BMPR-Ⅱ+/-). Lung tissues were saved for immunohistochemistry analysis and PASMCs extraction.(2) hPASMCs incubation, SiRNA knock down, RNA and protein analyses: Human pulmonary arterial smooth muscle cells (hPASMCs) were from patients who received graft operation. BMPR-II phenotypes were detected in all cell linages. Cells were incubated in DMEM medium with 10% FBS. SiRNA were purchased or produced from company.RESULTS1. Id protein induction by BMPs was BMPR-II dependent in PASMCs, while Id1 and Id3 showed the strongest responces;2. Id1 and Id3 protein expression were lower in PASMCs harboring BMPR-II mutants before and after BMPs incubation;3. Id proteins located at smooth muscle layer of pulmonary vessel in mice;4. Idl and Id3 expression were lower in BMPR-Ⅱ+/- mice than in wild type mice under nomoxia and hypoxia condition.;5. An early and late increase of Id1 and Id3 after BMPs incubation.CONCLUSIONId protein induction by BMPs was BMPR-II dependent in PASMCs. BMPs induce an early and late increase of Id1 and Id3 proteins. Part Two Rolse of Id proteins in regulation of proliferation and cell cycle in PASMCs by BMPsBACKGROUNDBMPs are important regulators of cell proliferation. Eighteen members of BMP family have been identified and their binding styles with receptors vary. Except BMPR-II, ActR-II was another type II receptor. Type I receptors included ALK2, ALK3 and ALK6 and so on. It has been recognized that id proteins are crucial factors on cell fate decision and closely related cell differentiation and proliferation. Generally, Id proteins highly expressed in proliferating cells while it was less or even absent in non-proliferating cells. It is reported that Id proteins regulate cell proliferation through cell cycle control. The regulation site is at the stage from G1 phase to S phase. Id proteins regulate cell cycle through two pathways. First way is to regulate CDK activity via inhibition of CDKI like p16, p21 and p27. Another one is to directly binding to pRb protein and diassociating E2F, then stimulate cell into S phase.It is unclear that what is the individual effect on PASMCs proliferation regulation by BMPs, and also the difference between four Id proteins is still need to claim. Here we will investigate the role of BMP/Id signaling in regulation of cell cycle and the implication for PASMCs proliferation.METHODSPulmonary arterial smooth muscle cells were cultured in DMEM medium with 10% FBS. Cell counting at day1,4 and day6 were used to determine the effect on proliferation by BMPs and serum starvation. Meanwhile, we also detect the expression of p21, p27 and pRb by BMPs. SiRNA of Id proteins were used to reveal the role Id proteins during regulation of CDKI and pRb by BMPs.RESULTS1. BMP4 inhibited BMPR-Ⅱwildtype PASMCs proliferation;2. BMP4 had no effect on PASMCs harboring BMPR-II mutants;3. BMP4 induced a different expression pattern of P21 in BMPR-II wildtype and BMPR-Ⅱmutants PASMCs;4. SiRNA silenced Idl and Id3 expression efficiently;5. Id1 and Id3 proteins were involved in regulation of P21 and pRb proteins by BMP4 in PASMCs. CONCLUSIONBMP4 inhibited PASMCs proliferation through regulating cell cycle related proteins. Idl and Id3 proteins played critical role during this processes.Chapter II Mechanism research of drug therapy targeted to BMP signalingPart One BMP signaling is involved in PAH therapy with treprostinilBACKGROUNDHigh mortality and Low survival rate exist in patients with PAH. There is still no specific treatment program. In addition to symptomatic treatment in clinic to ease the patients breathing, heart function and to relieve symptoms and improve quality of life purposes, the drug therapy is the primary means of treatment of PAH. The commonly used drugs include prostacyclin analogues, phosphodiesterase 5 (PDE5) inhibitors, endothelin receptor inhibitors and calcium channel blockers and so on. All these durgs are mainly used to controll blood pressure, improve blood vessel dilation, and delay vascular remodeling. Prostacyclin analogues such as Treprostinil can activate in vivo adenylate cyclase (AC), upregulate cyclic adenosine monophosphate (cAMP) generation, and finally achieve relaxation of blood vessels.Several studies found that prostacyclin analogues may also have ease of vascular remodeling, but its mechanism remains unclear. In this study, treprostinil treatment was given to the monocrotaline-induced PAH rats. We observed its effect on pulmonary artery pressure, right ventricular function, vascular remodeling and muscularization. Based on the discovery that BMP/Id signaling pathway played a role in the regulation of pulmonary artery smooth muscle cell proliferation, we also investigated the role of treprostinil regulation of the BMP/Id signaling pathway and its significance. Our Research suggests that the effect of treprostinil on regulating pulmonary artery pressure and pulmonary vascular remodeling may be related to the activation of BMP signaling pathway, while inhibition of TGFβsignaling pathwayMETHODS(1) Monocrotaline-induced PAH rats and treprostinil treatmentMale Sprague-Dawley rats (Charles River, Sulzfeld, Germany) either received a single injection of monocrotaline (MCT,60 mg/kg, s.c.) or saline. Three weeks after MCT injection, osmotic minipumps containing treprostinil (0.15 mg/mL; to deliver an approximate rate of 45 ng/kg/min) or saline vehicle were implanted for a further 2 weeks. Six animals were used per group.(2) Surgen and tissue preparementAt the end of the treatment protocol, rats were anesthetized for hemodynamic assessment and then euthanized for measurement of right ventricular hypertrophy and lung tissue collection. Slides of lung tissue were stained by hematoxylin and eosin techniques and examined by light microscopy. Slides were analyzed in a blinded fashion without knowledge of treatment groups. In each rat,30 to 40 intra-acinar arteries were categorized as muscular, partially muscular, or nonmuscular. All experiments were performed according to institutional guidelines that complied with national and international regulations.RESULTS1. In monocrotaline-induced PAH rats, pulmonary artery pressure, right ventricular systolic pressure and pulmonary vascular resistance increased significantly, significant vascular remodeling and right ventricular hypertrophy were observed in PAH rats. Treprostinil therapy reduced pulmonary vascular resistance and right ventricular systolic pressure, also improved vascular remodeling and muscularization and right ventricular hypertrophy.2. Immunohistochemical analysis showed that in monocrotaline-induced PAH rats, phosphorylation of Smad1/5, Id1 expression decreased significantly, while the proliferation of cells increased. Treprostinil treatment increased the Smadl/5 phosphorylation and Id1 expression, significantly reduced the number of proliferating cells.3. Westernblot experiments showed that in monocrotaline-induced PAH rats, BMPR-II protein expression and Smadl/5 phosphorylation were significantly reduced. Treprostinil treatment did not restore the expression of BMPR-II, but increased Smadl/5 phosphorylation and Idl expression.4. Westernblot experiments showed in monocrotaline-induced PAH rats, Smad3 phosphorylation level reduced while PAI-1 expression was upregulated. Treprostinil treatment reduced Smad3 phosphorylation and inhibited the expression of PAI-1.CONCLUSIONTreprostinil treatment inhibits vascular remodeling and PAH through restoring BMP signaling and inhibition of TGFβsignaling in pulmonary arterial smooth muscle cells. BMP signaling may be an important therapeutic target for the treatment of PAH. Part Two Regulation of BMP signaling by Sildenafil in PASMCsBACKGROUNDRegulation of BMP signaling pathway has been focused, but so far little is known. A recent study found that cGMP-dependent kinase I (cGKI) is an important regulator of BMP signaling pathway. cGKI can directly bind to BMPR-II, promote the receptor-ligand association. It also can combine with the Smad and promote its phosphorylation and then together transmit into the nucleus and trigger downstream gene expression. cGKI is mainly activated by cGMP in vivo. Nitic oxide (NO) can activate guanylate cyclase (GC) and increase production of cGMP, while cGMP is mainly degradated by phosphodiesterase (PDE).PDE inhibitors such as Sildenafil has been widely used for treatment of PAH, its pharmacological mechanism is based on the inhibition of degradation and increased concentration of cGMP, and fially achieve the purpose of relaxing blood vessels. As the regulation of BMP signaling by cGKI, we explored the effect and significance on BMP/Id signaling regulation by phosphodiesterase inhibitor Sildenafil in this section. We found that Sildenafil inhibited the proliferation of human pulmonary artery smooth muscle cells, accompany with increased cGMP production, activation of cGKI, thereby regulation of the BMP signaling pathway. Our research suggested that BMP signaling pathway may be important targets for drug therapy.METHODS(1) Effect on PASMCs proliferation by Sildenafil:Human PASMCs were divided into 5 groups (n=3):Serum starvation control group:DMEM medium with 0.1% FBS, Normal control group:DMEM medium with 10% FBS, BMP4 incubation group:BMP4 concentration 10ng/ml, Sildenafil incubation group: Sildenafil concentration 1μM, BMP4 plus Sildenafil incubation group. Cells were plated at density 20000/well in 24 wells plate. Cell counting were carried out at day1,4 and day6. All medium was changed every two days.(2) Effect on BMP signaling by Sildenafil in PASMCs:Sildanfil (1-3μM) was used with or without BMP4 (lOng/ml) to incubate PASMCs. Related proteins in BMP signaling pathway were detected using Real-time PCR or Westernblot techniques.(3) The role of cGMP/cGKI in regulation of PASMCs proliferation and BMP siganlin by Sildenafil:cGKI inhibitor Rp-8-Br-PET-cGMPs and SiRNA were used to knoct down the expression or activity of cGKI. The effect on PASMCs proliferation and BMP signaling by Sildenafil were determined befor and after cGKI inhibition. RESULTS1. Sildenafil inhibited PASMCs proliferation and enhanced inhibition of PASMCs proliferation by BMP4;2. Sildenafil enhanced activation of BMP downstream signaling by BMP4 in PASMCs;3. cGMP analogue 8-br-cGMP enhanced activation of BMP signaling by BMP4 in PASMCs;4. The enhanced activation of BMP signaling by Sildenafil and BMP4 was inhibited by cGKI inhibitor Rp-8-Br-PET-cGMPs and SiRNA;5. The enhanced activation of BMP signaling by Sildenafil and BMP4 was inhibited by cGKI knock down using SiRNA。CONCLUSIONSildenafil regulated BMP signaling and inhibited proliferation in PASMCs through activation of cGMP/cGKI pathway. cGKI may be an important target during BMP signaling regulation.更多还原