The Role And Related Molecular Mechanisms Of BMP Signaling About Vascular Remodeling In Pulmonary Arterial Hypertension

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-?+/-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-?knock out group(BMPR-?+/-), Wild type under hypoxia group (hypoxia+WT), and BMPR-?knock out under hypoxia group (hypoxia+BMPR-?+/-). 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-?+/- 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-?wildtype 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-?mutants 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 TGF?signaling 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 TGF?signaling 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 1?M, 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 (1-3?M) 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.