Mechanisms Of Protein Kinase D In The Hypertensive Aortic Remodeling

BackgroundHypertension is the most important risk factor for cardiovascular diseases. Vascular remodeling (VR) in hypertensive patients is an independent risk factor for cardiovascular events. Changes of the structure and function companied with hypertension is defined as VR in hypertension, which consist of thickness of the wall, increase of medium/lumen ratio, decrease of arterioles and consequent dysfunction of the vessel. VR is not only an adaptive response, but also the main foundation of hypertension pathology, which contributes to serious vascular complications and dysfunction of the circulation. It's a prime contributor to the pathogenesis of a number of clinical disorders, including hypertension, atherosclerosis and restenosis. Current evidence shows that pathological remodeling of aorta in hypertension is one major mechanism responsible for morbidity and mortality. Thus the prevention and reversal of VR is the major objective for treatment of hypertension and as an important indicator of treatment evaluation. The study on the pathogenesis and control of VR has become a worldwide hot topic. VR is a complex course, and the pathogenesis mechanism still remains unclear. It is well-known that cell hypertrophy, hyperplasia, apoptosis, remodeling, inflammation, oxidative stress, cytokines and extracellular matrix changes are involved in VR. The main feature of aortic remodeling is thickness of medium, due to hypertrophy of vascular smooth muscle cells (VSMC).The protein kinase D (PKD) family is a recent addition to the calcium/calmodulin-dependent protein kinase group of serine/threonine kinases. PKD family consists of 3 isoforms PKD1, PKD2 and PKD3. It's structural and enzymatic properties distinguish it from PKC isoforms. Increasing evidence now points toward important roles for PKD-mediated signaling pathways in the cardiovascular system, particularly in the regulation of myocardial contraction, hypertrophy and remodeling. In recent years, evidence has suggested that vascular smooth muscle cells also express PKD, and PKD activity in them has been shown to be increased by physiologically important stimuli, such as angiotensinⅡ, platelet-derived growth factor, and thrombin. Functionally, PKD activity appears to regulate hypertrophy in VSMCs. Studies have indicated that PKD may control hypertrophy of VSMCs via the AT1/PKCδpathway by Ang II. Most recently, it has been proposed that PKD mediated HDAC5 phosphorylation may facilitate angiotensinⅡ-induced hypertrophy in VSMCs. However, the activation of PKD and it's possible mechanisms in hypertensive aortic remodeling remain unclear.As hypertension is often accompanied by dyslipidemia, the treatment frequently involves statins. More and more evidences established that statins not only effectively reduced serum cholesterol level, but also exerted pleiotropic beneficial effects on cardiovascular disease, including improvement of endothelial function, stabilization of atherosclerotic plaques, antioxidant properties, inhibition of inflammatory responses and prevention of cardiac hypertrophy or remodeling. Statins reduce cardiovascular morbidity and mortality in patients with high and moderate hypercholesterolemia or even normal cholesterol levels. Recently, atorvastatin have been shown to inhibit cardiac hypertrophy and fibrosis. However, the precise mechanism of vascular protective effects of atorvastatin remains to be fully clarified. This study was therefore designed to observe the expression and activation of PKD; to analyze its relationship with hypertensive aortic remodeling; to study the effects and the mechanism of atorvastatin in the prevention and treatment of aortic remodeling. The purpose of the study is to elucidate the cellular and molecular mechanisms of vascular remodeling associated with hypertension and the interventional effects of atorvastatin on it, and provide novel theoretical evidences and strategy for prevention and treatment of hypertensive vascular remodeling.Objectives1. To study the development of aortic remodeling in SHR.2. To investigate the involvement of PKD in aortic remodeling in SHR3. To evaluate the effects and the mechanisms of atorvastatin in the prevention and treatment of aortic remodeling.Methods8-week-old male SHRs were obtained from Genetic Models Inc. (Indianapolis, IN). Age-matched normotensive male WKY rats were purchased from animal center of Shandong University as the control. Rats were divided into the following groups:A Group:WKY-8W; B Group:WKY-16W; C Group:WKY-24W; G Group: WKY-placebo; D Group:SHR-8W; E Group:SHR-16W; F Group:SHR-24W; H Group:SHR-placebo; I Group:SHR-atorvastatin. Each group has 10 rats for 16 weeks treatment. WKY placebo, SHR placebo and SHR atorvastatin received distilled water or atorvastatin at 50 mg/kg/day by intragastric administration from 16 weeks to 24 weeks. Animals were killed when they were 8 weeks,16 weeks and 24 weeks old by decapitation. The aortas were immediately harvested and weighed. The following parameters were measured during the study:(1) All the rats have their body weight, heart rate and tail blood pressure measured once per 2 week; (2) Blood was collected at 8 weeks,16 weeks and 24 weeks respectively. Plasma lipids were determined using routine method; (3) Changes of vascular morphology and histology were measured by special H-E, tricrome Masson and Sirius red staining methods. Thickness of the media, collagen fibers and elastic fibers were used for the detection of remodeling. (4)Media thickness (MT), luminal diameter (LD), ratio of media to lumen (M/L) and volume fraction of collagen (VFC) were measured. (5) Hydroxyproline, an indicator to reflect collagen amount in mammalian tissues was measured. (6) Measure IL-6 and TNF-a by ELISA. (7) Nitrotyrosine is considered to be an indirect marker of oxidative stress, Western blot for the expression of nitrotyrosine. (8) Western blot for the expression of PKD,p-PKD744/748,p-PKD916.Results1. The experimental animalsTwo rats of SHR group(E and F) died in the entire experiment. A total of 88 rats finished the study,40 rats in WKY group,48 rats in SHR group.2. Comparisons of HR, SBP and Lipids level between WKY and SHR groupsThere were no significant differences in terms of heart rat and lipids at the beginning of the experiment. SBPs in SHRs at 8.16 and 24 weeks are higher than those in WKY rats (P<0.05). In SHR group, SBP increased during the course, while the one in WKY group remained unchanged. After treatment with atorvastatin, SBP decreased significantly (P<0.05). The serum lipid levels of SHRs and those of WKY rats were not significantly different at the beginning of the experiment. Atorvastatin at 50 mg/kg/day significantly reduced the serum cholesterol and LDL-cholesterol of SHRs (P<0.05). However, no significant changes were found in TG and HDL-cholesterol levels with atorvastatin treatment.3. Changes of vascular morphology and histologyH-E staining:In SHR group, aortic media were thickening; VSMCs in media were hypertrophy and disordered; elastic fibers were decreased and unregular; More smoothing aortic tunica intima, thinner vascular wall, less media VSMCs hypertrophy, more regular array of media VSMCs and elastic fibers were observed in atorvastatin group.Masson staining:Aortic elastic fibers of WKY rats arranged in order and no hyperplasia of collagen; Aortic wall of untreated SHR was thickening; collagen fibers in media were hyperplastic; elastic fibers were decreased, disordered, and even ruptured; Aortic elastic fibers in Atorvastatin group were fairly ordered, collagen fibers were not hyperplastic significantly compared with SHR group.Sirius red staining:Under polarization microscope, type I collagen is in colour red or orange, and type III collagen is in colour green. Much more collagen I and III existing in the vascular wall of untreated SHR group compared with WKY rats, which decreased significantly in the atorvastatin group.4. Aortic remodeling parametersArterial MT of untreated SHR were significantly higher than WKY rats, and atorvastatin treated animals had lower MT compared with untreated animals in the model group. Luminal diameter (LD) in each group was no significant difference. The ratio of MT to LD in untreated SHR was significantly higher than WKY rats, whereas animals in the atorvastatin group had lower ratio of MT to LD compared with untreated SHR. Volume fraction of collagen (VFC) was significantly higher in untreated SHR than it in WKY rats, and it decreased in the atorvastatin group.5. Effect of atorvastatin on hydroxyprolineCompared to WKY group, the groups of SHRs in 16W and 24W showed a significant rise in hydroxyproline content in aorta tissue (P<0.05), which were reduced by atorvastatin treatment.6. Effect of atorvastatin on inflammatory cytokinesCompared to WKY group, IL-6 and TNF-a in SHRs increased significantly(P<0.05), which were reduced by atorvastatin treatment.7. Effect of atorvastatin on oxidative stress in SHRsNitrotyrosine is considered to be an indirect marker of oxidative stress. In SHR group, nitrotyrosine increased during the course (P<0.05). while these in WKY group remained unchanged. These changes were attenuated by atorvastatin (P<0.05).8. Expression of PKD, p-PKD744/748 and p-PKD916 by Western-blotCompared to WKY group, the groups of SHRs in 16W and 24W showed a significant rise in p-PKD protein in aorta tissue (P<0.05). In SHR group, expression of p-PKD increased during the course (P<0.05). while these in WKY group remained unchanged. These changes were attenuated by atorvastatin (P<0.05).Conclusions1. The age-related aortic remodeling occured with the development of hypertension in SHRs, which offers reliable animal model for the researches on the mechanisms of hypertensive aortic remodeling.2. The over-expression of p-PKD has been confirmed in the aorta tissue of SHR, which suggested that activation of PKD was involved in the development and process of aortic remodeling in SHR.3. Atorvastatin could partially reverse the hypertension-induced aortic remodeling through its anti-inflammatory and anti-oxidant effects. Another mechanism for atorvastatins to reverse remodeling is down-regulation of PKD activation. BackgroundIn hypertension small and large arteries undergo structural, mechanical and functional changes that contribute to vascular remodeling and increased cardiovascular risk. Increase of vascular media thickness due to hypertrophy of vascular smooth muscle cell is the most important pathological change in hypertensive aortic remodeling. It is well established that vascular hypertrophy and remodeling in hypertension is an adaptive process in response to chronic changes in hemodynamic conditions during development and vascular pathologies. The cellular events underlying vascular hypertrophy during hypertension involve VSMC hypertrophy, hyperplasia, migration, apoptosis, inflammation, oxidative stress and fibrosis.The renin-angiotensin system (RAS)-plays a key role in the development and pathophysiology of hypertension and cardiovascular disease (CVD). Evidences show that activation of local RAS and levels of angiotensinⅡ(AngⅡ) are elevated in the development of vascular remodeling in hypertension.The central part of RAS is recognizing of different receptors by AngⅡand then carrying out its functions through different signal pathways. AngⅡ, the main effector of the renin-angiotensin system (RAS), is one of the major mediators of vascular remodeling in hypertension. Ang II, a potent vasoactive peptide, induces vascular remodeling and endothelial dysfunction in association with increases in levels of BP. However, AngⅡis able to induce vascular remodeling independently of its haemodynamic effects. Mounting evidence shows that AngⅡactivation contributes to pathological vascular remodeling, largely by stimulating VSMCs hypertrophy. Ang II also is crucially involved in fibrosis, Ang II induced vascular fibrosis via both TGF-β-dependent and-independent Smad signaling pathways. Furthermore, Ang II is a strong modulator of ROS production and proinflammatory transcription factors in the vasculature.ERK5 has recently been identified as a new member of the MAPK family, the subcellular localization of ERK5 is cell type specific. Various stimuli, including nerve growth factor, epidermal growth factor, platelet-derived growth factor, and angiotensin II (Ang II), activate ERK5. The central part of ERK5 cascades involved in signal transduction from the membrane to the nucleus is structured by three sequentially activated kinases:a MAPKKK family, or MEKK2/MEKK3; a MAPKK family, or MEK.5; and a MAP kinase, or ERK5. On stimulation, these kinases are consecutively phosphorylated and thus activated, leading to the activation of downstream effectors including MEF2. ERK5 has a potential role in the transcriptional regulation of hypertrophic genes, in particular MEF2-dependent genes such as c-jun gene through direct phosphorylation and activation of MEF2C, and govern the hypertrophic growth. ERK5 was recently found to interact with MEF2C on Ang II stimulation in rat aortic SMCs.As a hypertrophic modulator, PKD was involved in cardiac and vascular hypertrophy. PKD regulates the signal pathway of the MAPK family. For example, PKD upregulates Ras activity and ERK1/2 signaling by phosphorylating the Ras-binding protein RIN1, and downregulates JNK activity, possibly by phosphorylating c-Jun. But, to our knowledge, little is known about the effect of PKD on ERK5. Therefore the aim of this study is to investigate the role of PKD/ERK5/MEF2C signaling pathway in Ang II induced hypertrophy of VSMC, which may provide a new target for reversal of hypertensive vascular remodeling.Objectives1. To observe the hypertrophy of HASMCs stimulated by Ang II.2. To explore the AT1/PKCδ/PKD/ERK5/MEF2C pathway in Ang II-induced hypertrophy of HASMCs.Methods 1. Cell cultureThe human aortic smooth muscle cells, which was purchased from ScienCell (San Diego, CA), was cultured in MEM growth medium at 37℃and 5% CO2. Cells in generation 7-15 were used in the treatment.2. Cells were divided into several groups:1) Control Group:no stmulation factor;2) AngⅡstmulation Group:different dosges of AngⅡwith different times;3) AngⅡstmulation +DMSO Group;4) AngⅡstmulation+AT1 antagonist Losartan Group;5) AngⅡstmulation+AT2 antagonist PD123319 Group;6) AngⅡstmulation+PKC inhibitor Go6983 Group;7) AngⅡstmulation+Control siRNA Group;8) AngⅡstmulation+PKCδsiRNA Group;9) AngⅡstmulation+PKD siRNA Group;10) AngⅡstmulation+ERK5 siRNA Group.3. Detecting1) Observing changes of HASMCs form by electron microscope;2) 3H-leu incorporation rate, evaluated the level of cell hypertrophy;3) Western blot dectected expression of p-PKCα/β,ζ,ε,δ,PKD,p-PKD744/748,p-PKD916,ERK1/2,p-ERK1/2,ERK5,p-ERK5,MEF2C,p-MEF2C;4) Detecting the expression of PKD,ERK5 and MEF2C and EKR5 translocation between cytoplasm and nucelus by immunofluorescence.Results1. Morphological changes of HASMCs stimulated by AngⅡAngⅡgroup:the final concentration is 100nM; Control group:HASMCs were incubated in serum-free medium. Morphological changes were observed by electron microscope after stimulation by AngⅡ. Hypertrophy phenomenon of HASMCs was significantly greater after stimulation with AngⅡ.2. Measurement of [3H]-Leu incorporation HASMCs which were incubated in 24 orifice plates were made quiescent by incubation in serum-free DMEM medium for 24 h. Cells were stimulated by different concentrations of AngⅡ(0 nM, 1nM, 10nM,100 nM and 1000nM). [3H]-Leu incorporation were determined using a scintillation counter. [3H]-Leu incorporation was concentration dependent, beginning at 10 nmol/l AngⅡand with maximum effect at 1000 nmol/l AngⅡ.100nM AngⅡrapidly increased [3H]-Leu incorporation with peak incorporation at 15 and 30 min.3. Western blot for AT1/PKCδ/PKD/ERK5/MEF2C signal-transduction pathway1) Activation of PKCs by AngⅡinHASMCs HASMCs were stimulated by AngⅡat 100nM for different times (0,5,15,30, 60min), AngⅡ(100 nmol/L) induced phosphorylation of PKCδafter 5 min, with peak phosphorylation between 15 and 30 min (P<0.01), which returned to base line after 1 hr. However, the phosphorylation of PKCaα/βand PKCεremain the same, PKCζphosphorylation was later than that of PKCδ.2) Activation of ERK1/2 and ERK5 by AngⅡinHASMCs HASMCs were stimulated by AngⅡat 100nM for different times (0,5,15,30, 60min), AngⅡ(100 nmol/L) induced phosphorylation of ERK5 after 5 min, with peak phosphorylation between 15 and 30 min (P<0.01), which returned to base line after 1 hr. However, the phosphorylation of ERK1/2 were earlier than that of ERK5, with peak phosphorylation at 5 min (P<0.01). Total protein level of ERK5 and ERK1/2 did not change.3) AngⅡstimulates ERK5 activation through a AT1-dependent pathway AngⅡreceptor has two subtypes:AT1 and AT2, cells were pretreated for 30 mins with losartan (0.3,1.0,3.0μmol/L, a specific antagonist for AT1, or PD123319 (5, 10,20μmol/L), an antagonist for AT2, then stimulated with AngⅡ(100 nmol/L) for 15 mins. Losartan inhibited AngⅡ-induced ERK5 activation in a dose-dependent manner, whereas PD123319 had no effect on AngⅡactivation of ERK5.4) Activation of ERK5 by AngⅡis PKCδ-dependent Cells were pretreated with the general PKC inhibitor Go6983 (0.3,1,3μmol/L) or PKCδsiRNA before exposure to AngⅡ(100 nmol/L) for 15 mins. Go6983 blocked ERK5 phosphorylation in a dose-dependent manner, PKC8 siRNA also inhibited the activation of ERK5, which suggests that PKC8 is involved in AngⅡ-stimulated ERK5 phosphorylation in HASMCs.5) Ang II induces AT1/PKCδ-dependent activation of PKD in HASMCs AngⅡtime-and dose-dependently induces PKD phosphorylation both at Ser744/748 and at Ser916. Losartan (3 mmol/L) but not PD123319 (10 mmol/L) abolished AngⅡ-induced PKD phosphorylation, suggesting that AngⅡinduces activation of PKD via ATI receptor. Go 6983 also dose-dependently inhibited AngⅡ-stimulated PKD activation, PKC8 siRNA blocked PKD phosphorylation, which demonstrated that PKD activation is PKCδdependent.6) Knockdown of PKD by siRNA inhibited AngⅡ-induced activation of ERK5 in HASMCs HASMCs were transfected with PKD siRNA and then stimulated with AngⅡfor the time indicated. Treatment of HASMCs with PKD siRNA significantly reduced endogenous PKD expression, whereas control siRNA had no effect. Decreasing PKD expression by its siRNA significantly inhibited AngⅡ-induced ERK5 activation in HASMCs.7) PKCδ/PKD/ERK5 pathway is involved in Ang II-induced activation of MEF2CAngⅡsignificantly (P<0.05) stimulated the phosphorylation of MEF2C by 15 min of treatment and returned to basal levels by 30 min. Go 6983 markedly blocked MEF2C activation while knocking down PKCδ, PKD and ERK5 expression by siRNA significantly attenuated activation of MEF2C.8) PKCδ/PKD/ERK5 pathway is implicated in AngⅡ-stimulated HASMCs hypertrophy [3H]-leucine incorporation were significantly greater in AngⅡ(100 nM) treated cells than in controls (P<0.05). PKCs inhibitor, PKCδ, PKD and ERK5 siRNA greatly suppressed AngⅡ-induced [3H]-leucine incorporation (P<0.05).3. Immunofluorescence staining1) Immunofluorescence for expression of all molecules The expression of p-PKD744/748,p-PKD916,p-ERK5,p-MEF2C was observed by immunofluorescence staining. After stimulation by AngⅡfor 15 min, P-PKD744/748 and p-PKD916 were expressed in cytoplasm while p-ERK5 and p-MEF2C were in the nucleus.2) Translocation of ERK5Before stimulation of AngⅡ, ERK5 was located primarily in the cytoplasm of HASMCs, ERK5 nuclear entry was seen at 5 min after AngⅡstimulation, striking translocation from the cytoplasm to the nucleus was observed by 15 min after addition of AngⅡ, after 60 min of AngⅡtreatment, ERK5 gradually shuttled back to the cytoplasma.3) Ang II stimulates ERK5 translocation via ATl/PKCδ/PKD-dependent PathwayThe pharmacological inhibitors for AT1 and PKC significantly inhibited AngⅡ-induced ERK5 translocation. Knocking down PKCδand PKD expression by siRNA also greatly attenuated AngⅡ-induced translocation of ERK5 in HASMCs, which suggest that ERK5 phosphorylation plays an important role in ERK5 translocation.Conclusions1. AngⅡinduced hypertrophy of HASMCs.2. AT1/PKC8/PKD/ERK5/MEF2C signal pathway was involved in hypertrophy of HASMCs induced by AngⅡ.