The Mechanism Of Profilin-1on Hypertension-Induced Artery Remodeling

Essential hypertension is one of the most common cardiovascular disease in our country, it is also a worldwide public health problem. The major damage caused by hypertension is its effect on the target organs including heart, brain, kidney and vessels. Vascular remodeling is an important mechanism of hypertension-induced target organs damage.The pathogenesis mechanism of vascular remodeling is complicated, it is reported that cell hypertrophy, apoptosis, inflammation, oxidative stress, cytokines and extracellular matrix changes are involved in vascular remodeling, but the exact mechanism remained unclear. With the development of proteomic techniques, a quantitative proteomics-isobaric tags for relative and absolute quantification (iTRAQ) was introduced, which provides an opportunity to solve this problem. iTRAQ enables the detection and quantitation of differentially expressed proteins, and analyzes the protein function. It has been widely used in finding the pathogenesis, disease markers, the differentially expressed proteins in diseases. We resorted to iTRAQ technique of the aorta using spontaneous hypertensive rats (SHRs), SHRs are well-established animal model to study hypertension vascular remodeling. This experiment explore the protective mechanisms of vascular remodeling by searching the differential protein among SHR group and WKY group, then validate for Profilin-1, a markedly differential protein’s effect in vascular remodeling, thus provide candidate targets for clinical treatment of hypertension. Part OneThe proteomic analysis of aortic tissue of different week-old spontaneous hypertensive rats BackgroundProtein is the ultimate embodiment of vital movement, proteomics is a discipline which focus on all the proteins and their dynamic regularity in tissue or cell. Proteomics explicates the essence and regularity of vital movement on the whole through analyzing the composition, expression and modification of all the proteins and their interactions in tissue and cell. Proteomics is a new research field and it is more complex than genomics. The expression of protein has spatiality and regulatability, and when the protein functions, doesn’t like the genome who is almost invariable, it is dynamic, and many proteins are involved in one kind of vital movement at the same time. Proteomics have provided new methods and ideas for the research on life science.Comparative proteomics is a kind of approach that compares and analyzes the proteome to find the differences of expression of proteins. In recent years, the comparative proteomics is used in medical research more and more frequent. By comparing with the normal individual, we can find the specific proteins which only express in the abnormal individual and these specific proteins can be utilized in the early diagnosis and treatment of diseases. Isobaric tags for relative and absolute quantification (iTRAQ) is a new quantitative technique for proteomics analysis. It can be used for separating up to8different samples at the same time, with high repeatability and good quantitative effect.It is reported that cell hypertrophy, apoptosis, inflammation, oxidative stress, cytokines and extracellular matrix changes are involved in vascular remodeling, but the exact mechanism remained unclear. iTRAQ provides an opportunity to solve this problem, which enables the detection and quantitation of differentially expressed proteins, and analyzes the protein function. We resorted to iTRAQ technique of the aorta using SHRs, explored the protective mechanisms of vascular remodeling by searching the differential protein among SHR group and WKY group. Objectives1. To research the characteristics of aortic pathological changes of SHR, to observe the blood pressure, weight and aortic pathological changes of five-week-old and seventeen-week-old SHR.2. To explore the differentially expressed proteins of aorta in five-week-old and seventeen-week-old SHR and the same week-old WKY rats by iTRAQ.3. To find out the aortic differentially expressed proteins of five-week-old and seventeen-week-old rats, further confirm the target proteins and potential mechanism of vascular remodeling under hypertension.MethodsMale SHRs (n=6,5weeks old; n=6,17weeks old), male WKY (n=6,5weeks old; n=6,17weeks old) assigned to4groups respectively:S5group, S17group, W5group, W17group. After3days adaptive feeding, blood pressure and weight were measured.1. Aortic morphology:After anesthetized, aortic tissue of rats were extracted, and fixed in10%formalin, then embedded in paraffin for HE staining, the internal circumference and wall thickness of the thoracic aorta were determined using Image-Pro Plus. Aortic fibrosis was shown on histological pictures, where red and yellow fibers represent collagen I and green fibers show collagen Ⅲ. Picrosirius red staining was observed by polarized light and measured by Image-Pro Plus to obtain the percentage of collagen per media area. Or fix the aortic tissue in glutaraldehyde for ultrastructure observation using electron microscope.2. Proteomic analysis:The proteins-enriched fractions were digested by tripsin. The resulting complex peptide mixtures were labeled with iTRAQ reagents (113for the W5group,114for the S5group,115for the W17group and118for the S17group respectively), All the labeled samples were finally mixed together, separated by Strong Cation Exchange (SCX) chromatography into10fractions and finally desalted by an offline fraction collector and C18cartridges. After being labeled, an amount of peptides among each group were performed on mass spectrometric analysis using MALDI-TOF/TOF to evaluate the effectiveness of reagents labeling. Panther software was applied to analyze protein functions.3. Western blot and PCR:4differentially expressed proteins were validated using western blot and PCR. Results1. Aortic MorphologyAortic remodeling and proliferation of vascular smooth cells (VSMC) and endothelial injury were observed in the aorta of5-week SHRs and17-week WKY, and17-week SHRs showed significantly greater lumen size (internal circumference), wall thickness and wall-lumen ratio (wall thickness:internal diameter), accompanied by evidence of broken elastic membranes. Compared with17-week WKYs,17-week SHRs showed increased collagen deposition in the media.Under electron microscopy, normal ultrastructure was observed in the aortic tissue of W5, and the rupture of vascular elastic membrane and the increased collagen fibers were observed in the aorta of W17and S5rats. Moreover, ultrastructure changed more obviously in S17rats.2. Mass Spectrometry Identify the Differentially Expressed ProteinsWe identified92differential proteins between W5group and S5group, among which the expression of8proteins was significantly changed;94differential proteins between W17group and S17group, among which the expression of18proteins was significantly changed.3. Subcellular localization analysis vascular remodeling associated proteinsWe performed the localization analysis of the identified proteins by Panther software. Cellular component of differentially expressed proteins among W17group and S17group mainly included cell part, extracellular region, organelle.4. Protein function analysis vascular remodeling associated proteinsMolecular function of differentially expressed proteins among W17group and S17group mainly included catalytic activity, binding and structural molecular activity, they were mainly involved in metabolic processes, cellular processes, developmental processes and cellular component organization or biogenesis.5. Differentially expressed proteins in aortaProtein and mRNA levels of Profilin-1and Cofilin-1were higher in SHRs than in same aged WKYs, protein level of AC AD VL was lower in SHRs.Conclusions1. As a new technique of quantitative proteomics, iTRAQ is more accurate and reproducible, enables comparative analysis for many groups. We obtained reliable differentially expressed proteins from different aged SHRs and WKYs, and find potential new drug targets for hypertension and its complications.2. Molecular function of differential proteins we identified mainly included catalytic activity, binding and structural molecular activity, they were mainly involved in metabolic processes, cellular processes, developmental processes and cellular component organization or biogenesis.Part TwoProfilin-1promotes the development of hypertension-induced artery remodelingBackgroundVascular hypertrophy and remodeling in hypertension is an adaptive process in response to chronic changes in hemodynamic conditions during the development of vascular diseases. The ensuing changes in the size and/or composition of a remodeling blood vessel allow blood vessels to adapt and heal but they also underlie the pathogenesis of major cardiovascular diseases.Profilin-1is a ubiquitous, small (12-15kD) actin-binding protein that plays an important role in the regulation of actin polymerization and cytoskeleton remodeling by activating hypertrophic signaling cascades, such as mitogen activated protein kinase (MAPK) signaling, to thus contribute to vascular hypertrophy and hypertension. Numerous studies have provided strong evidence for the important role of profilin-1in vascular inflammation and vascular remodeling, and have linked profilin-1to inducible nitric oxide synthase (iNOS) and peroxynitrite production. Also, peroxynitrite has been linked to vascular remodeling because it activates MAPKs (c-Jun N-terminal kinase [JNK], extracellular signal-regulated kinase1/2[ERK1/2]). However, the relationship between profilin-1, iNOS and the associated remodeling in SHRs remains to be fully clarified. Here, we studied the effects of profilin-1on hypertension-induced vascular remodeling and its potential relationship with iNOS. We used SHRs as a classical, genetic hypertensive animal model. Overexpression of profilin-1significantly promoted arterial structural remodeling in SHRs through a p38-iNOS-peroxynitrite-pathway. Profilin-1may be a potential target for genetic therapy of hypertension-induced arterial structural remodeling.Objective1. To investigate the effect of Profilin-1on vascular remodeling by transfection of pAd-profilin-1-IRES-EGFP and pAd-miR-profilin-1into SHRs;2. To study the effect of Profilin-1on vascular inflammation and oxidative stress, and the relationship between oxidative stress and vascular remodeling.3. To explore the mechanism of Profilin-1on vascular remodeling in molecular, histological and functional levels.MethodsFive-month-old male SHRs (n=75) and Wistar-Kyoto rats (WKYs)(n=20) were obtained from Slac Laboratory Animal Co. SHRs were divided into3groups (n=25each) for treatment:profilin-1overexpression adenovirus vector (SHR-E), profilin-1miRNA adenovirus vector (SHR-I) or negative control adenovirus (SHR-C) after3days adaptation. WKYs (n=20) were treated with negative control adenovirus at the same time. The adenoviruses were injected twice into the tail vein at3x109infectious units per rat with an interval of6weeks between injections. At3weeks after the first administration,15SHRs from each of the three SHR groups (n=5each) were killed: At6weeks after the second administration of adenovirus, the remaining SHRs (n=60) and WKYs were killed.1. Systolic blood pressure (SBP) and body weight were measured in conscious animals before the start of treatment and weekly during treatment. Systolic blood pressure (SBP) was measured by means of a tail-cuff sphygmomanometer.2. Six days after the second injection of adenovirus, five rats in each group were killed and thoracic aortas were removed to determine transfection efficiency. Thoracic aorta specimens were sectioned at5μm for enhanced green fluorescent protein (EGFP) observation. Tissues underwent sky blue staining to shield the autofluorescence of the vascular tissues and DAPI staining was used to locate the nuclei. EGFP expression in the vascular tissues was observed by fluorescence microscopy.3. Changes of vascular morphology and histology were measured by HE staining, Picrosirius-red-staining. 4. Nitrotyrosine is considered to be an indirect marker of oxidative stress, measured by immunohistochemical analysis.5. Measure IL-6and NO by ELISA.6. Western blot for the expression of p-p38, Profilin-1and iNOS, RT-PCR for mRNA expression of Profilin-1and iNOS.Results1. Transfection Efficiency of Adenovirus VectorThe high transfection efficiency was confirmed by observing EGFP expression under fluorescence microscopy and detecting the mRNA and protein expression of Profilin-1. EGFP expression was observed in more than90%cells for each transduction group. Profilin-1expression was higher in HUVECs and aortic tissues after profilin-1overexpression by adenovirus vector transfection, but was reduced by profilin-l miRNA vector treatment.2. Effect of Profilin-1on Body Weight and SBP of SHRsBody weight did not differ among the four groups during the12weeks of treatment. SBP for the three SHR groups increased with age, no significant difference between SHR-C and the other SHR-groups (p>0.05).3. Effect of profilin-1on3-week-treatment SHRsAt3weeks after the administration of the adenovirus, we found that the lumen size (internal circumference), wall thickness and wall to lumen ratio (wall thickness: internal diameter) did not differ among the three SHRs groups. Protein level of iNOS and phosphorylation of p38were higher in SHR-E than in SHR-C tissues, but were decreased by the knockdown of profilin-1. We also found an increase in peroxynitrite expression (as determined by nitrotyrosine staining) in SHR-E (0.530±0.028) and a decrease in SHR-I (0.072±0.016), as compared with SHR-C.4. Profilin-1Promotes Vascular Hypertrophy and FibrosisThis increased Profilin-1expression was accompanied by vascular remodeling in SHRs, as evidenced by a36.8%increase in wall thickness, a12%increase in wall-lumen ratio (the wall thickness:internal diameter) and increased vascular fibrosis. Compared with SHR-C, SHR-E showed significantly greater lumen size (internal circumference), wall thickness and wall-lumen ratio (wall thickness:internal diameter), accompanied by evidence of broken elastic membranes. 5. Effect of Profilin-1on Remodeling in SHR via the p38iNOS and Peroxynitrite-dependent MechanismTotal p38protein expression did not differ among the four treatment groups, but phosphorylation of p38was higher in SHR-E than in SHR-C. In contrast, phosphorylation of p38was decreased by the knockdown of profilin-1. Thoracic aorta expression of iNOS showed a similar trend to that of p38phosphorylation.Our results showed a significant increase in peroxynitrite expression (as determined by nitrotyrosine staining) in SHR-E tissues but a significant decrease in SHR-I tissues, as compared with SHR-C. IL-6level was significantly higher in SHR-E as compared with SHR-I and SHR-C.Next, we investigated the production of NO in aortic tissues and found a lower level of NO in SHR-E as compared with SHR-I (1.45±0.15vs.2.34±0.16μmol/g protein nitrites, p<0.05) and as compared with SHR-C (1.88±0.14μmol/g protein nitrites). We also tested the NO levels in the serum of rats and found no significant difference among the three SHR groups, indicating that changes in NO levels are a local phenomenon rather than a systemic reaction.Conclusions1. Vascular remodeling of SHRs was companied with oxidative stress reactions and over expression of Profilin-1.2. Over expression of Profilin-1could facilitate the vascular remodeling and oxidative stress reactions, and knockdown of Profilin-1reversed those changes.3. Prohypertrophic effects of profilin-1are mediated, at least in part, by suppression of the p38-iNOS-peroxynitrite pathway.