AcSDKP对TGF-β1诱导大鼠MSCs向肌成纤维细胞分化的抑制作用

目的:研究N-乙酰基-丝氨酰-天冬氨酰-赖氨酰-脯氨酸(N-acetyl-seryl-aspartly-lysyl-proline,Ac SDKP)对转化生长因子β1(Transforming growth factor beta 1,TGF-β1)诱导大鼠骨髓间充质干细胞(mesenchymal stem cells,MSCs)向肌成纤维细胞(myofibroblast,MF)分化的影响,探讨Ac SDKP抗纤维化作用的可能机制。方法:全骨髓贴壁法分离培养大鼠骨髓MSCs。使用免疫组化,Western blotting技术分析α-SMA蛋白的表达以及Smad2/3,ERK1/2蛋白磷酸化的变化情况。结果:和对照组相比,TGF-β1诱导的MSC中α-SMA、磷酸化-Smad2/3及磷酸化-ERK1/2的表达大大增强,使用Ac SDKP干预细胞则三者的表达量明显下降且呈一定的剂量依赖性。结论:Ac SDKP可以显著抑制TGF-β1诱导的大鼠MSCs向MF分化,可能通过抑制TGF-β/Smad/ERK1/2信号通路的激活,从而发挥其抗器官纤维化作用。     

Stanford A型主动脉夹层血管壁及外周血中热休克蛋白90的表达及意义

目的:探索热休克蛋白90(Heat shock protein 90, HSP90)在Stanford A型主动脉夹层(Aortic dissection, AD)血管壁组织中的表达及其与平滑肌细胞(Smooth muscle cells, SMCs)表型标志物的相关性。方法:收集本院急性Stanford A型主动脉夹层患者病变主动脉壁组织和外周血标本(AD组,20例),心脏移植供体等来源的正常主动脉壁组织和外周血标本(正常组,10例);ELISA法检测血浆HSP90含量;免疫组化染色检测HSP90的表达差异及定位;Western blot检测组织标本中蛋白的表达差异;应用Spearman分析HSP90和SMCs表型标志物表达的相关性。结果:AD患者血浆中HSP90的含量显著高于正常组(142.38±40.16ng/mL vs. 54.99±24.46 ng/mL,P<0.01)。相对于正常主动脉壁组织,主动脉夹层血管壁组织中HSP90表达明显升高,且主要分布在血管壁中层细胞的胞浆中,分泌型SMCs标志物OPN的表达明显增多,而收缩型标志物α-SMA表达则显著减少。HSP90和α-SMA的蛋白表达呈负相关(R2=0.677,P<0.01),和OPN呈正相关(R²=0.572,P<0.01)。结论:主动脉夹层患者的血管壁组织及外周血中的HSP90含量明显升高,参与了主动脉夹层的发生发展。     

Downregulation of HDAC1 suppresses media degeneration by inhibiting the migration and phenotypic switch of aortic vascular smooth muscle cells in aortic dissection

Although much progress has been made in the diagnosis and treatment of thoracic aortic dissection (TAD), the overall morbidity and mortality rates of TAD are still high. Therefore, the molecular pathogenesis and etiology of TAD need to be elucidated. In this study, we found that histone deacetylase 1 (HDAC1) expression is dramatically higher in the aortic wall of patients with TAD (than that in a normal group) and negatively correlates with the levels of the vascular smooth muscle cell (SMC) contractile-phenotype markers. Knockdown of HDAC1 upregulated both smooth muscle 22 α (SM22α) and α-smooth muscle actin (α-SMA) in platelet-derived growth factor (PDGF)-BB-treated and -untreated SMCs. In addition, the knockdown of HDAC1 markedly decreased SMC viability and migration in contrast to the control group under the conditions of quiescence and PDGF-BB treatment. We also showed that the expression of polycystic kidney disease 1 (PKD1) is decreased in the aortic wall of patients with TAD and negatively correlates with HDAC1 expression. Overexpressed PKD1 obviously increased SM22α and α-SMA expression and reduced the viability and migration of SMCs, but these effects were attenuated by HDAC1. Furthermore, we demonstrated that HDAC1 serves as an important modulator of the migration and phenotypic switch of SMCs by suppressing the PKD1– mammalian target of the rapamycin signaling pathway. HDAC1 downregulation inhibited media degeneration and attenuated the loss of elastic–fiber integrity in a mouse model of TAD. Our results suggest that HDAC1 might be a new target for the treatment of a macrovascular disease such as TAD.     

Silencing CCL8 inhibited the proliferation and migration of PDGF-BB-stimulated human aortic smooth muscle cells

ABSTRACT

C-C motif Chemokine ligand 8 (CCL8) has been found in diseases’ pathogenesis. But its molecular mechanism in atherosclerosis (AS) remains to be elucidated. Human aortic smooth muscle cells (HASMCs) were stimulated by PDGF-BB to establish cell model. α-SMA in PDGF-BB-stimulated HASMCs was measured by immunofluorescence staining. Relative gene expressions in PDGF-BB-stimulated HASMCs were detected by quantitative real-time polymerase chain reaction and western blot. HASMCs proliferation, migration, and cell cycle were assessed by cell counting kit-8, wound-healing assay, and flow cytometry. HASMCs viability was increased after PDGF-BB stimulation, with α-SMA downregulation yet CCL8 upregulation. Silencing CCL8 inhibited PDGF-BB-stimulated HASMCs proliferation and migration, and increased cells percentage in G1 phases but decreased those in S phase. Also, silencing CCL8 decreased OPN and cyclinD1 expressions and AKT and ERK1/2 phosphorylation while increased those of α-SMA and Sm22α. However, upregulating CCL8 led to opposite effects, suggesting CCL8 could be an atherosclerosis therapeutic target.     

Suppression of 12-O-Tetradecanoylphorbol-13-Acetate-Induced MCF-7 Breast Adenocarcinoma Cells Invasion/Migration by α-Tomatine Through Activating PKCα/ERK/NF-κB-Dependent MMP-2/MMP-9 Expressions

α-Tomatine, isolated from Lycopersicon esculentum Linn., is a naturally occurring glycoalkaloids in immature green tomatoes. Some reports demonstrated that α-tomatine had various anti-carcinogenic properties. First, the result demonstrated α-tomatine could inhibit TPA-induced the abilities of the adhesion, morphology/actin cytoskeleton arrangement, invasion, and migration by cell–matrix adhesion assay, immunofluorescence stain assay, Boyden chamber invasion assay, and wound-healing assay. Data also showed α-tomatine could inhibit the activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and protein kinase C-α (PKCα) involved in the downregulation of the enzyme activities and messenger RNA levels of matrix metalloproteinase-2/9 (MMP-2/MMP-9) induced by TPA. Next, α-tomatine also strongly inhibited TPA-induced the activation of nuclear factor kappa B (NF-κB) and phospho-inhibitor of kappa Bα (phospho-IκBα). In addition, TPA-induced translocation of PKC-α from cytosol to membranes, and suppression of TPA elicited the expression of PKC-α by adding the PKC-α inhibitors, GF-109203X and Gö-6983. The treatment of specific inhibitor for ERK (U0126) to MCF-7 cells could inhibit TPA-induced MMP-2/MMP-9 and phospho-ERK along with an inhibition on cell invasion and migration. Application of α-tomatine to prevent the invasion/migration of MCF-7 cells through blocking PKCα/ERK/NF-κB activation is first demonstrated herein.     

Phenotypic characterization of culture expanded rabbit limbal corneal keratocytes

The in vivo quiescent corneal stroma keratocytes need to be transformed to activated state in order to obtain sufficient number of cells either for monolayer evaluation or corneal stroma reconstruction. This study aimed to investigate the phenotypic characterization of corneal stromal cells during culture expansion from the limbal region of the cornea. Isolated corneal keratocytes from limbal tissue of New Zealand White Strain rabbits’ corneas (n = 6) were culture expanded until three passages. Keratocytes morphology was examined daily with viability, growth rate, number of cell doubling and population doubling time were recorded at each passage. The expression of collagen type 1, aldehyde dehydrogenase (ALDH), lumican and alpha smooth muscle actin (α-SMA) were detected by RT-PCR. Immunocytochemistry was also used to detect ALDH, α-SMA, collagen type I and Cytokeratin-3 (CK3). Growth kinetic study revealed that the growth rate was low at the initial passage but increase to about two folds with concomitant reduction in population doubling time in later passages. Freshly isolated and cultured keratocytes expressed collagen type 1, ALDH and lumican but α-SMA expression was absent. However, α-SMA was expressed along with the other genes during culture expansion. Keratocytes at P1 expressed all the proteins except CK3. These results suggest that cultured keratocytes maintained most of the gene expression profile of native keratocytes while the emergence of α-SMA in serial passages showed a mix population of various phenotypes. The phenotypic characterization of monolayer keratocytes provides useful information before reconstruction of bioengineered tissue or in vitro pharmaceutical applications.     

Taurine suppresses osteoblastic differentiation of aortic valve interstitial cells induced by beta-glycerophosphate disodium, dexamethasone and ascorbic acid via the ERK pathway

Aortic valve calcification (AVC) is an active process characterized by osteoblastic differentiation of the aortic valve interstitial cells (AVICs). Taurine is a free β-amino acid and plays important physiological roles including protective effect of cardiovascular events. To evaluate the possible role of taurine in AVC, we isolated human AVICs from patients with type A dissection without leaflet disease. We demonstrated that the cultured AVICs express SM α-actin, vimentin and taurine transporter (TAUT), but not CD31, SM-myosin or desmin. We also established the osteoblastic differentiation model of the AVICs induced by pro-calcific medium (PCM) containing β-glycerophosphate disodium, dexamethasone and ascorbic acid in vitro. The results showed that taurine attenuated the PCM-induced osteoblastic differentiation of AVICs by decreasing the alkaline phosphate (ALP) activity/expression and the expression of the core binding factor α1 (Cbfα1) in a dose-dependent manner (reaching the maximum protective effect at 10 mM), and taurine (10 mM) inhibited the mineralization level of AVICs in the form of calcium content significantly. Furthermore, taurine activated the extracellular signal-regulated protein kinase (ERK) pathway via TAUT, and the inhibitor of ERK (PD98059) abolished the effect of taurine on both ALP activity/expression and Cbfα1 expression. These results suggested that taurine could inhibit osteoblastic differentiation of AVIC via the ERK pathway.     

The impact of pericytes on the blood-brain barrier integrity depends critically on the pericyte differentiation stage

The blood-brain barrier consists of the cerebral microvascular endothelium, pericytes, astrocytes and neurons. In this study we analyzed the differentiation stage dependent influence of primary porcine brain capillary pericytes on the barrier integrity of primary porcine brain capillary endothelial cells. At first, we were able to induce two distinct differentiation stages of the primary pericytes in vitro. TGFβ treated pericytes expressed more α-SMA and actin while desmin, vimentin and nestin expression was decreased when compared to bFGF induced cells. Further analysis of α-SMA revealed that most of the pericytes differentiated with TGFβ expressed functional α-SMA while only few cells expressed functional α-SMA in the presence of bFGF. In addition the permeability factors VEGF, MMP-2 and MMP-9 were higher secreted by the α-SMA positive phenotype indicating a proangiogenic role of this TGFβ induced pericyte differentiation stage. Higher level of VEGF, MMP-2 and MMP-9 were as well detected in the TGFβ pretreated pericyte coculture with endothelial cells when compared to the influence of the bFGF pretreated pericytes. The TEER measurement of the barrier integrity of endothelial cells revealed that bFGF induced α-SMA negative pericytes stabilize the barrier integrity while α-SMA positive pericytes differentiated by TGFβ decrease the barrier integrity. These results together reveal the potential of pericytes to regulate the endothelial barrier integrity in a differentiation stage dependant pathway.     

Extracellular matrix composition and remodeling in human abdominal aortic aneurysms: a proteomics approach

Abdominal aortic aneurysms (AAA) are characterized by pathological remodeling of the aortic extracellular matrix (ECM). However, besides the well-characterized elastolysis and collagenolysis little is known about changes in other ECM proteins. Previous proteomics studies on AAA focused on cellular changes without emphasis on the ECM. In the present study, ECM proteins and their degradation products were selectively extracted from aneurysmal and control aortas using a solubility-based subfractionation methodology and analyzed by gel-liquid chromatography-tandem MS and label-free quantitation. The proteomics analysis revealed novel changes in the ECM of AAA, including increased expression as well as degradation of collagen XII, thrombospondin 2, aortic carboxypeptidase-like protein, periostin, fibronectin and tenascin. Proteomics also confirmed the accumulation of macrophage metalloelastase (MMP-12). Incubation of control aortic tissue with recombinant MMP-12 resulted in the extensive fragmentation of these glycoproteins, most of which are novel substrates of MMP-12. In conclusion, our proteomics methodology allowed the first detailed analysis of the ECM in AAA and identified markers of pathological ECM remodeling related to MMP-12 activity.     

LncRNA H19 regulates smooth muscle cell functions and participates in the development of aortic dissection through sponging miR-193b-3p

Background: Multiple studies showed that long-chain noncoding RNA H19 (LncRNA H19) is high-expressed in human and mouse abdominal aortic aneurysms (AAAs). We speculated that it plays an important role in arterial disease, and therefore studied the role and mechanism of H19 in aortic dissection (AD).Methods: The expressions of related genes in human aortic smooth muscle cells (HASMCs) induced by platelet-derived growth factor BB (PDGF-BB) or in the aortic tissue of AD patients/mice were identified by Western blot and quantitative real-time polymerase chain reaction. The targeting relationship between H19 and miR-193b-3p was predicted and verified by bioinformatics analysis, dual luciferase assay, RNA pull-down assay, RNA immunoprecipitation (RIP), and Pearson correlation coefficient. The H19 and miR-193b-3p effects on the biological functions of tissues and cells were examined by MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, thiazolyl blue tetrazolium bromide) assay, wound-healing assay, and Hematoxylin–Eosin (HE) staining.Results: LncRNA H19 was abnormally high-expressed in thoracic aorta tissues of AD patients, and it could competitively bind to and inhibit miR-193b-3p. In the PDGF-BB group, the expressions of H19, matrix metallopeptidase (MMP) 2 (MMP-2) and MMP-9 were up-regulated and the expressions of miR-193b-3p, α-SMA, and SM22α were down-regulated; moreover, the proliferation and migration rate of HASMCs were increased. However, H19 silencing reversed the regulation of PDGF-BB on HASMCs. More interestingly, miR-193b-3p inhibitor could partially reverse the effect of H19 silencing. In addition, the above results were verified by animal experiments, showing that shH19 and up-regulated miR-193b-3p could significantly reduce the thoracic aorta pathological damage in AD mice.Conclusion: LncRNA H19 regulated smooth muscle cell function by sponging miR-193b-3p and it participated in the development of AD.     

Biomarkers and heterogeneous fibroblast phenotype associated with incisional hernia

Development of incisional hernia (IH) is multifactorial but inflammation and abdominal wall ECM (extracellular matrix) disorganization are key pathological events. We investigated if the differential expression of fibroblast biomarkers reflects the cellular milieu and the dysregulated ECM in IH tissues. Expression of fibroblast biomarkers, including connective tissue growth factor, alpha-smooth muscle actin (α-SMA), CD34 (cluster of differentiation 34), cadherin-11 and fibroblast specific protein 1 (FSP1), was examined by histology and immunofluorescence in the hernial-fascial ring/neck tissue (HRT) and hernia sack tissue (HST) harvested from the patients undergoing hernia surgery and compared with normal fascia (FT) and peritoneum (PT) harvested from brain-dead healthy subjects undergoing organ procurement for transplantation. The H&E staining revealed alterations in tissue architecture, fibroblast morphology, and ECM organization in the IH tissues compared to control. The biomarker for undifferentiated fibroblasts, CD34, was significantly higher in HST and decreased in HRT than the respective FT and PT controls. Also, the findings revealed an increased level of CTGF (connective tissue growth factor) with decrease in α-SMA in both HRT and HST compared to the controls. In addition, an increased level of FSP1 (fibroblast specific protein 1) and cadherin-11 in HRT with decreased level in HST were observed relative to the respective controls (FT and PT). Hence, these findings support the heterogeneity of fibroblast population at the laparotomy site that could contribute to the development of IH. Understanding the mechanisms causing the phenotype switch of these fibroblasts would open novel strategies to prevent the development of IH following laparotomy.

     

Rapid expression and activation of MMP-2 and MMP-9 upon exposure of human breast cancer cells (MCF-7) to fibronectin in serum free medium

Interactions between tumour cells and the extracellular matrix (ECM) strongly influence tumour development, affecting cell survival, proliferation and migration. Many of these interactions are mediated through a family of cell surface receptors named integrins. Fibronectin and its integrin receptors play important roles in tumour development. The alpha5beta 1 integrin interacts with the central cell adhesive region of fibronectin and requires both the RGD and synergy sites for maximal binding. Matrix metalloproteinases (MMPs) are a family of zinc dependent endopeptidases. They are capable of digesting the different components of the ECM and basement membrane. The ECM gives structural support to cells and plays a central role in cell adhesion, differentiation, proliferation and migration. Binding of ECM to integrins modulates expression and activity of the different MMPs. Our experimental findings demonstrate that cultivation of human breast cancer cells, MCF-7, in serum free medium in the presence of fibronectin upregulates the activity of MMP-2 and MMP-9. Blocking of alpha5beta 1 integrin with anti-alpha5 monoclonal antibody inhibits the fibronectin-induced MMP activation response appreciably. This strongly indicates alpha5beta 1 mediated signalling events in activation of MMP-2 and MMP-9. Phosphorylation of FAK and PI-3 kinase and the nuclear translocation of ERK and NF-kappaB upon fibronectin binding demonstrate possible participation of the FAK/PI-3K/ERK signalling pathways in the regulation of MMP-2 activity.     

Loss of LRP1 promotes acquisition of contractile-myofibroblast phenotype and release of active TGF-β1 from ECM stores

In healing tissue, fibroblasts differentiate to α-smooth muscle actin (SMA)-expressing contractile-myofibroblasts, which pull the wound edges together ensuring proper tissue repair. Uncontrolled expansion of the myofibroblast population may, however, lead to excessive tissue scarring and finally to organ dysfunction. Here, we demonstrate that the loss of low-density lipoprotein receptor-related protein (LRP) 1 overactivates the JNK1/2-c-Jun-Fra-2 signaling pathway leading to the induction of α-SMA and periostin expression in human lung fibroblasts (hLF). These changes are accompanied by increased contractility of the cells and the integrin- and protease-dependent release of active transforming growth factor (TGF)-β1 from the extracellular matrix (ECM) stores. Liberation of active TGF-β1 from the ECM further enhances α-SMA and periostin expression thus accelerating the phenotypic switch of hLF. Global gene expression profiling of LRP1-depleted hLF revealed that the loss of LRP1 affects cytoskeleton reorganization, cell-ECM contacts, and ECM production. In line with these findings, fibrotic changes in the skin and lung of Fra-2 transgenic mice were associated with LRP1 depletion and c-Jun overexpression. Altogether, our results suggest that dysregulation of LRP1 expression in fibroblasts in healing tissue may lead to the unrestrained expansion of contractile myofibroblasts and thereby to fibrosis development. Further studies identifying molecules, which regulate LRP1 expression, may provide new therapeutic options for largely untreatable human fibrotic diseases.