HTRAl Gene Mutations Influence The Function Of Vascular Adventitial Fibroblasts And Its Association With TGF-β/Smad/CTGF Signaling Pathway

HtrA is a heat shock induced membrane serine proteases, HtrAl is a secreted protein which is first found in human HtrA serine proteases family. HtrAl can degrade the extracellular matrix (ECM) proteins, including fibronectin, laminin, collagen and elastin, etc. HtrAl can hydrolyse matrix protein through association with proteoglycans, I, II, III collagen. HtrAl inhibits fibroblasts phenotypic transformation and cell growth and proliferation, and inhibits tumor expression. HtrAlprotein binds to transforming growth factor β (TGF-β) proteins family and inhibits signal transduction mediated by TGF-β/Smads signaling pathway. HtrAl gene was found to cause a rare clinical disease-cerebral autosomal recessive arteriopathy with subcortical infarcts and leucoencephalopathy(CARASIL) in2009. HtrAl gene has9exons,6mutation sites were found and located at HtrAl serine protease activity areas. The gene mutation can reduce HtrAl protease activity, the protein expression, and inhibition on TGF-β signaling pathway. TGF-/Smads was up-regulated, resulting in overproduction and excessive deposition of ECM. CARASIL is a recessive hereditary cerebrovascular disease onset of adolescence. Early onset of dementia, stroke, lumbar joint disease, hair loss is the main clinical manifestations. Brain CT or MRI scan showed symmetry lesions in bilateral cerebral hemisphere deep white matter beside ventricular and multiple cortical infarction. pathological changes in CARASIL include small cerebral artery intimal thickening, elastic fiber breakage, hyaline tunica media, smooth muscle cells lost, adventitia thining and concentric lumen stenosis. The specific pathogenesis of CARASIL caused by gene mutations is unclear.In2006we reported the first domestic CARASIL pedigrees, and found a new gene mutation HTRA1sites:1091T> C through genetic testing analysis, which was a highly pathogenic missense mutation. This was a good platform for research on the pathogenesis of CARASIL. Previous research found that HTRA1mutant gene transfected into vascular smooth muscle cells can lead to a reduction expression of HTRA1mRNA and protein, and can cause an up-regulation expression of TGF-β1/Smads signaling pathway. For further research on the pathogenisis of pathological changes in CARASIL We study the structure and the secretion function of vascular adventitia fibroblasts in CARASIL caused by HTRA1gene mutations and establish a vascular adventitia fibroblasts model of HTRA1wild-type and mutant gene. We observe changes in TGF-β1/Smads/CTGF signaling pathway, and further explore the pathogenesis of CARASIL at the cellular level and the molecular level.Part Ⅰ Construction and packaging of lentiviral expression vector of HTRA1geneObjective:To construct lentiviral vector HTRA1genes and HTRA11091T> C mutation (Mut) lentiviral expression vector and transfected into vascular adventitia fibroblasts.Methods:Design a pair of primers according to HTRA1human gene sequence, Primer Forword:5-GGAATTCATGCAGATCCCGCGCGCC-3, Primer Reverse:5-CGGGATC CCTATGGGTC AATTTCTT-3. Target gene was amplification through PCR. PCR product of pLenO-GTP vector, HTRA1gene and HTRA1-Mut gene was digested using EcoR I and BamH I enzyme, the digestion product of pLenO-GTP vector and PCR products of HTRA1gene and HTRA1-Mut gene were loaded onto1%agarose gel, recovered by agarose gel containing the DNA, the target DNA was eluted. HTRA1gene and HTRA1-Mut gene fragment were connected with lentiviral vector (pLenO-GTP) and transformed into E. Coli. The plasmid was purified and verified by EcoR I and BamH I double-enzyme cleavage and gene sequencing and constructed HTRA1gene and HTRA1-Mut gene lentivirus expression vector. Lentiviral vector system include four plasmid system(Tronolab), four plasmids vectorswere high purity extracted without endotoxin. Lentiviral packaging plasmids were transfected into293T cells with the calcium phosphate-DNA coprecipitation method and tested virus titer with doubling dilution.Results:The gene fragment of wild-type HTRA1and mutant HTRA1gene were obtained by PCR amplification, HTRA1was cloned into the lentiviral expression vector pLenO-GTP and packaged lentiviral particles in293T cells. Viral titer:GTP-HTRA1titer of5.1×108TU/ml, GTP-HTRA1-Mut titer of8.6×108TU/ml.Conclusion:HTRA1wild-type gene and mutant HTRA1gene lentiviral vector were successfully constructed and laid the foundation for further study on HTRA1protein function at the molecular level, and also laid the foundation for vascular adventitial fibroblasts model of HTRA1mutant gene. Part Ⅱ Model of vascular Adventitial Fibroblasts transfected with lentiviral expression vector of HTRA1geneObjective:To observe the influence of HTRA1gene mutations on the proliferation activity and migration activity of fibroblasts, the constructed HTRA1wild-type and mutant gene lentiviral gene expression vector was transfected into the adventitia fibroblasts.Methods:The human cerebral vascular adventitial fibroblasts (AF) were subcultured with FM medium. The first generation AF culture was obsreved on cell size, morphology, growth characteristics and arrangement through microscope. Fiberoptic connexin (FN) vimentin and a-smooth muscle actin(a-SMA)were used by immunofluorescence cytochemistry staining techniques to identify cultured cells. The fouth generation cell cultures were transfected with lentivirus.Transfection was divided into three groups:(1) the wild-type HTRA1gene;(2) the mutant HTRA1-Mut gene;(3) empty vector control group. The cell proliferation activity was tested with CCK-8assay. The cell migration activity was tested with Transwell small room.Results:Digestion subculture fusiform fibroblasts, star, or irregular polygonal shape, showing a typical fibroblast-like morphology. FN and vimentin antibody positive identification results and a-SMA antibody negative identification results confirmed vascular adventitial fibroblasts. Green fluorescence was obsreved at48-72hours after lentiviral vector transfection, indicating successful transfection. Cell proliferative activity and cell migration activity in the wild-type gene group were lower than that in mutant gene group.Conclusion:1,HTRA1wild-type gene and mutant gene lentiviral expression vector were successfully transfected into vascular adventitia fibroblasts, which can establish HTRA1gene expression model cells.2, HTRA1mutations result in a decrease on the inhibition of AF proliferation activity and migration activity. Part Ⅲ The expression of HTRA1genes and its influence on the secretion function of vascular adventitial fibroblastsObjective:To test the expression of HTRA1mRNA and HtrAl protein after the wild-type and mutant HTRA1gene transfected into vascular adventitial fibroblasts and its influence on the.secretion function of vascular adventitial fibroblasts.Methods:Collected cell culture supernatant from the wild-type gene, mutant gene and control group at48hours after cell transfection. The content of extracellular matrix (ECM: fibronectin, laminin, collagen type Ⅰ, Ⅲ type, Ⅳ type) in cell culture supernatant was measured by ELISA. Collected the total RNA and protein of three groups. The expression of HTRA1gene, a-SMAmRNA and OPNmRNA in the three groups was detected through real-time PCR and Western blot method.Results:Data of RT-PCR showed the expression of HtrA1mRNA was higher in the wild-type gene than that in the mutant gene group, the control group was the lowest. The expression of a-SMAmRNA and OPNmRNA was higher in the mutant gene group than that in the wild-type gene, the control group was the lowest. Data of Western blot showed the expression of HtrAl protein was higher in the wild-type gene than that in the mutant gene group, the control group was the lowest.Conclusion:HTRA1gene was successfully expressed in vascular adventitia fibroblasts after transfection. The mutant HTRA1gene (1091T>C) result in low expression of HTRA1mRNA and HtrAl protein and high expression of a-SMAmRNA and OPNmRNA. Part Ⅳ Influence of HTRA1gene matations on TGF-β1/Smads/CTGF signaling pathway in vascular adventitia fibroblastsObjective:To test the influence on TGF-β1/Smads/CTGF signaling pathway after the wild and mutant HTRA1gene transfected into vascular adventitia fibroblasts.Methods:Collected the total RNA of three groups:the wild-type gene, mutant gene and control group at48hours after cell transfection. TGF-β1、Smad2、Smad3and CTGF were detected through real-time PCR.Results:Data of RT-PCR showed mRNA level of TGF-β1、Smad2、Smad3and CTGF were higher in mutant gene group than that in wild-type group.Conclusion:The inhibition effect on TGF-β/Smads/CTGF signaling pathway was weakened after HTRA1mutant gene (1091T> C) transfected into vascular adventitia fibroblasts, causing up-regulated expression of TGF-1/Smads/CTGF signal pathway.