Association Of Htra1Gene Mutation In A Family With CARASIL And TGF-β Signaling In Vascular Smooth Muscle Cells

Cerebral autosomal recessive arteriopathy with subcortical infarcts andleucoencephalopathy (CARASIL) is an inherited vascular diseases characterized by youngadult-onset non-hypertension stroke, progressive motor and cognitive impairment, alopeciaand lumbago. Brain MRI demonstrated diffuse leukoencephalopathy and multiple subcorticalinfarcts. Brain autopsy showed concentric thickening of vascular wall, narrowing of thelumen, mild fibrous proliferation of the intima and extensive loss of smooth muscle cells,which was similar with arteriosclerosis. About50%cases were born of the consanguineousparents. The patients would die after about10years from the onset. The first case wasreported by Japanese in1965. And then there were only a few cases reported including a fewfamilies and some sporadic cases because the understanding of the disease was insufficientand the diagnostic tools were not perfect. Up to now, only about50cases have been reportedand most of them were from Japan. In2009, some Japanese scholars collected5families ofCARASIL and found CARASIL was associated with mutations in the HTRA1gene which islocated on chromosome10q. There are9exons in HTRA1gene. To date,4missensemutations and2nonsense mutations had been reported and they were distributed in exon3,4and6where located the HtrA1protease domain. So HTRA1gene mutation might lead to thechange of its activity. But studies about that was insufficient. HtrA1serine protease waswidespread in the body and acted with a lot of target proteins, most of them were extracellularmatrix components. HtrA1serine protease was closely associated with osteoarthritis andmany types of cancers. TGF-β/Smads signaling was associated with HTRA1gene. HtrA1serine protease can inhibit the signaling. HTRA1gene mutations reduced the protease activity,and then followed with the over-expression of TGF-β/Smads, including TGF-β1,BMP-2,BMP-4. That was the cause of lesions on brain small artery and symptom of extra-nervoussystem.Extensive loss of vascular smooth muscle cells can be found pathologically, so studies onthe structure and function of vascular smooth muscle cells are important to explore thepathological mechanism of CARASIL. Up to date, there are no investigations about HTRA1gene transfection to vascular smooth muscle cells. We had reported a CARASIL family inChina for the first time. Our study was mainly on the detection of the HTRA1gene mutationsite, then we intend to build a model of wild and mutant HTRA1gene expressed in vascularsmooth muscle cells, and study on the TGF-β/Smads signaling which is helpful to explor thepathological mechanism of CARASIL. Part Ⅰ Study on the clinicalpathological and genetic characteristicof a CARASIL familyObjective: To explorer the clinicopathological characteristics of a CARASIL family, andanalyze the causative gene.Methods: The clinical, imaging and pathological characteristics were described. Thesequence of the exons from1to33on NOTCH3in chromosome19and that of exons from1to9on HTRA1gene in chromosome10were detected. Meaningwhile the healthy control wasdetected the HTRA1gene.Results: Two patients were born from the consanguineous parents. The age of onset wasabout20to25years old. Electroencephalogram showed diffuse slow waves. Brain MRIdemonstrated diffuse leukoencephalopathy and multiple subcortical infarcts. Skin and suralnerve biopsy showed concentric thickening of vascular wall, narrowing of the lumen and mildfibrous proliferation of the intima. There were no amyloid, PAS granular deposition andultrastructural granular osmiophilic materials (GOMs) on the vascular wall. No mutationswere found in exons1-33of NOTCH3gene. we identified a homozygous T to C missensemutation (c.1091T>C) in exon6in the HTRA1gene of the two patients. Both parents and theproband's daughter had the heterozygous c.1091T>C mutation. We included100healthycontrols admitted to the sequencing of the corresponding exon and didn't find the samemutation.Conclusion: we analyzed the first CARASIL family genetically, and found a new mutation ofHTRA1gene. Part Ⅱ Model of vascular smooth muscle cells transfected witheukaryotic expression vector of HTRA1geneObjective: To build vector of wild HTRA1gene with pEGFP-N1plasmid and mutantHTRA1gene (1091T>C) with pEGFP-N1plasmid. Then the two plasmid vectors weretransfected to vascular smooth muscle cell.Methods: Design one pairs of primer according to the CDS of HTRA1gene, the forwardprimer is5'–CTCAAGCTTCG AATTCATGCAGATCCCGCGCGCCGCTCTTC-3', the reverse primer is5'-GGCGACCGGTGGATCC CG TGGG TCAATTTCTTCGGGAA-3'.Target gene was amplification through PCR. Join the PCR product with the p-EGFP-N1vector, then transform to E.coli and purified the plasmids. Verified by double-enzyme leavageand gene sequencing. This is wild plasmid vector(WT). In another experimental group, wedesign two primers containing the new mutation site (1091T>C) that we have already foundand use the site-directed mutagenesis method to ampliate two pieces of PCR productscontaining the new mutation site, then join the PCR products with the p-EGFP-N1vector andthe mutant plasmid vector (L364P) is obtained. Transform to E.coli and purified the plasmids.Verified by double-enzyme cleavage and gene sequencing. Human vascular smooth musclecells were cultured with SMCM. The cells were detected by immunocytochemical stainingwith α-SM-actin antibody. VSMC were divided into three groups and transiently transfectedwith plasmid by means of DNAfect.(1) WT group: transfected with wild HTRA1-pEGFP-N1plasmid.(2) L364P group: transfected with mutant HTRA1-pEGFP-N1plasmid.(3)controlgroup: transfected with pEGFP-N1plasmid. Estimate the transfection efficiency by flowcytometry.Results:(1)The OD of the wild and mutant plasmid is between1.8to2.0, the concentrition isbetween300to400ng/ul. Two products of4.7kb and1400bp were observed after doubledouble-enzyme cleavage which were corresponding with the length of pEGFP-N1vector andHTRA1gene, and were verified by sequencing.(2) The cultured cells appeared fusiform,triangle and star shapes. The characteristic peak and valley features were evident, and theywere positive to α-SM-actin antibody which indicated the cells were smooth muscle cells.The green fluorescence were observed in three group cells in24to48hours after transfection.The transfection efficiency was about33%.Conclusions: We successfully built the wild and mutant HTRA1gene plasmid vector andtransfected the vector into vascular smooth muscle cells by transient transfection method.Model of vascular smooth muscle cell with HTRA1gene was built. Part Ⅲ Expression of HTRA1gene in vascular smooth musclecellsObjective: To test the HTRA1mRNA and HtrA1expression after the wild and mutantHTRA1gene transfected into vascular smooth muscle cells. Methods: Collected the cells of three groups48hours after transfection and extrated the totalRNA and proteins. The expression of HTRA1gene was detected through real-time PCR andWestern blot method.Results: The HTRA1gene expression can be detected in the three groups. Data of RT-PCRshowed HTRA1mRNA level in L364P group and WT group were higher than that in thecontrol group, and HTRA1mRNA in L364P was lower than that in WT group. Results ofWestern blot demonstrated that the HtrA1protein expression of the two transfection groupswere high than that of the control group and the expression was less in L364group than thatin WT group.Conclusions: HTRA1gene was successfully expressed in vascular smooth muscle cells aftertransfection. The mutant HTRA1gene (1091T>C) result in the low expression of HTRA1mRNA and HtrA1protein. Part Ⅳ Fluence on TGF-β1/Smads signaling of HTRA1gene invascular smooth muscle cellsObjective: To test the change of TGF-β1/Smads signaling after the wild and mutant HTRA1gene transfected into vascular smooth muscle cells.Methods: Collected the total RNA and protein of three groups (control group, WT group andL364P group)48hours after transfection and extrated the total RNA and proteins. TGF-β1,Smad2/3/4and phosphorylated Smad2/3/were detected through real-time PCR and Westernblot method.Results: Data of RT-PCR showed mRNA level of TGF-β1and Smad2/3were higher inL364P group than that in WT group, and the differences were significant(P<0.05). The smad4mRNA level was not different in the two groups. Results of Western blot demonstrated thatthe Smad2/3protein expression in L364P group were high than that in WT group andphosphorylated Smad2/3/were detected only in L364P group. Smad4of L364P decreased alittle and the different was not significant.Conclusion: The mutant HTRA1gene (1091T>C) might increase the TGF-β1/Smadssignaling.