Effects Of FGF13 On Blood Pressure Of Mice And The Phenotypic Transformation Of Vascular Smooth Muscle Cell

Vascular smooth muscle cell（VSMC）is the main cell type of vascular wall,which plays an important role in maintaining vascular morphology and function and the stability of blood pressure.In normal blood vessels,VSMC is mostly maintained in contractile static morphology,high expression of the corresponding smooth muscle cell specific contractile markers,such as smooth muscle 22kd calcium binding protein（SM22α）,serum response factor（SRF）,myosin heavy chain 11（MYH-11）,etc.However,when blood vessels are damaged or stimulated by pathophysiological factors,VSMC will undergo phenotypic transformation,from contractile static morphology（differentiationphenotype）toproliferativesynthesismorphology（dedifferentiation phenotype）,and the proliferation and migration ability of VSMC will become stronger.The expression of the specific contractile marker of smooth muscle cells will decrease.The occurrence of vascular diseases,such as hypertension,atherosclerosis,pulmonary hypertension,postoperative restenosis and so on,are accompanied by the transformation of VSMC phenotype.Through reading the literature,we found that the research of FHF subfamily is more focused on the nervous system,heart and tumorigenesis,but less on the vascular system.Recently,FGF12 has been found to maintain the contractile static phenotype of VSMC,and can inhibit intimal thickening caused by the model of arterial injury,which can be used as a potential therapeutic target for vascular diseases such as atherosclerosis in clinical.FGF13 and FGF12 belong to the same subfamily.We speculate that FGF13will also have an impact on the vascular system and the phenotypic transformation of VSMC.In order to verify this conjecture,we have carried on the preliminary exploration in the animal model and the cell level respectively.Part Ⅰ Construction of vascular smooth muscle cell specific knockout FGF13 mice and detection of blood pressure and vascular structureObjective:To prepare and use vascular smooth muscle cell specific knockout FGF13 mice to study the effect of FGF13 deletion on blood pressure and vascular structure.Methods:（1）Vascular smooth muscle cell specific knockout mice were obtained by mating FGF13-loxp mice with Tagln-Cre mice.The knockout mice were identified by PCR and the knockout efficiency of FGF13 was verified by qPCR techniques.（2）The blood pressure of mice was measured by noninvasive tail sleeve method and carotid artery catheterization,and to observe the effect of knockout FGF13 on blood pressure.（3）Doppler ultrasound was used to detect the related indexes of blood flow velocity and vascular wall thickness,and to observe the effect of knockout FGF13 on vascular structure.Results:1.Vascular smooth muscle specific knockout FGF13 mice were successfully obtained.qPCR results showed that FGF13 decreased significantly in knockout mice,suggesting that the knockout effect was good.2.The non-invasive tail sleeve method showed that the vascular smooth muscle cell specific knockout FGF13 mice（KO）which systolic blood pressure（SBP）,diastolic blood pressure（DBP）,mean arterial pressure（MAP）,pulse pressure（PP）were significantly increased compared with control mice(WT,Cre,FGF13^+/-).Carotid artery catheterization showed that SBP,DBP,MAP in KO mice was significantly higer than in WT mice.3.Doppler vascular ultrasound showed that under normal physiological conditions,KO mice compared with WT mice,there was no significant change in the related indexes of blood velocity（VTI,mean Vel）and the related indexes of vascular wall thickness（Ao Diam（d）,Ao Diam（s）,Depth）.Conclusion:vascular smooth muscle cell specific knockout of FGF13can significantly increase blood pressure,and has no significant effect on the structure of blood vessels under normal physiological conditions.Part Ⅱ Effect of FGF13 on phenotypic transformation of VSMC.Objective:To investigate the effect of FGF13 on the phenotypic transformation of VSMC at the cellular level,and to observe the effect of overexpression and knockdown of FGF13 on the proliferation and migration of VSMC.Method:（1）The primary VSMC of SD rats were cultured by tissue block method.（2）qPCR technique was used to detect the expression of the specific contractile markers（SM22α,MYH-11）in KO mice and WT mice;qPCR technique was used to detect the expression of SM22αand SRF in SD rat’s VSMC which overexpressed and knocked down FGF13 respectively.（3）MTS kit was used to detect the optimal stimulation concentration of PDGB-BB,to detect the change of FGF13 expression level of VSMC stimulated by PDGF-BB,and to detect the expression of related specific contractile markers.（4）MTS proliferation assay was used to detect the effect of overexpression and knock down of FGF13 on the proliferation of VSMC.（5）The effect of overexpression and knock down of FGF13 on the migration of VSMC were detected by cell scratch test,and the overexpression efficiency was verified by qPCR and western blot techniques.Results:1.VSMC were cultured for about 7 days,about 80%of them climbed out of the vascular tissue,recorded as P0.The cells grew well and continued to pass,it can be seen that the typical"valley-peak"growth state of VSMC,recorded as P3,The 3-5 generations of desirable cells were be used.2.The results of qPCR showed that the expression of SM22αin KO mice was significantly lower than that in WT mice,but the expression of MYH-11was not significantly affected;The expression level of SM22αin SD rat’s VSMC overexpressed FGF13（FGF13-OE）group was significantly higher than Vector group,but the expression of SRF was not significantly affected;The expression of SM22αin knocked down FGF13（FGF13-KD）group was significantly lower than Vector group,but the expression of SRF was not significantly affected.3.The optimal concentration of PDGF-BB was 30ng/ml by MTS assay.The results of qPCR showed that the expression of FGF13 was significantly decreased under the stimulation of PDGF-BB,the expression level of SM22ɑand SRF were decreased significantly.4.The results of MTS cell proliferation assay showed that overexpression of FGF13 significantly inhibited the proliferation of VSMC induced by PDGF-BB,and knockdown of FGF13 significantly increased the proliferation of VSMC induced by PDGF-BB5.The results of cell scratch test showed that the wound healing ability of VSMC was significantly lower than that of Control group and Vector group after overexpression of FGF13 and the wound healing ability of VSMC group was significantly higher than that of Vector group after VSMC knocked down FGF13.Conclusion:Knockdown of FGF13 promoted the transformation of VSMC from contractile static morphology to proliferative and synthetic morphology;in the proliferative phase of VSMC,the expression level of FGF13 decreases significantly;FGF13 regulates the proliferation and migration of VSMC.