| Part one Microlymphatic vessel contractility and relaxation in SHR mesentery.ObjectiveTo explore the microlymphatic vessel contractility and relaxation in SHR mesentery.MethodsAnimals included male SHR and Wistar rats of 3,8 and 13 weeks age. The rats were fixed before the exteriorization of the mesenteric tissue. And then, microscope was used for recording a video of vasomotion of mesenteric microlymphatic vessels. VasTrack was used to assay the relative amplitude and frequency of mesenteric microlymphatic vasomotion and to record diameter of the mesenteric microlymphatic vessels. And two index representing the contractility of mesenteric microlymphatic vessels were calculated.Results(1) Differences in microlymphatic vasomotion between SHR and Wistar rats at various weeks age were observed. No difference in microlymphatic vasomotion was observed between SHR and Wistar rats of 3 weeks age (all P values>0.05). The relative amplitude, contraction amplitude, contraction time, contraction speed, relaxation amplitude, relaxation speed and overall contractile activity in SHR were significantly decreased than that of Wistar rats at 8 weeks age (P＜0.001, P<0.01,P＜0.05, P<0.01, P<0.001, P<0.01, P<0.05). And the differences in frequency, contraction time and contractile fraction between SHR and Wistar rats at 8 weeks age were not significant (all P values>0.05). Compared with age-matched Wistar rats, SHR at 13 weeks age had increased frequency and relaxation speed (both P values<0.05) and decreased relative amplitude, contraction amplitude, contraction time, relaxation amplitude, relaxation time and contractile fraction (all P values<0.001), while the differences in contraction speed and overall contractile activity were not significant (all P values>0.05). (2) Differences in microlymphatic vasomotion between various weeks age of SHR were also observed. Compared with SHR of 3 weeks age, SHR of 8 weeks age had decreased relative amplitude, frequency, relaxation amplitude, relaxation speed and overall contractile activity (P＜0.05, P＜0.05,P＜0.05,P＜0.01, P<0.01). Differences in contraction amplitude, contraction time, contraction speed, relaxation time and contractile fraction between SHR of 3 weeks age and SHR of 8 weeks age were not significant (all P values>0.05). Compared with SHR of 8 weeks age, SHR of 13 weeks age had increased frequency, contraction speed, relaxation speed and overall contractile activity (P＜0.05, P<0.05, P<0.01, P<0.01) and decreased contraction time, relaxation time and contractile fraction(P<0.05, P＜0.01,P＜0.05), while the differences in relative amplitude, contraction amplitude and relaxation amplitude between the two groups were not significant (all P values>0.05).Conclusion(1) Microlymphatic vasomotion is prevalent in mesentery of both SHR and Wistar rats.(2) Hypertension is probably a critical factor causing dysfunction of microlymphatic vasomotion in SHR. (3) Once hypertension arises in SHR, activity in diastole is reduced and contractivity is compromised in the mesenteric microlymphatic vasomotion. (4) In the progression of hypertension, the characterizations of dysfuction of microlymphatic vasomotion is an enchanced contractivity which has an important role in the homeostasis of the body.Part two Matrix metalloproteinases promote calcification of vascular smooth muscle cells by up-regulating bone morphogenetic protein-2 and inducing apoptosisObjectiveTo investigate the role of matrix metalloproteinases (MMPs) in vascular calcification and its relationship with bone morphogenetic protein-2 (BMP-2) in the course of vascular calcification.MethodsThe explant method was applied to culture vascular smooth muscle cells (VSMCs) from the aorta of wistar rat with 8 weeks age. And morphological and immunofluorescent observation were made to identify VSMCs. An in vitro model of vascular calcification was used by treating VSMCs with beta-glycerophosphate (P-GP). To confirm the occurrence of VSMCs calcification, von kossa staining and assay of VSMCs calcium were performed. In order to obseve the phenotype transition of VSMCs, expression of ALP, α-SMA and desmin were detected by western blot. Western blot was applied to investigate the effects of doxycycline (Doxy) on the expression of MMP-2 and MMP-9, and gelatin zymography was applied to observe the effects of Doxy on the activity of MMP-2 and MMP-9. After MMPs inhibition by Doxy, expression of bone morphogenetic protein-2 (BMP-2), Runt-related transcription factor 2 (RUNX2) and msh homeobox homolog 2 (Msx-2) were detected by western blot. To clarify whether the decrease in expressions of RUNX2 and Msx-2 in β-GP plus Doxy group was dependent on BMP-2 down-regulation induced by MMPs inhibition, cells were treated with 100 ng/mL BMP-2 or 100 ng/mL noggin (an antagonist of BMP-2) for 4 hours and incubated with 50μM Doxy in calcification medium for 24 hours, and then expressions of RUNX2 and Msx-2 were analyzed by western blot analysis. To investigate the role of VSMCs apoptosis in vascular calcification, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was applied. And caspase-3 activity and expression level were investigated as well as expression of Bax and Bcl-2.Results(1) Morphological and immunofluorescent observation confirmed the cells cultured are VSMCs. (2) VSMCs treated with β-GP had an elevated calcium content and larger calcification area (P<0.001, P<0.001) compared with control group, demonstrating the presence of VSMCs calcification which can be inhibited by Doxy (P<0.001). And phenotype transition of VSMCs was observed in the P-GP group which was attenuated by Doxy. (3) Western blot showed MMPs inhibition lead to decreased expressions of MMP-2 and MMP-9 (P＜0.01, P＜0.01), and gelatin zymography showed MMPs inhibition lead to decreased activity of MMP-2 and MMP-9 (P<0.01,P＜0.01). (4) MMPs inhibition lead to decreased expressions of BMP-2, RUNX2 and Msx-2 (P＜0.01, P＜0.001, P＜0.01) compared with P-GP group. (5) Interventions with BMP-2 and noggin showed that BMP-2 group had increased expressions of RUNX2 and Msx-2 compared with control group (P＜0.01, P＜0.01), while noggin decreased their expressions compared with control group (P＜0.05, P＜0.05). (6) The ratio of apoptotic cells in β-GP group was higher than that of control group (P＜0.001), and Doxy reduced this increase (P＜0.001). The P-GP group had activated caspase-3 and increased expression of cleaved-caspase-3 compared with control group (P＜0.001, P<0.001), as well as increased expression of Bax and decreased expression of Bcl-2 (P＜0.01,P＜0.05).Conclusion(1) Beta-GP induces calcification of VSMC in vitro. (2) MMPs inhibiton lead to attenuation of VSMCs calcification and phenotype transition of VSMCs in the process of vascular calcification. (3) MMPs inhibition attenuates VSMCs calcification by down-regulating BMP-2 which causes an decreased expression of RUNX2 and Msx-2, two critical proteins in phenotype transition of VSMCs in the process of vascular calcification. (4) MMPs inhibiton attenuates vascular calcification probably by reducing VSMCs apoptosis which is an initial factor in vascular calcification.
|
PDF Full Text Request