| Objective:Part1:To dynamically monitor and quantitively analyze the red cells velocity in true capillary and leukocyte rolling respectively of Wistar rat and SHR mesentery. The characteristic of mesentery microlymphatic vessel vasomotion in SHR was to be explored.Part2:To establish the method of primary isolation, cultivation and identification of rat aortic endothelial cells (RAECs), investigate the cell biological characteristics between Wistar RAECs and SHR RAECs and explore the effect of matrix metalloproteinase-2(MMP-2) in microvascular rarefaction in vitro deprived from in vivo factors.Methods:Part1:Mesentery exteriorization model was performed on8weeks-aged male Wistar Rat and age-matched SHR. The dynamic courses of microlymphatic vessel vasomotion, leukocyte rolling in microvascular, mast cell morphology and red cells velocity in true capillary were continuously visualized, and the parameters were analyzed by VasTrack automatic detection system to accomplish the quantitive description and analysis.Part2:(1) Aortic arch to iliac arteries of8weeks-aged male Wistar Rat and age-matched SHR under sterile conditions was dissected and intima tunica was exposed to be placed on25cm2cell culture flasks. RAECs were indentified by morphological observation and detection of vWF related antigen by immunoflurescence. Transwell system,"scratch assay" and capillary-like tube formation assay were adopted to determine the permeability, migration and angiogenesis respectively.(2) Primary RAECs were isolated, cultured and indentified with immunoflurescence. The enzymatic activities and proteins expressions of MMP-2and MMP-9in Wistar RAECs and SHR RAECs were examined by gelatin zymography and western blot respectively. Endothelial permeability assay of Wistar RAECs, SHR RAECs and SHR RAECs co-cultured with50μM doxycycline was evaluated with Transwell system by measuring the diffusion of fluorescein isothiocyanate (FITC)-dextran through the RAECs monolayer reaching optical confluence. To detect the location changes of VEGFR2, VE-cadherin and β-catenin, the groups above were incubated with anti-VEGFR2, anti-VE-cadherin and anti-β-catenin, and then observed by fluorescence microscope. The proteins expression of VE-cadherin and β-catenin were quantitatively analyzed. The change in mitochondrial transmembrane potential (MTP) was determined to evaluate the apoptosis of different groups using a cationic fluorescent indicator JC-1.Results:Part1:(1) The red cells velocity in true capillary of SHR mesentery was1402±864μm/s, dramatically increased (p<0.01), compared with Wistar Rat (982±560μm/s).(2) Leukocyte rolling velocity in micro vein of SHR exhibited increasing speed significantly than Wistar (p<0.001), and significantly accelerated leukocyte rolling velocity up to75.10±31.50μm/s in SHR, mainly appeared only in micro vein with diameter30～40μm. The morphology of mast cells in SHR mesentery showed overt degranulation and out of shape to some extent.(3) There was lymphatic vasomotion on most of lymphatic vessels in Wistar rat and SHR mesentery. Relative amplitude of mesenteric microlymph vessel vasomotion i.e., rhythmic oscillation changes of microlymphatic vessel diameter, was attenuated to29.2%±13.11%significantly in SHR compared with that of43.30%±12.50%in Wistar rat (p<0.001). The frequency of SHR mesenteric microlymphatic vasomotion was6.50±0.70cycles/min, slightly lower than that of Wistar rats (8.80±2.60cycles/min) with no statistical significance. During the contraction period, the amplitude, spreading speed and time interval of microlymphatic vessel in SHR were36.00±21.96μm,25.00±8.63μm/s and1.34±0.51s respectively compared with48.00±14.30μm,32.00±11.30μm/s and1.58±0.30s in Wistar rat with statistical significance (p<0.01). During the dilatation period, the amplitude, spreading speed and time interval of microlymphatic vessel in SHR were11.30±4.36μm,11.30±4.36μm/s and2.67±1.30s respectively compared with46.00±14.30μm,16.70±7.70μm/s and3.24±1.60s in Wistar rat with dramatically decreased in the amplitude and spreading speed.Part2:(1) Wistar RAECs and SHR RAECs were isolated successfully. Optically confluent Wistar RAECs showed "cobblestone-like" shape under phase-contrast microscopy, SHR RAECs during the same period were observed to be irregular and non-confluence in shape with interendothelial fenestrations and denuded areas; vWF by immunofluorescence staining of RAECs was positive.(2) There were significant biological difference between Wistar RAECs and SHR RAECs. Compared with Wistar RAECs, SHR RAECs presented hyper-permeability and declined migration and angiogenesis.(3) Compared with Wistar RAECs, the selective MMP-2enzymatic activity of SHR RAECs was significantly elevated (p<0.01), but no change of MMP-9enzymatic activity in SHR RAECs (p>0.05). The elevated MMP-2protein expression in SHR RAECs was further confirmed by western blotting. Incubation of SHR RAECs with50μM doxycycline inhibited MMP-2protein expression effectively (p<0.05). SHR RAECs showed increased permeability significantly at different time interval compared with Wistar RAECs, while doxycycline (50μM) prevented SHR RAECs hyper-permeability dramatically. The extracellular domain density of VEGFR2was reduced in the SHR, which could be protected by doxycycline (50μM) pretreatment and an obvious disruption of VE-cadherin and β-catenin in SHR RAECs. Protein expressions of VE-cadherin and β-catenin in SHR were reduced conspicuously (p<0.01), compared with Wistar RAECs by Western blotting, and the reduction could be reversed by co-culture with50μM doxycycline. There was a decrease in red mitochondrial fluorescence which showed a loss of mitochondrial transmembrane potential in SHR RAECs and could be partially reversed to red fluorescence indicating protection of MTP co-cultured by doxycycline (50μM).Conclusion:Part1:The red cells velocity in true capillary was increased compensatively in8-aged SHR mesentery. Leukocyte rolling in microvein of8weeks-aged SHR was accelerated significantly on microveins with diameters in30-40μm, which could be attributed to the lower inflammation degree than compensively accelerated blood flow rate in microvein with diameter in30～40μm. Microlymphatic vasomotion dysfunction with declined frequency, amplitude, and attenuated constriction activities might be related with hemorheologic changes and inflammation in SHR.Part2:The isolation method of primary RAECs was established and SHR RAECs exhibited lower ability of migration and angiogenesis but hyper-permeability. Enhanced MMP-2activated endothelial hyperpermeabity via VEGFR2, VE-cadherin and β-catenin cleavage resulting apoptosis, which might be a mechanism of microvascular rarefaction in SHR. |
PDF Full Text Request