| Part OneThe alterations of microcirculation in mesentery of spontaneously hypertensive ratsObjective:To dynamically monitor and quantitively analyze leukocytotic rheological properties of venule, the red blood cells velocity (RBCV) of true capillary, degranulation of mast cell and microvascular tortuosity in different age of spontaneously hypertensive rats (SHR) mesentery.Methods:Mesentery exteriorization model was performed on 3,8 and 13 weeks male Wistar Rats and age-matched SHR respectively. The leukocytotic rheological properties of venule, the RBCV of true capillary, degranulation of mast cell and microvascular tortuosity were continuously visualized, and the parameters were analyzed by VasTrack automatic detection system to accomplish the quantitive description and analysis.Results:(1) Leukocytotic rheological properties:there were no statistical differences in leukocyte rolling velocity, number of rolling leukocyte and total leukocyte-endothelium contact time (TLECT) between 3-week-old of SHR and Wistar rats. However, leukocyte rolling velocity exhibited significantly increasing speed (8 W:72.73±36.12μm/s vs 36.09±23.39 μm/s, P<0.01; 13 W:81.33±24.37 ⊙m/s vs 35.89±11.37 μm/s, P<0.01), the number of rolling leukocyte were significantly decreased (8 W:41.38±21.79 cells/min vs 60.83±28.87 cells/min, P<0.01;13 W:38.73±22.79 cells/min vs 56.33±29.54 cells/min, P<0.01), and TLECT were prolonged (8 W:142.67±27.51 s/min·100μm vs 116.24±17.33 s/min·100μm, P<0.01; 13 W:327.29±56.44s/min·100μm vs 150.34±35.81s/min·100μm, P<0.01) in both 8 and 13 weeks age of SHR compared with the same weeks age of Wistar rats. Furthermore, the number of rolling leukocyte was gradually increased and TLECT was prolonged with age in SHR. (2) The RBCV in true capillary:There was no significantly difference in 3-week-old of SHR compared with the same weeks age of Wistar rats. RBCV was significantly higher in 8-week-old of SHR than the same weeks age of Wistar rats (1402±684μm/s vs 982±560 μm/s, P<0.01). Nevertheless, in 13-week-old of SHR, the RBCV was lower that Wistar rats (874±335μm/s vs 1153±617μm/s, P<0.05). (3) Degranulation of mast cells:There was no statistical difference in the number of mast cells between 3-week-old of SHR and Wistar rats. However, the number of mast cells in 8 and 13-week-old of SHR was obviously more than that in the same age of Wistar rats (8W:10.51±3.8 vs 8.4±2.2, P<0.01;13W: 15.6±3.8 vs 10.13±3.3, P<0.01). The degranulation percentage in the three groups of SHR were significantly increased compared with the same age of Wistar rats (3W: 75%±14% vs 25%±18%,P<0.01; 8W:83%±9% vs 33%±8%, P<0.01; 13W:90%±4% vs 46%±20%, P<0.01). (4) Microvascular tortuosity:Tortuosity was no significant change in arteriole of SHR including 3W,8W and 13W compared with Wistar rats. There was statistical change only in 3 weeks age of SHR venule (1.83±0.49 vs 1.29±0.16, P<0.01), and in 8W,13W of SHR, there were no significant changes compared with Wistar rats.Conclusion:With the progession of hypertension, mesenteric microcirculation of SHR manifested that a:leukocyte rolling velocity was speeded up. b:the number of rolling leukocyte was reduced.c:TLECT was prolonged; RBCV was firstly compensatory increase, and then decompensatory decrease.d:the number of mast cells around of microvascular was increased, and the degranulation percentage was increased.e: increase of microvascular tortuosity mainly occurred venule in the early stage of hypertension.Part TwoEndothelial cells promote calcification in aortic smooth muscle cells from spontaneously hypertensive ratsObjective:To investigate the effects of endothelial cells (ECs) on calcification of smooth muscle cells (SMCs) isolated from aortas of spontaneously hypertensive rats (SHR).Methods:ECs and SMCs were isolated from aortas of SHR and age-matched Wistar rats respectively, and identified by morphology and immunofluorescence of α-SMA and vWF. ECs and SMCs from each type of rats were divided into three groups:co-culture of SMCs with ECs, conditioned culture of SMCs with the medium of ECs, and control culture without any treatment. The alterations of calcium deposition, osteogenic transition, the expressions of BMP2, Runx2, Msx2, MMP-2, MMP-9, OPN and MGP in SMCs, and the expressions of BMP2, OPN, MGP, MMP-2 and MMP-9 in ECs of SHR were compared with those of Wistar rats by von Kossa staining, calcium content, ALP activity and western blot, respectively.Results:(1) RASMCs and RAECs were isolated successfully from aortas of Wistar rats and SHR, and identified by morphology and immunofluorescence. (2) Calcium deposition:Both calcification area and calcium content increased in SHR RASMCs than the control culture SMCs of SHR (calcium content:58.38±7.95μg/mg protein vs 24.30±3.22μg/mg protein, P<0.01) when SMCs were co-cultured with ECs for 6 days. The calcification deposition in SMCs of SHR (calcium content:30.15±5.42μg/mg protein) were still higher than the control culture (P<0.01) after treated with the medium of ECs for 6 days. However, there were no statistical difference both in co-culture and conditioned culture of Wistar RASMCs. (3) ALP activity:ALP activity was significantly higher than the control culture SHR RASMCs (106.28±16.63 unit/mg protein vs 75.87±6.06 unit/mg protein,P<0.01) after RASMCs were co-cultured with RAECs for 6 days. Compared with the control culture, ALP activity in SHR RASMCs was still increased (86.73±9.15, P<0.05) when SMCs treated with the medium of ECs for 6 days. Nevertheless, there was no statistical change in Wistar RASMCs when RASMCs were co-cultured with RAECs. (4) The expressions of BMP2, Runx2 and Msx2:The expression levels (vs. control) of BMP2, Msx2 and Runx2 in SHR RASMCs were significantly higher than those of Wistar RASMCs (BMP2:2.88±0.12 fold vs 1.43±0.21 fold, P<0.01; Msx2:3.10±0.45 fold vs 1.53±0.48 fold, P<0.01; Runx2:1.76±0.09 fold vs 1.26±0.08 fold, P<0.01) after 6 days of co-culture. The levels of those protein in SHR RASMCs were still increased compared with Wistar RASMCs (BMP2:1.84±0.37 fold vs 1.22±0.22 fold, P<0.05; Msx2:1.94±0.34 fold vs 1.26±0.19 fold, P<0.01; Runx2:1.39±0.02 fold vs 1.07±0.13 fold, P<0.05) when SMCs were cultured with the medium of ECs for 6 days. The expression level of BMP2 in SHR RAECs was significantly enhanced than that of Wistar rats (3.03±0.52 fold vs 1.19±0.29 fold, P<0.01) after co-culture for 6 days. (5) The expressions of MMP-2 and MMP-9:Compared with control culture SMCs, there were no significant change of expressions of MMP-2 and MMP-9 in both RASMCs of SHR and Wistar rats when SMCs were co-cultured or conditionally cultured for 6 days. However, the expression levels of MMP-2 and MMP-9 in co-cultured RAECs of SHR were remarkably higher than that of Wistar rats (MMP-2:2.18±0.48 fold vs 1.38±0.38 fold, P<0.01; MMP-9:1.84±0.40 fold vs 1.16±0.21 fold, P<0.01). (6) The expressions of MGP and OPN:The expression levels of MGP and OPN in RASMCs of SHR were increased compared with those protein expressed in Wistar rats (MGP: 3.13±0.51 fold vs 1.51±0.37 fold, P<0.01; OPN:1.91±0.16 fold vs 1.56±0.23 fold, P<0.05) after SMCs were co-cultured with ECs for 6 days. Furthermore, the levels of those proteins in RAECs of SHR were higher than Wistar rats (MGP:2.33±0.34 fold vs 1.26±0.48 fold, P<0.01; OPN:1.74±0.09 fold vs 1.28±0.32 fold, P<0.05). There were no statistical differences in MGP and OPN of RASMCs between SHR and Wistar rats when SMCs were cultured alone with the medium of ECs. However, the expressions of MGP and OPN in both RASMCs of SHR and Wistar rats were still upregulated compared with the control.Conclusion:RAECs of SHR have the ability to drive the calcification of aortic SMCs isolated from SHR via BMP2-Msx2/Runx2 signaling pathway. MMP-2 and MMP-9 excreted from SHR RAECs facilitate the calcification of SHR RASMCs. The compensatory increases of MGP and OPN fail to interrupt the calcification-promoting effect of SHR RAECs on SHR RASMCs. |
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