Differentiation Of Vascular Smooth Cells Research Articles

Effect of Bushen Huoxue decoction on inhibiting osteogenic differentiation of vascular smooth cells by regulating OPG/RANK/RANKL system in vascular calcification.

To investigate the effects of Bushen Huoxue Decoction (BSHXD) and its underlying molecular mechanisms on inhibiting osteogenic differentiation of vascular smooth muscle cells (VSMCs) in vascular calcification via regulating the mRNA expression of osteoprotegerin (OPG) and the receptor activator of the nuclear factor-kappa B ligand (RANKL). VSMCs from the aortas of rats were cultured in vitro. Osteogenic differentiation of VSMCs was induced by high levels of an inorganic phosphate medium (2.4 mM). BSHXD-containing serum was prepared using the serum-pharmacological method. VSMCs were plated using 6-well plates at an approximate density of 4.0×104 cells/mL and cultured for 10 days. This was followed by the application of different concentrations of BSHXD-containing serum. The percentage of concentrations of BSHXD-containing serum in high, middle and low dosage group was 20%, 10% and 5%, respectively. Calcium nodules were evaluated by alizarin red S staining, and alkaline phosphatase (ALP) activity and calcium deposition were both examined as per the instruction of the test kits on the 3rd, 6th, and 10th days. Protein expression level of ALP and α-smooth muscle actin (α-SMA) were detected by Western blot on the 3rd, 6th, and 10th days. The mRNA expression of the OPG and RANKL were also detected by real-time PCR on the 3rd, 6th, and 10th days. Compared with the control group, BSHXD significantly attenuated the calcium nodules that were examined by alizarin red-S staining. Protein expression levels of α-SMA were up-regulated and ALP were down-regulated on the BSHXD group (P<0.05). BSHXD also attenuated the ALP activity and calcium deposition of the VSMCs (P<0.05). These changes were associated with the effect of BSHXD on up-regulating the expression of OPG mRNA and down-regulating the expression of RANKL mRNA in the process of osteogenic differentiation of VSMCs. BSHXD has a beneficial effect on inhibiting osteogenic differentiation of VSMCs induced by high levels of phosphate. The underlying mechanism appears to be related to the modulation of expressions of OPG mRNA and RANKL mRNA in the VSMCs, thereby preventing the phenotypic changes of VSMCs to an osteogenic phenotype.

Effects of core-binding factor α1 gene silenced by siRNA on calcification of vascular smooth muscle cells induced by high phosphate

To explore the effects of core-binding factor α1 (Cbfα-1) gene silenced by siRNA on osteogenic differentiation and calcification of vascular smooth muscle cells (VSMC) induced by high phosphate in vitro. VSMC were cultured in vitro and passaged 3 to 8 times. Four pairs of Cbfα-1 siRNA were designed and synthesized. Transfection was performed with cationic lipid vectors (Lipofectamine 2000). Transfection conditions were optimized by the FAM fluorescent labeling-siRNA to screen effective siRNA sequences by reverse transcription-polymerase chain reaction (RT-PCR). After transfection with effective siRNA sequences, VSMCs were divided into 4 groups: (1) normal phosphate (Pi 1.3 mmol/L); (2) high phosphate (Pi 2.6 mmol/L); (3) siRNA transfection: high phosphate+Cbfα-1-siRNA; (4) negative transfection control: high phosphate+negative control siRNA. Cbfα-1 and osteopontin (OPN) mRNA and protein expression were detected by RT-PCR and Western blotting. Calcium deposition was visualized by Alizarin stain method. The transfection efficiency was around 55% with a concentration of Cbfα-1 siRNA 100 nmol/L and Lipo 8 µl/ well. Cbfα-1 siRNA 1952 was chosen as the effective sequence with a suppression ratio up to 81.8%. At 24 and 48 h post-transfection, the expression of Cbfα-1 mRNA was significantly lower in siRNA transfection group than that in high phosphate group (0.335 ± 0.059 vs 0.714 ± 0.106, 0.574 ± 0.036 vs 0.726 ± 0.086, all P < 0.01) . At 48 and 72 h post-transfection, the expression of Cbfα-1 protein in siRNA transfection group was significantly lower than that in high phosphate group (both P < 0.01) . While Cbfα-1 gene was silenced by siRNA in siRNA transfection group, the mRNA and protein expression of OPN significantly declined (all P < 0.05) and calcium deposition in cell layers decreased. Cbfα-1 siRNA can effectively inhibit the expression of Cbfα-1 mRNA and protein in VSMC and thus suppress the transformation of VSMC into osteoblast-like cells and calcification induced by high phosphate. Cbfα-1 may become a potential therapeutic target in vascular calcification of chronic kidney disease.

Clinical relevance of coronary artery calcification, as a risk factor for plaque rupture: viewpoint from pathology

Atherosclerotic calcification is usually associated with long - standing atherosclerosis. There is accumulating evidence that both apoptosis and osteogenic differentiation of vascular smooth muscle cells contribute to arterial calcification. Our long experience of cardiovascular pathology over many years revealed that ruptured plaques were closely associated with areas of small calcium deposits. To further clarify the association between patterns of coronary arterial calcification and morphology of atherosclerotic plaques, we studied preinterventional intravascular ultrasound (IVUS) images at the site of culprit lesions in patients with acute myocardial infarction (AMI), unstable angina pectoris, or stable angina pectoris. Our study demonstrated that small calcium deposits within an arc of < 90° significantly more frequent in the culprit lesion segments of AMI patients. Thus, our study revealed that the spotty calcification pattern of the coronary culprit segment is associated with unstable plaques in AMI patients.

Mechanism of vascular calcification

Vascular calcification is clinically important in the development of cardiovascular disease. It has been suggested that apoptosis and chondro/osteogenic differentiation of vascular smooth muscle cells (VSMC) play important roles in the initiation and progression of vascular calcification. During the process of vascular calcification, growth arrest-specific gene 6 (Gas6) mediated-survival pathway is downregulated in VSMC, leading to apoptosis. Chondro/osteogenic differentiation regulated by transcription factors such as core binding factor alpha1 (Cbfa1) or msh homeo box homolog 2 (Msx2), promotes VSMC calcification. It is clear that vascular calcification is an active process regulated by various factors including Gas6, Cbfa1, or Msx2.