Effect Of Uremic Serum On Calcification And The Expression Of Type â…  Collagenin Vascular Smooth Muscle Cells

Objective: Cardiovascular disease (CVD) is the leading cause of death inpatients with chronic kidney disease (CKD), accounted for more than50%ofthe mortality in uremic patients. Researches showed that vascular calcificationand its progression is the independent risk factor for CVD. Recent studiesdemonstrate that vascular calcification is an active, cell-mediated, highlyregulated, programmed process, resembling the ossification in bone andcartilage formation. Vascular smooth muscle cells (VSMCs) is the major celltype in vessel medial layer. Once transdifferentiating into osteochondrogenicphenotype, the expression of the key transcription factors (such ascore-binding factor alpha-1, Cbfa1/Runx2) increase, and then VSMCs cansecrete collagen (such as typeⅠcollagen, ColⅠ), non-collagen protein, whichplays a very important role in vascular calcification. In this study, we use theuremic serum to create uremia environment, and stimulate VSMCs withdifferent concentrations. We observe the changes of VSMCs calcification andthe expression of type Ⅰcollagen to explore the influence of uremic serum onVSMCs calcification and analyze the role of typeⅠcollagen during theprocess. Furthermore we try to demonstrate the role and mechanism of typeⅠcollagen in uremia vascular calcification.Methods:1Serum preparation:12patients with maintenance hemodialysis from bloodpurification center of the Fourth Hospital of Hebei Medical Universityfrom June2010to July2011and10normal healthy volunteers wereselected.4ml fasting venous blood was collected in the morning beforedialysis, and then centrifuged (1800rpm×15min), collected supernatant in1.5ml Eppendof tube,56℃deactivated, use0.22μm membrane filter toremove bacteria and stored at-80℃. 2Primary VSMCs were obtained from rat vessel wall by mass of tissueadherence method, building a cell line, and confirmed with morphologicaland immunocytochemistry methods.3Groups and the treatment: VSMCs were randomly divided into thenegative control group, the normal serum groups and the uremic serumgroup three groups, the negative control group were cultured with normalmedium containing10%fetal bovine serum given high phosphate (10mmol/L forβ–glycerolphosphate). On the basis of this, the other twogroups were divided into normal serum and uremic serum groups, eachgroup set up three serum concentrations of5%,10%,15%(low, medium,high). After6days stimulation, we examined the expression of ColⅠandCbfa1and calcifications.4Using Alizarin red staining and Von Kossa staining to detect thecalcification of VSMCs. Levels of calcium in cell cultures were determinedcolorimetrically by the o-cresolphthalein complexone method.5Using RT-PCR and Western-Blot respectively to detect the effect ofdifferent concentrations of uremic serum on Cbfa1mRNA and type Icollagen mRNA and protein levels in VSMCs.6Statistical methods: Analyses were performed using SPSS13.0software.The measurement data were described in (x±S). Differences between thetwo groups were compared by t test, differences among multi-groupswere compared with one-way analysis of variance (ANOVA) andStudent-Newman-Keuls (SNK) was used as pairwise comparison.Relationships between variables were tested by Spearman correlationanalysis. Statistical significance was defined as P<0.05.Results:1Mass of tissue adherence method: Cells could be seen to climb out oftissue on the3rd day in primary culture. On the6th to7th day, when cellsbecame schistic, they could be subcultured. Using inverted phase contrastmicroscope, HE staining and Switzerland-Giemsa staining, we observed thatcells showed a typical "peak-valley" appearance with spindle (in major), star or irregular shape with abundant cytoplasm and round or ellipse nucleus. Toidentify these cells, we performed S-P immunohistochemical stainings fora-smooth muscle actin (a-SMA actin). a-SMA actin were strong positivelyexpressed in cytoplasm with brown yellow immune reaction product.2Alizarin red stain and Von Kossa stain showed that compared with normalgroup, VSMCs in all concentrations of uremic groups showed significantlycalcification. And as time went on, the calcification became worse.3Calcium content test results revealed that compared with the control group,no significant changes in the calcium content after serum stimulation ofhealthy people. Uremic serum stimulation increased calcium content (P>0.05).Compared with the control group, the content of calcium in VSMCs increasedin uremic groups in a dose dependent manner (P>0.05).4RT-PCR for Cbfa1mRNA showed that, compared with negative controlgroup and the normal serum groups, Cbfa1mRNA in uremic group wasincreased in a dose-dependent manner (P>0.05). The pairwise comparison ofuremic serum of low, medium, and high concentration, the differences werestatistically significant (P>0.05) and also in dose-dependent manner.5RT-PCR for ColⅠ mRNA showed that, compared with negative controlgroup and the normal serum groups, ColⅠ mRNA in uremic group wasincreased in a dose-dependent manner (P>0.05). The pairwise comparison ofuremic serum of low, medium, and high concentration, the differences werestatistically significant (P>0.05) and also in dose-dependent manner.6Western-Blot for ColⅠprotein showed that, the was no ColⅠ proteinexpression in the cells in all the groups of the negative control group,thenormal group and the uremic serum group,other wise it was expression in thecells supernatant. compared with negative control group and the normal serumgroups, ColⅠprotein in uremic group was increased in a dose-dependentmanner (P>0.05). The pairwise comparison of uremic serum of low, medium,and high concentration, the differences were statistically significant (P>0.05)and also in dose-dependent manner.7The Cbfa1mRNA was positively related with type I collagen mRNA in uremic groups(r=0.784, P=0.012).8The type I collagen protein was positively related with calcium content inuremic groups(r=0.878, P=0.002).Conclusion:1We successfully build a vascular smooth muscle cell line with mass oftissue adherence method.2Uremic serum can aggravate VSMCs calcification in aconcentration-dependent manner. It may be associated with the level of uremiatoxin and VSMCs phenotype transformation.3Uremic serum can elevate the expression of typeⅠcollagen at genetic andpost-transcriptional levels in a dose-dependent manner. It may be associatedwith uremic toxin level and increased Cbfa1levels.