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Vascular calcification is the major cause of cardiovascular disease in patients with CKD and is shown to be a strong independent predictor of mortality in the dialysis population. Using cell culture models we investigated the potential for uremic serum from patients on haemodialysis to induce intercellular signalling that has a potential to induce calcification.
Following informed consent blood was collected from 10 patients and 6 healthy control and serum prepared. Human Umbilical Vein Endothelial Cells (HUVEC) were treated with the subjects serum (10% v/v) for 24 and 72 hours. After 24 hours cell culture medium was removed and stored. The cells were lysed and mRNA extracted. Following reverse transcription real time quantitative PCR was carried out. After 72 hours cell culture medium was removed and stored. The cells were lysed and total cellular protein was collected for analysis by Polyacrylamide Gel Electrophoresis(PAGE) followed by Western blotting. Bovine aortic smooth muscle cells were incubated with medium collected from treated HUVEC, described above. After 72 hours Alizarin Red S was used for the quantitative analysis of calcification in the smooth muscle cell.
Gene expression of Transforming Growth Factor beta (TGFβ1), CCN2/CTGF (Connective Tissue Growth Factor) and CCN3/Nov were determined by real time PCR. No statistically significant differences were observed between the two groups, i.e. HUVEC treated with control serum versus HUVEC treated with patient serum. Protein expression of vascular endothelial (VE) cadherin was significantly reduced in HUVEC treated with patient serum compared to controls (p<0.05), indicating some loss of phenotype. There was also a significant reduction in MMP9 (Matrix Metalloproteinase 9) protein in HUVEC treated with patient serum (p<0.05). There was an increase in the amount of full length CCN2/CTGF secreted from cells treated with a patients serum with a corresponding decrease in fragments derived by enzymatic degradation. The medium derived from patient serum-treated HUVEC induced significantly more calcification in the smooth muscle cell than the medium from the control group, 0.015 vs 0.011 AU (p< 0.05)
Our data demonstrates the endothelial cells treated with uremic serum from patients on haemodialysis secrete a factor that induces calcification in aortic smooth muscle cells. This is associated with a reduced endothelial cell phenotype. In addition these cells produce less MMP9 and consequently maintain an environment where there is more intact CCN2/CTGF. We hypothesise that CCN2/CTGF is the endothelial derived factor that drives the calcification response. MMP9 enzymatically cleaves CCN2/CTGF between the third and fourth domains. Hence a reduction in MMP9 could result in more of the intact form. Understanding the regulation of this intercellular communication system may allow us to develop targeted therapy to treat vascular calcification in patients with CKD.