tram-34 and Vascular-Calcification

To observe the role of TRAM-34 (1-((2-chlorophenyl)diphenylmethyl)-1H-pyrazole), the blocker of intermediate conductance calcium-activated potassium channel (KCa3.1), on β-glycerophosphate induced vascular calcification in vitro.. Vascular smooth muscle cells(VSMCs) were obtained from rat thoracic aorta, and VSMCs after the fourth passage and aortic rings were divided into control group (cultured in DMEM with 10% fetal bovine serum), high phosphorus group (cultured in DMEM with 10% fetal bovine serum and 10% β-glycerophosphate) and TRAM-34 group(20 nmol/L TRAM-34 was added into high phosphorus DMEM). Calcium deposition of VSMCs and aortic rings were measured by o-cresolphthalein complexone method.Calcium influx of VSMCs was measured by immunofluorescence probe Fluo-3 AM.The expression of runt-related transcription factor 2(Runx2)was detected by RT-PCR and Western blot for cells and immunohistochemistry for aortic rings.ALP activity was measured by alkaline phosphatase activity detection kit.. (1) Compared with control group, calcification was significantly increased in high phosphorus group ((121.67±6.17) mg/g vs. (84.38±8.17) mg/g, P<0.05) and this effect could be attenuated by TRAM-34 ((93.31±11.36) mg/g, P<0.05 vs. high phosphorus group) after 12 days culture. Similar results were found in aortic rings cultured for 12 days-high phosphorus group: (7.17±0.57) mg/g vs.. (1.18±0.13) mg/g (P<0.05) and TRAM-34: (4.71±0.42) mg/g, P<0.05 vs. high phosphorus group.(2) Compared with control group, the calcium influx was higher in high phosphorus group (349.22±40.47 vs. 151.67±16.94, P<0.05) and reduced in TRAM-34 group (194.67±22.21, P<0.05 vs. high phosphorus group) in VSMCs simulated for 4 days. (3) Both mRNA and protein expressions of Runx2 in high phosphorus groups were higher than in control group (0.630±0.033 vs.0.340±0.058 and 0.865±0.031 vs.0.414±0.011, both P<0.05) and lower in TRAM-34 group (0.399±0.023 and 0.575±0.014, both P<0.05 vs. high phosphorus group) in VSMCs simulated for 4 days.Besides, compared with high phosphorus group, the expression of Runx2 was decreased in control group(0.113±0.010 vs.0.067±0.008, P<0.05) and TRAM-34 group (0.069±0.006, P<0.05) after aortic rings were cultured for 4 days. (4) Compared with control group, the activity of ALP was significantly increased in high phosphorus group (96.56±9.84 vs.46.92±4.60, P<0.05) and decreased in TRAM-34 group(70.20±8.41, P<0.05 vs. high phosphorus group) in VSMCs simulated for 12 days.. KCa3.1 blocker TRAM-34 can inhibit β-glycerophosphate induced VSMCs and aortic ring calcification through inhibiting calcium influx, downregulating Runx2 expression and attenuating osteogenic differentiation.

Topics: Animals; Aorta, Thoracic; Calcinosis; Calcium; Cell Differentiation; Cells, Cultured; Core Binding Factor Alpha 1 Subunit; Glycerophosphates; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Osteogenesis; Potassium Channels, Calcium-Activated; Pyrazoles; Rats; Vascular Calcification

Vascular calcifications are a hallmark of advanced cardiovascular disease in patients with chronic kidney disease. A key event is the transition of contractile vascular smooth muscle cells (VSMC) into an osteoblast-like phenotype, promoting a coordinated process of vascular remodeling resembling bone mineralization. Intermediate-conductance calcium-activated potassium channels (KCa3.1) are expressed in various tissues including VSMC. Aiming for novel therapeutic targets in vascular calcification, we here studied effects of KCa3.1-inhibition on VSMC calcification by the specific KCa3.1 inhibitor TRAM-34. Calcification in the murine VSMC cell line MOVAS-1 and primary rat VSMC was induced by calcification medium (CM) containing elevated levels of PO4(3-) and Ca(2+). Cell signaling, calcification markers, and release of nitric oxide and alkaline phosphatase were assessed by luciferase reporter plasmids, RT-PCR and specific enzymatic assays, respectively. KCa3.1 gene silencing was achieved by siRNA experiments. TRAM-34 at 10nmol/l, decreased CM-induced calcification and induced NO release of VSMC accompanied by decreased TGF-β signaling. The CM-induced mRNA expressions of osterix, osteocalcin, matrix-metalloproteinases (MMP)-2/-9 were reduced by TRAM-34 while osteopontin expression was increased. Further, TRAM-34 attenuated the CM- and TNF-α-induced activation of NF-κB and reduced the release of MMP-2/-9 by VSMC. Finally, TRAM-34 abrogated CM-induced apoptosis and KCa3.1 gene silencing protected VSMC from CM-induced onset of calcification. In summary, TRAM-34 interferes with calcification relevant signaling of NF-κB and TGF-β thereby blocking the phenotypic transition/calcification of VSMC. We conclude that the results provide a rationale for further studies regarding a possible therapeutic role of KCa3.1 inhibition by TRAM-34 or other inhibitors in vascular calcification.

Topics: Alkaline Phosphatase; Animals; Aorta, Thoracic; Apoptosis; Calcium; Cell Line; Cells, Cultured; Gene Expression; Gene Silencing; Intermediate-Conductance Calcium-Activated Potassium Channels; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Myocytes, Smooth Muscle; Nitric Oxide; Osteocalcin; Osteopontin; Phosphates; Potassium Channel Blockers; Pyrazoles; Rats; Transcription Factors; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Vascular Calcification