title: Inhibition of vascular calcification by block of intermediate conductance calcium-activated potassium channels with TRAM-34.
abstract: 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.
authors.affiliation: Department of Pediatric Nephrology and Center for Cardiovascular Research, Charité - University Medicine, Campus Virchow Clinic, Berlin, Germany. Electronic address: christian.freise@charite.de.
authors.forename: Christian
authors.initials: C
authors.lastname: Freise
authors.affiliation: Department of Pediatric Nephrology and Center for Cardiovascular Research, Charité - University Medicine, Campus Virchow Clinic, Berlin, Germany; Department of Pediatric Nephrology, Charité - University Medicine, Campus Virchow Clinic, Augustenburger Platz 1, 13353 Berlin, Germany. Electronic address: uwe.querfeld@charite.de.
authors.forename: Uwe
authors.initials: U
authors.lastname: Querfeld
mesh_terms: D000469:Alkaline Phosphatase; D000818:Animals; D001013:Aorta, Thoracic / Q000166:cytology; D017209:Apoptosis / Q000187:drug effects; D002118:Calcium; D002460:Cell Line; D002478:Cells, Cultured; D015870:Gene Expression / Q000187:drug effects; D020868:Gene Silencing; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels / Q000037:antagonists & inhibitors* / Q000235:genetics; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D051379:Mice; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D015675:Osteocalcin / Q000235:genetics; D053495:Osteopontin / Q000235:genetics; D010710:Phosphates; D026902:Potassium Channel Blockers / Q000494:pharmacology*; D011720:Pyrazoles / Q000494:pharmacology*; D051381:Rats; D014157:Transcription Factors / Q000235:genetics; D016212:Transforming Growth Factor beta / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D061205:Vascular Calcification / Q000139:chemically induced / Q000188:drug therapy / Q000378:metabolism*
chemical_list: D051660:Intermediate-Conductance Calcium-Activated Potassium Channels; D010710:Phosphates; D026902:Potassium Channel Blockers; D011720:Pyrazoles; C495181:Sp7 protein, rat; C411671:TRAM 34; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D014409:Tumor Necrosis Factor-alpha; D015675:Osteocalcin; D053495:Osteopontin; D009569:Nitric Oxide; D000469:Alkaline Phosphatase; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9; D002118:Calcium
keywords: KCa3.1; NF-κB; TNFα; TRAM-34; VSMC; Vascular calcification