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Analysis Of The Retinoblastoma Protein In Aortic Valve Calcification
Author(s) -
Freytsis Marina,
Hinds Phil,
Huggins Gordon
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.764.1
Subject(s) - calcification , western blot , aortic valve , gene knockdown , pathology , chemistry , medicine , microbiology and biotechnology , biology , gene , biochemistry
Calcific aortic valve disease (CAVD) shares several features with osteogenesis. This pathological process is similar to normal development of bone, phenotypically and transcriptionally. Because the retinoblastoma protein (Rb) pathway is critical to bone development, we hypothesized that the Rb pathway also regulates development and progression of CAVD. To test this hypothesis we generated a mouse model with cre‐mediated knockout of Rb in the heart valves. These mice were maintained for a year on high fat diet to induce valve disease. Experimental animals (Rb Flox/Flox ;Tie2Cre) showed significantly more aortic valve regurgitation, a manifestation of CAVD in mice, assessed by echocardiography, compared with control animals(Rb Flox/+ ;Tie2Cre). To investigate the molecular mechanism behind this, we infected cultured porcine aortic valve interstitial cells (pAVICs) with lentivirus‐expressed shRNA constructs targeting Rb mRNA. Knockdown of the Rb transcript was confirmed by qRT‐PCR (p<0.005) and we consistently observed reduced Rb protein by Western blot compared to a non‐targeting (NT) shRNA control. Calcification was induced by culturing pAVICs in medium containing ascorbic acid, β‐glycerophosphate, and dexamethasone. After three weeks, we evaluated gene expression by qRT‐PCR. Rb‐deficient pAVICs showed reduced induction of bone‐specific transcripts, as well as the AVIC activation marker α‐SMA. Calcification, detected by alizarin red S staining, was significantly higher in cells treated with osteogenic media for one week (p<0.05), but no difference between pRb deficient and normal pAVICs was observed. These studies support an important role for Rb in aortic valve pathology in mice.