Background: Emerging evidence indicates that an important paradox exists in diseases such as renal failure, diabetes, and osteoporosis: decreased bone calcification correlates with increased vascular calcification (VC) and cardiovascular risk. Retinoids, the metabolites of vitamin A, control gene expression through promoter- specific transcriptional and epigenetic mechanisms. However, studies examining retinoid regulation of these processes during calcification in vessels and bone are lacking. Our data show that the retinoid retinaldehyde (Rald) limits calcification in vascular smooth muscle cells (VSMC) while promoting calcification in bone marrow mesenchymal stem cells (MSC) through differential effects on bone morphogenetic protein 2 (BMP2). These findings frame the central hypothesis of this research proposal: Rald differentially modulates vascular and skeletal calcification through cell-specific transcriptional and epigenetic effects at the BMP2 promoter.
Aim 1 : Determine the transcriptional and epigenetic basis of Rald's divergent effects on the induction of BMP2 in VSMC and MSC. Chromatin immunoprecipitation (ChIP) and methylation specific PCR (MSP) will be used to study Rald's effects on: 1) transcription factor and coregulator recruitment to the BMP2 promoter, 2) DNA methylation at BMP2 promoter and enhancer DNA elements, and 3) Histone methylation and acetylation at BMP2 promoter and enhancer DNA elements. Two models of high Rald levels will be used: 1) Primary wild type VSMC and MSC treated with Rald, and 2) Primary VSMC and MSC isolated from mice deficient in retinaldehyde dehydrogenase 1 (Aldh1a1), the enzyme that metabolizes Rald.
Aim 2 : Determine the in vivo effects of Rald on VC and bone loss induced by a high fat diet (HFD) in LDL receptor deficient (LDLR-/-) mice. Two strategies will be employed to increase Rald levels in LDLR-/- mice: 1) generating Aldh1a1-/-/LDLR-/- mice, and 2) Aldh1a1 antisense oligonucleotide treatment. BMP2 expression and BMP2 promoter events studied in Aim 1 will be assessed using real time PCR, ChIP, and MSP in bone and aorta. In addition, VC and bone loss will be assessed using fluorescence imaging and histology (VC and bone calcification) and micro CT (bone density). Significance: These studies hold implications for understanding links between vessels and bone and developing novel therapies that target dysregulated vascular and skeletal calcification in a coordinated manner.
Emerging data indicate that vascular and skeletal health are closely coupled. The central aim of this research proposal is to determine how retinoids; which are derivatives of vitamin A; coordinately regulate mineralization in blood vessels and bone.
