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Altered programs of cellular differentiation (or phenotypic modulation) underlie most complex diseases. Within the vasculature, smooth muscle cells (SMC) exhibit phenotypic modulation in which their normal differentiated program, defined as the expression of genes encoding for contractile/cytoskeletal proteins that assemble myofilaments (A), is subverted to one of growth, migration, and matrix secretion with concomitant loss in normal myofilament array (B). The latter occurs in such vascular complications as atherosclerosis or restenosis following balloon angioplasty (C). Current efforts are devoted to an understanding of the biology of five genes expressed in differentiated vascular SMC (A): serum response factor (SRF) and its potent coactivator, myocardin (MYOCD) which constitute a master switch for SMC gene expression; a direct target of this transcriptional switch called SMC calponin (CNN1); a tumor suppressor gene, AKAP12; and a novel orphan vascular serine carboxypeptidase we call SCPEP1. Several of these genes’ expression is attenuated in the setting of vascular disease although we recently have observed elevated expression of SMC differentiation genes in the setting of Alzheimer’s angiopathy.
A major effort in the lab is directed towards studying the transcriptional regulation of SMC gene expression. Gene promoters are cloned and assayed in vitro and ultimately in transgenic mice. Our transgenic mouse models of SMC gene promoters encompass conventional transgenic approaches using the bacterial lacZ reporter gene or large genomic sequences contained in bacterial artificial chromosomes (BACs). Panel D shows how mutating critical SRF-binding CArG boxes within a BAC nullify human CNN1 expression in the aorta of mice. SMC-restricted promoters such as SM22[alpha] have been exploited in tissue-specific knockout studies where we have inactivated SRF in the cardiovascular system and observe profound defects in vascular SMC recruitment and differentiation (E). Defining the activity of SMC gene promoters in vivo provides an opportunity to interrogate DNA sequence elements critical for such regulation. We use a variety of bioinformatic tools to define conserved DNA sequences that may be critical for SMC gene activity. For example, VISTA plots are generated through a process known as comparative genomics (F). The completion of numerous genomes has greatly facilitated this analysis. Regulatory element discovery and identification of variants (SNPs) that may alter function is a major goal in the Miano Lab. One regulatory element we are particularly interested in is the CArG element (G), whose consensus sequence is CC(A or T)6GG. The CArG element binds SRF, which “toggles” between disparate gene programs based on its association with a variety of cofactors. One such cofactor we have been studying is called myocardin. Together SRF-myocardin coordinate biochemical, structural, and physiological attributes of a differentiated SMC. We hypothesize that SRF-myocardin activity is altered in vascular diseases leading to a compromise in expression of many CArG-containing genes such as those encoding for contractile/cytoskeletal proteins. The pipeline of discovery is completed with the evaluation of new SRF-Myocardin target genes (H) in the setting of various diseases such as vascular occlusive disease or Alzheimer’s angiopathy.
Our ideas and efforts span the spectrum from computer to DNA to cells to whole animals. We intend to elucidate the regulation of genes and/or their functions during normal or pathological processes involving, but not limited to, the cardiovascular system. The work in the Miano Lab is necessarily multi-disciplinary and provides ample opportunities for trainees to embrace state-of-the-art technologies in genomics, genetics, bioinformatics, vascular pathobiology, and gene transcription control.Miano's suggested Interesting Links:
Advancing our understanding of the basic mechanisms responsible for normal and pathological function of the cardiovascular system.
