Although we are interested in many areas  of cardiovascular physiology and pathophysiology, the main focus of the laboratory  is on the regulation of smooth muscle cell (SMC) growth and differentiation.  The SMCs that line the blood vessels and many of the hollow organs of the GI tract and airways are very important for maintaining the structure and integrity of these organs and for regulating flow (i.e. blood, food, or air)  through these systems.  SMC differentiation is very important during the formation of new blood vessels, and alterations in the differentiation state of SMC have been shown to contribute to the development of atherosclerosis and hypertension.  Therefore, it will be critical to identify the mechanisms that regulate this process.

Experiments in our lab are designed to identifying the following;

   1) the transcriptional mechanisms that regulate SMC-specific gene expression - Since a SMC’s  main function is that of contraction, it expresses a range of SMC-specific contractile associated proteins (including SMC-specific isoforms of actin and myosin) that allow it to perform this specialized task.  We study how these "differentiation marker genes" are regulated to get a better  understanding  of the transcription pathways that ultimately govern SMC differentiation.   

   2) the signaling pathways by which environmental cues regulate SMC differentiation - It is well established that SMC phenotype is regulated by a complex array of local environmental cues including growth factors, cell-cell and cell-matrix interactions, inflammatory stimuli, and mechanical stresses. However, the mechanisms by which these diverse signals are integrated to regulate SMC phenotype are largely unknown.

   We use a large number of molecular and genetic approaches to study SMC differentiation.  Experiments in primary SMC culture models have demonstrated that the expression of nearly all of the SMC differentiation  marker genes is regulated by the transcription factor, serum response factor (SRF), and we have developed several transgenic mouse models to show that SRF is important for regulating SMC-specific transcription  in  vivo (see figure below).

   These images were taken from mice containing a Lac Z transgene driven by the SM alpha-actin promoter regions from -2.6 Kb to +2.7 Kb. Notice that in adult organs (lungs and vessels shown at right), this transgene is only expressed in SMC. Besides giving us important information about the mechansisms involved in regulating SMC-specific gene expression, these mice are also extremely useful for studying SMC differentiation in vivo.  The 13.5d mouse embryos shown on the left demonstrate that the the transcription factor, serum response factor (SRF) is important  for regulating SM  alpha-actin  expression  in vivo. Mutations to one SRF binding element within  the SM alpha-actin promoter inhibited transgene expression in all tissues while mutation to a second inhibited expression only in SMC. Mechanisms  in  addition to the presence and activity of SRF are probably involved, and  we  are currently attempting to identify and clone additional transcription  factors  that interact with SRF to regulate SMC-specific transcription.

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   Our studies on SMC signaling are currently focused on the small GTPase, Rho, which is an important regulator of the cytoskeleton. We have recently shown that Rho plays a role in the regulation of SMC-specific  transcription, and interestingly, that such regulation is probably mediated by activation of SRF in a gene-specific and cell-type-specific manner (see figure below). Importantly, many of the environmental  factors that stimulate SMC differentiation have also been shown to activate Rho, suggesting that this signaling pathway may be a common mechanism by which these factors contribute to SMC phenotype.

    The micrographs to the left demonstrate that Rho is an important regulator of the cytoskeleton in SMC. A) SMCs have been transfected with constitutively active Rho. The overexpressed  Rho was c-myc tagged and can be visualized by immunostaining with an anti c-myc antibody. B) SMC expressing constitutively active Rho (large arrows) have substantially increased levels  of stress fibers which can be visualized with the actin binding compound,  phalloidin.   

    Rho also affects SMC-specific transcription. C) Inhibition of Rho with C3 transferase abolished the expression of  the SMC-specific marker genes,  SM22 and SM alpha-actin. D) Transfection of a constitutively active form of Rho into SMC increased expression of the SMC markers. Interestingly, constitutively active Rho did not increase expression of c-fos which is a ubiquitously expressed gene that is also regulated by SRF. This indicates that the effects of Rho may be selective in SMC.

    We are continuing to use cell culture models to further define Rho signaling  in SMC, and we are using and developing transgenic and knockout mouse models to test the importance of rho signaling in vivo. The promoter technology that we have developed will allow us to express  proteins  specifically in SMC which makes this technology a very powerful  tool for studying SMC differentiation.  In addition, our transgenic mice are excellent models for studying transcriptional regulation in vivo which is important when studying gene expression in a cell-type that must integrate a large number of environmental signals. 

    A third project in the lab is an  ongoing  collaboration with my wife, Joan Taylor, who also became a member of the Pathology department at UNC in February 2001.  Joan and I have been studying the role of extracellular matrix signaling on SMC differentiation.    Matrix components signal through the integrin family of cell surface  receptors leading to the formation of complex multiprotein signaling structures  called focal adhesions.  A very important protein present in this complex  is a tyrosine kinase called Focal Adhesion Kinase (FAK) which becomes activated  upon integrin occupation.  Interestingly, SMC in the vasculature express  high levels of a protein that is thought to be an endogenous inhibitor of  FAK.  This protein is called FRNK (FAK-Related Non-Kinase) and is specifically expressed  in large arteries which suggests that it may be important for SMC function.  We have recently  shown that FRNK expression in vascular SMC is regulated during embryonic development  and following vessel injury. We have also shown that overexpression of FRNK decreases SMC growth and migration which suggests that this protein is an  important regulator of SMC differentiation. We are currently studying  FRNK expression patterns in the vasculature during development and atherosclerosis, and we are attempting to determine the role that FRNK plays in SMC. As with the studies on Rho, we are using knockout strategies and our ability to express  proteins specifically  in SMC to study this pathway  in vivo.

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