The overall goal of my research is to understand the signaling mechanisms that regulate growth and development  in the cardiovascular system.  The recent focus of my studies has been on the convergence of growth factor and extracellular matrix (ECM) signaling pathways leading to normal and pathophysiological growth responses in two specialized muscle cell types, cardiomyocytes and vascular smooth muscle cells (SMC).  Cardiomyocytes are the differentiated muscle cells in the heart that constitute the bulk of the ventricle wall, whereas vascular smooth muscle cells line blood vessels.  Although both are muscle cell types, cardiomyocytes and vascular SMC vary in their mechanisms of contraction, growth and differentiation.  Notably, cardiomyocytes become terminally differentiated shortly after heart formation and thus loose the capacity to divide; whereas vascular SMC are continualy undergoing modulation from the contractile to proliferative phenotype.  Under various pathophysiological stresses such as hypertension, baloon angioplasty, valvular disease and myocardial infarction, the heart and vessels undergo morphologic growth-related alterations that can reduce cardiac function and eventually manifest in heart failure.  Understanding the precise mechanisms that control growth and differentiation in these cell types is an important step towards developing therapeutic interventions for the treatment of cardiovascular disease, the leading cause of mortality in men and women in the United States.
       
                                                                              

Cardiomyocyte Project:

BACKGROUND:            
Cardiomyocyte growth (hypertrophy), characterized by increased volume and myofibrillar protein content, is important for the normal developmental growth of the heart.  However, in the fully developed adult heart, pressure or volume overload, myocardial infarction or hormonal imbalance can lead to chronic pathological hypertrophy characterized by induction of immediate early genes, re-activation of an embryonic gene program, and reorganization of myocyte cytoskeletal architecture.  Although these changes are compensatory and initially lead to enhanced cardiac output, the final outcome results in heart failure.

The precise mechanisms whereby cardiomyocytes undergo pathological hypertrophy are not completely understood.  However, many of the growth factor signaling cascades that result in aberrant cell proliferation in other cell types (i.e. enhanced activation of Ras/Raf/MEK/ERK) lead to cellular hypertrophy in cardiomyocytes. Studies have shown that many of these growth factor regulated pathways are modified by signaling through integrins, receptors for extracellular matrix (ECM) proteins such as fibronectin and collagen.  Importantly, the expression levels and profile of ECM components and cardiac integrin expression is modulated during the development of cardiac hypertrophy.  Therefore, we hypothesize that altered integrin signaling may play a critical role in the development of pathological hypertrophy.
     
FINDINGS:                                                              
We have found using a cultured cardiomyocyte  cell  model  that indeed agonist-induced hypertrophy is modulated  by plating cells  on various ECM components.  For instance, plating  cultured cardiomyocytes  on laminin and fibronectin supports hypertrophic  signaling whereas plating  on type I collagen does not.  Since signaling through the protein tyrosine  kinase focal adhesion kinase (FAK) appears central to integrin-dependent activation of downstream signals, we hypothesized that FAK might also play a role in hypertrophic signaling.  We showed that FAK activity was elevated following agonist treatment of cardiomyocytes  when cells were    plated on productive ECM.  Also, using a dominant-negative strategy,  we showed that FAK activation was necessary for agonist-induced hypertrophy.    These data support a role for integrin-mediated, FAK-dependent signaling  for induction of cardiac hypertrophy.
        
FUTURE DIRECTIONS:
Our future directions involve the use of biochemical and genetic approaches to identify mechanisms by which FAK influences myocardial growth and development.  One of our major goals is to develop genetically engineered mice that have cardiac-specific deletion of FAK.  We will study the growth and development of the heart in these animals using both physical and histological methods.  In addition, using cultured FAK null cardiomyocytes, we will identify the point of convergence between integrin- and growth factor- signaling.  We propose that a novel cardiac-selective FAK binding partner termed GRAF (GTPase Regulator Associated with FAK) which we recently cloned and characterized might be a key regulator in the coordination of these signaling pathways.  We are currently using overexpression of activated and dominant-interfering GRAF mutants to address this question.  In addition, we are in the process of screening for additonal novel cardiac-specific FAK binding partners that might be involved in adhesion-dependent hypertrophic signaling using either yeast 2-hybrid analysis or pull downs coupled with MALDI-mass spec sequencing.
     
The studies outlined above were previously supported by an American Cancer Society IRG seed grant and an American Heart Affiliate Grant-In-Aid and are currently funded by an American Heart Scientist Development Grant.
     
     

Smooth Muscle  Project:

BACKGROUND:   
Large blood vessels are organized into three distinct layers, the intima, which consists largely of a layer of endothelial cells that lines the lumen of the vessel; the media (the bulk of the vessel wall), which consists of several SMC layers surrounded by ECM; and the adventitia, made up of connective tissue and fibroblasts.  After completion of vessel formation during which the SMC population increases dramatically, SMC typically change from a synthetic (proliferative) to a more contractile (non-proliferative) phenotype.  However, even in the adult, vascular SMC can revert to the synthetic state under certain pathological conditions such as atherosclerosis, whereby vessel injury stimulates aberrant SMC migration and subsequent proliferation in the intima.  Therefore, understanding the precise mechanisms that control SMC proliferation and migration is crucial to our understanding of both vascular development and disease.

A variety of growth factors, contractile agonists, cytokines have been shown to be important for SMC proliferation, migration, and differentiation during normal vascular development and also under pathologic conditions.  Evidence suggests that the activity of these mitogens may be regulated by the interactions of SMC with medial ECM proteins and that the profile of ECM and integrins are dramatically modulated during vascular development and disease.  Specifically, it has been shown that plating SMC on fibronectin (the ECM enriched in both developing vessels and diseased adult arteries) is permisive for the proliferative phenotype, whereas plating SMC on polymerized collagen (the ECM enriched in adult vessels) supports the differentiated contractile phenotype.  As a collaborative effort with my husband, Chris Mack (a vascular biologist who is also a faculty member in the department of Pathology at UNC) we embarked on a study to examine a potential role for FAK in the regulation of SMC functions. 
          
Although FAK is relatively  ubiquitously  expressed,  we discovered that a dominant-inhibitory  form of FAK comprising   the C-terminal  binding motif termed FRNK (FAK-Related Non Kinase) is expressed specifically   in  smooth muscle cells (SMC) with particularly high levels observed  in large   blood vessels.  FRNK is expressed as a "gene within a gene" whereby promotor sequences within an intron of the FAK gene drive it's tissue-specific expression.  We showed that expression of FRNK in large arterioles was tightly regulated during development; FRNK was significantly  upregulated in the postnatal period and was also dramatically upregulated  following balloon-induced carotid    artery injury.  In cultured  rat aortic smooth muscle cells, overexpression of FRNK attenuated PDGF-BB-induced  migration and also dramatically inhibited  PDGFBB-induced cell proliferation. Taken together, these data suggest  that increased FRNK expression following  vascular injury or during development  may alter smooth muscle cell phenotype by negatively regulating proliferative  and migratory signals.     

Chris has developed some neat animal models that direct expression of target genes specifically to viceral and vascular SMC populations. We are currently using this transgenic approach to overexpress FRNK in vivo to assess the capacity of FRNK to regulate SMC functions in the intact vessels. As with the studies in the heart, we plan use tissue specific knockout strategies to determine the precise role for FAK/FRNK signaling in vascular development  and differentiation. We are also in the process of screening for  specific FAK/FRNK binding partners in the vasculature by genetic and biochemical  means to aid in determining signaling pathways that might be regulated by these proteins in SMC.           

   

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