ABSTRACT
Formation of the epicardium requires interactions between alpha(4)beta(1) integrin, and the extracellular matrix. We investigated the role of other integrins expressed by epicardial cells. We detected transcripts for alpha(5), alpha(8), alpha(v), beta(1), beta(3), and beta(5) integrins in the chick proepicardial organ (PE). We demonstrate that alpha(5)beta(1), alpha(8)beta(1), and alpha(v)beta(3) integrins are expressed by chick epicardial mesothelial cells (EMCs). Migration of EMCs in vitro was reduced by RGD-containing peptides. Using adenoviruses expressing an antisense to chick alpha(4) (AdGFPalpha4AS), full-length (Adhalpha4V5), and C-terminal deleted alpha(4) (Adhalpha4DeltaCV5), we found that EMCs were less able to adhere to vitronectin and fibronectin(120) indicating that alpha(4)beta(1) plays a role in regulating EMC adhesion to ligands of alpha(5)beta(1), alpha(8)beta(1), and alpha(v)beta(3). In Adhalpha4DeltaCV5-infected EMCs, alpha(5)beta(1) was diminished in fibrillar adhesions and new FN matrix assembly was abnormal. We propose that cooperation between alpha(4)beta(1) and RGD integrins is important for EMC adhesion and subepicardial matrix formation.
Subject(s)
Cell Adhesion/physiology , Extracellular Matrix/metabolism , Integrin alpha4beta1/metabolism , Integrin alpha5beta1/metabolism , Pericardium/cytology , Pericardium/embryology , Animals , Cell Movement/physiology , Chick Embryo/anatomy & histology , Chick Embryo/metabolism , Coronary Vessels/anatomy & histology , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Epithelium/anatomy & histology , Epithelium/metabolism , Extracellular Matrix/chemistry , Fibronectins/metabolism , Humans , Integrin alpha4beta1/genetics , Integrin alpha5beta1/genetics , Oligopeptides/metabolism , Pericardium/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Vitronectin/metabolismABSTRACT
Epithelial-mesenchymal transformation of the embryonic epicardium produces the subepicardial mesenchyme that is essential for normal coronary vascular development. Gene targeting experiments in mice have demonstrated an essential role for alpha4-integrin in normal epicardial development, but the precise cellular consequences of alpha4-integrin loss remain uncertain. To better understand the function of alpha4-integrin in epicardial development, we constructed a replication-incompetent adenovirus (AdlacZalpha4AS) that expresses antisense chicken alpha4-integrin as the 3' untranslated region of a lacZ reporter gene. This construct effectively labeled cells while greatly reducing levels of alpha4-integrin mRNA and protein. In quail chick chimeras, transplanted epicardial cells infected with AdlacZalpha4AS adhered to the heart and were incorporated into the epicardium, but 4 days after grafting, were largely absent from the epicardial epithelium, recapitulating the defect in alpha4-null mice. This did not result from epicardial cell apoptosis or anomalous migration of epicardial cells to extracardiac sites. Rather, AdlacZalpha4AS-infected epicardial cells were particularly invasive, being three to four times more likely to migrate to the interstitium of the myocardium than AdlacZ-infected epicardial cells. Accelerated epicardial-mesenchymal transformation and migration of alpha4-negative epicardium was observed in an organ culture system that does not require prior culture of epicardial cells. Remarkably, AdlacZalpha4AS infection also prevented targeting of epicardially derived mesenchyme to the media of developing coronary vasculature in the myocardial interstitium. This study provides evidence that epicardial alpha4-integrin normally restrains epicardial-mesenchymal transformation, invasion, and migration and is essential for correct targeting of epicardially derived mesenchyme to the developing coronary vasculature.
