a5b1 Integrin Expression and Luminal Edge Fibronectin Matrix Assembly by Smooth Muscle Cells after Arterial Injury

Fibronectin is secreted from the cell as a soluble protein that must then polymerize to regulate cell function. To elucidate the process of fibronectin matrix assembly in vascular disease, we immunostained sections of balloon-injured rat carotid artery for the fibronectin-binding a5b1 integrin. Whereas a5b1 integrin was not evident in the normal carotid artery, its expression was induced after a vascular injury. By 14 days, the a5b1 integrin was localized exclusively to the less differentiated smooth muscle cells (SMCs) at the luminal surface of the neointima. Platelet-derived growth factor-BB, dominant in neointimal formation, selectively increased the expression of the a5b1 integrin by human SMCs in culture. To track the assembly of fibronectin fibers, fluorescence-labeled soluble fibronectin protomers were added to cultured SMCs and to fresh segments of normal and balloon-injured rat carotid arteries. Fibronectin fiber formation in cultured SMCs could be detected within 10 minutes, and was blocked by an RGD peptide, an anti-b1 integrin antibody, and an anti-a5b1 integrin antibody, but not by an anti-b3 integrin antibody. En face confocal microscopy of arterial segments revealed that soluble fibronectin had polymerized on the a5b1 integrin-expressing SMCs of the luminal surface of the injured arterial neointima, but not on the a5b1 integrin-negative neointimal SMCs below this or on the endothelial cells of uninjured arteries. Furthermore, in situ fibronectin assembly by the neointimal SMCs was inhibited by an RGD peptide and by an anti-b1 integrin antibody. These studies indicate that a subpopulation of SMCs in the repairing artery wall orchestrates integrin-mediated fibronectin assembly. (Am J Pathol 2000, 156:453–465) Fibronectin is an extracellular glycoprotein that has important roles in cell adhesion, migration, growth, and differentiation. In the normal adult artery wall, fibronectin comprises a small fraction of the extracellular matrix (ECM); however, in the diseased artery wall, fibronectin is abundant. An increased expression of arterial fibronectin is observed in the vascular lesions of atherosclerosis, restenosis after angioplasty, and transplant arteriopathy, suggesting an important pathophysiological role for fibronectin in these conditions. In vitro studies suggest that such roles may include the regulation of the vascular smooth muscle cell (SMC) phenotype, SMC and endothelial-cell migration and proliferation, and leukocyte trafficking. After balloon-mediated arterial injury, there is rapid accumulation of fibronectin at the site of the injury, in association with neointimal formation. There are two potential sources for this newly deposited fibronectin. Some fibronectin may be derived from the circulatory system, where it exists as a plasma protein originally synthesized by the liver. In addition, fibronectin is synthesized locally, as a specific response to an injury, by resident vascular SMCs. Locally derived or cellular fibronectin is distinct from plasma fibronectin by virtue of the unique fibronectin domains that arise through alternative splicing. Cellular fibronectin contains the type III fibronectin modules ED-A and AD-B, whereas plasma fibronectin does not contain either of these splice variants. Regardless of its origin, fibronectin is initially secreted from the cell as a soluble dimeric protein. Within the ECM, soluble fibronectin protomers polymerize to form insoluble, multimeric fibronectin. This assembly process is of paramount importance because only insoluble, fibrillar fibronectin can act as an adhesive ligand and regulate cell function. Moreover, fibronectin polymerization is not a spontaneous process, but requires specific cellular interaction. This contrasts with other ECM components,