alpha5beta1 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 alpha5beta1 integrin. Whereas alpha5beta1 integrin was not evident in the normal carotid artery, its expression was induced after a vascular injury. By 14 days, the alpha5beta1 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 alpha5beta1 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-beta1 integrin antibody, and an anti-alpha5beta1 integrin antibody, but not by an anti-beta3 integrin antibody. En face confocal microscopy of arterial segments revealed that soluble fibronectin had polymerized on the alpha5beta1 integrin-expressing SMCs of the luminal surface of the injured arterial neointima, but not on the alpha5beta1 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-beta1 integrin antibody. These studies indicate that a subpopulation of SMCs in the repairing artery wall orchestrates integrin-mediated fibronectin assembly.

Evidence for a role of collagen synthesis in arterial smooth muscle cell migration.

Migration of smooth muscle cells (SMCs) and collagen synthesis by SMCs are central to the pathophysiology of vascular disease. Both processes can be induced shortly after vascular injury; however, a functional relationship between them has not been established. In this study, we determined if collagen synthesis was required for SMC migration, using ethyl-3,4-dihydroxybenzoate (EDHB), an inhibitor of prolyl-4-hydroxylase, and 3,4-DL-dehydroproline (DHP), a proline analogue, which we demonstrate inhibit collagen elaboration by porcine arterial SMCs. SMCs exposed to EDHB or DHP attached normally to collagen- and vitronectin-coated substrates; however, spreading on collagen but not vitronectin was inhibited. SMC migration speed, quantified by digital time-lapse video microscopy, was significantly and reversibly reduced by EDHB and DHP. Flow cytometry revealed that expression of beta1 integrins, through which SMCs interact with collagen, was unaffected by EDHB or DHP. However, both inhibitors prevented normal clustering of beta1 integrins on the surface of SMCs, consistent with a lack of appropriate matrix ligands for integrin engagement. Moreover, there was impaired recruitment of vinculin into focal adhesion complexes of spreading SMCs and disassembly of the smooth muscle alpha-actin-containing cytoskeleton. These findings suggest that de novo collagen synthesis plays a role in SMC migration and implicates a mechanism whereby newly synthesized collagen may be necessary to maintain the transcellular traction system required for effective locomotion.

Processing of chimeric antisense oligonucleotides by human vascular smooth muscle cells and human atherosclerotic plaque. Implications for antisense therapy of restenosis after angioplasty.

BACKGROUND: Antisense oligonucleotides have been used in animals to inhibit the accumulation of vascular smooth muscle cells (VSMCs) after arterial injury. This has raised prospects for an oligonucleotide-mediated approach to prevent restenosis in patients undergoing angioplasty. However, little is known about the processing of oligonucleotides by human VSMCs or their bioavailability in human atherosclerotic tissue. METHODS AND RESULTS: Oligonucleotides were synthesized with a mixture of unmodified and sulfur-modified linkages (S-chimeric oligonucleotides). These were more stable than unmodified oligonucleotides and could be recovered from within human VSMCs after 36 hours. Oligonucleotide antisense to human proliferating cell nuclear antigen mRNA specifically reduced DNA synthesis (P < .01) and proliferating cell nuclear antigen protein content (P < .05) in human VSMCs. Confocal microscopy of both live and fixed cells showed modest oligonucleotide uptake that was primarily nuclear. Surprisingly, cationic liposomes did not enhance nuclear uptake but led to extensive, punctated cytoplasmic loading without an enhanced antisense effect. Oligonucleotides incubated with human coronary atherosclerosis fragments associated with cells within 1 hour, despite the presence of abundant extracellular matrix. CONCLUSIONS: S-chimeric oligonucleotides are stable and can specifically inhibit gene expression in human VSMCs. Nuclear transport is a feature of oligonucleotide processing by human VSMCs, indicating a potential influence at the nuclear level rather than with cytoplasmic mRNA. Cationic liposomes increased oligonucleotide uptake but not intracellular bioavailability, and S-chimeric oligonucleotides can be incorporated into cells within human atherosclerotic plaque, despite the presence of a dense extracellular matrix.