Expression of Extracellular Matrix Proteins Accompanies Lesion Growth in a Model of Intimal Reinjury

Abstract: Fibrillar collagen is a critical component of atherosclerotic lesions. Uncontrolled collagen accumulation leads to arterial stenosis, while excessive collagen breakdown combined with inadequate synthesis weakens plaques thereby making them prone to rupture. This review discusses cellular sources of collagen synthesis in atherosclerosis, local and systemic factors modulating collagen gene expression, as well as temporal and spatial patterns of collagen production in human and experimental atherosclerotic lesions.

Abstract: Smooth muscle cells in the atherosclerotic lesions of diseased arteries produce new extracellular matrix, largely collagenous in nature, which is responsible in part for the occlusion of the vessel lumen by the atherosclerotic plaque. These smooth muscle cells express a different phenotype, responsive to growth factors, to that of the differentiated, nondividing contractile cell in the media. Specific collagens may be involved in the regulation of phenotype and in the migration of the cells to the site of lesion growth. Collagens may also be involved in the calcification of lesions, in the retention of low-density lipoprotein in the vessel wall and in smooth muscle cell survival. Glycation of collagen may promote atherogenesis. Effects as summarized in this short review, are not always, at first sight, consistent. The following points should be kept in mind, though, when considering the response of a cell to collagen. Any effect may be governed not just by the identity of the collagen type as such but by its state of polymerization: monomeric collagen, for instance, whether in solution or immobilized on plastic, may express different effects to the same collagen type when presented in its native polymerized state, e.g., as fibers. The precise identity of the cell and its location may be important: SMCs in secondary culture may not necessarily respond to any given collagen exactly as SMCs within the lesion or possess precisely the same properties, albeit both types are regarded as expressing the same (synthetic) phenotype. Effects may not necessarily be directly attributable to collagen, but to some other matrix constituent bound to collagen.

Abstract: Hypothesis Deep venous thrombosis (DVT) confers vein wall injury associated with fibrosis and extracellular matrix (ECM) turnover, likely mediated by matrix proteases. This study investigated the expression of proteases and collagen involved in early vein wall remodeling. Methods In the mouse, DVT was produced by ligation of the infrarenal inferior vena cava (IVC) or sham operation, and tissue was harvested at 4, 8, and 12 days. The vein wall tissue was processed for real-time reverse transcriptase-polymerase chain reaction (6 to 8 per time point), Western immunoblotting (5 per time point), and gelatin zymography (5 per time point). Analysis of variance was used for multiple comparisons, and a P Results Thrombus resolution was documented by a 38% decrease in the thrombosed IVC weight from day 4 to day 12 ( P = .007). Total vein wall collagen increased over time, with a corresponding increase in procollagen I and III, and expression peaked at 12 days (24-fold and 6.1-fold, respectively, P P P P P Conclusions Vein wall remodeling after DVT is similar to wound healing and is associated with increased procollagen gene expression and total collagen. It is also associated with increased early MMP-9 expression, followed by MMP-2 expression and activity after DVT resolution. Clinical Relevance Deep vein thrombosis is an often neglected problem that long term is associated with the postphlebitic syndrome of limb swelling, pain, and often ulceration. The basic mechanisms of the vein wall damage that results have not been delineated. The following study describes the vein wall matrix metalloproteinase gene and activity response induced over time in the vein wall after DVT. Additionally, the corresponding collagen upregulation and proximate plasmin system mediators are determined. With this knowledge, potential therapies to reduce vein wall injury directly might be possible.

Abstract: Aims MicroRNA (miR)-92a is an important regulator of endothelial proliferation and angiogenesis after ischaemia, but the effects of miR-92a on re-endothelialization and neointimal lesion formation after vascular injury remain elusive. We tested the effects of lowering miR-92a levels using specific locked nucleic acid (LNA)-based antimiRs as well as endothelial-specific knock out of miR-92a on re-endothelialization and neointimal formation after wire-induced injury of the femoral artery in mice. Methods and results MiR-92a was significantly up-regulated in neointimal lesions following wire-induced injury. Pre-miR-92a overexpression resulted in repression of the direct miR-92a target genes integrin α5 and sirtuin1, and reduced eNOS expression in vitro . MiR-92a impaired proliferation and migration of endothelial cells but not smooth muscle cells. In vivo , systemic inhibition of miR-92a expression with LNA-modified antisense molecules resulted in a significant acceleration of re-endothelialization of the denuded vessel area. Genetic deletion of miR-92a in Tie2-expressing cells, representing mainly endothelial cells, enhanced re-endothelialization, whereas no phenotype was observed in mice lacking miR-92a expression in haematopoietic cells. The enhanced endothelial recovery was associated with reduced accumulation of leucocytes and inhibition of neointimal formation 21 days after injury and led to the de-repression of the miR-92a targets integrin α5 and sirtuin1. Conclusion Our data indicate that inhibition of endothelial miR-92a attenuates neointimal lesion formation by accelerating re-endothelialization and thus represents a putative novel mechanism to enhance the functional recovery following vascular injury.

Abstract: Intimal smooth muscle (SMC) proliferation was examined in the rat left carotid in regions lacking endothelium for prolonged periods of time Arteries of animals injected with tritiated thymidine and Evans blue were examined at intervals between 0 and 12 weeks The endothelial layer was regenerated from the ends of the denuded segment but failed to cover the central third of the artery by 12 weeks Autoradiography on samples from this central region (stained blue) and the endothelialized ends (white) showed that SMC proliferation reached a maximum at 48 hours in the media (46%) and at 96 hours in the intima (73%) Subsequently, the thymidine index declined to baseline (006%) by 4 weeks throughout the media and by 8 weeks in the intima covered by endothelium SMC proliferation persisted at a high level (38%) at the surface of the intima lacking endothelium even at 12 weeks Despite continued proliferation of luminal SMC, total arterial SMC number was the same at 2 and 12 weeks These results support the concept that intimal SMC proliferation after arterial injury is an acute event related to the initial injury process Persistent proliferation of luminal SMC does not result in an increase in intimal cell number

Abstract: Approximately 30 to 40 percent of atherosclerotic coronary arteries treated by angioplasty or by bypass surgery occlude as a result of restenosis. This restenosis is due principally to the accumulation of neointimal smooth muscle cells, which is also a prominent feature of the advanced lesions of atherosclerosis. The factors responsible for the accumulation of intimal smooth muscle cells have not been identified. Platelet-derived growth factor (PDGF) is a potent smooth muscle chemoattractant and mitogen. It is present in platelets and can be formed by endothelium, smooth muscle, and monocyte-derived macrophages. The development of an intimal lesion in the carotid artery of athymic nude rats induced by intraarterial balloon catheter deendothelialization was inhibited by a polyclonal antibody to PDGF. These data demonstrate that endogenous PDGF is involved in the accumulation of neointimal smooth muscle cells associated with balloon injury and may be involved in restenosis after angioplasty, and perhaps in atherogenesis as well.

Abstract: To further understand the temporal mode and mechanisms of coronary restenosis, 229 patients were studied by prospective angiographic follow-up on day 1 and at 1, 3 and 6 months and 1 year after successful percutaneous transluminal coronary angioplasty. Quantitative measurement of coronary stenosis was achieved by cinevideodensitometric analysis. Actuarial restenosis rate was 12.7% at 1 month, 43.0% at 3 months, 49.4% at 6 months and 52.5% at 1 year. In 219 patients followed up for ≥3 months, mean stenosis diameter was 1.91 ± 0.53 mm immediately after coronary angioplasty, 1.72 ± 0.52 mm on day 1, 1.86 ± 0.58 mm at 1 month and 1.43 ± 0.67 mm at 3 months. In 149 patients followed up for ≥6 months, mean stenosis diameter was 1.66 ± 0.58 mm at 3 months and 1.66 ± 0.62 mm at 6 months. In 73 patients followed up for 1 year, mean stenosis diameter was 1.65 ± 0.56 mm at 6 months and 1.66 ± 0.57 mm at 1 year. Thus, stenosis diameter decreased markedly between 1 month and 3 months after coronary angioplasty and reached a plateau thereafter. In conclusion, restenosis is most prevalent between 1 and 3 months and rarely occurs beyond 3 months after coronary angioplasty.

Abstract: Medium conditioned by Chinese hamster ovary (CHO) cells transfected with the simian pre-pro-TGF beta 1 cDNA contains high levels of latent TGF beta 1. The amino-terminal region of the TGF beta 1 precursor is secreted and can be detected in the conditioned medium by immunoblotting using peptide antibodies specific for amino-terminal peptides. Chemical cross-linking of CHO-conditioned medium using bis-(sulfosuccinimidyl)-suberate (BS3) followed by immunoblot analyses indicates that latent recombinant TGF beta 1 contains both the cleaved amino-terminal glycopeptide and mature TGF beta 1 polypeptide in a noncovalent association and that this association confers latency. The data presented here do not support the involvement of a unique TGF beta binding protein(s) in latent recombinant TGF beta 1. Plasmin treatment of CHO-conditioned medium resulted in the appearance of TGF beta competing activity. In addition, immunoblot analysis of plasmin-treated CHO-conditioned medium indicates that the amino-terminal glycopeptide is partially degraded and that mature TGF beta 1 is released. Thus, activation of latent TGF beta 1 may occur by proteolytic nicking within the amino-terminal glycopeptide thereby causing a disruption of tertiary structure and noncovalent bonds, which results in the release of active, mature TGF beta 1. Acid activation of latent TGF beta, in comparison, appears to be due to dissociation of the amino-terminal glycopeptide from the mature polypeptide.

Abstract: We report the isolation of cDNA clones for fibronectin from a rat liver library prepared in the expression vector, lambda gt11. Restriction mapping and DNA sequencing of these clones establish the sequence of the C-terminal 35% of rat fibronectin, covering the cell-, heparin-, and fibrin-binding domains. The cell- and heparin-binding regions have homologous repeating sequences. Based on the sequence data and S1 nuclease mapping, we conclude that there are at least three different fibronectin mRNAs in rat liver which differ in coding potential. The three RNAs appear to arise by alternative splicing within the coding region and are probably all encoded by a single gene. The implications of these results for the structure and function of fibronectin and the differences between various types of fibronectin are discussed.