Induction of collagenase and stromelysin gene expression by mechanical injury in a vascular smooth muscle‐derived cell line

Abstract

We describe a novel system for studying the production of matrix metalloproteinases types I and III (collagenase and stromelysin) by a vascular smooth muscle cell line (Rb-1 cells) in response to mechanical injury. Highly confluent Rb-1 cells are disrupted by passing a plastic comb around the plate to clear a series of circumferential paths, which are bordered by two ridges of displaced cells. Over the next 24 hours, cells migrate into the cleared areas. Northern blot analysis demonstrates the accumulation of mRNAs for collagenase and stromelysin during this process, although they are undetectable prior to injury. Cotreatment with all-trans-retinoic acid (10(-6) M) markedly decreases the level of mRNAs induced by injury, whereas dexamethasone (10(-7) M) causes only a slight reduction. In situ hybridization studies for stromelysin mRNA and immunohistochemical staining for collagenase protein on plates of injured cells showed the highest levels of stromelysin mRNA in cells in the ridges left by the injury; lower levels were observed in some cells migrating into the clear region. The same pattern of expression was observed when cells were stained with antiserum to collagenase protein. Nuclear run-on assays demonstrated increases in transcription of stromelysin and collagenase genes following injury. Transient transfection of cells with a vector containing the luciferase gene driven by a wild-type promoter comprising 1.8 kb of the 5'-flanking region of the rabbit collagenase gene showed increased activity associated with injury. We conclude that: (1) mechanical injury is associated with induction of mRNAs for the metalloproteinases collagenase and stromelysin, (2) retinoic acid effectively antagonizes this responses, and (3) the increase in steady-state mRNA levels is, at least in part, transcriptionally mediated. Thus our data suggest a role for mechanical forces in initiating the changes in gene expression in vascular smooth muscle cells following arterial injury in vivo.

p38 mitogen-activated kinase is a bidirectional regulator of human fibroblast collagenase-1 induction by three-dimensional collagen lattices

“…fl-coll [3], as well as other signals that induce changes in cell morphology and actin reorganization, stimulate MMP-1 expression. The other signals include mechanical strain [4] and direct mechanical wounding [5] in human vascular smooth-muscle cells, exposure to cytochalasin D in human and rabbit synovial fibroblasts [6,7] and specific cell-matrix interactions [8].…”

all- Trans -Retinoic Acid Reduces Neointimal Formation and Promotes Favorable Geometric Remodeling of the Rat Carotid Artery After Balloon Withdrawal Injury

“…For example, a wealth of data supports an important role for SMC migration in the evolution of a neointima, 47,48 and atRA has been shown to suppress SMC migration in vitro, apparently through an inhibition of the AP-1-dependent proteases collagenase and stromelysin. 19 Because there is mounting evidence supporting a critical role for retinoid receptors in modulating such gene expression, 18 an examination of retinoid receptors within the normal and injured vessel wall should be a future goal. Another possible mechanism for atRA-mediated reduced intimal mass may relate to accelerated cell death.…”

“…15 Many effects elicited by retinoids are of relevance to the pathogenesis of human restenosis. For example, all-trans-retinoic acid (atRA) promotes differentiation 16 and fibrinolysis 17 and inhibits cell proliferation, 18 migration, 19 thrombosis, 20 angiogenesis, 21 platelet aggregation, 22 and inflammation. 23 Although their clinical efficacy has been documented for some proliferative disorders, 24,25 virtually nothing is known with respect to retinoids and vascular occlusive disease.…”

Retinoids and Arterial Smooth Muscle Cells

“…RA has been shown to reduce SMC migration, possibly by decreasing collagenase and stromelysin transcription, 2 MMPs induced by mechanical injury. 84 However, we have observed that RA increases migration of both medial and IT15 SMCs, probably by stimulating tissue-type plasminogen activator (tPA) activity. 28 tPA is the main PA involved in the proteolytic activities of SMCs in vitro, and the higher proteolytic activity of IT15 cells is mainly due to increased expression of tPA.…”