Alexander W. Clowes

Platelet-derived growth factor promotes smooth muscle migration and intimal thickening in a rat model of balloon angioplasty.

Platelet-derived growth factor (PDGF) is a mitogen and chemoattractant for vascular smooth muscle cells (SMC) in vitro, but its activities in vivo remain largely undefined. We infused recombinant PDGF-BB (0.01-0.30 mg/kg per d i.v.) into rats subjected to carotid injury. PDGF-BB produced a small increase (two- to threefold) in medial SMC proliferation. More importantly, PDGF-BB greatly increased (20-fold) the intimal thickening and the migration of SMC from the media to the intima during the first 7 d after injury. These data provide support for the hypothesis that PDGF, and perhaps other platelet factors, might play an important role in the movement of mesenchymal cells into zones of injury undergoing repair.

Smooth muscle cell migration and matrix metalloproteinase expression after arterial injury in the rat.

We have characterized matrix metalloproteinase expression in the rat carotid artery after two forms of arterial injury, balloon catheter denudation and nylon filament denudation. Gelatinolytic enzymes with molecular masses of 70 and 62 kD were produced constitutively in the rat carotid. Production of an 88-kD gelatinase was induced after balloon catheter injury, and proteinase production continued during the period of migration of smooth muscle cells from the media to the intima, from 6 hours to 6 days after balloon catheter injury. In addition, a marked increase in 62-kD gelatinolytic activity was observed between 4 and 14 days after arterial injury. Gelatinase activities (88 and 62 kD) were also increased after nylon filament denudation but were markedly less after this injury than after balloon catheter injury. These results suggested a correlation between gelatinase activity and smooth muscle cell migration after arterial injury. Administration of a metalloproteinase inhibitor after balloon catheter injury resulted in a 97% reduction in the number of smooth muscle cells migrating into the intima. Therefore, we hypothesize that gelatinase expression directly facilitates smooth muscle cell migration within the media and into the intima. These results suggest that gelatinases are involved in the vascular smooth muscle cell activation and neointimal formation that characterize arterial tissue remodeling after injury.

Interstitial Collagenase (MMP-1) Expression in Human Carotid Atherosclerosis

BACKGROUND In human atherosclerosis, most clinical events occur when plaque integrity is compromised and hemorrhage and thrombosis result. One mechanism for this might be the release by plaque cells of matrix-degrading proteases, such as interstitial collagenase (matrix metalloproteinase-1, MMP-1), which degrades two major plaque structural proteins, types I and III collagen. This study was undertaken to determine whether MMP-1 is expressed in human atherosclerotic plaques. METHODS AND RESULTS To determine the cellular source and location of MMP-1 in human carotid atherosclerotic lesions, in situ hybridization and immunohistochemistry were performed on 20 endarterectomy specimens. Six nonatherosclerotic carotid arteries also were studied. Intense MMP-1 expression (mRNA and protein) was detected in a subset of plaque macrophages located at the borders of the lipid cores adjacent to fibrous caps and shoulder regions. Subsets of plaque smooth muscle cells and endothelial cells also expressed MMP-1. There was a strong correlation between the percentage of the lipid core occupied by hemorrhage and the percentage of the lipid core perimeter positive for MMP-1 (r = .823, P = .0001). MMP-1 was not detected in any cell type in nonatherosclerotic carotid arteries. CONCLUSIONS This study demonstrates that MMP-1 is expressed by several cell types in human carotid atherosclerosis and that there is a correlation between the expression of the protease and histopathological evidence of plaque instability. Since MMP-1 may degrade the major structural collagens of the plaque, expression of the protease by macrophages in regions critical to plaque integrity could contribute to plaque expansion, rupture, and hemorrhage.

Smooth muscle cells express urokinase during mitogenesis and tissue-type plasminogen activator during migration in injured rat carotid artery.

Although the level of plasminogen activator (PA) expression has been correlated with cellular proliferation and migration in vitro, this relation has not been established in tissue undergoing repair. In a rat model of arterial injury, we have measured the expression of PAs by vascular smooth muscle cells (SMCs) during entry into the growth cycle (0-24 hours) and subsequent migration from the media to the intima (starting at approximately 4 days). In normal rat carotid, low levels of urokinase-type PA (uPA) and tissue-type PA (tPA) are present; after removal of the endothelium, only uPA is detected in the media. uPA activity in extracts of carotid arteries increases and reaches a maximum between 16 and 24 hours after injury; uPA mRNA increases steadily and is maximal at 7 days. tPA activity appears at 3 days and is maximal at 7 days; tPA mRNA is present in normal vessels and reaches a maximum by 7 days. Most of the tPA in the media is associated with SMC and not with regenerating endothelium. Furthermore, tPA is present in the media before the SMCs migrate into the intima. These results demonstrate that PA expression by vascular SMCs is differentially regulated, with uPA present during mitogenesis and tPA during migration.

Matrix metalloproteinases of vascular wall cells are increased in balloon-injured rat carotid artery * ** *

PURPOSE Although matrix metalloproteinase (MMP) expression has been correlated with proliferation and migration of various tumor cells, the relation between MMP expression and smooth muscle cell (SMC) proliferation and migration has not been established. METHODS We measured MMP expression (gelatin, casein, and elastin zymography) by vascular wall cells in balloon-injured carotid artery during the period of medial SMC proliferation, migration of SMC from the media to the intima, and subsequent intimal SMC proliferation. RESULTS The 72 and 64-kd gelatinases (presumably 72 kd type IV collagenase or MMP 2) were constitutively expressed in normal carotid arteries, and the activated (59 and 54 kd) forms of this enzyme were increased at 5 days when SMCs start to migrate. A 92 kd gelatinase (presumably 92 kd type IV collagenase or MMP 9) was increased at 24 hours, when SMCs entered the growth cycle, and decreased thereafter. A low-molecular-weight metalloproteinase with elastolytic activity was present in the adventitia, and the activity was increased at 5 days after surgery. CONCLUSIONS These results suggest that the 72 kd and 92 kd gelatinases may be involved in basement membrane and matrix degradation in the media in relation to SMC proliferation and migration, whereas the low-molecular-weight metalloproteinase may have a role in elastin turnover in the adventitia.

Local overexpression of TIMP-1 prevents aortic aneurysm degeneration and rupture in a rat model.

Although matrix metalloproteinases (MMPs) are expressed in abundance in arterial aneurysms, their contribution to arterial wall degeneration, dilation, and rupture has not been determined. We investigated MMP function in a rat model of aneurysm associated with arterial dilation, elastin loss, medial invasion by mononuclear inflammatory cells, and MMP upregulation. Rupture was correlated with increased gelatinase B (MMP-9) and activated gelatinase A (MMP-2). Syngeneic rat smooth muscle cells retrovirally transfected with tissue inhibitor of matrix metalloproteinases (TIMP)-1 cDNA (LTSN) or with the vector alone as a control (LXSN) were seeded onto the luminal surface of the vessels. The seeding of LTSN cells resulted in TIMP-1 local overexpression. The seeding with LTSN cells, but not LXSN cells, decreased MMP-9, activated MMP-2 and 28-kD caseinase and elastase activity, preserved elastin in the media, and prevented aneurysmal degeneration and rupture. We conclude that MMP overexpression is responsible for aneurysmal degeneration and rupture in this rat model and that local pharmacological blockade might be a reasonable strategy for controlling the formation of aneurysms in humans.

Increased blood flow inhibits neointimal hyperplasia in endothelialized vascular grafts.

Intimal hyperplasia is a primary cause of failure after vascular reconstruction and may be affected by blood flow. We have studied the effects of increased blood flow on intimal hyperplasia in porous polytetrafluoroethylene grafts implanted in baboons. These grafts develop an endothelial lining by 2 weeks and neointimal thickening due to proliferation of underlying smooth muscle cells by 1 month. Creation of a distal arteriovenous fistula increased flow (from 230 +/- 35 to 785 +/- 101 ml/min, p less than 0.001) and mean shear (from 26 +/- 4 to 78 +/- 10 dynes/cm2, p less than 0.001) without causing a drop in pressure across the grafts. Fistula flow did not alter the pattern of endothelial coverage but did cause a marked reduction in the cross-sectional area of the neointima (from 2.60 +/- 0.52 to 0.42 +/- 0.07 mm2 at 3 months, p less than 0.01). Detailed morphometric analysis revealed an equivalent percentage decrease in smooth muscle cells and matrix content, suggesting that the primary effect of increased flow was to reduce smooth muscle cell number without affecting the amount of matrix produced by individual cells. The neointima remained sensitive to changes in flow at late times; ligation of the fistula after 2 months resulted in a rapid increase in neointimal thickness (from 0.60 +/- 0.03 mm2 after 2 months of fistula flow to 3.88 +/- 0.55 mm2 1 month after ligation of fistula, p less than 0.01). These results support the hypothesis that changes in blood flow affect the structure of diseased as well as normal vessels.

Matrix Metalloproteinase-9 Overexpression Enhances Vascular Smooth Muscle Cell Migration and Alters Remodeling in the Injured Rat Carotid Artery

Matrix metalloproteinase-9 (MMP-9) has been implicated in the pathogenesis of atherosclerosis as well as intimal hyperplasia after vascular injury. We used Fischer rat smooth muscle cells (SMCs) overexpressing MMP-9 to determine the role of MMP-9 in migration and proliferation as well as in vessel remodeling after balloon denudation. Fischer rat SMCs were stably transfected with a cDNA for rat MMP-9 under the control of a tetracycline-regulatable promoter. In this system, MMP-9 was overexpressed in the absence, but not in the presence, of tetracycline. In vitro SMC migration was determined using a collagen invasion assay as well as a Boyden chamber assay. In vivo migration was determined by measuring the invasion into the medial and intimal layers of transduced SMCs seeded on the outside of the artery. Transduced SMCs were also seeded on the luminal surface, and the effect of local MMP-9 overexpression on vascular structure was measured morphometrically at intervals up to 28 days. MMP-9 overexpression enhanced SMC migration in both the collagen invasion assay and Boyden chamber in vitro, increased SMC migration into an arterial matrix in vivo, and altered vessel remodeling by increasing the vessel circumference, thinning the vessel wall and decreasing intimal matrix content. These results demonstrate that MMP-9 enhances vascular SMC migration in vitro and in vivo and alters postinjury vascular remodeling.

Heparin regulates smooth muscle S phase entry in the injured rat carotid artery.

Smooth muscle cell (SMC) proliferation in injured arteries is inhibited by heparin, but the mechanism of inhibition is unknown. In particular, it is not clear whether heparin prevents exit of quiescent SMC from the resting state, inhibits progression through the prereplicative (G1) sequence, or acts during DNA synthesis itself. In this study, induction of ornithine decarboxylase (ODC) activity was used as a marker of SMC entry into the cell cycle in an attempt to localize the site of heparin action during the initial hours after rat carotid injury. Rapid and transient induction of ODC activity was observed that reached a maximum (twenty-three-fold) 6 hours after wounding. Heparin failed to prevent ODC induction but greatly reduced frequencies of [3H]thymidine-labelled SMC nuclei 33 hours after injury. Moreover, heparin infusion could be delayed for up to 18 hours after the injury event with no significant loss of antiproliferative effect. Further delays resulted in marked loss of growth inhibition. The results of these studies show that SMC rapidly and synchronously leave the resting state after injury and suggest that heparin acts late in the prereplicative (G1) sequence or early in S phase to inhibit SMC proliferation in damaged arteries.

Aortic aneurysm repair. Reduced operative mortality associated with maintenance of optimal cardiac performance.

Recent advances in the operative management of aortic aneurysms have resulted in a decreased rate of morbidity and mortality. In 1972, we hypothesized that a further reduction in operative mortality might be obtained with controlled perioperative fluid management based on data provided by the thermistor-tipped pulmonary artery balloon catheter. From 1972 to 1979 a flow directed pulmonary artery catheter was inserted in each of 110 consecutive patients prior to elective or urgent repair of nonruptured infrarenal aortic aneurysms. The slope of the left ventricular performance curve was determined preoperatively by incremental infusions of salt-poor albumin and Ringers lactate solution. With each increase in the pulmonary arterial wedge pressure (PAWP), the cardiac index (CI) was measured. The PAWP was then maintained intra- and postoperatively at levels providing optimal left ventricular performance for the individual patient. There were no 30-day operative deaths among the patients in this series and only one in-hospital mortality (0.9%), four months following surgery. The five-year cumulative survival rate for patients in the present series was 84%, a rate which does not differ significantly from that expected for a normal age-corrected population. Since the patient population was unselected and there were no substantial alterations in operative technique during the present period, these improved results support the hypothesis that operative mortality attending the elective or urgent repair of abdominal aortic aneurysm can be minimized by maintenance of optimal cardiac performance with careful attention to fluid therapy during the perioperative period.