Cultured arterial smooth muscle cells maintain distinct phenotypes when implanted into carotid artery.

Cultured arterial smooth muscle cells (SMCs) with distinct phenotypic features have been described by several laboratories; however, it is not presently known whether this phenotypic heterogeneity can be maintained within an in vivo environment. To answer this question, we have seeded into the intima of denuded rat carotid artery 2 SMC populations with well-established distinct biological features, ie, spindle-shaped, not growing in the absence of serum, and well differentiated versus epithelioid, growing in the absence of serum, and relatively undifferentiated, derived from the aortic media of newborn rats (aged 4 days) and old rats (aged >18 months), respectively. We show that these 2 populations maintain their distinct biochemical features (ie, expression of alpha-smooth muscle actin, smooth muscle myosin heavy chains, and cellular retinol binding protein-1) in the in vivo environment. The old rat media-derived SMCs continue to produce cellular retinol binding protein-1 but little alpha-smooth muscle actin and smooth muscle myosin heavy chains, whereas the newborn rat media-derived SMCs continue to express alpha-smooth muscle actin and smooth muscle myosin heavy chains but no cellular retinol binding protein-1. Our results reinforce the notion of arterial SMC phenotypic heterogeneity and suggest that in our model, heterogeneity is controlled genetically and not by the local environment.

Local expression of bovine decorin by cell-mediated gene transfer reduces neointimal formation after balloon injury in rats.

Decorin is an extracellular matrix (ECM) proteoglycan that may modify vascular smooth muscle cell (SMC) function by altering the response to growth factors and the accumulation of ECM proteins during vascular injury. To investigate these possibilities in vivo, decorin was overexpressed at the site of arterial injury by cell-mediated gene transfer. Fischer rat SMCs were transduced in vitro with a retroviral construct that contained the bovine decorin gene and were subsequently seeded into injured rat carotid arteries. A species-specific antibody to bovine decorin and polymerase chain reaction primers were used to detect bovine decorin and distinguish it from endogenous rat decorin. Immunohistochemical and Northern analyses of rat carotid arteries revealed only low levels of rat decorin expression up to 8 weeks after balloon injury. However, after cell-mediated transfer of bovine decorin, strong expression of bovine decorin was verified by immunohistochemistry and reverse transcriptase-polymerase chain reaction. Four weeks after injury, the intimal area in vessels seeded with bovine decorin-overexpressing SMCs was significantly reduced by 35+/-4% (mean+/-SEM, n=9; P<0.01). Decorin overexpression also induced a higher intimal nuclear density and decreased volume of ECM. Specifically, immunostaining for versican and fibronectin was markedly reduced. In contrast, immunostaining for collagen type I was increased, and electron microscopy confirmed that collagen accumulation was altered. Bromodeoxyuridine labeling indicated that intimal SMC proliferation was not affected by the expression of bovine decorin. In summary, we demonstrate that gene transfer of the ECM proteoglycan, decorin, into the injured arterial wall reduces intimal ECM volume and alters the composition of the ECM.

Prevention of aneurysm development and rupture by local overexpression of plasminogen activator inhibitor-1.

BACKGROUND: Arterial aneurysms exhibit a loss of elastin and an increase in the plasminogen activators urokinase plasminogen activator (u-PA) and tissue plasminogen activator (t-PA). Because u-PA, t-PA, and plasmin have a limited proteolytic activity against elastin, the role of plasminogen activators in the aneurysmal disease is unclear. To investigate this question, we overexpressed plasminogen activator inhibitor-1 (PAI-1), an inhibitor of t-PA and u-PA, in a rat model of aortic aneurysm. METHODS AND RESULTS: Guinea pig-to-rat aortic xenografts were seeded with syngeneic Fischer 344 rat smooth muscle cells retrovirally transduced with the rat PAI-1 gene (LPSN group) or the vector alone (LXSN group). Some grafts were not seeded with cells (NO group). Western blots showed increased PAI-1 in grafts from the LPSN group compared with LXSN and NO groups. All grafts in the NO group (n=8) and 40% in the LXSN group ruptured between days 4 and 14. At 4 weeks in the LXSN group, the remaining unruptured grafts (n=6) were aneurysmal (diameter increase > or =100%), whereas in the LPSN group (n=6) none of the grafts had ruptured or were aneurysmal. Elastin was preserved in the LPSN group. t-PA, the major PA expressed in the model, was decreased in the LPSN group compared with the other groups, as determined by zymography. Quantitative zymography showed decreased levels of two matrix metalloproteinases (MMPs), a 28-kD caseinase, and activated MMP-9 in the LPSN group. CONCLUSIONS: The blockade of plasminogen activators prevents formation of aneurysms and arterial rupture by inhibiting MMP activation.

The regulation of p42/p44 mitogen-activated protein kinases in the injured rat carotid artery.

UNLABELLED: Arterial smooth muscle cell (SMC) proliferation is an important factor in the development of atherosclerotic plaques and restenotic lesions following arterial reconstruction. Basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), and thrombin are known to induce SMC proliferation and migration in vitro and in vivo. In cultured cells the proliferative responses to these mitogens depend on the activation of the p42/p44 mitogen-activated protein kinases (MAPKs), whereas the role of these kinases in vivo has yet to be established. We tested whether MAPK activity is induced following vessel injury and whether activity is dependent on the release of bFGF, PDGF, and thrombin. Following balloon injury of the left carotid of male Sprague-Dawley rats, arteries were removed and analyzed with respect to MAPK activity, BrdU-labeled nuclei, and/or luminal, medial, and intimal areas. MAPK activity is induced in the rat carotid artery following balloon-catheter injury with a maximum activation at 30 min with a return to just above baseline at 11 hr after injury. Intravenous administration of heparin or neutralizing antibodies to bFGF or PDGF prior to injury reduced SMC proliferation and neointimal lesional formation but did not affect the early induction of MAPK activity. Administration of a tissue factor inhibitor or thrombin inhibitor also did not affect MAPK activity, although it impaired the initiation of the coagulation cascade. IN CONCLUSION: (1) MAPK is activated in a time-dependent manner in response to injury; (2) the antiproliferative effect of heparin in vivo is not mediated through the inhibition of MAPK activity induced 30 min after injury; (3) the activation of MAPK after 30 min is not dependent on PDGF, bFGF, or thrombin following vessel injury in the rat.

Gene therapy for long-term expression of erythropoietin in rats.

The injection of recombinant erythropoietin (Epo) is now widely used for long-term treatment of anemia associated with chronic renal failure, cancer, and human immunodeficiency virus infections. The ability to deliver this hormone by gene therapy rather than by repeated injections could provide substantial clinical and economic benefits. As a preliminary approach, we investigated in rats the expression and biological effects of transplanting autologous vascular smooth muscle cells transduced with a retroviral vector encoding rat Epo cDNA. Vector-derived Epo secretion caused increases in reticulocytes, with peak levels of 7.8-9.6% around day 10 after implantation. The initial elevation in reticulocytes was followed by clinically significant increases in hematocrit and hemoglobin for up to 11 weeks. Ten control and treated animals showed mean hematocrits of 44.9 +/- 0.4% and 58.7 +/- 3.1%, respectively (P < 0.001), and hemoglobin values of 15.6 +/- 0.1 g/dl and 19.8 +/- 0.9 g/dl, respectively (P < 0.001). There were no significant differences between control and treated animals in the number of white blood cells and platelets. Kidney and to a lesser extent liver are specific organs that synthesize Epo in response to tissue oxygenation. In the treated animals, endogenous Epo mRNA was largely down regulated in kidney and absent from liver. These results indicate that vascular smooth muscle cells can be genetically modified to provide treatment of anemias due to Epo deficiency and suggest that this cell type may be targeted in the treatment of other diseases requiring systemic therapeutic protein delivery.

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.

Long-term biological response of injured rat carotid artery seeded with smooth muscle cells expressing retrovirally introduced human genes.

Cultured vascular smooth muscle cells (SMCs) containing retrovirally introduced genes are a potential vehicle for gene replacement therapy. Because the cultured SMCs are selected for their ability to proliferate in vitro, it is possible that the SMCs might be permanently altered and lose their capacity to respond to growth-suppressing conditions after being seeded back into blood vessels. To investigate this possibility we measured SMC proliferation and intimal thickening in balloon-injured Fischer 344 rat carotid arteries seeded with SMCs stained with the fluorescent marker 1,1'-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate (DiI) and infected with replication-defective retrovirus expressing human adenosine deaminase or human placental alkaline phosphatase. The majority of the seeded SMCs remained in the intima while a few of the cells appeared to migrate into the first layer of the media. Intimal SMC proliferation returned to background levels (< 0.1% thymidine labeling index) by 28 d. At late times (1 and 12 mo) the morphological appearance of the intima was the same for balloon-injured arteries with or without seeded SMC, except that the seeded arteries continued to express human adenosine deaminase or alkaline phosphatase. These results support the conclusion that cultured SMC infected with a replication-defective virus containing human adenosine deaminase or alkaline phosphatase are not phenotypically altered and do not become transformed. After seeding onto the surface of an injured artery, they stop replicating but continue to express the introduced human genes even over the long term.

Heparin increased cell membrane-associated heparan sulfate proteoglycan in balloon-injured rat carotid artery.

The effect of heparin on the amount of membrane-associated heparan sulfate proteoglycan (HSPG) was examined biochemically in balloon-injured rat carotid arteries. Heparin (Choay 1772) was administered at the rate of 1 mg/kg/h.i.v. by an osmotic infusion pump for 10 days. Arterial tissues were incubated ex vivo with Na2(35)SO4 and 3H-glucosamine for 6 or 24 h. The arteries from heparin-treated rats were also incubated with heparin (100 micrograms/ml) during the radiolabeling. Membrane-associated proteoglycans were extracted with 4 M guanidine-HCl and isolated by a series of chromatographic steps using Sephadex G-50, DEAE-Sephacel and Octyl-Sepharose CL-4B. Membrane-associated proteoglycans were eluted with 0.8% Triton X-100 from Octyl-Sepharose columns. Heparin treatment significantly increased membrane-associated HSPG by 64% in the arteries incubated for 24 h, while heparin hardly increased the HSPG in 6 h incubated arteries. Since the 24-hour data seem to reflect a combination of biosynthesis and degradation of proteoglycans while the 6-hour data primarily reflect biosynthesis, these results suggest that inhibition of degradation of membrane-associated HSPG is involved in the mechanism of heparin action. This speculation is supported by the observation that membrane-associated HSPG was elevated in balloon-injured arteries by treatment with protease inhibitors.

Mechanisms of inhibition by heparin of vascular smooth muscle cell proliferation and migration.

Heparin, an inhibitor of vascular smooth muscle cell proliferation and migration, affects a number of other cell functions. These effects include inhibition of growth factor binding, deposition of matrix proteins and gene expression. Various mechanisms have been proposed and, yet, how heparin works as an inhibitor remains unclear. We have postulated that heparin inhibits smooth muscle cell growth and migration by suppressing the expression of matrix-degrading enzymes such as plasminogen activators and interstitial collagenase. The molecular mechanism of heparin's inhibitory action on these proteases is currently under investigation.

Pituitary factors in blood plasma are necessary for smooth muscle cell proliferation in response to injury in vivo.

Intimal thickening in response to vascular injury is inhibited in animals previously subjected to hypophysectomy. We have investigated the nature and cell kinetics of this effect in a balloon catheter model of injury to the rat carotid artery. The ability of injury to stimulate [3H]thymidine labeling 48 hours after injury was almost completely eliminated in hypophysectomized (hypox) compared with control animals (0.1% versus 32.1%). Total DNA content of the developing neointima 14 days after injury was only 30% of the values found in ballooned carotid arteries of normal rats. If hypox rats were treated with recombinant human growth hormone, the proliferative response was not restored. There are two possible general explanations for the reduction of proliferative response in hypox animals: 1) that smooth muscle cells in the hypox animals have lost the ability to respond to the stimulus of injury or 2) that the ability of the smooth muscle cells to respond has not been reduced by prior hypophysectomy, but that the response itself requires the presence of pituitary-dependent factors. Transplantation experiments were performed in vivo to distinguish between these possibilities. Carotid arteries in inbred Lewis rats were excised 1 hour after balloon injury to give platelets the opportunity to adhere. These vessels were then transplanted from hypox into control animals and vice versa. At 48 hours, proliferation of smooth muscle cells in "control-to-hypox" transplants was 0.3% compared with 14.3% in "control-to-control" transplants, whereas vessels from hypox rats increased their indices to 4.8% if transplanted into control animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Heparin inhibits the expression of tissue-type plasminogen activator by smooth muscle cells in injured rat carotid artery.

Smooth muscle cells (SMCs) in balloon-injured rat carotid artery express tissue-type plasminogen activator (t-PA) at a time when they are migrating from the media to the intima. Since heparin inhibits SMC migration and intimal thickening, we have examined the possibility that heparin might also inhibit t-PA expression. Heparin (nonanticoagulant fraction; molecular weight, approximately 6,000) was administered by continuous intravenous infusion (1.0 mg/kg per hour) to Sprague-Dawley rats subjected to balloon injury of the left common carotid artery. At various times up to 14 days after injury, plasminogen activator expression was analyzed by zymography, plasmin generation, enzyme-linked immunosorbent assay, Northern blotting, and in situ hybridization. This dose of heparin inhibited SMC accumulation at 14 days by 60%. Both urokinase plasminogen activator (u-PA) and t-PA activity increased in injured arteries and reached a maximum at 7 days. Heparin treatment decreased t-PA, but not u-PA, activity. Total t-PA protein was decreased by treatment with heparin but not chondroitin sulfate, and the decrease in t-PA protein was associated with decreased t-PA mRNA in the media. These results in the injured rat carotid artery agree with our earlier observations that heparin inhibits t-PA gene expression in cultured baboon aortic SMCs. They also provide support for the hypothesis that heparin interferes with the expression of certain proteases required for SMC migration and proliferation.

Long-term expression of human adenosine deaminase in vascular smooth muscle cells of rats: a model for gene therapy.

Gene transfer into vascular smooth muscle cells in animals was examined by using recombinant retroviral vectors containing an Escherichia coli beta-galactosidase gene or a human adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) gene. Direct gene transfer by infusion of virus into rat carotid arteries was not observed. However, gene transfer by infection of smooth muscle cells in culture and seeding of the transduced cells onto arteries that had been denuded of endothelial cells was successful. Potentially therapeutic levels of human adenosine deaminase activity were detected over 6 months of observation, indicating the utility of vascular smooth muscle cells for gene therapy in humans.

Heparin and cilazapril together inhibit injury-induced intimal hyperplasia.

Both heparin and the angiotensin converting enzyme inhibitor cilazapril inhibit intimal thickening in rat carotid arteries injured by the passage of a balloon catheter. The purpose of this study was to determine if combinations of the two drugs were more effective than either drug alone and whether the effect could be accounted for by inhibition of smooth muscle cell proliferation. Heparin (0.1-0.3 mg/kg/hr) administered by continuous intravenous infusion with or without cilazapril (0-25 mg/kg/day p.o.) produced a dose-dependent inhibition of smooth muscle accumulation at 14 days after rat carotid ballooning. At the lower doses, the inhibitory effects of heparin and cilazapril were additive when the drugs were used together. This overall effect on growth was reflected in decreased smooth muscle cell proliferation at 2 and 7 days. A 7-day course of heparin combined with cilazapril, a regimen that might be applicable in the clinical setting, produced an 80% inhibition of intimal thickening at 28 days. These results provide evidence that heparin and cilazapril together might prove to be more effective than either drug alone in the control of intimal hyperplasia after arterial injury.

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.

Kinetics of cellular proliferation after arterial injury. V. Role of acute distension in the induction of smooth muscle proliferation.

Arteries denuded by the passage of a balloon catheter demonstrate marked smooth muscle cell (SMC) proliferation, whereas arteries denuded by a fine wire do not. We examined the possibility that vessel distension in addition to endothelial loss accounted for the SMC proliferation in the balloon model. Segments of rat left carotid isolated by temporary ligatures were distended hydrostatically with tissue culture medium at 300 mm Hg pressure via an external carotid cannula. Animals received either continuous [3H]thymidine infusion (intraperitoneally, Alzet pump, Alz Corp., Palo Alza, California) for 14 days or pulsed with [3H]thymidine at 1, 2, 3, or 7 days, and SMC proliferation was assessed by autoradiography. Distended vessels demonstrated patchy denudation acutely and complete endothelial regeneration by 3 days. No SMC were present in the intima at later times. Nevertheless, cumulative SMC proliferation over 14 days was 21 +/- 3% (perfused but not distended control: 0.8 +/- 0.6%; partially de-endothelialized but not distended control: 0.3 +/- 0.1%). Pulse-labeling demonstrated a peak of SMC proliferation between 2 and 3 days after injury. These results suggest that acute distension can contribute to the induction of SMC proliferation after endothelial denudation.

Regulation of smooth muscle cell growth in injured artery.

The process of intimal thickening after arterial injury can be divided into several steps, namely, initiation of smooth muscle cell proliferation, migration, and further intimal proliferation and deposition of matrix. Factors controlling the initiation of proliferation include de-endothelialization and vessel distension but not platelet adherence. Migration and subsequent intimal proliferation are controlled by factors from platelets, re-endothelialization, and endogenously released gamma-interferon.

Arterial smooth muscle cells in vivo: relationship between actin isoform expression and mitogenesis and their modulation by heparin.

Quiescent smooth muscle cells (SMC) in normal artery express a pattern of actin isoforms with alpha-smooth muscle (alpha SM) predominance that switches to beta predominance when the cells are proliferating. We have examined the relationship between the change in actin isoforms and entry of SMC into the growth cycle in an in vivo model of SMC proliferation (balloon injured rat carotid artery). alpha SM actin mRNA declined and cytoplasmic (beta + gamma) actin mRNAs increased in early G0/G1 (between 1 and 8 h after injury). In vivo synthesis and in vitro translation experiments demonstrated that functional alpha SM mRNA is decreased 24 h after injury and is proportional to the amount of mRNA present. At 36 h after injury, SMC prepared by enzymatic digestion were sorted into G0/G1 and S/G2 populations; only the SMC committed to proliferate (S/G2 fraction) showed a relative slight decrease in alpha SM actin and, more importantly, a large decrease in alpha SM actin mRNA. A switch from alpha SM predominance to beta predominance was present in the whole SMC population 5 d after injury. To determine if the change in actin isoforms was associated with proliferation, we inhibited SMC proliferation by approximately 80% with heparin, which has previously been shown to block SMC in late G0/G1 and to reduce the growth fraction. The switch in actin mRNAs and synthesis at 24 h was not prevented; however, alpha SM mRNA and protein were reinduced at 5 d in the heparin-treated animals compared to saline-treated controls. These results suggest that in vivo the synthesis of actin isoforms in arterial SMC depends on the mRNA levels and changes after injury in early G0/G1 whether or not the cells subsequently proliferate. The early changes in actin isoforms are not prevented by heparin, but they are eventually reversed if the SMC are kept in the resting state by the heparin treatment.

Gamma-interferon regulates vascular smooth muscle proliferation and Ia antigen expression in vivo and in vitro.

A significant fraction of the arterial smooth muscle cells in atherosclerotic plaques and injury-induced intimal thickenings express class II major histocompatibility complex (Ia) antigens. This might be the consequence of gamma-interferon secretion by T lymphocytes also present in these lesions. We have therefore analyzed the effects of gamma-interferon on cultured rat aortic smooth muscle cells. Recombinant gamma-interferon inhibited smooth muscle proliferation in vitro in a dose-response relation; inhibition was detectable down to a concentration of 1 unit/ml. In similar concentrations, gamma-interferon also induced Ia expression by the cells. This suggested that Ia antigens might be selectively expressed by nonproliferating smooth muscle cells. In vivo, there was a strong negative correlation between Ia expression and 3H-thymidine labeling of smooth muscle cells in intimal thickenings induced by balloon catheter injury. In rats receiving continuous infusions of 3H-thymidine for two weeks after injury, Ia-positive 3H-positive cells had undergone fewer rounds of replication than Ia-negative ones. This indicates that Ia-expression both in vivo and in vitro is associated with a reduced proliferative capacity. These results suggest that gamma-interferon, a secretory product of activated T lymphocytes, acts as a natural regulator of smooth muscle cell growth and Ia expression in injury-induced intimal thickenings and atherosclerotic plaques.

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.