Vascular Smooth Muscle Cell Neointima Research Articles

Balloon catheter vascular injury of the alloxan-induced diabetic rabbit: the role of insulin-like growth factor-1.

Neointimal thickening following catheter injury is characterized, in part, by growth factor-induced vascular smooth muscle cell (VSMC) proliferation. It was hypothesized that a reduction in serum insulin-like growth factor-1 (IGF-1), characteristic of chemically-induced diabetes, would result in decreased VSMC proliferation and attenuate neointimal thickening. It was found that alloxan-treated New Zealand White rabbits exhibit varying degrees of glycemia. Rabbits classified as diabetic (glucose > or = 400 mg/dL) had significantly decreased serum concentration of IGF-1 (87.4+/-14 nmol/L vs. 170+/-14 nmol/L) and significantly decreased intimal/medial (I/M) ratios 2, 4, and 8 weeks after aortic injury compared to euglycemic rabbits (13.7+/-2, 21.1+/-2, 32.4+/-3 in euglycemics and 6.6+/-1, 14+/-2, 19+/-5 in diabetics, respectively). The I/M for high hyperglycemic animals (glucose 286-399 mg/dL) was comparable to diabetic animals yet their serum IGF-1 levels were normal rather than depressed. Vascular IGF-1 content similarly increased upon injury in both diabetic and euglycemic animals. In diabetic animals, proliferating cell nuclear antigen (PCNA) immunostaining was present by day 1 peaked by day 5 and returned to control by day 14. In euglycemic animals, staining by day 1 continued to increase through day 14. A similar increase in mitogen-activated protein kinase (MAPK) activity occurred from day 1 through day 5 in both diabetic and euglycemic animals. This is the first demonstration of an association between MAPK activity and VSMC proliferation following vascular injury in diabetic animals as previously reported in euglycemic animals. In conclusion, this study provides evidence against a direct effect of IGF-1 in the reduction in neointimal thickening, VSMC proliferation, and MAPK activity upon catheter injury in chemically-induced diabetic rabbits.

Distribution of endothelin-1 (ET) receptors (ET A and ET B) and immunoreactive ET-1 in porcine saphenous vein–carotid artery interposition grafts

Proliferation of vascular smooth muscle cells (VSMC) is a principal event in neointima formation in saphenous vein–coronary artery bypass grafts. Since endothelin-1 (ET-1) promotes VSMC replication and ET-1 receptor antagonists inhibit neointima formation in arterial injury models, it is reasonable to propose that ET-1 may be involved in neointima formation in vein grafts. However, it is not known what alterations of ET-1 and its receptors (if any) occur in vein grafts. The objective of this study, therefore, was to investigate the distribution of ET-1 and ET-1 receptor subtypes (ET A and ET B) in porcine vein grafts. Unilateral interposition saphenous vein grafting was performed by end to end anastomosis after excision of a segment of carotid artery in Landrace pigs. One month after surgery, vein grafts, ungrafted saphenous veins and carotid arteries were excised, ET-1 immunoreactivity identified by immunocytochemistry and ET A and ET B receptor subtypes studied using autoradiography. In vein grafts, there was a greater density of ET A compared to ET B receptors in both the tunica media and neointima. ET A binding in the tunica media of ungrafted saphenous vein was greater than that in the carotid artery or vein grafts, but greater in the vein graft compared to the carotid artery. Immunoreactive ET-1 was located in endothelial cells and throughout the neointima of the vein graft. Dense ET-1 binding (to both ETA and ET B receptors) was also associated with microvessels in the adventitia within the graft. In vein grafts, there was strong ET B binding to neutrophils which were present in high numbers at the subendothelium and within the adventitia. It is concluded ET A receptors may play a role in vein graft thickening at the medial and neointimal VSMC level, whereas ET B receptors may play a role in microangiogenesis. The higher levels of ET A receptors in the tunica media of ungrafted saphenous vein relative to the carotid artery and vein graft may also render this conduit susceptible to neointima formation. These data indicate that studies on the effect of ET receptor antagonists on the pathobiology of vein graft disease is warranted.

Endothelin-1 and endothelin receptors in porcine saphenous vein-carotid artery grafts.

The regional distribution of endothelin-1 (ET-1) and its receptor subtypes (ETA and ETB) in porcine saphenous vein into carotid artery interposition grafts was studied 1 month after surgery and compared to ungrafted saphenous vein and carotid artery. ET-1 immunoreactivity was identified by immunohistochemistry and ET receptor subtypes were studied using in vitro autoradiography. In vein grafts, there was a higher density of ETA compared to ETB receptor binding in both the tunica media and the neointima. ETA binding to the tunica media of ungrafted saphenous vein was greater than that in the carotid artery or vein grafts, but greater in the vein graft compared to the carotid artery. Immunoreactive ET-1 was located in endothelial cells and throughout the neointima of the vein graft. Dense ETA and ETB binding was also associated with adventitial microvessels in the graft, and ETB binding was also identified to neutrophils, which accumulated at the subendothelium and within the adventitia. ETA receptors may play a role in vein graft thickening at the medial and neointimal vascular smooth-muscle cell level, whereas ETB receptors may play a role in microangiogenesis. The high levels of ETA receptors in the tunica media of ungrafted saphenous vein, relative to the carotid artery and vein graft, may also render this conduit susceptible to neointimal formation. These data indicate that studies of the effect of ET receptor antagonists on the pathobiology of vein graft disease are warranted.

An autocrine system for C-type natriuretic peptide within rat carotid neointima during arterial repair.

C-type natriuretic peptides (CNPs) are produced by endothelium and inhibit vascular smooth muscle cell (VSMC) proliferation. However, endothelial damage stimulates only transient VSMC proliferation in arteries. Here we report that a new source of CNP develops in rat carotid neointima 14 days after balloon angioplasty, when VSMC replication is subsiding despite continued absence of endothelium. CNP was detected immunohistochemically in neointimal but not medial VSMC. No other natriuretic peptides were detected immunohistochemically. CNP immunoreactivity (0.036 +/- 0.010 fmol/mg wet wt) was found in damaged arteries by radioimmunoassay, but none was detected in normal media. Reverse-phase chromatography suggested that this immunoreactivity consisted of CNP(1-53) and perhaps CNP(1-22). CNP transcript was identified by reverse transcription and polymerase chain reaction in carotid segments that had been stripped of endothelium but only once neointima had formed. Moreover, neointima expressed the NPR-C type of natriuretic peptide receptor at the same time as it synthesized CNP. Thus neointima develops an autocrine system for CNP that could regulate neointimal growth. Furthermore, the findings establish novel phenotypic differences between medial and neointimal VSMC in vivo.

Increased secretion of gelatinases A and B from the aortas of cholesterol fed rabbits: relationship to lesion severity

Basement membrane degrading metalloproteinases (gelatinases) have been implicated in the regulation of vascular smooth muscle cell migration and proliferation in culture and during neointima formation in vivo. We compared the expression and activation of gelatinases A and B in explants derived from the arch, mid and distal portions of thoracic aortas of normal rabbits and those given a 1% cholesterol-containing diet for 8 weeks. Neointimal/medial ratio was less than 0.01 in normal rabbits but was significantly increased by cholesterol feeding in the arch (1.08±0.26), mid (0.75±0.28) and distal (0.32±0.12) portions of the aorta (mean±S.E.M., n=6), and to a significantly ( P<0.05) greater extent in the arch and mid than distal portions. Secretion of gelatinase B measured by densitometric scanning of zymograms was undetectable from normal aortas, but was significantly increased by cholesterol feeding in the arch (0.16±0.06), mid (0.26±0.08) and distal (0.11±0.05) portions (optical density units, n=6, each P<0.05 versus normal diet). The increase in gelatinase B expression was localised by in situ hybridisation to neointimal vascular smooth muscle cells, macrophages and endothelial cells. Secretion of pro-gelatinase A was detected from normal aortas; it was increased by cholesterol feeding from the arch (4.0 versus 2.8, P<0.05) and mid (3.6 versus 2.8, P<0.05) but not distal portions of the aorta (1.8 versus 1.2, n.s.). Similar results were obtained for active gelatinase A secretion from the arch (0.50 versus 0.28, P<0.05) and mid (0.47 versus 0.23, P<0.05) but not distal portions (0.19 versus 0.20, n.s.). Increases in pro- and active gelatinase A secretion therefore paralleled the severity of atheroma formation. The results imply that increased basement membrane turnover mediated by gelatinases occurs during cholesterol induced atherosclerosis formation.

Chapter 7 Vascular Smooth Muscle Cell Cultures

Publisher Summary Vascular smooth muscle cells (VSMCs) are the predominant cell type in blood vessels of the arterial tree. They are responsible for maintaining vascular tone in response to various hormonal and hemodynamic stimuli and they are the major cell type involved in the process of vascular repair, growth, and response to injury. The development of cell culture systems has greatly aided in this pursuit by providing a controlled environment to study the unique characteristics of these cells in isolation from other influences. When the blood vessel is mechanically injured and its endothelial lining removed, there is significant remodeling of the vessel. Medial VSMCs migrate across the elastic laminae and basement membrane and accumulate in the intima. There they proliferate and secrete increased amounts of interstitial matrix proteins. This new intimal structure is appropriately referred to as the neointima and the cells within it are neointimal VSMCs. This chapter discusses methods for culturing both medial and neointimal VSMCs as well as for culturing VSMCs obtained from atheromatous lesions.

Vascular smooth muscle cell phenotype influences glycosaminoglycan composition and growth effects of extracellular matrix.

Rat neonatal and neointimal vascular smooth muscle cells differ dramatically from adult medical vascular smooth muscle cells in their growth properties, with the neonatal and neointimal cells exhibiting growth in the absence of exogenously added growth factors. Since it has been hypothesized that extra-cellular matrix proteoglycans may influence the growth and differentiation of vascular smooth muscle cells, we examined the ability of matrix derived from these cells to influence vascular smooth muscle cell proliferation. To produce test matrices, cells were grown to confluence and removed by brief alkali treatment. Test cells were seeded onto these matrices and the rates of growth in a growth-factor-deficient medium determined. Compared to plastic wells, matrix from neonatal or neointimal cells stimulated the growth of vascular smooth muscle cells. Interestingly, matrix from adult cells was less efficient at promoting growth. Enzymatic digestion of extracellular matrix heparan sulfate, but not of other glycosaminoglycans, further increased the growth-stimulatory effect of extracellular matrix, suggesting that matrix heparan sulfate acts as a growth inhibitor. Consistent with this, biochemical analysis showed that the adult matrix contained a higher percentage of heparan sulfate compared with neonatal or neointimal matrix. These results suggest that autocrine production of heparan sulfate proteoglycans may play an important role in growth regulation of vascular smooth muscle cells during normal vascular development and differentiation as well as in pathological response to injury.

Elaboration of type-1 plasminogen activator inhibitor from adipocytes. A potential pathogenetic link between obesity and cardiovascular disease.

Obesity is known to predispose to attenuated fibrinolysis attributable to increased concentrations in plasma of type-1 plasminogen activator inhibitor (PAI-1), the primary physiological inhibitor of endogenous fibrinolysis. PAI-1 is present in neointimal vascular smooth muscle cells and lipid-laden macrophages. The present study was designed to determine whether PAI-1 expression occurs in adipose tissue as well, thereby potentially contributing to increased cardiovascular risk associated with obesity. 3T3-L1 preadipocytes were differentiated into adipocytes by exposing them to isobutylxanthine (0.5 mmol/L) and dexamethasone (0.25 mumol/L) over 7 days and incubated for 24 hours with transforming growth factor-beta (TGF-beta), known to augment PAI-1 synthesis in several cell types and to be released from platelets when they are activated. TGF-beta increased PAI-1 activity in the conditioned media of the 3T3-L1-derived cells in a concentration-dependent fashion without significantly affecting cell proliferation. Western blotting and immunoprecipitation of 35S-labeled PAI-1 showed that the increased PAI-1 activity paralleled increased PAI-1 protein. Northern blotting showed that increased PAI-1 mRNA preceded increased accumulation of PAI-1 activity and protein in the conditioned media. Furthermore, TGF-beta (10 ng/g body wt) administered in vivo increased PAI-1 activity in mouse plasma and PAI-1 mRNA expression in mouse adipose tissue. Increased plasma PAI-1 activity in obese human subjects may result from PAI-1 release from an increased mass of adipose tissue, particularly in association with thrombosis and elaboration of TGF-beta from platelet alpha-granules into the circulation. The increased PAI-1 may exacerbate vascular disease by shifting the balance between thrombosis and thrombolysis toward thrombosis and consequently exposing luminal surfaces of vessels to mitogens associated with microthrombi over protracted intervals.

Expression of Local Hepatocyte Growth Factor System in Vascular Tissues

Since endothelial cells (EC) are known to secrete various anti-proliferative and vasodilating factors, an agent that promotes seeding or regeneration of EC may have potential therapeutic value against vascular smooth muscle cell (VSMC) proliferation. For the above purpose, we have found that hepatocyte growth factor (HGF) fulfills such conditions. However, the local HGF system has not yet clarified. Therefore, we investigated endogenous HGF production and its specific receptor (c-met) in vascular tissues. Our results revealed the expressions of HGF and its receptor (c-met) mRNA in endothelial cells, VSMC and neointimal VSMC of rat assessed by RT-PCR. HGF and c-met mRNA were also detected in human aortic endothelial cells and VSMC. Endothelial cells and VSMC of both rat and human produce and secrete immunoreactive HGF as assessed by ELISA. Of importance, the existence of local HGF system (HGF and c-met) was also observed in vivo in intact abdominal aorta of SD and Wistar rats. Overall, this study demonstrated that HGF is expressed and secreted from endothelial cells and VSMC of both rat and human in vitro as well as in vivo. Since HGF has characteristics of an endothelium-specific growth factor, locally synthesized HGF in endothelial cells and VSMC may have a role in vascular functions in autocrine-paracrine manner.

Taxol inhibits neointimal smooth muscle cell accumulation after angioplasty in the rat.

Despite significant improvements in the primary success rate of the medical and surgical treatments for atherosclerotic disease, including angioplasty, bypass grafting, and endarterectomy, secondary failure due to late restenosis continues to occur in 30-50% of individuals. Restenosis and the later stages in atherosclerotic lesions are due to a complex series of fibroproliferative responses to vascular injury involving potent growth-regulatory molecules (such as platelet-derived growth factor and basic fibroblast growth factor) and resulting in vascular smooth muscle cell (VSMC) proliferation, migration, and neointimal accumulation. We show here, based on experiments with both taxol and deuterium oxide, that microtubules are necessary for VSMCs to undergo the multiple transformations contributing to the development of the neointimal fibroproliferative lesion. Taxol was found to interfere both with platelet-derived growth factor-stimulated VSMC migration and with VSMC migration and with VSMC proliferation, at nanomolar levels in vitro. In vivo, taxol prevented medial VSMC proliferation and the neointimal VSMC accumulation in the rat carotid artery after balloon dilatation and endothelial denudation injury. This effect occurred at plasma levels approximately two orders of magnitude lower than that used clinically to treat human malignancy (peak levels achieved in this model were approximately 50-60 nM). Taxol may therefore be of therapeutic value in preventing human restenosis with minimal toxicity.

Fibroblast growth factor stimulates angiotensin converting enzyme expression in vascular smooth muscle cells. Possible mediator of the response to vascular injury.

Angiotensin converting enzyme (ACE) activity contributes to the vascular response to injury because ACE inhibition limits neointima formation in rat carotid arteries after balloon injury. To investigate the mechanisms by which ACE may contribute to vascular smooth muscle cell (VSMC) proliferation, we studied expression of ACE in vivo after injury and in vitro after growth factor stimulation. ACE activity 14 d after injury was increased 3.6-fold in the injured vessel. ACE expression, measured by immunohistochemistry, became apparent at 7 d in the neointima and at 14 d was primarily in the most luminal neointimal cells. To characterize hormones that induce ACE in vivo, cultured VSMC were exposed to steroids and growth factors. Among steroids, only glucocorticoids stimulated ACE expression with an 8.0 +/- 2.1-fold increase in activity and a 6.5-fold increase in mRNA (30 nM dexamethasone for 72 h). Among growth factors tested, only fibroblast growth factor (FGF) stimulated ACE expression (4.2 +/- 0.7-fold increase in activity and 1.6-fold increase in mRNA in response to 10 ng/ml FGF for 24 h). Dexamethasone and FGF were synergistic at the indicated concentrations inducing 50.6 +/- 12.4-fold and 32.5-fold increases in activity and mRNA expression, respectively. In addition, when porcine iliac arteries were transfected with recombinant FGF-1 (in the absence of injury), ACE expression increased in neointimal VSMC, to the same extent as injured, nontransfected arteries. The data suggest a temporal sequence for the response to injury in which FGF induces ACE, ACE generates angiotensin II, and angiotensin II stimulates VSMC growth in concert with FGF.

Increased intramural expression of plasminogen activator inhibitor type 1 after balloon injury: A potential progenitor of restenosis

Objectives. This study was performed to determine whether altered gene expression of plasminogen activator inhibitor type I (PAI-1) occurs within the arterial wall after experimentally induced balloon injury.Background. PAI-1, known to inhibit fibrinolysis in the circulation and to be present within atherosclerotic vessels, may influence proteolysis in the arterial wall and neointimal formation after angioplasty.Methods. In rabbit carotid arteries subjected to balloon injury, both PAI-1 gene and protein expression were assayed sequentially with the use of Northern blotting, in situ hybridization and immunohistochemical studies.Results. In uninjured, normal vessels PAI-1 messenger ribonucleic acid (mRNA) was not detectable by Northern blotting or in situ hybridization. However, injury was followed within 3 h by increases in PAI-1 mRNA (3.2 kb) of 5.9-fold compared with that in contralateral control carotid arteries (Northern blots). PAI-1 mRNA was detectable by in situ hybridization early after injury first in adventitia; after 24 h it was particularly prominent in the media. From 1 to 4 weeks after injury it was consistently detectable and was localized in neointimal vascular smooth muscle and endothelial cells at a time when neointimal thickening was marked. Cells of both types exhibited PAI-1 protein detected immunohistochemically. In vessels maintained in organ culture after balloon injury in vivo, sustained increases in PAI-1 activity appeared in conditioned media as well.Conclusions. Our results indicate that balloon injury simulating angioplasty in patients induces intramural expression of PAI-1 in vascular smooth muscle and endothelial cells. The decreased cell surface fibrinolytic activity likely to result from the increased PAI-1 expression may initiate or exacerbate mural thrombosis. Accordingly, excessive stimulation with clot-associated mitogens may stimulate vascular smooth muscle cell proliferation, which, coupled with increased accumulation of extracellular matrix attributable to decreased plasmin-mediated degradation, may contribute to restenosis.

Mitogenic effects of prolonged angiotensin II infusion on medial and neointimal vascular smooth muscle cell in vivo

Mitogenic effects of prolonged angiotensin II infusion on medial and neointimal vascular smooth muscle cell in vivo