Sphingosine 1-phosphate: a regulator of arterial lesions.

Sphingosine-1 phosphate (S1P) is a bioactive sphingolipid that is critical in the development of blood vessels, and in the adult regulates vascular functions including vascular tone, endothelial integrity, and angiogenesis. Further, S1P may regulate arterial lesions in disease and after injury by controlling leukocyte recruitment and smooth muscle cell functions.

PDGF ligand and receptor gene expression during repair of arterial injury.

Smooth muscle cells (SMC) in rat carotid artery leave the quiescent state and proliferate after balloon catheter injury, but the signals for mitogenesis are not known. In this study, the possibility that cells within damaged arteries produce a growth factor that could act locally to stimulate SMC replication and repair was examined. We found that the genes for PDGF-A and -B (ligand) and PDGF receptor (alpha and beta subunits) were expressed in normal and injured carotid arteries and were independently regulated during repair of carotid injury. Two phases of PDGF ligand and receptor gene expression were observed: (a) In the early stage, a large decrease in PDGF beta-receptor mRNA levels preceded 10- to 12-fold increases in PDGF-A transcript abundance in the first 6 h after wounding. No change in PDGF alpha-receptor or PDGF-B gene expression was found at these times. (b) In the chronic phase, 2 wk after injury, neointimal tissue had lower levels of PDGF alpha-receptor mRNA (threefold) and higher levels of PDGF beta-receptor mRNA (three- to fivefold) than did restored media. Moreover, in situ hybridization studies identified a subpopulation of neointimal SMC localized at or near the luminal surface with a different pattern of gene expression than the underlying carotid SMC. Luminal SMC were strongly positive for PDGF-A and PDGF beta-receptor transcripts, while showing little or no hybridization for PDGF-B or PDGF alpha-receptor. Immunohistochemical studies showed strongly positive staining for PDGF-A in SMC along the luminal surface. These data show that changes in PDGF ligand and receptor expression occur at specific times and locations in injured carotid artery and suggest that these changes may play a role in regulating arterial wound repair.

Inhibition of neointimal smooth muscle accumulation after angioplasty by an antibody to PDGF.

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.

Basic fibroblast growth factor stimulates endothelial regrowth and proliferation in denuded arteries.

A large percentage of vascular reconstructions, endarterectomies, and angioplasties fail postoperatively due to thrombosis and restenosis. Many of these failures are thought to result from an inability of the vascular endothelium to adequately regenerate and cover the denuded area. After balloon catheter denudation of the rat carotid artery, regrowth of endothelium ceases after approximately 6 wk, leaving a large area devoid of endothelium. Here we show that this cessation of reendothelialization can be overcome by the systemic administration of basic fibroblast growth factor (bFGF). Administration of 120 micrograms bFGF over an 8-h period caused a highly significant increase in the replication rate of endothelial cells at the leading edge of 38.5 vs. 2.1% in controls, and, when given over a longer period of time (12 micrograms daily for 12 d), resulted in a significant increase in the extent of endothelial outgrowth onto the denuded surface. Furthermore, total regrowth could be achieved within 10 wk after balloon catheter denudation when 12 micrograms bFGF was injected twice per week for a period of 8 wk. Endothelium in unmanipulated arteries responded to bFGF with a significant increase in replication, but no increase in endothelial cell density was observed in these arteries. These data demonstrate that bFGF can act as a potent mitogen for vascular endothelial cells in vivo, and add considerably to our understanding of the mechanism underlying endothelial repair after in vivo vascular injuries.

Inhibition of smooth muscle cell proliferation in injured rat arteries. Interaction of heparin with basic fibroblast growth factor.

Heparin inhibits smooth muscle cell (SMC) proliferation after arterial injury by mechanisms that have yet to be defined. Since the initiation of SMC proliferation is mediated by basic fibroblast growth factor (bFGF), we have investigated the possibility that heparin inhibits SMC proliferation by displacing bFGF from the arterial wall. Using a rat carotid artery model of balloon catheter injury, we demonstrate that a bolus injection of heparin depletes the arterial wall of both systemically administered bFGF and of endogenous bFGF. Heparin, however, does not reduce the bFGF content of unmanipulated arteries. Further, a single injection of heparin given at the time of balloon injury reduces SMC proliferation by 55% but has no effect when given 6 h after injury. SMC proliferation induced in a denuded artery by injection of bFGF is inhibited almost completely by a bolus injection of heparin; however, pretreatment with a bolus of heparin does not prevent SMC from responding to a subsequent bolus of bFGF. These experiments suggest that heparin can inhibit SMC proliferation in part by removal of released bFGF from sites of injury.

Production of transforming growth factor beta 1 during repair of arterial injury.

Repair of arterial injury produced by balloon angioplasty leads to the formation of a neointima and a narrowing of the vascular lumen. In this study, we examined the possibility that smooth muscle cells (SMC) in injured rat carotid arteries are stimulated to produce type-1 transforming growth factor-beta (TGF-beta 1) during neointima formation in vivo. Levels of TGF-beta 1 transcripts (2.4 kb) were significantly increased within 6 h after carotid injury and reached a maximum (five to sevenfold) by 24 h. Regenerating left carotids had sustained increases in TGF-beta 1 mRNA levels (about fivefold) over the next 2 wk, during which time a substantial neointimal thickening was formed. No changes in basal TGF-beta 1 mRNA levels were found in contralateral uninjured carotids at any of the times examined. Immunohistochemical studies showed that a large majority of neointimal SMC were stained for TGF-beta 1 protein in an intracellular pattern, consistent with active TGF-beta 1 synthesis in this tissue. Neointima formation and TGF-beta 1 immunoreactivity were correlated with increases in fibronectin, collagen alpha 2(I), and collagen alpha 1(III) gene expression. Infusion of purified, recombinant TGF-beta 1 into rats with a preexisting neointima produced a significant stimulation of carotid neointimal SMC DNA synthesis. These results suggest that TGF-beta 1 plays an important role as an endogenous growth regulatory factor produced by neointimal SMC themselves during progressive neointimal thickening after balloon angioplasty.

Rat glomerular mesangial cells synthesize basic fibroblast growth factor. Release, upregulated synthesis, and mitogenicity in mesangial proliferative glomerulonephritis.

Mesangial injury and cell proliferation are frequent findings in various glomerular diseases in man. Previous studies have demonstrated that basic fibroblast growth factor (bFGF) is a potent mesangial cell mitogen in vitro. To further elucidate the role of bFGF in rat mesangial cell (RMC) proliferation, we examined whether RMC synthesize bFGF in vitro and whether bFGF is involved in mesangial proliferation in vivo. Cultured RMC expressed bFGF protein (23, 21.5, and 18 kD forms) and bFGF mRNA, and released biologically active bFGF into the culture medium after antibody- and complement-mediated injury. Normal rat glomeruli in vivo contained no detectable bFGF mRNA, but bFGF protein (23 and 21.5 kD) could be demonstrated, which immunolocalized to the mesangium. Glomerular bFGF decreased markedly during the acute phase of glomerulonephritis induced by anti-Thy 1.1 antibody, compatible with mesangial bFGF release after complement-mediated mesangiolysis. During the subsequent mesangial proliferative phase, glomerular bFGF protein and mRNA increased above normal. Intrarenal infusion of heparin did not affect the bFGF immunostaining of glomeruli at this stage, indicating a predominantly intracellular localization of the bFGF. The capability of bFGF to mediate proliferation in the anti-Thy 1.1 model was further supported by experiments in which intravenous bFGF given 24 h after a subnephritogenic dose of anti-Thy 1.1 antibody led to a 4.9- to 5.1-fold increase in glomerular cell proliferation (with > 60% of the cells identified as mesangial cells by double immunolabeling). No such increase was observed in normal rats injected with bFGF. These data show that mesangial cells produce and release bFGF after injury and that bFGF is mitogenic for injured mesangial cells in vivo. Release of mesangial cell bFGF thus may be an important mechanism involved in the initiation of mesangial cell proliferation in vivo.

Mitogen-activated protein kinases mediate matrix metalloproteinase-9 expression in vascular smooth muscle cells.

Expression of matrix metalloproteinase (MMP)-9 has been linked to the progression of plaque rupture and intimal formation in arterial lesions. In this study, we determined which factors and signaling pathways are involved in regulating the MMP-9 gene. Rat carotid arterial smooth muscle cells treated with tumor necrosis factor (TNF)-alpha showed a marked increase in MMP-9 activity and mRNA level, whereas platelet-derived growth factor (PDGF) showed a slight induction of the MMP-9 mRNA level. TNF-alpha treatment caused an increase in c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38 MAPK), and extracellular signal-regulated kinase (ERK) activities, whereas PDGF treatment caused an increase in ERKs and p38 MAPK activities without any effect on JNK activity. Treatment with either SB203580 (inhibitor of p38 MAPK) or U0126 (inhibitor of the ERK pathway) downregulated the TNF-alpha-induced MMP-9 expression in a dose-dependent manner. Treatment of cells with TNF-alpha and PDGF together stimulated the MMP-9 expression at a level higher than that observed with either factor alone, suggesting that TNF-alpha and PDGF have a synergistic effect on MMP-9 expression in arterial smooth muscle cells. Furthermore, suboptimal inhibitory concentrations of SB203580 and U0126 together almost completely inhibited the MMP-9 expression. These results suggest that p38 MAPK and ERK pathways contribute to the transcriptional regulation of MMP-9 in arterial smooth muscle cells.

Phosphatidylinositol 3-kinase signaling is important for smooth muscle cell replication after arterial injury.

The phosphoinositide 3-kinase [PI(3)K] pathway is a key signaling pathway important for replication of mammalian cells. In this study, we examined the role of PI(3)K in smooth muscle cell (SMC) replication after balloon catheter injury of rat carotid arteries. Protein kinase B (PKB), a downstream target of PI(3)K, was phosphorylated at 30 and 60 minutes after injury and to a lesser degree after 6 hours and 1 and 2 days but not after 7 days. Wortmannin (10 microgram per rat), a PI(3)K inhibitor, given to rats 60 and 5 minutes before and 11 hours after balloon injury, reduced the levels of phosphorylated PKB. SMC replication quantified between 24 to 48 hours was significantly reduced compared with control replication, as were the levels of cyclin D(1). Wortmannin was also administered to rats between days 7 and 8 and between days 7 and 9 after balloon catheter injury. A reduction in levels of phosphorylated PKB was detected, but no decrease in the replication of intimal SMCs was observed in either experiment. These data demonstrate that the PI(3)K signal transduction pathway plays an important role in medial but not intimal SMC replication.

Distortion of endothelial repair. The effect of hypercholesterolaemia on regeneration of aortic endothelium following injury by endotoxin. A scanning electron microscope study.

Five young male New Zealand White rabbits were fed a semi-synthetic diet containing 0.2% cholesterol for 2 weeks and a control group of 5 animals was fed a normal stock diet. All animals were then injected intravenously with a single dose of endotoxin from Serratia marcescens (200 microgram/kg body weight) and continued on their respective diets for a further 4 weeks. The aortas were then stained with silver nitrate and fixed under pressure for Scanning Electron Microscopy (SEM). Argyrophilic endothelial cells were present in both groups of animals 4 weeks after endotoxin injections. In the cholesterol-fed animals, however, these cells were often covered with pits and craters. These findings suggest that the hypercholesterolaemia may affect the regeneration of arterial endothelial cells.

A scanning electron microscopic study of arterial endothelial cells using vascular casts.

Vascular casts were made of rabbit aortas by infusing Batson's No. 17 anatomical corrosion compound into the artery at physiological pressure. The arterial tissue was then digested with sodium hydroxide and the cast viewed by scanning electron microscopy (SEM). Outlines of the endothelial cells and their silver stained boundaries were clearly visible. Cell nuclei and fine surface detail were also discernible. In EDTA damaged arteries, injured endothelial cells and platelets could also be observed in the vascular casts.

Scanning electron microscopy: morphology of aortic endothelium following injury by endotoxin and during subsequent repair.

A single injection of endotoxin P45 Poly Serratia marcescens was used to induce endothelial injury in rabbits. The aortic endothelium was examined by Scanning Electron Microscopy (SEM), at various times after administration of endotoxin, using the technique of silver staining and pressure fixation. Within one hour after injection, some endothelial cells were curled-up and spindle-shaped in appearance. Areas of aorta devoid of endothelial cover were occasionally observed and platelets were sometimes found adhering to these sites. Two and four weeks after initial injury no spindle-shaped cells were found. Instead, some endothelial cells were heavily stained with silver. Small denuded zones were still found and these were surrounded by brightly silver-stained cells. This study confirms that endotoxin rapidly causes endothelial injury and suggests that regenerating endothelial cells which were formed following injury are avidly stained by silver salts and appear as bright cells by SEM.

Scanning electron microscopy of arteries. The morphology of aortic endothelium in haemodynamically stressed areas associated with branches.

The endothelial surface around branches of the normal rabbit aorta was examined by scanning electron microscopy (SEM). Focal endothelial damage was consistently found both on, and distal to, aortic flow dividers. In some case small areas were denuded of endothelial cover. Similar injury to endothelal cells was also occasionally observed proximal to the ostium of a branch. Haemodynamic forces, expecially high shear stress, may be responsible for these morphological changes. This altered endothelial integrity probably underlies the susceptibility of these sites to the formation of induced atherosclerotic lesions.

Intimal cell mass-derived atherosclerotic lesions in the abdominal aorta of hyperlipidemic swine. Part 2. Investigation of endothelial cell changes and leukocyte adherence associated with early smooth muscle cell proliferative activity.

We have investigated several aspects of endothelial cell (EC) behavior during the initiation and early development of intimal cell mass (ICM)-derived atherosclerotic lesions in the distal abdominal aortas of young swine fed hyperlipidemic (HL) diets for 0, 14, 49, or 90 days. By scanning electron microscopy no breaks in endothelial integrity or other abnormalities were observed even at 90 days on diet after lesions were well established. Also, counts of leukocytes adherent to the endothelium by both scanning and light microscopy revealed no greater numbers in HL than in mash control swine. Estimates of individual EC losses over ICM-lesions in the HL swine (based on calculations from tritiated thymidine labeling indices and EC growth rates determined by counts) suggested a loss of approximately one per 100 EC/day. This loss over ICM lesion was not significantly greater than that over ICM in mash controls, but was significantly greater than that of EC not over ICM lesions in the same HL animal. In any event, the estimated loss seems to be too trivial for the endothelial barrier to be compromised even transiently in a biologically significant fashion. A significant correlation was observed between tritiated thymidine labeling indices of cells within the ICM lesions and of those of the overlying EC that was not observed with the ICM in the controls. Possibly the positive correlation may be a result of the abluminal surface of the overlying EC being exposed to the abnormal milieu of the ICM lesion. It is emphasized that results reported here apply only to ICM-derived lesions at an early stage of development and that they do not contradict results obtained by others with other lesion types such as those derived from monocytes. Furthermore, in later stages of development of ICM-derived lesions in the same model we know that extensive endothelial cell damage can be demonstrated. Also, functional changes in endothelial permeability may have been present in early stages that would not have been detected with the methods used in this study.

Scanning electron microscopy in the evaluation of endothelial integrity of the fatty lesion in atherosclerosis.

The luminal surface of fatty lesions of atherosclerosis was viewed by scanning electron microscopy (SEM). Endothelial cells were outlined by staining intercellular junctions with silver and the aortas were fixed in situ at physiological pressure. When aortas were dehydrated by passage through organic solvents followed by critical point drying from liquid CO2, there was considerable disruption of the luminal surface and it was not possible to correctly interpret the morphological integrity of the endothelium. In contrast, simple air-drying of aortas, without solvent dehydration after fixation, allowed the integrity of the cell layer overlying the lesion to be evaluated. The success of this technique was attributed to the retention of arterial lipids during dehydration of the tissue.

Aortic endothelial cell morphology observed in situ by scanning electron microscopy during atherogenesis in the rabbit.

The morphology of endothelial cells during the induction of atherosclerosis in the descending aortic arch of the hypercholesterol rabbit was studied in situ by scanning electron microscopy (SEM) following silver staining, fixation at physiological pressure, and air-drying of specimens- The earliest deviations from normal endothelial morphology were observed 3 weeks after starting to feed a semi-synthetic diet containing 20% beef fat and 0.2% cholesterol. These were (1) the occurrence of brightly silver stained (argyrophilic) cells, (2) areas of irregularly shaped cells which were often larger and more weakly stained than normal cells and (3) increased incidence of stigmata and stomata associated with the irregular cells. After 6 weeks of hypercholesterolaemia, similar changes were present in the endothelium, but were often also associated with sub-endothelial swelling. These represented the first atherosclerotic lesions. Following 12, 20 and 24 weeks of hypercholesterolaemia, larger raised macroscopic lesions were observed which were always endothelialized. Endothelial morphology and lesion topography suggested that early fatty streaks were composed of numerous focal swellings. In addition to the abnormal endothelial morphology noted at 6 weeks, endothelial cells overlying more advanced lesions became rounded in outline.

Common mechanisms of proliferation of smooth muscle in atherosclerosis and hypertension.

At least two exogenous sources of agents able to control vascular smooth muscle proliferation can be identified. Platelets contain and release mitogens as well as a factor, TGF-beta, that inhibits cell growth on plastic surfaces while stimulating it when cells are grown in suspension in soft agar. Macrophages release mitogens, including PGDF, and macrophage invasion is characteristic of early experimental lesions in fat-fed animals. Finally, it is at least possible that endothelial cell production of mitogens may represent a response to some as yet undefined external injury. The vessel wall also offers sources of growth control endogenous to the smooth muscle cell layers. The vessel wall contains heparan sulfate able to inhibit cell growth of smooth muscle cells, which by themselves can synthesize PDGF. This provides possible positive and negative control of replication intrinsic to the smooth muscle cells themselves. The role of these intrinsic or extrinsic factors in the smooth muscle proliferation of hypertension and atherosclerosis remains hypothetical. It is intriguing to implicate platelets and/or macrophages in the denuding injuries seen in small hypertensive vessels and in advancing atherosclerotic plaques. At least for the latter case, however, there seem to be other critical factors. Simple denudation and thrombosis, for example, are not sufficient to stimulate smooth muscle growth, and the kinetics of proliferation after balloon denudation imply the presence of some other event required to initiate smooth muscle proliferation. Similarly, smooth muscle replication in large vessels of hypertensive animals occurs without loss of endothelial continuity. This implies that replication in response to hypertension depends on factors intrinsic to the vessel wall. Benditt's observation of monoclonality also implies some intrinsic mechanism allowing cells to grow in a focal manner. It is intriguing to consider the possibility that this commitment process could require the release of cells from the intrinsic inhibitory effects of heparan sulfate located around the cells or the synthesis of growth factors secreted by the smooth muscle cells themselves. If we add the hypothesis that only some cells are capable of such a response, we would expect the sort of oligodense phenomenon demonstrated by Benditt. Proof of such a hypothesis, however, will have to await development of methods to explore these mechanisms directly in the vessel wall responding to injury.

The role of plasminogen activation in smooth muscle cell migration after arterial injury.

The migration of smooth muscle cells from the media to the intima that occurs after balloon catheter injury to the rat common carotid artery has been quantified by an electron microscopic surveying technique. These vessels have also been assayed for plasminogen-activator activity, which was found to rise sharply 4 days after balloon injury. At this time point smooth muscle cells begin to migrate in appreciable numbers. In order to investigate whether there is a causal relationship between plasminogen-activator activity and smooth muscle cell migration, animals were dosed with tranexamic acid. This synthetic inhibitor of plasmin activity reduced smooth muscle cell migration by 73% (p < 0.05), indicating that plasmin activity is necessary for migration after balloon injury. Lisinopril, an inhibitor of angiotensin-converting enzyme, inhibited smooth muscle cell migration after balloon injury by 78% (p < 0.01) but did not influence plasminogen-activator activity. Taken together, these results show that plasmin is a necessary but not sufficient component in the pathway that leads to smooth muscle cell migration after balloon catheter injury in the rat.

Factors controlling smooth-muscle cell proliferation.

Injury to the arterial wall normally elicits a rapid and significant increase in smooth-muscle cell (SMC) replication with the subsequent development of intimal lesions. A variety of factors have been proposed to control SMC replication, but recent work has highlighted the role of basic fibroblast growth factor (bFGF) and platelet-derived growth factor in this process. In the carotid artery of the uninjured rat, we have shown that the SMCs express mRNA for bFGF and that bFGF can be readily extracted from these arteries. Following mechanical injury to the artery, ie, after balloon injury, we suggested that bFGF is released from damaged cells and then stimulates adjacent SMCs. In support of this concept, the infusion of a blocking antibody to bFGF was found to significantly inhibit the early SMC replication induced by use of a balloon catheter. The addition of the antibody at the time of injury, however, did not inhibit the development of intimal lesions. In contrast, studies by us and other investigators have shown that platelet-derived growth factor is not directly important for SMC replication after balloon injury, but that it plays a key role in stimulating the migration of SMCs into the intima. Intimal SMC replication was not inhibited with antibodies to either bFGF or platelet-derived growth factor. Therefore, while significant inroads have been made in understanding the initial events, we still do not fully understand all the processes involved in the proliferation of arterial intimal lesions.

A simple technique for the frequent intravenous infusion of fluid without heparin into the rabbit.

A technique is described for the daily intravenous injection over 16 weeks in the rabbit. The system consists of a Teflon cannula passed down the external jugular vein with the tip in the superior vena cava. The cannula is joined at the point of entry to the jugular vein to a length of silicon rubber tubing, which is then passed subcutaneously to the forehead. The silicon tubing is terminated on a Luer needle hub, which is held in a simple Perspex plate secured subcutaneously between the rabbit's ears. The Luer hub is covered with a replaceable rubber cap through which injections may be made. With this system cannulae were maintained patent for 16 weeks by flushing once a day with Hanks' balanced salt solution, pH 7-4.