The role of the pericytes of the adventitial microcirculation in the arterial intimal thickening

Segments of rat femoral arteries, with one collateral each, occluded between ligatures and dissected from surrounding tissue, developed intimal thickening, with or without ligation of their collaterals. Numerous newly-formed capillaries from the surrounding arterial microcirculation growing into the adventitia, tunica media and intimal thickening were demonstrated by means of serial longitudinal sections, predominantly in the ostium of the collateral. When the ligatures were applied without damaging the microcirculation surrounding the artery and the normal continuity of the adventitial vessels was unchanged, earlier presence of intimal thickening was observed. When the fibrous layers of the adventitia were removed at the moment of the arterial ligation, the continuity between newly-formed vessels of the neoadventitia and those growing into the media and neointima was much more evident. It was then noted that the pericytes constituted a major component of the intimal thickening. The introduction of contrast material in microcirculation confirmed the connections between newly-formed adventitial and intimal vessels. At the beginning of the experiment, autoradiographic studies showed an increased DNA synthesis in the cells of preformed postcapillary venules and capillaries of surrounding arterial microcirculation and later in those of the newly-formed vessels growing into the arterial wall. These results indicate that newly-formed capillaries derived from surrounding arterial microcirculation penetrate the wall of the occluded arterial segments and contribute to the intimal thickening formation. It is likely that the pericytes and endothelial cells (EC) of these ingrowing vessels are sources of myointimal cells at the intimal thickening and of endothelium at the luminal surface, respectively.     

Cell contribution of vasa-vasorum to early arterial intimal thickening formation

In occluded femoral artery segments, intimal thickening occurred and abundant neovascularization from the surrounding microcirculation developed. Under these conditions, the contribution of vasa-vasorum as a source of supplementary population of cells during the early intimal thickening formation was studied. Using a technique that specifically labels venules, predominantly postcapillary venules, a marker-Monastral Blue B-was used as a tracer to follow the pericyte, endothelial cell and monocyte/macrophage lineages. In the first two days of the experiment, the marker was restricted to the wall of the periarterial microcirculation, being incorporated by endothelial cells, pericytes and some monocytes/macrophages crossing the venule walls. Later, the marker continues to be observed in some of the following cells: endothelial cells and pericytes of the newly-formed vessels, fibroblast-like cells, transitional cells between pericytes and fibroblast-like cells, macrophages migrating into the interstitium, myointimal cells and neoendothelial cells of the arterial lumen. These findings provide evidence that, during arterial intimal thickening formation in occluded arterial segments, the periarterial microvascularization contributes, in addition to recruited macrophages, newly-formed endothelial cells and a supplementary population of fibroblast-like cells and myointimal cells.     

Biphasic response of intimal prostacyclin production during the development of experimental atherosclerosis

Feeding a cholesterol rich diet (0.3%) to rabbits for up to 10 weeks resulted in morphological changes of the vascular wall. Microscopic evaluation of the aorta revealed a lipid infiltration and an intimal thickening containing foam cells, which both became more pronounced as the cholesterol feeding was more prolonged. The intimal prostacyclin production showed a transient increase after 2 weeks, but was significantly decreased after 6 weeks of diet and remained at this low level during the rest of the experiment. No significant changes in formation of thromboxane B2 by the platelets could be observed, whereas the production of 12-HETE was enhanced.     

Exogenous heat shock protein-70 inhibits cigarette smoke-induced intimal thickening

Cigarette smoke is associated with increased carotid intimal thickening or stroke. Preliminary work showed that exposure to smoke resulted in a 4.5-fold reduction of heat shock protein-70 (HSP70) expression in spleens of mice using gene microarray analysis. In the current study, we investigated the role of extracellular HSP70 in carotid intimal thickening of mice exposed to cigarette smoke. Intimal thickening was induced by placement of a cuff around the right carotid artery of mice. Cuff injury resulted in increased HSP70 mRNA expression in carotid arteries that persisted for 21 days. Cigarette smoke exposure decreased arterial HSP70 expression and significantly increased intimal thickening compared with mice exposed to air. Treatment of mice exposed to cigarette smoke with intravenous recombinant HSP70 attenuated intimal thickening through reduced phosphorylated extracellular signal-regulated kinase (pERK) expression in the arterial wall. In vitro experiments with rat aortic smooth muscle cells confirmed that recombinant HSP70 decreases pERK and proliferating cell nuclear antigen (PCNA) expression in cells exposed to cigarette smoke extract and H(2)O(2). Our study suggests that decreased expression of arterial HSP70 is an important mechanism by which exposure to cigarette smoke augments intimal thickening. The effects of recombinant HSP70 suggest a role for extracellular HSP70.     

Vitamin C increases the prostacyclin production and decreases the vascular lesions in experimental atherosclerosis in rabbits

Feeding a cholesterol rich diet (0.3%) to rabbits for up to 10 weeks resulted in morphological changes of the vascular wall. Microscopic evaluation of the aorta revealed a lipid infiltration and an intimal thickening containing foam cells, which both became more pronounced as the cholesterol feeding was more prolonged. The intimal prostacyclin production showed a transient increase after 2 weeks, but was significantly decreased after 6 weeks of diet and remained at this low level during the rest of the experiment. No significant changes in formation of thromboxane B₂ by the platelets could be observed, whereas the production of 12-HETE was enhanced.     

Carotid artery ligation induced intimal thickening and proliferation is unaffected by ageing

Following interventions to treat atherosclerosis, such as coronary artery bypass graft surgery, restenosis occurs in approximately 40% of patients. Identification of proteins regulating intimal thickening could represent targets to prevent restenosis. Our group previously demonstrated that in a murine model of vascular occlusion, Wnt4 protein expression and β-catenin signalling was upregulated which promoted vascular smooth muscle cell (VSMC) proliferation and intimal thickening. In this study, the effect of age on VSMC proliferation, intimal hyperplasia and Wnt4 expression was investigated. In vitro proliferation of VSMCs isolated from young (2 month) or old (18–20 month) C57BL6/J mice was assessed by immunocytochemistry for EdU incorporation. As previously reported, 400 ng/mL recombinant Wnt4 protein increased proliferation of VSMCs from young mice. However, this response was absent in VSMCs from old mice. As our group previously reported reduced intimal hyperplasia in Wnt4^+/? mice compared to wildtype controls, we hypothesised that impaired Wnt4 signalling with age may result in reduced neointimal formation. To investigate this, carotid artery ligation was performed in young and old mice and neointimal area was assessed 21 days later. Surprisingly, neointimal area and percentage lumen occlusion were not significantly affected by age. Furthermore, neointimal cell density and proliferation were also unchanged. These data suggest that although Wnt4-mediated proliferation was impaired with age in primary VSMCs, carotid artery ligation induced neointimal formation and proliferation were unchanged in old mice. These results imply that Wnt4-mediated proliferation is unaffected by age in vivo, suggesting that therapeutic Wnt4 inhibition could inhibit restenosis in patients of all ages.     

Effects of soluble factors and extracellular matrix components on vascular cell behavior in vitro and in vivo: models of de-endothelialization and repair.

Vessel walls are comprised of several different cell populations residing in and on complex extracellular matrices. Each of the vascular cell types has diverse and sometimes unique functions and morphologies, and each has roles in repair processes following injury. Large vessel endothelial cells are known to respond to denudation injury by sheet migration and proliferation. This is in contrast to the migration through soft tissues with tube formation and subsequent lumen formation exhibited by microvascular endothelial cells in response to injury. Vascular smooth muscle cells of larger vessels respond to injury by migration from the arterial media into the intima, proliferation, and matrix biosynthesis, ultimately causing intimal thickening. Both these cell types exhibit "dysfunctional" phenotypes during their responses to injury. Microvascular cell responses to injury, while extremely variable, are less well documented. Specifically, responses to injury by microvascular endothelial vascular cells appear to be modulated, in part, by the composition and organization of the surrounding matrix as well as by the various soluble factors and cytokines found at sites of injury, suggesting that the extracellular matrix and soluble factors modulate each other's effects on local vascular cell populations following injury.     

Increased neointimal thickening in dystrophin-deficient mdx mice

Background

The dystrophin gene, which is mutated in Duchenne muscular dystrophy (DMD), encodes a large cytoskeletal protein present in muscle fibers. While dystrophin in skeletal muscle has been extensively studied, the function of dystrophin in vascular smooth muscle is less clear. Here, we have analyzed the role of dystrophin in injury-induced arterial neointima formation.

Methodology/Principal Findings

We detected a down-regulation of dystrophin, dystroglycan and β-sarcoglycan mRNA expression when vascular smooth muscle cells de-differentiate in vitro. To further mimic development of intimal lesions, we performed a collar-induced injury of the carotid artery in the mdx mouse, a model for DMD. As compared with control mice, mdx mice develop larger lesions with increased numbers of proliferating cells. In vitro experiments demonstrate increased migration of vascular smooth muscle cells from mdx mice whereas the rate of proliferation was similar in cells isolated from wild-type and mdx mice.

Conclusions/Significance

These results show that dystrophin deficiency stimulates neointima formation and suggest that expression of dystrophin in vascular smooth muscle cells may protect the artery wall against injury-induced intimal thickening.     

Exendin-4, a glucagon-like peptide-1 receptor agonist, reduces intimal thickening after vascular injury

Glucagon-like peptide-1 is a hormone secreted by L cells of the small intestine and stimulates glucose-dependent insulin response. Glucagon-like peptide-1 receptor agonists such as exendin-4 are currently used in type 2 diabetes, and considered to have beneficial effects on the cardiovascular system. To further elucidate the effect of glucagon-like peptide-1 receptor agonists on cardiovascular diseases, we investigated the effects of exendin-4 on intimal thickening after endothelial injury. Under continuous infusion of exendin-4 at 24 nmol/kg/day, C57BL/6 mice were subjected to endothelial denudation injury of the femoral artery. Treatment of mice with exendin-4 reduced neointimal formation at 4 weeks after arterial injury without altering body weight or various metabolic parameters. In addition, in vitro studies of isolated murine, rat and human aortic vascular smooth muscle cells showed the expression of GLP-1 receptor. The addition of 10 nM exendin-4 to cultured smooth muscle cells significantly reduced their proliferation induced by platelet-derived growth factor. Our results suggested that exendin-4 reduced intimal thickening after vascular injury at least in part by the suppression of platelet-derived growth factor-induced smooth muscle cells proliferation.     

Vitamin C increases the prostacyclin production and decreases the vascular lesions in experimental atherosclerosis in rabbits

Feeding a cholesterol-rich diet (0.3%) to rabbits resulted in an intimal thickening and lipid infiltration of the aorta. The prostacyclin production by the vascular endothelium was significantly decreased, after a transient increase after 2 weeks of diet. The arachidonic acid metabolism in platelets was hardly changed. Addition of a low dose vitamin C (150 mg/day) to the cholesterol rich diet resulted in decreased lipid infiltration and intimal thickening and the transient increase of the prostacyclin production was postponed to the 4th week. Although this dose of vitamin C could not restore the decreased prostacyclin production observed after 6 weeks diet, a higher dose of vitamin C (600 mg/day), besides its beneficial effect on the lipid infiltration and the intimal thickening in the thoracic aorta, kept the intimal prostacyclin production at normal levels for at least 8 weeks.     

Regulatory effects of platelet-derived growth factor-AA homodimer on migration of vascular smooth muscle cells.

Migration of medial smooth muscle cells (SMC) into the intima is important in intimal thickening of atherosclerotic tissues. To study the functions of three isoforms of platelet-derived growth factor (PDGF) in atherosclerosis, we investigated their effects on SMC migration by Boyden's chamber method. Although PDGF-AB and PDGF-BB enhanced SMC migration dose-dependently, PDGF-AA did not enhance SMC migration, but instead inhibited SMC migration induced by PDGF-AB or PDGF-BB. PDGF-AA also inhibited SMC migration induced by two other migration factors, fibronectin and SMC-derived migration factor. PDGF-AA is considered to be coexpressed with transforming growth factor (TGF)-beta 1 in atherosclerotic tissues. Treatment of SMC with TGF-beta 1 reduced an autocrine migration activity from SMC. Studies using anti-PDGF antibody revealed that an increased secretion of PDGF-AA by TGF-beta 1 caused the reduced migration activity. cAMP increase by forskolin and dibutyryl cAMP suppressed SMC migration, whereas cAMP decrease by pertussis toxin had no effects on PDGF-AA-suppressed migration. In contrast, staurosporine, an inhibitor of protein kinase C, enhanced SMC migration and neutralized the inhibitory effect of PDGF-AA. These findings suggest that PDGF-AA regulates SMC migration in intimal thickening in atheroma formation and that protein kinase C may play an important role in the inhibitory mechanism of PDGF-AA.     

Reduced Intimal Thickening Following αvβ 3 Blockade is Associated with Smooth Muscle Cell Apoptosis

The adhesion integrin αvβ3 is expressed by both activated endothelial cells (ECs) and smooth muscle cells (SMCs). Peptide and antibody antagonists of αvβ 3 have been shown to block angiogenesis by initiating unscheduled programmed cell death of proliferating ECs. The present study was designed to determine if antagonism of αvβ 3 immediately following balloon injury might similarly lead to programmed cell death among activated SMCs, and thereby inhibit intimal thickening. LM609, a monoclonal antibody antagonist of αvβ 3, was administered locally and/or systemically immediately after balloon angioplasty in a rabbit model of vascular injury. Immunohistochemical studies documented that LM609, even when administered systemically, localized to sites of vascular injury. LM609 administered immediately following balloon injury of the external iliac artery markedly reduced intimal thickening at 2 and 4 wk post-injury. Apoptosis was abundant where balloon injury resulted in expression of αvβ 3. At both 2 and 4wk, re-endothelialization at the site of balloon injury was not retarded in LM609-treated rabbits versus controls. Thus, blockade of αvβ 3 inhibits intimal thickening when administered immediately following balloon injury. This favorable impact on neointimal thickening is associated with apoptosis of activated SMCs expressing αvβ 3. These findings may explain the reduction in restenosis observed clinically following,93 integrin blockade.     

Systemic and Pulmonary Vascular Remodelling in Chronic Obstructive Pulmonary Disease

BackgroundChronic Obstructive Pulmonary Disease (COPD) is associated with subclinical systemic atherosclerosis and pulmonary vascular remodelling characterized by intimal hyperplasia and luminal narrowing. We aimed to determine differences in the intimal thickening of systemic and pulmonary arteries in COPD subjects and smokers. Secondary aims include comparisons with a non-smokers group; determining the clinical variables associated with systemic and pulmonary intimal thickening, and the correlations between systemic and pulmonary remodelling changes.MethodsAll consecutive subjects undergoing lung resection were included and divided into 3 groups: 1) COPD, 2) smokers, and 3) non-smokers. Sections of the 5th intercostal artery and muscular pulmonary arteries were measured by histo-morphometry. Four parameters of intimal thickening were evaluated: 1) percentage of intimal area (%IA), 2) percentage of luminal narrowing, 3) intimal thickness index, and 4) intima-to-media ratio.ResultsIn the adjusted analysis, the systemic arteries of COPD subjects showed greater intimal thickening (%IA) than those of smokers (15.6±1.5% vs. 14.2±1.6%, p = 0.038). In the pulmonary arteries, significant differences were observed for %IA between the 2 groups (37.3±2.2% vs. 29.3±2.3%, p = 0.016). Among clinical factors, metabolic syndrome, gender and COPD status were associated with the systemic intimal thickening, while only COPD status was associated with pulmonary intimal thickening. A correlation between the %IA of the systemic and pulmonary arteries was observed (Spearman’s rho = 0.46, p = 0.008).ConclusionsGreater intimal thickening in systemic and pulmonary arteries is observed in COPD patients than in smokers. There is a correlation between systemic and pulmonary vascular remodelling in the overall population.     

Thrombin and its protease-activated receptor-1 (PAR1) participate in the endothelial-mesenchymal transdifferentiation process

The serine protease thrombin, independently of its participation in hemostasis and thrombosis, has been involved in tissue repair and remodeling, embryogenesis, angiogenesis, and development and progression of atherosclerosis. Many of these functions appear to be mediated by specific thrombin receptors, particularly the protease-activated receptor-1 (PAR1). In this study, we investigated whether both thrombin and PAR1 were present in the aortic wall of chicken embryos at days 11 and 12 of development. We found that PAR1 was limited to some cells of the intimal thickening and the inner media, whereas thrombin appeared distributed across the aortic wall. We also investigated whether PAR1 was present during endothelial-mesenchymal transdifferentiation (EMT) in vitro. A moderate immunoreactivity was detected in the monolayer of endothelial cells. In contrast, a strong cytoplasmic immunoreactivity was observed in the detaching and migrating cells and those that had acquired mesenchymal characteristics. This PAR1 expression was confirmed by flow cytometry. In this study, the addition of thrombin to arrested endothelial cell cultures was assessed. We found that thrombin stimulated endothelial cell spreading and migration, as no migrating cells were observed in serum-free medium (SFM) condition. Immunolocalization of PAR1 in the thrombin-treated cultures showed strong cytoplasmic immunoreactivity in the monolayers and in spreading and migrating cells, whereas in the SFM condition undetectable PAR1 immunoreactivity was observed. Flow cytometry of these cultures revealed an elevated expression of PAR1 in the presence of thrombin, in contrast to that detected in SFM and complete medium. These data indicate that both thrombin and PAR1 are involved in the remodeling of the aortic wall and intimal thickening formation, and in the endothelial-mesenchymal transdifferentiation process.     

Intimal thickenings in the inferior epigastric artery and coronary bypass

OBJECTIVE: To investigate the relationship between the inferior epigastric artery and the development of pathological intimal thickenings. MATERIAL AND METHODS: The inferior epigastric artery structure was studied with conventional histology techniques, histochemistry, immunohistochemistry and morphometry. RESULTS: The inferior epigastric artery develops 2 types of intimal thickenings: physiological and pathological. In our study, the pathological or atherosclerotic thickenings in this artery were not very common: they were present in 28% of the analysed cases. Vessel flow was not affected as shown by the intimal thickness index (I.T.I.) and the lumen reduction index (L.R.I.). The alpha-actin technique confirmed the role of the smooth muscle cells in the genesis of the thickening. Furthermore, histochemistry demonstrated that the extracellular matrix is rich in highly sulphated acid mucopolysaccharides. CONCLUSION: The inferior epigastric artery seems to be a good candidate for grafting in coronary revascularization because of the low susceptibility to thickening of its intima and the minimal repercussion on its blood flow.     

Reduced intimal thickening following alpha(v)beta3 blockade is associated with smooth muscle cell apoptosis

The adhesion integrin alpha(v)beta3 is expressed by both activated endothelial cells (ECs) and smooth muscle cells (SMCs). Peptide and antibody antagonists of alpha(v)beta3 have been shown to block angiogenesis by initiating unscheduled programmed cell death of proliferating ECs. The present study was designed to determine if antagonism of alpha(v)beta3 immediately following balloon injury might similarly lead to programmed cell death among activated SMCs, and thereby inhibit intimal thickening. LM609, a monoclonal antibody antagonist of alpha(v)beta3, was administered locally and/or systemically immediately after balloon angioplasty in a rabbit model of vascular injury. Immunohistochemical studies documented that LM609, even when administered systemically, localized to sites of vascular injury. LM609 administered immediately following balloon injury of the external iliac artery markedly reduced intimal thickening at 2 and 4 wk post-injury. Apoptosis was abundant where balloon injury resulted in expression of alpha(v)beta3. At both 2 and 4 wk, re-endothelialization at the site of balloon injury was not retarded in LM609-treated rabbits versus controls. Thus, blockade of alpha(v)beta3 inhibits intimal thickening when administered immediately following balloon injury. This favorable impact on neointimal thickening is associated with apoptosis of activated SMCs expressing alpha(v)beta3. These findings may explain the reduction in restenosis observed clinically following beta3 integrin blockade.