Intimal Lesion Formation Research Articles

Disease-Relevant Single Cell Photonic Signatures Identify S100\u03b2 Stem Cells and their Myogenic Progeny in Vascular Lesions

A hallmark of subclinical atherosclerosis is the accumulation of vascular smooth muscle cell (SMC)-like cells leading to intimal thickening and lesion formation. While medial SMCs contribute to vascular lesions, the involvement of resident vascular stem cells (vSCs) remains unclear. We evaluated single cell photonics as a discriminator of cell phenotype in vitro before the presence of vSC within vascular lesions was assessed ex vivo using supervised machine learning and further validated using lineage tracing analysis. Using a novel lab-on-a-Disk(Load) platform, label-free single cell photonic emissions from normal and injured vessels ex vivo were interrogated and compared to freshly isolated aortic SMCs, cultured Movas SMCs, macrophages, B-cells, S100β+ mVSc, bone marrow derived mesenchymal stem cells (MSC) and their respective myogenic progeny across five broadband light wavelengths (λ465 - λ670 ± 20 nm). We found that profiles were of sufficient coverage, specificity, and quality to clearly distinguish medial SMCs from different vascular beds (carotid vs aorta), discriminate normal carotid medial SMCs from lesional SMC-like cells ex vivo following flow restriction, and identify SMC differentiation of a series of multipotent stem cells following treatment with transforming growth factor beta 1 (TGF- β1), the Notch ligand Jagged1, and Sonic Hedgehog using multivariate analysis, in part, due to photonic emissions from enhanced collagen III and elastin expression. Supervised machine learning supported genetic lineage tracing analysis of S100β+ vSCs and identified the presence of S100β+vSC-derived myogenic progeny within vascular lesions. We conclude disease-relevant photonic signatures may have predictive value for vascular disease.Graphical abstract

Dichloroacetate prevents but not reverses the formation of neointimal lesions in a rat model of severe pulmonary arterial hypertension.

The formation of neointimal lesions is one of the characteristic pathological alterations of the pulmonary vasculature in response to sustained pulmonary arterial hypertension (PAH). To date, the effect of dichloroacetate (DCA) on these intimal lesions had remained to be investigated. In the present study, the beneficial impact of DCA on the formation of neointimal lesions was examined in rats treated with monocrotaline following pneumonectomy. The rats were subjected to monocrotaline subcutaneous injection seven days following left pneumonectomy and received DCA by gastric gavage (80mg/kg/day) for 20days. At the end of the experiments, hemodynamic measurement was performed prior to the excision of the rats' right lungs for further morphometric and immunoblot analysis. Furthermore, the activity of reactive oxygen species (ROS), superoxide dismutase (SOD) in the lungs was examined by a colorimetric assay. The results revealed that DCA treatment from day 8-28following pneumonectomy caused a marked reduction in pulmonary arterial pressure and amelioration in right ventricle hypertrophy (mean pulmonary arterial pressure, 24±2.8 vs. 33±5.5mmHg; right ventricle-to-left ventricle + intra-ventricle septum ratio, 29±2.8 vs. 43±3.3%; P<0.05) and pulmonary arterioles intimal proliferation (grade 1 and 2 occlusion, 24 and 20% vs. 44 and 40%; P<0.05) in pulmonary hypertensive rats. By contrast, DCA treatment initiated 29days following pneumonectomy did not result in any improvement in pulmonary circulatory parameters or regression of occlusive neointimal lesions. Immunohistochemical and immunoblot assays demonstrated markedly reduced hypoxia inducible factor (HIF-1α) levels, and increased voltage-dependent potassium channel subtype 1.5 (Kv1.5) expression levels were observed in those neointimal regions in rats receiving DCA preventive therapy. Furthermore, preventive treatment with DCA significantly increased the activity of copper/zinc superoxide dismutase (Cu/Zn SOD activity, 88±4.2 vs. 53±2.7U/mgprot; P<0.05) and promoted the degradation of ROS (106±4.7 vs. 79±13.3U/mgprot; P<0.05), which was compromised in the delayed intervention group. Therefore, DCA is effective to prevent the formation of intimal lesions, which may be attributed to the induction of the upregulation of Cu/Zn SOD activity and the suppression of HIF-1α activation.

Non-endothelial cell endothelin-B receptors limit neointima formation following vascular injury

The potent vasoconstrictor endothelin-1 (ET-1), acting on the endothelin-A (ETA) receptor, promotes intimal lesion formation following vascular injury. The endothelin-B (ETB) receptor, which mediates nitric oxide release and ET-1 clearance in endothelial cells, may moderate lesion formation, but this is less clear. We used selective ET receptor antagonists and cell-specific deletion to address the hypothesis that ETB receptors in the endothelium inhibit lesion formation following arterial injury. Neointimal proliferation was induced by wire or ligation injury to the femoral artery in mice treated with selective ETA (ABT-627) and/or ETB antagonists (A192621). Measurement of lesion formation by optical projection tomography and histology indicated that ETA blockade reduced lesion burden in both models. Although ETB blockade had little effect on ligation injury-induced lesion formation, after wire injury, blockade of the ETB receptor increased lesion burden (184% of vehicle; P < 0.05) and reversed the protective effects of an ETA antagonist. Selective deletion of ETB receptors from the endothelium, however, had no effect on neointimal lesion size. These results are consistent with ETB receptor activation playing an important role in limiting neointimal lesion formation following acute vascular injury, but indicate that this protective effect is not mediated by those ETB receptors expressed by endothelial cells. These data support the proposal that selective ETA antagonists may be preferable to mixed ETA/ETB antagonists for targeting the arterial response to injury.

Changing topographic Hox expression in blood vessels results in regionally distinct vessel wall remodeling

SummaryThe distinct topographic Hox expression patterns observed in vascular smooth muscle cells (VSMCs) of the adult cardiovascular system suggest that these transcriptional regulators are critical for maintaining region-specific physiological properties of blood vessels. To test this proposition, we expanded the vascular Hoxc11 expression domain normally restricted to the lower limbs by utilizing an innovative integrated tetracycline regulatory system and Transgelin promoter elements to induce Hoxc11 expression universally in VSMCs of transgenic mice. Ectopic Hoxc11 expression in carotid arteries, aortic arch and descending aorta resulted in drastic vessel wall remodeling involving elastic laminae fragmentation, medial smooth muscle cell loss, and intimal lesion formation. None of these alterations were observed upon induction of Hoxc11 transgene expression in the femoral artery, i.e. the natural Hoxc11 activity domain, although this vessel was greatly enlarged, comparable to the topographically restricted vascular changes seen in Hoxc11−/− mice. To begin defining Hoxc11-controlled pathways of vascular remodeling, we performed immunolabeling studies in conjunction with co-transfection and chromatin immunoprecipitation (ChIP) assays using mouse vascular smooth muscle (MOVAS) cells. The results suggest direct transcriptional control of two members of the matrix metalloproteinase (Mmp) family, including Mmp2 and Mmp9 that are known as key players in the inception and progression of vascular remodeling events. In summary, the severe vascular abnormalities resulting from the induced dysregulated expression of a Hox gene with regional vascular patterning functions suggest that proper Hox function and regulation is critical for maintaining vascular functional integrity.

CC chemokine receptor-1 activates intimal smooth muscle-like cells in graft arterial disease.

Graft arterial disease (GAD) limits long-term solid-organ allograft survival. The thickened intima in GAD contains smooth muscle-like cells (SMLCs), leukocytes, and extracellular matrix. The intimal SMLCs in mouse GAD lesions differ from medial smooth muscle cells in their function and phenotype. Although intimal SMLCs may originate by migration and modulation of donor medial cells or by recruitment of host-derived precursors, the mechanisms that underlie their localization within grafts and the factors that drive these processes remain unclear. This study of aortic transplantation in mice demonstrated an important function for chemokines beyond their traditional role in leukocyte recruitment and activation. Intimal SMLCs, but not medial smooth muscle cells, express functional CC chemokine receptor-1 (CCR1) and respond to RANTES by increased migration and proliferation. Although RANTES infusion in vivo promoted inflammatory cell accumulation in the adventitia of aortic allografts of wild-type and CCR1-deficient recipients, it increased GAD intimal thickening with SMLC proliferation in only the wild-type hosts. Aortic allografts transplanted into CCR1-deficient mice after wild-type bone marrow transplantation did not develop intimal lesions, which indicates that CCR1-bearing inflammatory cells do not contribute to intimal lesion formation. Moreover, RANTES induced SMLC proliferation in vitro but did not promote medial smooth muscle cell growth. Blockade of CCR5 attenuated RANTES-induced T-cell and monocyte/macrophage proliferation but did not affect RANTES-induced SMLC proliferation, consistent with a larger role of CCR1-binding chemokines in SMLC migration and proliferation and GAD development. These studies provide a novel mechanistic insight into the formation of vascular intimal hyperplasia and suggest a novel therapeutic strategy for preventing allograft arteriopathy.

Arterial internal elastic lamina holes: relationship to function?

Internal elastic lamina (IEL) hole (fenestration) characteristics and myoendothelial gap junction (MEGJ) density were examined in selected resistance and conduit arteries of normal and diseased rat and mouse models, using conventional, ultrastructural and confocal microscopy methods. Selected vessels were those commonly used in functional studies: thoracic aorta, proximal and distal mesenteric, caudal, saphenous, middle-cerebral and caudal cerebellar artery. Rat and mouse strains and treatment groups examined were Dahl, Sprague Dawley, Wistar Kyoto, Wistar, spontaneously hypertensive (SHR), deoxycorticosterone (DOC) treated rat; and apolipoprotein E knockout, C57/BL6 and BALB/c mice. Vessel size (as IEL circumference), IEL hole and MEGJ density were quantified. In mesenteric arteries, the width of IEL holes and the percent of IEL occupied by holes were also determined. IEL hole density varied significantly within and between mesenteric artery beds, even among normotensive rat strains. Among the hypertensive rats (SHR and DOC), hole density in some vessels was higher in the normotensives than in the hypertensives within each strain, whereas in Dahl rats, hole density was similar between hypertensives and normotensives. Hole density was not correlated with the formation of intimal lesions in superior mesenteric artery. There was no positive general correlation between IEL hole and MEGJ density in resistance and conduit vessels. However, there was a positive correlation between the size of some resistance arteries and MEGJ density, although such a relationship did not hold for conduit vessels or during development, and there was no such relationship between vessel size and IEL hole density. Whilst IEL holes are obviously required for MEGJ communication, their presence is not an indication of contact-mediated communication, but rather may be related to the presence of sites for the low resistance passage of diffusion-mediated release of vasoactive endothelial and smooth muscle substances.

Integrin α vβ 3 as a target in the prevention of neointimal hyperplasia

Although major advances have been made in the prevention and treatment of restenosis following coronary and peripheral interventions, the persistent complications of thrombosis and reintervention remain a mainstay for repeat hospitalizations in this patient population. For many years, a ubiquitous cell surface receptor called alpha(v)beta(3) integrin was the target of investigators in the prevention of restenosis because its interaction with the extracellular matrix was believed to coordinate the migration of smooth muscle cells (SMCs) from the media to the intima, the seminal event in the formation of intimal occlusive lesion. After the publication of uniformly positive animal studies demonstrating that alpha(v)beta(3) integrin blockade led to a significant reduction in new intimal (neointimal) lesion formation, early clinical trials supported the association of avoidance of target lesion revascularization and the use of antagonists to the SMC integrin alpha(v)beta(3) and its related platelet integrin alpha(IIb)beta(3). However, a series of clinical trials subsequently demonstrated that these antagonists did not necessarily prevent revascularizations by inhibiting intimal hyperplasia per se. Additional animal studies subsequently showed that, indeed, in the setting of pre-existing SMCs in the intimal lesion (ie, atherosclerotic plaque, fatty streaks), inhibiting SMC migration by way of beta(3) integrin blockade was an ineffective approach in the prevention of intimal hyperplasia and restenosis. However, given the wealth of basic and clinical information on the alpha(v)beta(3) integrin and its antagonists, we discuss in this article our new approach to this old solution by targeting a new clinical problem of early failure arteriovenous access for hemodialysis. Given the uniqueness of arteriovenous access in that there are essentially no significant atherosclerotic lesions in the artery and vein prior to the anastomosis, the seminal event of the migration of SMCs from the media to the neointima could by targeted once again with beta(3) integrin antagonists.

Activation of adventitial fibroblasts contributes to the early development of atherosclerosis: A novel hypothesis that complements the “Response-to-Injury Hypothesis” and the “Inflammation Hypothesis”

The role of the adventitia in vascular function and vascular lesion formation has been largely ignored. This article introduces the hypothesis that the activation of the adventitia, specifically the fibroblasts, contributes to the formation of intimal atherosclerotic lesions. The evidence for this hypothesis includes: (a) the early proliferative changes seen in fibroblasts found in the adventitia; (b) the increase and the alteration of extracellular matrix deposition in the adventitia; (c) fibroblast differentiation into myofibroblasts and migration into the intima; and (d) fibroblast synthesis and release of cytokines that have potent effects on neighboring smooth muscle and endothelial cells prior to intimal lesion formation. In conclusion, the activation of adventitial fibroblasts is a key regulator of vascular function and structure from the "outside-in" and contributes to the development of atherosclerotic lesions. The outer location of the adventitia makes it a suitable location for drug delivery and gene therapy aimed at preventing and treating atherosclerosis.

Adventitial fibroblasts are activated in the early stages of atherosclerosis in the apolipoprotein E knockout mouse

The role of the adventitia in vascular function and vascular lesion formation has been largely ignored. This study observed the activation of the adventitia and specifically the fibroblasts in the development of atherosclerosis in the apoE(−/−) mouse. The results showed a gradual increase in expression of collagen types I and III after 2, 4, and 8 weeks of hyperlipidic diet. The earliest expression of monocyte chemoattractant protein-1 (MCP-1) protein and mRNA was detected in the adventitial fibroblast before the formation of intimal lesions. Proliferation, too, was first found in the adventitial fibroblasts. We hypothesize that the adventitial fibroblast is activated in the early stage of atherosclerosis. Adventitial inflammation may be an early event in the development of atherosclerotic lesions.

Femoral artery neointimal hyperplasia is reduced after wire injury in Ref-1+/− mice

Redox factor-1 (Ref-1) is a multifunctional protein that regulates redox, DNA repair, and the response to cell stress. We previously demonstrated that Ref-1(+/-) mice exhibit a significantly reduced Ref-1 mRNA and protein levels within the vasculature, which are associated with increased oxidative stress. The goal of this study was to test the hypothesis that partial loss of Ref-1 altered the cellular response to vascular injury. Fourteen days after femoral artery wire injury, we found that vessel intima-to-media ratio was significantly reduced in Ref-1(+/-) mice compared with that in wild-type mice (P < 0.01). Bromodeoxyuridine labeling and transferase-mediated dUTP nick-end labeling staining at 14 days did not differ in the Ref-1(+/-) mice. In vitro studies found no significant changes in either serum-induced proliferation or baseline apoptosis in Ref-1(+/-) vascular smooth muscle cells. Exposure to Fas ligand; however, did result in increased susceptibility of Ref-1(+/-) vascular smooth muscle cells to apoptosis (P < 0.001). Ref-1(+/-) mice exhibited an increase in circulating baseline levels of IL-10, IL-1alpha, and VEGF compared with those in wild-type mice but a marked impairment in these pathways in response to injury. In sum, loss of a single allele of Ref-1 is sufficient to reduce intimal lesion formation and to alter circulating cytokine and growth factor expression.

A TGF-β1-Dependent Autocrine Loop Regulates the Structure of Focal Adhesions in Hypertrophic Scar Fibroblasts

Following injury, fibroblasts migrate into wounds and differentiate into alpha smooth muscle cell actin (SMCA)-positive cells, termed myofibroblasts, that assemble and remodel the scar. Cultured myofibroblasts assemble larger focal adhesions than do normal dermal fibroblasts and these focal adhesions attach to alpha SMCA-rich stress fibers. Following severe traumatic or thermal injury to the dermis, hypertrophic scars (HTSs) often develop and these scar fibroblasts (HTSFs) express alpha SMCA persistently. We now report that HTSFs stably display large focal adhesions as a consequence of both the autocrine production and activation of transforming growth factor beta1 (TGF-beta1). We also observe that myofibroblasts elaborating larger focal adhesions adhere more tightly to fibronectin. Conditioned medium from HTSFs induces focal adhesion growth in normal fibroblasts and this is blocked by pre-incubation with a soluble TGF-beta1 receptor mimetic. Human foreskin fibroblasts transduced with a retrovirus encoding active TGF-beta1 elaborate large focal adhesions, whereas fibroblasts overexpressing normal, latent TGF-beta1 do not. We conclude that the large focal adhesions found in pathogenic myofibroblasts arise through an autocrine loop involving the production and activation of TGF-beta1; these adhesions likely mediate both tighter adhesion to wound matrix and the exuberant wound contraction observed in pathogenic scars.

Tu-P7:84 JE MRNA expression and monocyte chemoattractant protein-1 expression in the adventitial fibroblasts of aortic root in apolipoprotein gene knockout mice before the intimal lesion formation

Tu-P7:84 JE MRNA expression and monocyte chemoattractant protein-1 expression in the adventitial fibroblasts of aortic root in apolipoprotein gene knockout mice before the intimal lesion formation

Vascular Wall Remodeling in Patients with Supravalvular Aortic Stenosis and Williams Beuren Syndrome

Supravalvular aortic stenosis (SVAS) and Williams Beuren syndrome (WBS) can be considered as inherited diseases affecting the whole arterial tree and causing narrowing of the vessels. It has been reported that abnormal deposition of elastin in arterial walls of patients with SVAS and WBS leads to increased proliferation of arterial smooth muscle cells (SMC), which result in the formation of hyperplastic intimal lesions. In this work, we conducted morphological and morphometrical analysis with stenotic aortas from patients suffering from SVAS and WBS and from healthy control subjects and demonstrated that the amount of elastic fibers and the loss of integrity of vascular elastic fibers in the aortas reflect similar changes in the skin of patients with SVAS or WBS, as reported in our previous work conducted on skin in these pathological states. On the other hand, we conducted investigations on metalloproteinases (MMP2, MMP9, MMP7) and their specific tissue inhibitors TIMP1 and TIMP2 to verify their possible involvement in the etiopathogeny of SVAS and WBS. We particularly evidenced an altered MMP9/TIMP1 balance in favor of matrix degradation which could facilitate SMC migration and neointimal hyperplasia. Our findings suggest that elastinolytic enzymes secreted by arterial SMC, possibly including matrilysin 1, are critical for the development of arterial lesions in SVAS and WBS and contribute to perpetuate arterial stenosis in either SVAS or WBS.

Inflammatory signaling pathway containing TRAF6 contributes to neointimal formation via diverse mechanisms

The purpose of this study was to investigate the contribution of inflammatory signaling containing tumor necrosis factor receptor-associated factor 6 (TRAF6) to neointimal formation in a balloon injury model of rabbit carotid artery. Male Japanese white rabbits fed a normal diet were used. We transferred the dominant negative (DN) form of TRAF6 to a rabbit carotid artery that was subjected to balloon injury by in vivo electroporation method, and then evaluated its effect on intimal lesion formation after balloon injury. An expression plasmid vector containing the TRAF6 DN sequence was successfully transferred to arterial wall cells, and its inhibitory effect on inflammatory signaling was confirmed by the marked suppression of nuclear factor-kappaB (NFkappaB) activity after injury. Morphometric analyses revealed significant inhibition of intimal lesion formation at 7 days after injury. Cell replication and accumulation of macrophages in the media were significantly decreased, and apoptosis was enhanced on day 2. Cell migration to the intima was suppressed on day 4. Extracellular signal-regulated kinase1/2 (ERK1/2) activity at 2 h after injury was also down-regulated. Interestingly, intimal cell replication was significantly blocked when TRAF6 DN was transfected at 7 days after injury. TRAF6 plays important roles in cell replication and migration, besides promotion of inflammatory cell infiltration and suppression of apoptosis.

Inhibition of Vascular Oxidative Stress in Hypercholesterolemia by Eccentric Isosorbide Mononitrate

We sought to determine if the nitric oxide (NO) donor isosorbide mononitrate (ISMN) (200 mg/kg body weight/day) decreases vascular bioavailability of superoxide in atherosclerosis. Vascular oxidative stress limits the bioavailability of endothelial NO and promotes atherosclerosis, while NO itself exerts antioxidative effects. It is unknown if therapeutic NO impacts on vascular oxidative stress in atherosclerosis. New Zealand white rabbits (n = 10 each group) were fed either normal chow (control), cholesterol chow (CHOL) (0.75%), or cholesterol chow enriched with ISMN (CHOL-ISMN). Rabbits were fed twice daily. After 16 weeks we used aortic segments to measure vascular superoxide (5-microM lucigenin), intimal lesion formation, and vasoreactivity to acetylcholine (ACH) and ISMN. Plasma cholesterol increased by 40-fold in CHOL and CHOL-ISMN. The plasma concentration of ISMN in CHOL-ISMN was 1,529 +/- 447 ng/ml. Superoxide formation (control: 228 +/- 20 counts/20 min/mg) was strongly enhanced in CHOL (345 +/- 46 counts/20 min/mg, p = 0.02) but not in CHOL-ISMN (229 +/- 23 counts/20 min/mg) demonstrating antioxidative effects of eccentric ISMN in vivo. In parallel, intima-media thickness of thoracic aorta (159 +/- 4 microm in control) was reduced from 645 +/- 41 microm (CHOL) to 440 +/- 51 microm (CHOL-ISMN, p < 0.05). Likewise, eccentric ISMN partially restored vascular responses to the NO donor S-nitroso-N-acetyl-D,L-penicillamine and improved endothelium-dependent vasorelaxation. The maximal ACH relaxation increased from 26.3 +/- 9.6% in CHOL to 49.7 +/- 8.1% in CHOL-ISMN; ISMN treatment induced a moderate nitrate tolerance as evidenced by diminished ISMN-induced vasodilation. These data suggest that eccentric ISMN can completely inhibit the increase of vascular bioavailability of superoxide and partially prevent intimal lesion formation and endothelial dysfunction in hypercholesterolemia.

Increased intimal hyperplasia and smooth muscle cell proliferation in transgenic mice with heparan sulfate-deficient perlecan.

Smooth muscle cell (SMC) proliferation is a critical process in vascular disease. Heparan sulfate (HS) proteoglycans inhibit SMC growth, but the role of endogenous counterparts in the vessel wall in control of SMC function is not known in detail. Perlecan is the major HS proteoglycans in SMC basement membranes and in vessel wall extracellular matrix (ECM). In this study, transgenic mice with HS-deficient perlecan were analyzed with respect to vascular phenotype and intimal lesion formation. Furthermore, SMC cultures were established and characterized with respect to morphology, immunocytochemical features, proteoglycan synthesis, proliferative capacity, and ECM binding of basic fibroblast growth factor (FGF-2). In vitro, mutant SMCs formed basement membranes with perlecan core protein, but with decreased levels of HS, they showed diminished secretion of HS-containing perlecan into the medium and a defective ECM-binding capacity of FGF-2. In vitro, mutant SMCs showed increased proliferation compared with wild-type cells, and in vivo, enhanced SMC proliferation and intimal hyperplasia were observed after flow cessation of the carotid artery in mutant mice. The results indicate that the endogenous HS side-chains of perlecan contribute to SMC growth control both in vitro and during intimal hyperplasia, possibly by sequestering heparin-binding mitogens such as FGF-2.

Morphology of the Internal Elastic Lamina in Arteries from Pulmonary Hypertensive Patients: a Confocal Laser Microscopy Study

The development and progression of pulmonary hypertension lesions involve continuous remodeling of the arterial wall, including the extracellular matrix components. The integrity of the internal elastic lamina may represent a barrier to cell migration and formation of intimal proliferative lesions. Some patients with congenital cardiac shunts develop precocious intimal occlusive lesions,whereas others evolve with isolated medial hypertrophy. We studied the 2-D and 3-D morphology of the internal elastic lamina of peripheral pulmonary arteries to search for any difference regarding the type of histological lesion. Fifteen lung biopsies collected for diagnostic purposes from patients with congenital shunts and 6 control lungs (mean ages, 15.8 and 14.7 mo) were studied using the confocal laser scanning microscope, under predetermined conditions of laser intensity, brightness and contrast. We measured the thickness of the internal elastic lamina and determined the number of gaps and projections of elastic tissue towards the medial and intimal layers. The mean internal elastic lamina thickness was significantly higher in arteries from cases with isolated medial hypertrophy when compared with controls and to those with proliferative lesions (P < .05). The number of gaps of the internal elastic lamina was higher in arteries >100 μm in diameter from the group with intimal lesions when compared to the cases presenting with isolated hypertrophy, but did not differ from the controls. There was a positive linear correlation between the external arterial diameter and the thickness of the internal elastic lamina (r = .74, P < .001) in cases presenting isolated medial hypertrophy. The increased thickness and smaller number of gaps of the internal elastic lamina may act as a barrier that prevents smooth muscle cell migration in patients with pulmonary hypertension without intimal proliferative lesions. On the other hand, a greater number of gaps does not represent, by itself, unrestrained migration, because controls also showed fenestrated laminae.

Platelet-derived growth factor receptor inhibition reduces allograft arteriosclerosis of heart and aorta in cholesterol-fed rabbits.

Crosstalk between pro-inflammatory cytokines and platelet-derived growth factor (PDGF) regulates smooth-muscle-cell proliferation in cardiac-allograft arteriosclerosis. In this study, we tested the effect of STI 571, a novel orally active protein tyrosine kinase (PTK) inhibitor selective for PDGF receptor (PDGF-R) on transplant and accelerated arteriosclerosis in hypercholesterolemic rabbits. Cardiac allografts were transplanted heterotopically from Dutch Belted to New Zealand White rabbits. A 0.5% cholesterol diet was begun 4 days before transplantation. Recipients received STI 571 5 mg/kg per day or vehicle intraperitoneally throughout the study period of 6 weeks. Cyclosporine A was given as background immunosuppression. In cardiac allografts of vehicle-treated rabbits, 76.2+/-2.1% of medium-sized arteries were affected by intimal thickening, and the percentage of arterial occlusion was 45.0+/-5.0%. Treatment with STI 571 reduced the incidence of affected medium-sized arteries to 41.2+/-8.1% (P <0.05) and the arterial occlusion to 27.6+/-5.0% ( P<0.05). In addition, we observed that STI 571 treatment reduced intimal lesion formation in proximal ascending aorta of transplanted hearts from 72.3+/-19.9 to 12.7+/-1.9 microm ( P<0.05). Our results also show that STI 571 significantly inhibited accelerated arteriosclerosis in medium-sized arteries of recipients' own hearts. The results of the present study suggest that PDGF-R activation may regulate the development of transplant and accelerated arteriosclerosis in hypercholesterolemic rabbits. Thus, PTK inhibitors may provide new strategies for prevention of these fibroproliferative vascular disorders.

In vivo evidence for a role of toll-like receptor 4 in the development of intimal lesions.

Inflammation plays an important role in atherogenesis. The toll-like receptor 4 (TLR4) is the receptor for bacterial lipopolysaccharides and also recognizes cellular fibronectin and heat shock protein 60, endogenous peptides that are produced in response to tissue injury. To explore a possible role for this receptor in arterial obstructive disease, we determined the expression of TLR4 in the atherosclerotic arterial wall, including adventitia, and studied the effect of adventitial TLR4 activation on neointima formation in a mouse model. Localization of TLR4 was studied in human atherosclerotic coronary arteries by immunohistochemistry and detected in plaque and adventitia. In the adventitia, not all TLR4-positive cells colocalized with macrophages. In primary human adventitial fibroblasts, expression of TLR4 was demonstrated by immunofluorescence, Western blot, and reverse transcriptase-polymerase chain reaction. Adding lipopolysaccharide to these fibroblasts induced activation of NF-kappaB and an increase of mRNAs of various cytokines. The effect of adventitial stimulation of TLR4 was studied in a mouse model. A peri-adventitial cuff was placed around the femoral artery. Application of lipopolysaccharide between cuff and artery augmented neointima formation induced by the cuff (intimal area+/-SEM, 9134+/-1714 versus 2353+/-1076 microm(2), P<0.01). In TLR4-defective mice, application of cuff and lipopolysaccharide resulted in a smaller neointima than in wild-type mice (intimal area, 3859+/-904 microm(2), P=0.02 versus wild type). A functional TLR4 is expressed in human adventitial fibroblasts and macrophages. Adventitial TLR4 activation augmented neointima formation in a mouse model. These results provide evidence for a link between the immune receptor TLR4 and intimal lesion formation.

Pathogenetic heterogeneity of in-stent lesion formation in human peripheral arterial disease

Purpose: Intimal hyperplasia is known to promote in-stent restenosis after vascular stent implantation. Although previous studies have presented a variety of evidence that suggests the mechanisms of intimal lesion formation, it is still controversial which factor(s) predominantly contribute to the development of in-stent restenosis. In this study, we hypothesized that heterogenous mechanisms coexist in the same lesion and then assessed the validity with resected whole arteries with in-stent lesions. Methods: Whole arterial specimens with in-stent lesions were surgically resected from five patients who had undergone implantation of a Palmaz-Schatz stent 7 to 19 months previously and were histologically analyzed. For assessment of the pathogenetic heterogeneity of in-stent lesions, we divided each cross section into three parts: the inner intima within 250 μm from the luminal surface (zone A), the area surrounding the stent struts within 250 μm from the strut hole (zone B), and the remaining part of the intimal layer (zone C). We then evaluated cell density, cell replication, and cellular composition in each zone. Cell replication and cellular composition were analyzed with immunohistologic staining with antibodies against proliferating cell nuclear antigen and cell-specific antibodies. Each section was also stained with Alcian blue or Elastica van Gieson method for detection of matrix components. Results: In all samples, the cell density of zone A was significantly higher than that of zone B (P <.05). Proliferating cell nuclear antigen staining results showed significantly higher cell replication in zone A as compared with that in other zones (P <.05). To the contrary, cell-specific immunostaining results revealed marked accumulation of leukocytes, macrophages, and T lymphocytes in zone B (P <.05). Regarding matrix components, proteoglycan was predominantly stained around stent struts and in the inner intima. Conclusion: The data of this study showed that two different pathogenetic processes in different zones possibly contributed to in-stent lesion formation at the same time. One process was an increase of cell number in the inner intima, which was the result of a prolonged increase of cell replication. The other process was accumulation of matrix around stent struts, which was suggested to be linked to infiltration of inflammatory cells in the same zone. (J Vasc Surg 2002;35:672-8.)