A new concept of development of neointimal hyperplasia

The intima hyperplasia is a major morphological feature of various arterial pathologies such as atherosclerosis, postangioplasty restenosis and transplantation arteriopathy. It is commonly assumed that smooth muscle cells (SMC) comprising loci of the intima hyperplasia originate from arterial media. However, recent studies suggest that the bone marrow could also supply circulating vascular progenitor of SMCs and endothelial cells (EC). Such bone marrow progenitors participate in the formation of a cellular mass of neointima after experimental allotransplantation, mechanical vessel injury or hyperlipidemia induced experimental atherosclerosis. Circulating SMC and EC progenitors are also likely to be involved in the transplantation arteriopathy development in humans but their roles in the atherosclerosis and restenosis remain to be determined. Stages of the mobilization, defferentiation and proliferation of SMC progenitors could provide point of attack for new therapeutic strategies for the treatment of proliferative vascular diseases. The precise understanding of the neointima cells origin could provide a key for development of the optimal therapeutic strategy of treatnent of such disorders. This review is focused on the pathological significance of circulating progenitors of the bone marrow origin, particularly on the SMC progenitors, for development of vascular wall disorders.     

Endothelial cells are activated by angiopoeitin-1 gene transfer and produce coordinated sprouting in vitro and arteriogenesis in vivo

RATIONAL AND OBJECTIVES: Activation of fully differentiated vascular cells using angiogenic genes can lead to phenotypic changes resulting in formation of new blood vessels. We tested whether Ang-1 gene transfer to endothelial cells (EC) activates these cells. METHODS AND RESULTS: EC and SMC were transduced using retroviral or adenoviral vectors to produce Ang-1 or vascular endothelial growth factor (VEGF). EC Tie-2 receptor was phosphorilated by autologous secretion of Ang-1. Transduced EC and SMC sprouting capacity was tested using collagen embedded spheroids assay and capacity to produce arteriogenesis was tested in a hind limb model of ischemia. EC expressing Ang-1 in the presence of SMC expressing VEGF exhibited high levels of sprouting of the two cell types. Flow and numbers of arteries were increased after transduced cells implantation in vivo. CONCLUSIONS: Autologous secretion of Ang-1 by transduced EC resulted in Tie-2 activation and in the presence of SMC expressing VEGF resulted in coordinated sprouting in vitro and increase in flow and number of arteries in vivo.     

Uptake by vascular smooth muscle cells plays an important role in targeting of lipid microspheres incorporating prostaglandin E1 into a thickened intima

A study was done to determine how lipid microspheres (LM) containing prostaglandin E1 (lipo PGE1) accumulate in injured arterial tissue. After administration of lipo PGE1 labeled with a fluorescence probe, 1,1'-dioctadecyl-3, 3, 3',3'-tetramethyl-indocarbocyanine perchlorate (DiI-lipo PGE1) to rats 14 of days after balloon injury of the carotid artery, localization into the injured site was examined using a fluorescence confocal laser scanning microscope (CLSM). In contrast with the normal carotid artery, DiI-lipo PGE1 accumulated remarkably in neointima of the injured site which was occupied mainly by the migration of the proliferating and quiescent vascular smooth muscle cells (SMC). In vitro cellular uptake of DiI-lipo PGE1 was semi-quantitatively measured using CLSM, regarding differentiated and proliferative phenotypes of human vascular SMC, compared with the human endothelial cells (EC) and mouse fibroblasts. The differentiated SMC incorporated DiI-lipo PGE1 to equal or a higher level of the proliferative phenotype, and was significantly higher than EC and fibroblasts. The uptake of DiI-lipo PGE1 by both SMC and EC was inhibited at 4 degrees C, by dansylcadaverine and excessive LM, but was unaffected by cytochalasin B. These results suggest that the uptake of DiI-lipo PGE1 by SMC plays an important role in localization of DiI-lipo PGE1 at the injured site, and that the uptake seems to be a receptor mediated endocytosis.     

Stem cell marker expression,proliferation and apoptosis of vascular smooth muscle cells

Vascular endothelial Flt-1 and other stem cell markers are variably expressed in vascular smooth muscle cells (SMCs) during normal and pathological conditions, but their biological role remains uncertain. In normal rat aorta, rare flt-1+ and c-kit+ SMCs were detected. Fifteen days after injury, 61.8+3.8, 45.7+3% of the intimal cells resulted flt-1+ and c-kit+ and expressed low level of alpha-smooth muscle actin; CD133+ cells were 5.6+0.7%. BrDU+/flt-1+ largely predominated in the neointima, whereas BrDU+/CD133+ cells were rare. Forty-five and sixty days after injury, intimal proliferation such as BrDU+ cells was greatly reduced. After sixty days, intimal stem marker expression had almost disappeared whereas alpha-smooth muscle actin was restored. Flk-1 and Oct-4 SMC immunodection was consistently negative. In vitro, intimal cells obtained fifteen days after injury exhibited an epithelioid phenotype and increased flt-1 and c-kit protein and mRNA and low smooth muscle markers compared to spindle-shaped medial and intimal SMCs obtained after sixty days. Epithelioid clones, independently from layer of origin, were similar in stem cell marker expression. The anti-flt-1 blocking antibody added to epithelioid SMC cultures reduced serum-deprived apoptosis and migration but not PDGF-BB-induced proliferation, and increased cell-populated collagen lattice contraction. In conclusion, stem marker expression in vascular SMCs was variable, chronologically regulated and prevailed in epithelioid populations and clones; among stem markers, flt-1 expression critically regulates intimal SMC response to microenviromental changes.     

Inhibition of vascular smooth muscle cell proliferation and intimal hyperplasia by gene transfer of beta-interferon.

BACKGROUND: Balloon injury of the arterial wall induces increased vascular smooth cell proliferation, enhanced elastic recoil, and abnormalities in thrombosis, each of which contribute to regrowth of intima and the lesion of restenosis. Several gene transfer approaches have been used to inhibit such intimal smooth muscle cell growth. In this report, adenoviral gene transfer of beta-interferon (beta-IFN) was analyzed in a porcine model of balloon injury to determine whether a secreted growth inhibitory protein might affect the regrowth of vascular smooth muscle cells in vitro and in arteries. MATERIALS AND METHODS: An adenoviral vector encoding beta-interferon (ADV-beta-IFN) was prepared and used to infect porcine vascular smooth muscle cells in a porcine balloon injury model. Its antiproliferative effect was analyzed in vitro and in vivo. RESULTS: Expression of recombinant porcine beta-IFN in vascular smooth muscle cells reduced cell proliferation significantly in vitro, and supernatants derived from the beta-IFN vector inhibited vascular smooth muscle cell proliferation relative to controls. When introduced into porcine arteries after balloon injury, a reduction in cell proliferation was observed 7 days after gene transfer measured by BrdC incorporation (ADV-delta E1 arteries 14.5 +/- 1.2%, ADV-beta IFN 6.8 +/- 0.8%, p < 0.05, unpaired, two-tailed t-test). The intima-to-media area ratio was also reduced (nontransfected arteries, 0.70 +/- 0.05; ADV-delta E1 infected arteries, 0.69 +/- 0.06; ADV-beta-IFN infected arteries, 0.53 +/- 0.03; p < 0.05, ANOVA with Dunnett t-test). No evidence of organ toxicity was observed, and regrowth of the endothelial cell surface was observed 3-6 weeks after balloon injury. CONCLUSIONS: Gene transfer of an adenoviral vector encoding beta-IFN into balloon-injured arteries reduced vascular smooth muscle proliferation and intimal formation. Expression of this gene product may have potential application for the treatment of vascular proliferative diseases.     

Smooth muscle cells isolated from the neointima after vascular injury exhibit altered responses to platelet-derived growth factor and other stimuli

A variety of evidence suggests that vascular smooth muscle cells (SMC) exhibit a more immature phenotype when stimulated by injury to replicate in the adult. One growth characteristic common to immature (embryonic, fetal, and neonatal) SMC is a markedly reduced responsiveness to platelet-derived growth factor (PDGF) and other mitogenic stimuli. Here we demonstrate that SMC isolated from the 14-day neointima of experimentally injured carotid arteries exhibit a similar growth phenotype. The proliferative responses of neointimal cells to the BB homodimer of PDGF, which interacts with both forms of the PDGF receptor, were up to twenty-fold less (as assessed by BrdU immunocytochemistry) than that of adult control tunica media cells over a wide range of PDGF concentrations. Paradoxically, these cells expressed abundant mRNA for the α- and β-subunits of the PDGF receptor (by RT-PCR) and expressed abundant PDGF receptor protein (by Western blotting). Addition of PDGF-BB to neointimal SMC induced significant autophosphorylation of the PDGF receptor, suggesting that the PDGF receptors were fully functional. The chemotactic responses of neointimal SMC to PDGF, in in vitro migration assays, were identical to that of control medial cells. The data further establish the existence of vascular SMC phenotypes characterized by a refractoriness to growth stimulation by specific mitogens, and provide further evidence for the reiteration of developmentally regulated programs following vascular injury in vivo. © 1996 Wiley-Liss, Inc.     

Flow effects on cultured vascular endothelial and smooth muscle cell functions.

Cultured vascular endothelial cells were exposed to fluid shear stress by means of a rotary-disc shear-loading device, and the physiological effects of the conditioned medium (CM) and the homogenate (HM) of the cells on migration, adhesion and growth of endothelial cells (EC) or smooth muscle cells (SMC) were studied. Effects of shear stress on the production and secretion of collagen, one of the extracellular matrices of EC, were also studied. CM stimulated the adhesion and growth of SMC, but not of EC themselves. The ability to stimulate SMC adhesion and growth was similar in CM obtained from the static and shear-loaded cells. HM of the shear-loaded EC stimulated SMC migration. Further, HM of the shear-loaded EC contained increased amounts of collagen compared with the static EC. These results suggest that: 1) EC produce and secrete accelerators for the adhesion and growth of SMC, 2) EC react to the physical stimulus of fluid shear stress to produce stimulators of SMC migration, and 3) EC produce collagen, the production of which is enhanced by fluid shear stress.     

垂体腺苷酸环化酶激活肽对血管壁细胞成分的作用及其机制的实验研究——PACAP与动脉粥样硬化关系的探讨

以培养的猪主动脉内皮细胞（ＥＣ）和肺动脉平滑肌细胞（ＳＭＣ）为主要实验对象,研究了垂体腺苷酸环化酶激活肽（ＰＡＣＡＰ）对正常及高脂环境下培养的ＥＣ、ＳＭＣ形态和功能的影响,并对其作用机制进行了初步探讨。结果显示：ＰＡＣＡＰ可部分对抗高脂因素造成的ＥＣ、ＳＭＣ形态的损伤;能提高ＥＣ抗动脉粥样硬化（ＡＳ）物质的产生;抑制ＳＭＣ的增殖;并具有抗脂质过氧化作用。本研究表明,ＰＡＣＡＰ对ＥＣ、ＳＭＣ具有一定程度的细胞保护作用,因此提示ＰＡＣＡＰ可能具有一定的抗ＡＳ作用。     

Blood vessel maturation in a 3-dimensional spheroidal coculture model: direct contact with smooth muscle cells regulates endothelial cell quiescence and abrogates VEGF responsiveness.

Paracrine interactions between endothelial cells (EC) and mural cells act as critical regulators of vessel wall assembly, vessel maturation and define a plasticity window for vascular remodeling. The present study was aimed at studying blood vessel maturation processes in a novel 3-dimensional spheroidal coculture system of EC and smooth muscle cells (SMC). Coculture spheroids differentiate spontaneously in a calcium-dependent manner to organize into a core of SMC and a surface layer of EC, thus mimicking the physiological assembly of blood vessels with surface lining EC and underlying mural cells. Coculture of EC with SMC induces a mature, quiescent EC phenotype as evidenced by 1) a significant increase in the number of junctional complexes of the EC surface layer, 2) a down-regulation of PDGF-B expression by cocultured EC, and 3) an increased resistance of EC to undergo apoptosis. Furthermore, EC cocultured with SMC become refractory to stimulation with VEGF (lack of CD34 expression on VEGF stimulation; inability to form capillary-like sprouts in a VEGF-dependent manner in a 3-dimensional in gel angiogenesis assay). In contrast, costimulation with VEGF and Ang-2 induced sprouting angiogenesis originating from coculture spheroids consistent with a model of Ang-2-mediated vessel destabilization resulting in VEGF responsiveness. Ang-2 on its own was able to stimulate endothelial cells in the absence of Ang-1 producing SMC, inducing lateral sheet migration as well as in gel sprouting angiogenesis. Taken together, the data establish the spheroidal EC/SMC system as a powerful cell culture model to study paracrine interactions in the vessel wall and provide functional evidence for smooth muscle cell-mediated quiescence effects on endothelial cells.     

垂体腺苷酸环化酶激活肽对血管壁细胞成分的作用及其机制的实验研究

以培养的猪主动脉内皮细胞（ＥＣ）和肺动脉平滑肌细胞（ＳＭＣ）为主要实验对象, 垂体腺苷酸环化酶激活肽（ＰＡＣＡＰ）对正常及高脂环境下培养的ＥＣ、ＳＭＣ形态和功能的影响,并对其作用机制进行了初步探讨。结果显示：ＰＡＣＡＰ可部分对抗高脂因素造砀ＥＣ、ＳＭＣ形态的损伤;能提高ＥＣ抗动脉粥样硬化（ＡＳ）物质的产生;抑制ＳＭＣ的增殖;并具有抗脂质氧化作用。本研究表明,ＰＡＣＡＰ对ＥＣ、ＳＭＣ具有一定程度的细     

Platelet-derived growth factor-BB (PDGF-BB) induces differentiation of bone marrow endothelial progenitor cell-derived cell line TR-BME2 into mural cells, and changes the phenotype

Blood vessels are composed of endothelial cells (EC) and mural cells, and the interaction between EC and mural cells is essential for the development and maintenance of the vasculature. EC differentiate from bone marrow-derived endothelial progenitor cells (EPC). Recently, we established a conditionally immortalized bone marrow EPC-derived cell line, TR-BME2, and a brain capillary EC (BCEC) line, TR-BBB, from temperature-sensitive-SV40 T-antigen gene transgenic rats. To understand the function of EPC, it is important to analyze the difference between EPC and mature EC such as BCEC. In this study, we identified EPC-specific genes by means of subtractive hybridization between TR-BME2 and TR-BBB. There was no significant difference between TR-BME2 and TR-BBB in the mRNA level of annexin II, which is expressed in EC. In contrast, the mRNA level of smooth muscle cell (SMC) markers such as smooth muscle protein 22 (SM22), calvasculin, and platelet-derived growth factor (PDGF) receptor-beta, was higher in TR-BME2 than in TR-BBB. Moreover, the mRNA level of contractile SMC markers, such as smooth muscle alpha-actin and SM22, was increased in the absence of EC growth factors, such as vascular endothelial growth factor. The mRNA level of synthetic SMC markers, such as matrix Gla protein, was increased by the addition of PDGF-BB. The SMC derived from TR-BME2 showed an altered phenotype, from contractile-type to synthetic-type, when they were cultured in the absence of PDGF-BB. These results show that TR-BME2 cells have higher levels of SMC markers compared with mature EC, and can differentiate into contractile- or synthetic-type SMC.     

Microvascular pericytes contain muscle and nonmuscle actins

We have affinity-fractionated rabbit antiactin immunoglobulins (IgG) into classes that bind preferentially to either muscle or nonmuscle actins. The pools of muscle- and nonmuscle-specific actin antibodies were used in conjunction with fluorescence microscopy to characterize the actin in vascular pericytes, endothelial cells (EC), and smooth muscle cells (SMC) in vitro and in situ. Nonmuscle-specific antiactin IgG stained the stress fibers of cultured EC and pericytes but did not stain the stress fibers of cultured SMC, although the cortical cytoplasm associated with the plasma membrane of SMC did react with nonmuscle-specific antiactin. Whereas the muscle-specific antiactin IgG failed to stain EC stress fibers and only faintly stained their cortical cytoplasm, these antibodies reacted strongly with the fiber bundles of cultured SMC and pericytes. Similar results were obtained in situ. The muscle-specific antiactin reacted strongly with the vascular SMC of arteries and arterioles as well as with the perivascular cells (pericytes) associated with capillaries and post-capillary venules. The non-muscle-specific antiactin stained the endothelium and the pericytes but did not react with SMC. These findings indicate that pericytes in culture and in situ possess both muscle and nonmuscle isoactins and support the hypothesis that the pericyte may represent the capillary and venular correlate of the SMC.     

Water filtration rate and infiltration/accumulation of low density lipoproteins in 3 different modes of endothelial/smooth muscle cell co-cultures

Using different endothelial/smooth muscle cell co-culture modes to simulate the intimal structure of blood vessels, the water filtration rate and the infiltration/accumulation of LDL of the cultured cell layers were studied. The three cell culture modes of the study were: (i) The endothelial cell monolayer (EC/Φ); (ii) endothelial cells directly co-cultured on the smooth muscle cell monolayer (EC-SMC); (iii) endothelial cells and smooth muscle cells cultured on different sides of a Millicell-CM membrane (EC/SMC). It was found that under the same condition, the water filtration rate was the lowest for the EC/SMC mode and the highest for the EC/Φ mode, while the infiltration/accumulation of DiI-LDLs was the lowest in the EC/Φ mode and the highest in the EC-SMC mode. It was also found that DiI-LDL infiltration/accumulation in the cultured cell layers increased with the increasing water filtration rate. The results from the in vitro model study therefore suggest that the infiltration/accumulation of the lipids within the arterial wall is positively correlated with concentration polarization of atherogenic lipids, and the integrity of the endothelium plays an important role in the penetration and accumulation of atherogenic lipids in blood vessel walls.     

The cyclic adenosine monophosphate elevating medicine,forskolin, reduces neointimal formation and atherogenesis in mice

Neointimal formation and atherogenesis are major vascular complications following percutaneous coronary intervention, and there is lack of pharmacological therapy. This study was aimed to examine the effect of forskolin (FSK), a cyclic adenosine monophosphate (cAMP)‐elevating agent, on vascular response to angioplasty wire injury and on atherogenesis in mice. Forskolin treatment reduced neointima formation at 7 and 28 days after wire injury. Early morphometrics of the injured vessels revealed that FSK treatment enhanced endothelial repair and reduced inflammatory cell infiltration. In vitro treatment of primary aortic cells with FSK, at 3‐100 μmol/L, increased endothelial cell proliferation, whereas FSK, at 30‐100 μmol/L, inhibited smooth muscle cell proliferation. FSK inhibited lipopolysaccharide‐induced leucocyte‐endothelial interaction in vitro and in vivo. In a mouse model of atherosclerosis driven by dyslipidaemia and hypertension, FSK administration increased endothelial repair and reduced atherosclerotic plaque formation, without affecting blood pressure, plasma lipids or aortic aneurysms formation. In summary, FSK, at doses relevant to human therapeutic use, protects against neointimal hyperplasia and atherogenesis, and this is attributable to its activities on pro‐endothelial repair and anti‐inflammation. This study raises a potential of clinical use of FSK as an adjunct therapy to prevent restenosis and atherosclerosis after percutaneous coronary intervention.     

Adenovirus-Mediated Expression of a Ribozyme to c-myb mRNA Inhibits Smooth Muscle Cell Proliferation and Neointima Formation In Vivo

Smooth muscle cell (SMC) proliferation is an important component of restenosis in response to injury after balloon angioplasty. Inhibition of proliferation in vivo can limit neointima hyperplasia in animal models of restenosis. Ribozymes against c-myb mRNA have been shown to be effective inhibitors of SMC proliferation in vitro. The effectiveness of adenovirus as a gene therapy vector in animal models of restenosis is well documented. In order to test the utility of ribozymes to inhibit SMC proliferation by a gene therapy approach, recombinant adenovirus expressing ribozymes against c-myb mRNA was generated and tested both in vitro and in vivo. This adenovirus ribozyme vector is shown to inhibit SMC proliferation in culture and neointima formation in a rat carotid artery balloon injury model of restenosis.     

Antisense oligonucleotides to stromelysin mRNA inhibit injury-induced proliferation of arterial smooth muscle cells.

Smooth muscle cell migration and proliferation are important events in the formation of intimal lesions associated with atherosclerosis and restenosis following balloon angioplasty. To make this possible, the smooth muscle cell has to change from a contractile to an activated repair cell with capacity to synthesize DNA and extracellular matrix components. There is now considerable evidence that the extracellular matrix has important functions in modulating the phenotypic properties of smooth muscle cells, but less is known about the role of the matrix metalloproteinases. The present study investigates the role of stromelysin in the modulation of rat aortic smooth muscle cell morphology and function following mechanical injury in vitro and in vivo. Antisense mRNA oligonucleotides were used to investigate the role of stromelysin expression in injury-induced phenotypic modulation and the subsequent migration and proliferation of vascular smooth muscle cells. Cultured rat aortic smooth muscle cells and balloon-injured rat carotid arteries were used as experimental models. Light- and electron microscopy were used to follow changes in smooth muscle cell phenotype and lesion formation and incorporation of 3H-thymidine to detect DNA synthesis. Injury-induced DNA synthesis and migration in vitro were inhibited by 72% and 36%, respectively, by adding stromelysin antisense oligonucleotides to the medium prior to injury. In primary cultures, 67% of the smooth muscle cells treated with stromelysin antisense were retained in a contractile phenotype as judged by analysis of cell fine structure, compared to 15% untreated cells and 40% in cells treated with mismatched oligonucleotides. Examination of the carotid arteries one week after balloon injury likewise demonstrated a larger fraction of contractile cells in the inner parts of the media in vessels treated with antisense oligonucleotides compared to those treated with mismatched oligonucleotides. The neointima was also distinctly thinner in antisense-treated than in mismatched-treated and control arteries at this time. These findings indicate that stromelysin mRNA antisense oligonucleotides inhibited phenotypic modulation of rat arterial smooth muscle cells and so caused a decrease in migration and proliferation and neointima formation in response to vessel wall injury.     

Intra- and extracellular signaling by endothelial neuregulin-1

Suppression of tumor growth by inhibition of ErbB receptor signaling is well documented. However, relatively little is known about the ErbB signaling system in the regulation of angiogenesis, a process necessary for tumor growth. We have previously shown that heparin-binding EGF-like growth factor (HB-EGF) is expressed by vascular endothelial cells (EC) and promotes endothelial recruitment of vascular smooth muscle cells (SMC). To assess whether other members of the EGF-family regulate angiogenesis, the expression of 10 EGF-like growth factors in primary ECs and SMCs was analyzed. In addition to HB-EGF, neuregulin-1 (NRG-1) was expressed in ECs in vitro and in vivo. Endothelial NRG-1 was constitutively processed to soluble extracellular and intracellular signaling fragments, and its expression was induced by hypoxia. NRG-1 was angiogenic in vivo in mouse corneal pocket and chicken chorioallantoic membrane (CAM) assays. However, consistent with the lack of NRG-1 receptors in several primary EC lines, NRG-1 did not directly stimulate cellular responses in cultured ECs. In contrast, NRG-1 promoted EC responses in vitro and angiogenesis in CAM in vivo by mechanisms dependent on VEGF-A and VEGFR-2. These results indicate that NRG-1 is expressed by ECs and regulates angiogenesis by mechanisms involving paracrine up-regulation of VEGF-A.