Neointimal Smooth Muscle Cells Research Articles

Contribution of the Helix-Loop-Helix Factor Id2 to Regulation of Vascular Smooth Muscle Cell Proliferation

Smooth muscle cell (SMC) proliferation plays a key role in vascular proliferative disorders. The molecular mechanisms that control cell cycle entry of SMCs in response to vascular injury are not well understood. Id2 (inhibitor of DNA binding) is a member of the helix-loop-helix (HLH) family of transcription regulators that are known to promote cell cycle progression. Thus, we investigated the role of Id2 in SMC growth and cell cycle regulation. The results demonstrated that overexpression of Id2 resulted in a significant enhancement of SMC growth via increased S-phase entry. A possible mechanism of Id2-enhanced SMC growth is via regulation of p21 expression, as overexpression of Id2-inhibited transcriptional activity of a 2.3-kb p21 promoter/luciferase reporter construct as well as p21 protein levels. Id2 enhancement of SMC growth and inhibition of p21 expression were dependent on phosphorylation of Id2 by cyclin E/cdk2, as an Id2 cDNA containing a mutation in the cdk2 phosphorylation site (serine 5) failed to regulate SMC cell cycle progression or p21 promoter activity. The mechanism of cyclin E/cdk2 control of the Id2 effect may in part involve regulation of nuclear transport; unlike wild-type Id2, the Id2 mutant was not transported to the nucleus. Finally, in a rat carotid model of arterial injury, Id2 was expressed in a temporal pattern that parallels the kinetics of cellular proliferation. In summary, these results provide evidence that the Id2 protein is integrated into the cell cycle regulatory cascade that results in SMC proliferation following vascular injury and suggest that this effect is at least in part via a cdk2-dependent inhibition of p21 gene expression.

Systemic production of vascular endothelial growth factor and fms-like tyrosine kinase-1 receptor in acute Kawasaki disease.

Increased vascular permeability is an important event during the initial process of Kawasaki disease (KD). One potential responsible candidate for the induction of vascular hyperpermeability is vascular endothelial growth factor (VEGF). We investigated the expression of VEGF and its receptors (flt-1, KDR) in acute KD tissues at 7 days to 5 weeks of illness. Neuropilin-1, which enhances the binding of VEGF(165) to KDR, was also studied. Abundant expression of VEGF and flt-1 was documented immunohistochemically in many organs from acute KD, including heart and lung. VEGF and flt-1 were colocalized in all vessels that showed edema. These molecules resided in endothelium and vascular media and also in migrating smooth muscle cells in neointima and infiltrating macrophages. Compared with controls, coronary vessels of acute KD had upregulation of VEGF and flt-1 but not KDR or neuropilin-1. KDR was expressed by vessels at 7 days of illness but not later in the illness. Plasma proteins were more extensively bound to the extracellular matrix in coronary vessels in acute KD than controls. Furthermore, elevation of serum VEGF levels was correlated with low serum albumin in acute KD (n=220, r=-0.53, P<0.001). These findings suggest that VEGF and flt-1 are upregulated in blood vessels in many organs of acute KD. Expression of KDR was limited to the early stage of acute KD. The roles of VEGF in acute KD may involve promotion of vascular permeability and macrophage activation. Low serum albumin may indicate overproduction of VEGF in acute KD.

Upregulation of Nox-based NAD(P)H oxidases in restenosis after carotid injury.

Restenosis, a frequent complication of coronary angioplasty, is associated with increased superoxide (O2*(-)) production. Although the molecular identity of the responsible oxidase is unclear, an NAD(P)H oxidase appears to be involved. In smooth muscle, p22phox and 2 homologues of gp91phox, nox1 and nox4, are expressed, whereas fibroblasts contain gp91phox. To begin investigating the possibility that these oxidase components might contribute to the increased O2*(-) that accompanies neointimal formation, we measured their expression after balloon injury of the rat carotid artery. The increase in O2*(-) production 3 to 15 days after surgery was not due to inflammatory cell infiltration but appeared to be derived from medial and neointimal smooth muscle cells and adventitial fibroblasts. Nox1 and p22phox mRNAs were increased 2.7- and 3.6-fold, respectively, at day 3 after injury and remained elevated for 15 days. gp91Phox was increased 7 to 15 days after injury, and nox4 expression was increased 2-fold, but only at day 15 after surgery. These results confirm and extend our previous in vitro data and suggest that in the vasculature, the nox-based NAD(P)H oxidases serve different functions. This dynamic regulation of oxidase components may be critical to smooth muscle phenotypic modulation in restenosis and atherosclerosis.

The Expression of Tissue Factor and Tissue Factor Pathway Inhibitor in Aortic Smooth Muscle Cells Is Up-regulated in Synthetic Compared to Contractile Phenotype

Tissue factor (TF) and its specific inhibitor TF pathway inhibitor (TFPI) are produced by vascular smooth muscle cells (SMCs) in vitro and are increased in vivo in atherosclerotic compared to normal vessels. Besides local regulation of the hemostatic balance, this may be related to non-hemostatic TF/protease dependent functions such as SMC proliferation, adhesion and migration. The aim of the study was to compare the expression of both proteins between the contractile (normal adult) and synthetic (neo-intimal) SMC phenotypes. Primary cultures of SMCs isolated from rat thoracic aorta before and 10 days after balloon injury displayed stable characteristics of the contractile and synthetic phenotype, respectively. Synthetic SMCs expressed more TF mRNA than contractile SMCs, but released excess TF in the conditioned medium, so that the cell-associated TF activity measured by a factor Xa generating assay remained similar in the two subtypes. Accordingly, cell surface thrombogenicity measured under blood flow conditions was also similar. The production and release of functional TFPI was enhanced by a factor 3 to 6 (p < 0.01) in synthetic SMCs. A difference in the quantitative expression of TF and TFPI is a new distinctive feature of SMC phenotypes. Matrix-associated TFPI derived from synthetic SMCs may serve as an anchorage for their migration and regulate protease-activated processes during neo-intima formation.

Loss of p53 accelerates neointimal lesions of vein bypass grafts in mice.

The transcription factor p53 is essentially involved in regulation of cell death and proliferation. Recently, we have established a mouse model for vein graft arteriosclerosis by grafting autologous jugular veins or vena cava to carotid arteries. Using this model, we studied the role of p53 in the development of vein graft arteriosclerosis in p53(-/-) mice. Four weeks after grafting, neointimal hyperplasia of vein grafts in p53(-/-) mice was increased 2-fold compared with that of wild-type controls. Cell component analysis revealed that neointimal lesions in p53(-/-) mice consisted mainly of alpha-actin positive smooth muscle cells (SMCs), whereas the majority of cells in wild-type mice were MAC-1 (CD11b/18)-positive at 4 weeks. Importantly, SMC apoptosis as determined by TUNEL assay was significantly reduced in p53(-/-) vein grafts. TUNEL positive cells in wild-type vein grafts markedly increased from 0.5% to 6.4% of total cells 4 weeks postoperatively, but remained virtually unchanged in p53(-/-) grafts (0.8%). Immunofluorescence analysis revealed that increased p53 expression in neointimal SMCs of wild-type, but not p53(-/-), mice coincided with oxidative DNA damage in vein grafts. Interestingly, SMCs of p53(-/-) mice showed increased apoptosis in response to TNFalpha and decreased apoptosis in response to sodium nitroprusside. Additionally, p53-deficient SMCs showed a higher rate of proliferation and migration and expressed higher levels of matrix metalloproteinases. Thus, p53 deficiency accelerates neointima formation by facilitating SMC proliferation as well as abrogating cell apoptosis.

Urokinase plasminogen activator augments cell proliferation and neointima formation in injured arteries via proteolytic mechanisms

Urokinase plasminogen activator (uPA) has been implicated in the healing responses of injured arteries, but the importance of its various properties that influence smooth muscle cell (SMC) proliferation and migration in vivo is unclear. We used three recombinant (r-) forms of uPA, which differ markedly in their proteolytic activities and abilities to bind to the uPA receptor (uPAR), to determine, which property most influences the healing responses of balloon catheter injured rat carotid arteries. After injury, uPA and uPAR expression increased markedly throughout the period when medial SMCs were rapidly proliferating and migrating to form the neointima. Perivascular application of uPA neutralizing antibodies immediately after injury attenuated the healing response, significantly reducing neointima size and neointimal SMC numbers. Perivascular application of r-uPAwt (wild type uPA) or r-uPA/GDF (r-uPA with multiple mutations in its growth factor-like domain) doubled the size of the neointima. Four days after injury these two uPAs nearly doubled neointimal and medial SMC numbers in the vessels, and induced greater reductions in lumen size than injury alone. Proteolytically inactive r-uPA/H/Q (containing glutamine rather than histidine-204 in its catalytic site) did not affect neointima or lumen size. Also, in contrast to the actions of proteolytically active uPAs, tissue plasminogen activator (tPA) did not affect the rate of neointima development. We conclude that uPA is an important factor regulating the healing responses of balloon catheter injured arteries, and its proteolytic property, which cannot be mimicked by tPA, greatly influences SMC proliferation and early neointima formation.

Vascular cell adhesion molecule-1 up-regulation and phenotypic modulation of vascular smooth muscle cells predate mononuclear infiltration in transplant arteriopathy

Objective: Expression of embryonic myosin heavy chain isoforms and vascular cell adhesion molecule-1 by neointimal vascular smooth muscle cells are independent indicators of atherosclerotic plaque development in both human beings and experimental animal models. We examined the chronologic change in smooth muscle cell myosin heavy chain isoforms, vascular cell adhesion molecule-1 expression, and mononuclear cell infiltration in a carotid arterial transplant model to ascertain whether similar phenotypic changes would occur in transplant arteriopathy. Methods: Transplanted rabbit carotid arteries were examined at 7, 14, 21, and 35 days (n = 5, 7, 6, and 5, respectively). Lesion progression and the prevalence of smooth muscle cell myosin heavy chain isoforms, T-lymphocytes, macrophages, and vascular cell adhesion molecule-1 expression were evaluated immunohistochemically by computerized image analysis. Results: In this carotid arterial transplant model, the intima/media area ratio increased significantly by 35 days ( P = .01) as cell density decreased ( P = .01), suggesting extracellular matrix elaboration. Intimal smooth muscle cells expressing embryonic phenotypes were seen as early as 7 days, a phenotypic change that predated mononuclear cell infiltration of the graft by at least 7 days. By 35 days, up to 70% of intimal smooth muscle cells expressed the embryonic phenotype, coinciding with the transition from inflammatory to chronic lesions. Although, in early lesions, vascular cell adhesion molecule-1 was identified on luminal endothelium overlying mononuclear infiltrates, in advanced lesions vascular cell adhesion molecule-1 was identified primarily on intimal vascular smooth muscle cells. Conclusions: Overall, these vascular smooth muscle cell changes mark important early events in transplant arteriopathy that may not be ameliorated by immunosuppressive regimens in routine use.

Tc-99m-labeled endothelin derivative for imaging of experimentally induced atherosclerosis

Objective: to characterize the potential of an endothelin derivative labeled with technetium-99m (Tc-99m) for the imaging of experimentally induced atherosclerosis. Methods: neointima of different cellularity and severity of stenosis was induced in 32 rabbits by balloon denudation followed by distinct dietary regimens and drug application. Angiograms and scintigrams after injection of the Tc-99m-labeled endothelin derivative were obtained. The aorta was dissected for autoradiography, sudan-III-staining, morphometry, and immunohistology. Results: the lesions induced could be detected in vivo (whole body scintigram) in all the animals 15 min after the injection of the Tc-99m endothelin derivative. Autoradiography revealed a strong relationship between tracer accumulation and sudan-III-staining of lesions. Accumulation of the endothelin derivative correlated with the number of neointimal smooth muscle cells (SMC), but not with the number of medial SMC, neointimal macrophages, and neointimal area. Conclusions: the results indicate that in vivo imaging of atherosclerosis with an endothelin derivative is a feasible method of detecting and characterizing atherosclerotic arterial wall lesions at early stages.

Origin of neointimal endothelium and α-actin–positive smooth muscle cells in transplant arteriosclerosis

The development of transplant arteriosclerosis (TA) is today's most important problem in clinical organ transplantation. Histologically, TA is characterized by perivascular inflammation and progressive intimal thickening. Current thought on this process of vascular remodeling assumes that neointimal vascular smooth muscle (VSM) cells and endothelium in TA are graft-derived, holding that medial VSM cells proliferate and migrate into the subendothelial space in response to signals from inflammatory cells and damaged graft endothelium. Using MHC class I haplotype-specific immunohistochemical staining and single-cell PCR analyses, we show that the neointimal alpha-actin-positive VSM cells in rat aortic or cardiac allografts are of recipient and not of donor origin. In aortic but not in cardiac allografts, recipient-derived endothelial cells (ECs) replaced donor endothelium. Cyclosporine treatment prevents neointima formation and preserves the vascular media in aortic allografts. Recipient-derived ECs do not replace graft endothelium after cyclosporine treatment. We propose that, although it progresses beyond the needs of functional repair, TA reflects the activity of a normal healing process that restores vascular wall function following allograft-induced immunological injury.

Transcriptome Analysis Reveals a Role of Interferon-γ in Human Neointima Formation

The most effective immediate cure for coronary stenosis is stent-supported angioplasty. Restenosis due to neointima proliferation represents a major limitation. We investigated the expression of 2435 genes in atherectomy specimens and blood cells of patients with restenosis, normal coronary artery specimens, and cultured human smooth muscle cells (SMCs). Of the 223 differentially expressed genes, 37 genes indicated activation of interferon-γ (IFN-γ) signaling in neointimal SMCs. In cultured SMCs, IFN-γ inhibited apoptosis. Genetic disruption of IFN-γ signaling in a mouse model of restenosis significantly reduced the vascular proliferative response. Our data suggest an important role of IFN-γ in the control of neointima proliferation.

Marked neointimal lipoprotein lipase increase in distinct models of proclivity to atherosclerosis: a feature independent of endothelial layer integrity

Lipoprotein lipase (LPL) in the arterial wall has been proposed to enhance the retention of apoB-containing lipoproteins, an early event in atherosclerosis. As the neointima is considered the primary site of lipid accumulation in atherogenesis, the arterial expression and location of LPL was investigated in distinct experimental models of neointimal formation in normolipidemic rabbits and rats. Neointima elicited by balloon aortic denudation or raised beneath an anatomically intact endothelial layer by placing a silastic collar around the common carotid artery, both showed a striking LPL immunostaining that mostly co-localized with neointimal smooth muscle cells. Besides, increased LPL protein and mRNA in deendothelialized aortas was demonstrated by Western and Northern blot analysis, respectively, suggesting an enhanced expression of LPL in injured arteries. It was concluded that LPL is increased in neointima developed in either denuded vessels or arteries with a preserved endothelium, a finding which suggests that LPL abundance may be an attribute of the neointima, whatever the stimulus that promotes its formation. On the basis of former evidence concerning the role of LPL in lipid retention, this study provides a possible explanation for the injury-induced vessel susceptibility to atherosclerosis, and the particular proneness of the neointimal layer to lipid accretion.

Expression of a Novel RNA-Splicing Factor, RA301/Tra2β, in Vascular Lesions and Its Role in Smooth Muscle Cell Proliferation

RA301/Tra2beta, a sequence-specific RNA-binding protein, was first cloned as a stress molecule in re-oxygenated astrocytes. In human vascular tissues, we have found enhanced RA301/Tra2beta expression in coronary artery with intimal thickening, and atherosclerotic aorta. Balloon injury to the rat carotid artery induced RA301/Tra2beta transcripts followed by expression of the antigen, which was detected in medial and neointimal vascular smooth muscle cells (VSMCs). In cultured VSMCs, hypoxia/re-oxygenation caused induction of RA301/Tra2beta and was accompanied by cell proliferation, both of which were blocked by the addition of either diphenyl iodonium, a NADPH oxidase inhibitor, PD98059, a mitogen-activated protein kinase kinase inhibitor, or antisense oligonucleotide for RA301/Tra2beta. Consistent with a link between RA301/Tra2beta and cell proliferation, platelet-derived growth factor also induced expression of RA301/Tra2beta in cultured VSMCS: These data suggest a possible role for RA301/Tra2beta in the regulation of VSMC proliferation, especially in the setting of hypoxia/re-oxygenation-induced cell stress.

Vascular injury induces posttranscriptional regulation of the Id3 gene: cloning of a novel Id3 isoform expressed during vascular lesion formation in rat and human atherosclerosis.

The molecular mechanisms that regulate the proliferation of smooth muscle cells (SMCs) of the vasculature in response to injury are poorly understood. Members of the inhibitor of DNA binding (Id) class of helix-loop-helix transcription factors are known to regulate the growth of a variety of cell types; however, the expression of the various Id genes in SMCs and in vascular lesions has not been examined. In the present study, the yeast 2-hybrid system was used to clone Id genes from a cultured rat aortic SMC library. By use of ubiquitous E proteins as bait, Id3 and a novel isoform of Id3 (Id3a) were cloned. Id3a is the product of alternative splicing of the Id3 gene, resulting in inclusion of a 115-bp "coding intron," which encodes a unique 29-amino acid carboxyl terminus for the Id3a protein. Unlike Id3, Id3a mRNA was not detected in the normal rat carotid artery. However, after balloon injury, Id3a was abundantly expressed throughout the neointimal layer. In addition, mRNA of the human homologue of Id3a (Id3L) was detected in human carotid atherosclerotic plaques. Adenovirus-mediated overexpression of these Id3 isoforms in cultured rat aortic SMCs revealed that infection of SMCs with an adenovirus overexpressing Id3a (in contrast to Id3) resulted in a significant decrease in cell number versus AdLacZ-infected cells. DNA fragmentation analysis suggested that this decrease in SMC viability was due to increased apoptotic activity in cells infected with adenovirus overexpressing Id3a. These results provide evidence that alternative splicing of the Id3 gene may represent an important mechanism by which neointimal SMC growth is attenuated during vascular lesion formation.

Mechanisms Involved in the Inhibition of Neointimal Hyperplasia by Abciximab in a Rat Model of Balloon Angioplasty

Monoclonal antibodies raised against β 3 integrin are able to inhibit the binding of ligands to certain β 3 integrins such as α IIbβ 3 (glycoprotein IIb/IIIa complex) and α vβ 3 (vitronectin receptor) and as such are inhibitors of platelet aggregation and smooth muscle cell (SMC) migration, both of which are involved in neointimal hyperplasia. The present study was designed to explore the detailed mechanisms of abciximab (Reopro), a monoclonal antibody (mAb) raised against α IIbβ 3 integrin in neointimal hyperplasia. In this study, carotid arteries of Wistar rats were damaged, and neointimal hyperplasia and lumen occlusion was determined at different time points. Abciximab was administered intravenously by an implanted osmotic pump. Abciximab (0.25 mg/kg/day) time-dependently inhibited both neointimal hyperplasia and lumen occlusion after angioplasty in carotid arteries of rats. Furthermore, the electromicrographs highlighted that SMCs were phenotypically different from the typical contractile, spindle-shaped SMCs normally seen in uninjured vessel walls. Platelet-derived growth factor (PDGF)-BB was strongly produced in thrombus formation and neointimal SMCs after angioplasty, while abciximab significantly reduced PDGF-BB expression in vessel lumens and neointimal SMCs after angioplasty. Balloon angioplasty caused a significant increase of nitrate and cyclic GMP as compared with sham-operated rats. Infusion of abciximab (0.25 mg/kg/day) did not significantly change. Furthermore, the plasma level of thromboxane B 2 (TxB 2) obviously increased after angioplasty, while abciximab markedly suppressed the elevation of plasma TxB 2 concentration. The results indicate that abciximab effectively prevents neointimal hyperplasia, possibly through the following 2 mechanisms: (1) Abciximab binds to α IIbβ 3 integrin on platelet membranes resulting in inhibition of platelet adhesion, secretion, and aggregation in injured arteries, followed by inhibition of thromboxane A 2 formation and PDGF-BB release from platelets. (2) Abciximab may also bind to α vβ 3 integrin on SMCs, thus, subsequently inhibiting cell migration and proliferation.

The calcium inhibitor SR33805 reduces intimal formation following injury of the porcine carotid artery

We studied the effect of SR33805, a calcium channel blocker, in vitro on the proliferation of vascular smooth muscle cells (SMC) stimulated by foetal calf serum, basic fibroblast growth factor and platelet derived growth factor, and in vivo with regard to SMC migration and proliferation which occurred following injury of the porcine carotid artery. The intimal lesion was induced by a silasten collar surgically positioned around the carotid artery and by a stenosis reducing blood flow by 50% for 30 days. Animals received SR33805 (5 mg/kg/day) 8 days before the induction of the lesion and up to 30 days after. In vitro, SR33805 inhibited in a dose-dependent manner growth factor-induced proliferation of SMC (0.20<IC 50<0.46 μM). In vivo, SR33805 reduced the intima/media ratio of the cross sectional surface area (decrease of 60%, P<0.05) without affecting neointimal SMC density. The medial SMC density was 40% lower in treated than in control animals (upstream, P<0.05 and downstream to the stenosis, P<0.01). Thus, it appears that SR33805 significantly reduced intimal hyperplasia, which occurred after perivascular manipulation of the artery, an effect consistent with its in vitro proliferation inhibitory activity, suggesting that long-term treatment with SR33805 may reduce or delay SMC proliferation.

Association of thrombospondin-1 and cardiac allograft vasculopathy in human cardiac allografts.

Despite the expression of angiogenic growth factors in transplanted hearts, neovessel formation appears scant. We therefore hypothesized that cardiac allografts contain endogenous inhibitors of angiogenesis. In particular, we tested the involvement in cardiac allografts of thrombospondin-1 (TSP-1), a matrix glycoprotein that inhibits angiogenesis and facilitates smooth muscle cell (SMC) proliferation. Levels of TSP-1 mRNA in endomyocardial biopsy samples of human cardiac allografts substantially exceeded those in normal hearts. The ratio of TSP to GAPDH mRNA determined with quantitative RT-PCR was 6.54+/-1.6 in cardiac allografts versus 0.26+/-0.02 (P:=0.001) in normal hearts. Analysis in sequential biopsies revealed a strong association between persistent elevation of TSP-1 in allografts and the development of cardiac allograft vasculopathy (CAV). The CAV score was 2.4+/-0.8 in patients with persistent TSP-1 elevation compared with 0.2+/-0.2 in patients without elevation (P:=0.001). Immunohistochemistry demonstrated intense expression of TSP-1 in cardiac allografts, predominantly in cardiac myocytes and neointimal SMCs. In vitro experiments demonstrated that T cells expressed TSP-1, acidic fibroblast growth factor, and vascular endothelial cell growth factor on allogeneic stimulation. Cytokines known to be elevated in cardiac allografts (interleukin-1beta, interferon-gamma, and tumor necrosis factor-alpha) induced TSP-1 in SMCs but inhibited TSP-1 in endothelial cells. Persistent elevation of TSP-1 in cardiac allografts correlates with the development of CAV. Allogeneic stimulation induces angiogenic growth factors and TSP-1 in T cells. Cytokines differentially regulate TSP-1 expression in SMCs versus endothelial cells. Increased levels of TSP-1 in human cardiac allografts may alter vascular responses to angiogenic growth factors by inhibiting angiogenesis and promoting SMC proliferation characteristic of CAV.

HSP47 expression by smooth muscle cells is increased during arterial development and lesion formation and is inhibited by fibrillar collagen.

HSP47 is a heat-shock protein that interacts with intracellular procollagen. It has been found in fibrous atherosclerotic plaque, but its involvement in acute vascular restructuring is unknown. We analyzed the expression of HSP47 and its regulation in the developing rat aorta and after balloon injury to the adult rat carotid artery. HSP47 was strongly expressed in each layer of the maturing fetal aorta (embryonic day 17 to birth). Expression declined during the first 4 postnatal days but persisted at low abundance into adulthood. HSP47 expression was substantially upregulated in the injured carotid artery, with intense immunostaining in neointimal smooth muscle cells (SMCs). HSP47 expression in SMCs was correlated with the emergence of a less mature phenotype and with expression of type I procollagen. Interestingly, a precipitous decline in HSP47 expression was evident during aortic development and after carotid artery injury, in association with the appearance of collagen fibrils in the local extracellular matrix. Furthermore, type I collagen fibrils, but not collagen monomers, inhibited expression of HSP47 by SMCs. These findings indicate that upregulation of HSP47 is a feature of vascular restructuring, including acute neointimal formation, and that the constituents of the extracellular matrix regulate the duration of expression. This feedback control may be important for self-termination of vascular development and lesion growth.

Quantitation of cell migration in a rat carotid artery balloon injury model. Indications for a perivascular origin of the neointimal cells.

Medial and neointimal smooth muscle cells differ in phenotype. Adventitial cells have been shown to migrate to the intima and this could partly explain this difference. The aim of this study was to quantitate the kinetics of cell migration from the adventitial and medial layers to the intima after endothelial injury. Labelled proliferating cells at different periods post-injury in a rat carotid artery balloon injury model, were used to calculate the kinetics of migration using the alteration in cell populations and the ratio of proliferating cells. The increase in the number of neointimal cells was greater than the level of proliferation during the 30-day follow-up. Changes in the number and percentage of proliferating cells remained low after the appearance of the first neointimal cells. 28% of the neointimal cells were labelled during the first wave of migration, and in reverse at least 72% had migrated there. Of these migrating cells, 74% were non-proliferating. The formation of neointima was efficiently blocked with cyclophosphamide and batimastat (metalloproteinase inhibitor), which resulted in a decrease in the number of medial and intimal cells. The increase in the number of neointimal cells is mostly due to cells migrating from the outer layers through the media. The majority of these cells are not proliferating, but migration can still be efficiently blocked with antiproliferative drugs.

Gene Expression in Atherogenesis

It is conceivable that the extent and spatio-temperal expression of dozens or even a few hundred genes are significantly altered during the development and progression of atherosclerosis as compared to normal circumstances. Differential gene expression in vascular cells and in blood cells, due to gene-gene and gene-environment interactions can be considered the molecular basis for this disease. To comprehend the coherence of the complex genetic response to systemic and local atherosclerotic challenges, one needs accessible high through-put technologies to analyze a panel of differentially expressed genes and to describe the interactions between and among their gene products. Fortunately, new technologies have been developed which allow a complete inventory of differential gene expression, i.e. DD/RT-PCR, SAGE and DNA micro-array. The initial data on the application of these technologies in cardiovascular research are now being reported. This review summarizes a number of key observations. Special attention is paid to a few central transcription factors which are differentially expressed in endothelial cells, smooth muscle cells or monocytes/ macrophages. Recent data on the role of nuclear factor-kappaB (NF-kappaB) and peroxisome proliferation-activating receptors (PPARs) are discussed. Like the PPARs, the NGFI-B subfamily of orphan receptors (TR3, MINOR and NOT) also belongs to the steroid/thryroid hormone receptor superfamily of transcription factors. We report that this subfamily is specifically induced in a sub-population of neointimal smooth muscle cells. Furthermore, intriguing new data implicating the Sp/XKLF family of transcription factors in cell-cell communication and maintenance of the atherogenic phenotype are mentioned. A member of the Sp/XKLF family, the shear stress-regulated lung Krüppel-like factor (LKLF) is speculated to be instrumental for the communication between endothelial cells and smooth muscle cells. Taken together, the expectation is that the fundamental knowledge obtained on atherogenesis and the data that will be acquired during the coming decade with the new, powerful high through-put methodologies will lead to novel modalities to treat patients suffering from cardiovascular disease. In view of the phenotypic changes of vascular and blood-borne cells during atherogenesis, therapeutic interventions likely will focus on reversal of an acquired phenotype by gene therapy approach or by using specific drugs which interfere with aberrant gene expression.

Recipient origin of neointimal vascular smooth muscle cells in cardiac allografts with transplant arteriosclerosis

Background Coronary artery disease is today’s most important post–heart transplantation problem after the first perioperative year. Histologically, coronary artery disease is characterized by transplant arteriosclerosis. The current view on this vasculopathy is that vascular smooth muscle (VSM) cells from the media of affected arteries proliferate and migrate into the sub-endothelial space (intima) in response to signals from inflammatory cells and damaged graft endothelium. According to this model, the intimal VSM cells in transplant arteriosclerotic lesions should originate from donor tissue. Using recipient-specific polymerase chain reaction (PCR) analysis of microdissected, single, neointimal VSM nuclei, we recently showed that after allogeneic aorta transplantation the neointimal VSM cells are of recipient and not of donor origin. In this study, we analyzed whether VSM-cell replacement with recipient-derived cells also takes place after allogeneic heart transplantation. Methods Cardiac allografts, when transplanted from female donors to male immune-modulated recipient rats, eventually developed transplant arteriosclerosis. We microdissected α-actin positive neointimal VSM cells from tissue sections and determined the origin (donor or recipient) using recipient-specific (male), single-cell, PCR analysis. Results In total, we analyzed 35 VSM-cell nuclei from 3 allografts, and PCR analysis revealed that 30/35 (86%) of the samples displayed the male-specific 128 base pair DNA fragment. These results indicate that after allogeneic cardiac transplantation, at least 86% of VSM cells in transplant arteriosclerotic lesions are of recipient origin. Conclusions In contrast to current thought, the neointimal VSM cells in cardiac allografts that show transplant arteriosclerosis are of recipient and not of donor origin.