BackgroundScleroderma is an autoimmune disease with a characteristic vascular pathology. The vasculopathy associated with scleroderma is one of the major contributors to the clinical manifestations of the disease.Methodology/Principal FindingsWe used immunohistochemical and mRNA in situ hybridization techniques to characterize this vasculopathy and showed with morphometry that scleroderma has true capillary rarefaction. We compared skin biopsies from 23 scleroderma patients and 24 normal controls and 7 scleroderma patients who had undergone high dose immunosuppressive therapy followed by autologous hematopoietic cell transplant. Along with the loss of capillaries there was a dramatic change in endothelial phenotype in the residual vessels. The molecules defining this phenotype are: vascular endothelial cadherin, a supposedly universal endothelial marker required for tube formation (lost in the scleroderma tissue), antiangiogenic interferon α (overexpressed in the scleroderma dermis) and RGS5, a signaling molecule whose expression coincides with the end of branching morphogenesis during development and tumor angiogenesis (also overexpressed in scleroderma skin. Following high dose immunosuppressive therapy, patients experienced clinical improvement and 5 of the 7 patients with scleroderma had increased capillary counts. It was also observed in the same 5 patients, that the interferon α and vascular endothelial cadherin had returned to normal as other clinical signs in the skin regressed, and in all 7 patients, RGS5 had returned to normal.Conclusion/SignificanceThese data provide the first objective evidence for loss of vessels in scleroderma and show that this phenomenon is reversible. Coordinate changes in expression of three molecules already implicated in angiogenesis or anti-angiogenesis suggest that control of expression of these three molecules may be the underlying mechanism for at least the vascular component of this disease. Since rarefaction has been little studied, these data may have implications for other diseases characterized by loss of capillaries including hypertension, congestive heart failure and scar formation.

Abstract-We performed a systematic analysis of gene expression in arteries and veins by comparing message profiles of macaque aorta and vena cava media using a cDNA array containing 4048 known human genes, Ϸ35% of currently named human genes (Ϸ11 000). The data show extensive differences in RNA expression in artery versus vein media. Sixty-eight genes had consistent elevation in message expression by the aorta, but none were elevated in the vena cava. The most differentially expressed gene was regulator of G-protein signaling (RGS) 5, at an expression ratio of 46.5Ϯ12.6 (meanϮSEM). The data set also contained 2 genes already known to be expressed in the aorta, elastin at 5.0Ϯ1.4, and the aortic preferentially expressed gene 1 (APEG-1) at 2.3Ϯ0.6. We chose to analyze RGS5 expression further because of its high level of differential expression in the aorta. Levels of RGS5 mRNA were confirmed by Northern analysis and in situ hybridization. A human tissue RNA dot blot showed that RGS5 message is highest in aorta, followed by small intestine, stomach, and then heart. Northern analysis confirmed that RGS5 expression in human aorta is higher than in any region of the heart. RGS5 is a G-protein signaling regulator of unknown specificity most homologous to RGS4, an inhibitory regulator of pressure-induced cardiac hypertrophy. The expression pattern of the 68 differential genes as a whole is a start toward identifying the molecular phenotypes of arteries and veins on a systematic basis. (Circ Res. 2000;87:623-631.)Key Words: cDNA array Ⅲ aorta Ⅲ vena cava Ⅲ expression profile Ⅲ RGS5 W e have begun a systematic analysis of expression differences between vascular smooth muscle tissues. We chose to begin with an analysis of the media of aorta and vena cava because of the possibility of isolating relatively homogenous cellular populations and because of the distinct functional requirements, embryological origins, and hemodynamic environments of these 2 tissues.The present study, using a cDNA array containing 35% of human named genes (4048 of the Ϸ10 900 functionally identified human genes in UniGene Build No. 109 of the National Center for Biotechnology Information human Unigene collection), identified 68 genes that are consistently expressed at higher levels in the aorta. No genes were more consistently highly expressed by the vena cava. One of these aortic markers, regulator of G-protein signaling (RGS) 5, showed extreme levels of differential expression. Together, these genes offer a molecular definition of the phenotypes of these 2 types of smooth muscle. Materials and Methods Array HybridizationResearch Genetics GF211 cDNA arrays (4048 human genes) were used as specified. Equivalent amounts of RNA per vessel pair were used to synthesize cDNA probes, and equivalent counts per minute were added to hybridizations. Washed blots were exposed to phosphor image screens and scanned on a Storm phosphor imager (Molecular Dynamics). Expression was quantified from scans with similar intensity and background, using Pathways software ...

Apoptosis of smooth muscle cells is a common feature of vascular lesions but its pathophysiological significance is not known. We demonstrate that signals initiated by regulated Fas-associated death domain protein overexpression in rat vascular smooth muscle cells in the carotid artery induce expression of monocyte-chemoattractant protein-1 and interleukin-8, and cause massive immigration of macrophages in vivo. These chemokines, and a specific set of other pro-inflammatory genes, are also upregulated in human vascular smooth muscle cells during Fas-induced apoptosis, in part through a process that requires interleukin-1alpha activation. Induction of a pro-inflammatory program by apoptotic vascular smooth muscle cells may thus contribute to the pathogenesis of vascular disease.

Objective-This study represents the first in an effort to systematically characterize different intimas by using expression array analysis. Methods and Results-We compared smooth muscle cells (SMCs) of the neointima formed 4 weeks after aortic grafting with those from normal aorta and vena cava from cynomolgus monkeys. Hybridization to cDNA arrays identified subsets of 147 and 45 genes differentially expressed in the neointima versus the aorta and vena cava, respectively. The expression pattern differentiating neointima from aortic SMCs was characterized largely by suppression. Only 13 genes were induced in the neointima: 7 encoded matrix proteins (6 collagens and 1 versican) and 2 encoded inducers of matrix synthesis (osteoblast-specific factor-2/Cbfa1 and connective tissue growth factor). The genes suppressed most in the neointima included the regulator of G-protein signaling-5, SPARClike-1/hevin, and nonmuscle myosin heavy chain-B. A smaller gene set differentiated the neointima from the vena cava. Most were induced (39 of 45 genes), and overlap with the neointima-aorta set was significant (10 of 13 genes). Array results were validated with Northern analysis, in situ hybridization, or immunohistochemistry. Conclusions-These data underscore the importance of matrix synthesis in neointimal maturation, and novel genes, newly associated with neointimal SMCs (regulator of G-protein signaling-5 and osteoblast-specific factor-2/Cbfa1), have raised new hypotheses regarding the pathogenesis of intimal hyperplasia. (Arterioscler Thromb Vasc

These data identify RGS5 as a new member of a short list of genes uniquely expressed in peripheral arteries but not coronary arteries. Persistence of an arterial pattern of RGS5 expression in culture and lack of expression in coronary arteries support a unique SMC phenotype fixed by distinct lineage or differentiation pathways. The association between loss of expression and arterial wall disease has prompted the new hypothesis that prolonged inhibition by RGS5 of vasoactive or trophic G protein signaling is critical to normal peripheral artery function.

Background-The fibrous cap of the atherosclerotic lesion is believed to be critical to stability because disruption of the cap is the final event leading to plaque rupture. We have, therefore, used expression arrays to define the phenotype of the cap and other plaque components. Methods and Results-To identify unique expression programs able to distinguish the smooth muscle of the cap from other plaque smooth muscle cells, RNA profiles were determined in human carotid artery media, nonatherosclerotic adjacent intima, fibrous cap of advanced atherosclerotic plaques, and whole advanced plaque with cDNA arrays covering 21 000 or 26 000 Unigene clusters. The molecular signature of each tissue was dominated by a core gene-set with differential expression of Ͻ1% of clusters assayed. Conclusions-Both intima and cap expressed novel genes not previously associated with SMC pathology. If the cap is derived from a unique subpopulation, this pattern is the signature of that particular set of cells. Key Words: atherosclerosis Ⅲ cardiovascular diseases Ⅲ genes Ⅲ molecular biology Ⅲ plaque M ost discussions of clonal origins of the atherosclerotic plaque have neglected the obvious question: "Is the smooth muscle cell (SMC) clone a distinct cell type?" 1 ; for example, Williams and Tabas suggested that the matrix produced by intimal SMCs at preatherosclerotic sites may lay down proteoglycans able to bind LDL. 2 Others have suggested that plaque rupture might result from the high apoptotic rate or low replicative life spans of cap cells based on properties seen in cultured plaque SMCs. [3][4][5] Unfortunately, these properties cannot be confirmed in vivo. One way of analyzing differences between tissues is to perform transcript profiling using cDNA arrays with message derived from tissues of interest. We and others have done this to compare distinct vascular tissues in vivo. 6 -9 Many studies have been performed to examine differences in vivo between cancerous and normal tissues. 10 -12 To address this current issue of plaque SMC-specific phenotype, we have performed a largescale profiling of gene expression in normal and atherosclerotic arteries using human cDNA arrays of Ϸ21 000 or 26 000 Unigene clusters. Expression in the fibrous cap was contrasted with expression in normal carotid media, adjacent nonatherosclerotic intima, and the more complicated cellular milieu of the whole atherosclerotic plaque. Methods Isolation and Processing of Tissue SamplesCarotid plaques were removed by endarterectomy, separated into fibrous cap and adjacent nonatherosclerotic intima, or reserved whole. Media were obtained from transplant donors. To obtain sufficient RNA for arraying, cap and intima were distributed into pools. Please see http://atvb.ahajournals.org for detailed Methods and Table I. RNA Purification, Array Hybridization, and Analysis RNA was purified, P 33 -labeled cDNA probes were created from 2 to 4 micrograms of total RNA from each pool per sample, equivalent cpm probes were hybridized with GF200, GF201, GF202, GF203, and GF...

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Objective-We sought to identify differentially expressed genes in the athero-prone coronary artery and athero-resistant internal mammary arteries. Methods and Results-Using suppressive subtraction hybridization, we generated reciprocal cDNA collections of representative mRNAs specific to porcine coronary arteries versus porcine mammary arteries. We screened 1000 suppressive subtraction hybridization cDNA clones by dot blot array and sequenced 600 of those showing the most marked expression differences. Northern blot, in situ hybridization, and immunostaining confirmed the differential gene expression patterns identified by the dot blot arrays. Genes associated with mammary arteries included claudin-10 and h-cadherin, which are genes associated with tight junctions and intermediate junctions. In contrast, genes associated with proatherosclerotic processes, such as lipid retention and metabolism, inflammation, and cell growth, were preferentially expressed in coronary arteries. Conclusions-Normal coronary arteries have gene expression program that is significantly different than internal mammary arteries. These differences may partly explain the resistance of coronary arteries and internal mammary arteries to atherosclerosis.