Capillary regeneration in scleroderma: stem cell therapy reverses phenotype?

BACKGROUND: Scleroderma 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 FINDINGS: We 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 alpha (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 alpha 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/SIGNIFICANCE: These 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.

RGS5, RGS4, and RGS2 expression and aortic contractibility are dynamically co-regulated during aortic banding-induced hypertrophy.

Overexpression of regulator of G protein signaling 5 (RGS5) in arteries over veins is the most striking difference observed using microarray analysis. The obvious question is what arterial function might require RGS5. Based on functions of homologous proteins in regulating cardiac mass and G-protein-coupled receptor (GPCR) signaling, we proposed that RGS5 and vascular expressed RGS2 and RGS4 could participate in regulating arterial hypertrophy. We used the suprarenal abdominal aorta banding model to induce hypertension and hypertrophy. All 3 RGS messages were expressed in unmanipulated aorta with RGS5 predominating. After 2 days, thoracic aorta lost expression of RGS5, 4, and 2. At 1 week, all three returned to normal, and at 28 days, they increased many fold above normal. Valsartan blockade of angiotensin II (angII)/angII type 1 receptor signaling prevented upregulation of RGS messages but only delayed mass increases, implying wall mass regulation involves both angII-dependent and angII-independent pathways. The abdominal aorta showed less dramatic expression changes in RGS5 and 4, but not 2. Again, those changes were delayed by valsartan treatment with no mass changes. Thoracic aorta contraction to GPCR agonists was examined in aortic explant rings to identify vessel wall physiological changes. In 2-day aorta, the response to Galphaq/i agonists increased above normal, while 28-day aorta had attenuated induced contraction via Galphaq/i agonist, implicating a connection between RGS message levels and changes in GPCR-induced contraction. In vitro overexpression studies showed RGS5 inhibits angII-induced signaling in smooth muscle cells. This study is the first experimental evidence that changes in RGS expression and function correlate with vascular remodeling.

Expression profiling identifies smooth muscle cell diversity within human intima and plaque fibrous cap: loss of RGS5 distinguishes the cap.

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 <1% 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. The loss of RGS5 in the fibrous cap is of particular interest because of its role in vessel development and physiology.

Regulator of G protein signaling 5 marks peripheral arterial smooth muscle cells and is downregulated in atherosclerotic plaque.

OBJECTIVE: Regulator of G protein signaling 5 (RGS5), an inhibitor of Galpha(q) and Galpha(i) activation, was recently identified among genes highly expressed in smooth muscle cells (SMCs) of aorta but not vena cava. This finding prompted the hypothesis that RGS5 provides long-term G protein inhibition specific to normal arterial SMC populations and that loss of expression may in turn contribute to arterial disease. METHODS: To test this hypothesis we characterized RGS5 gene expression throughout the vasculature of nonhuman primates to determine whether RGS5 was restricted to arteries in other vascular beds and whether expression was altered in arterial disease. RESULTS: In situ hybridization localized RGS5 message to medial SMCs of peripheral arteries, including carotid, iliac, mammary, and renal arteries, but not accompanying veins. SMCs of many small arteries and arterioles also expressed RGS5, including glomerular afferent arterioles critical to blood pressure regulation. Differential expression persisted in culture, inasmuch as RGS5 message was significantly higher in SMCs derived from arteries than from veins at real-time polymerase chain reaction. It was remarkable that the only major arterial bed lacking RGS5 was the coronary circulation. In atherosclerotic peripheral arteries RGS5 was expressed in medial SMCs, but was sharply downregulated in plaque SMCs. CONCLUSION: 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.

Vascular failure: a hypothesis.

Although cardiac failure has been studied extensively, vascular failure is not a recognizable term. We suggest that it is reasonable to argue that failure of the vessel to control its mass, contractile capacity, and lumen will involve pathways similar to cardiac failure. Vascular failure, or perhaps more accurately arterial failure, has very different consequences. Failure to control mass and external diameter will result in hypertension or loss of lumen in atherosclerosis. We review what is known about this normal remodeling response and its failure, and propose directions for research.

Suppression subtractive hybridization identifies distinctive expression markers for coronary and internal mammary arteries.

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.

Expression profiling identifies 147 genes contributing to a unique primate neointimal smooth muscle cell phenotype.

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.