. .. Table 1. Molecular markers of microvascular phenotype, . .. inflammation and cell cycle in skin biopsies from scleroderma . . compared to controls. . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... Type of molecular marker N#opromsa/nl{ S#cpleorso/dne{rma P value { . ... A) Endothelial markers . ... Alkaline Phosphatase 4/4 0/8 0.002 ... VE Cadherin 4/6 0/10 0.008 . ... CD31 13/13 13/13 1 . ... Von Willebrand Factor 3/3 1/7 0.033 .... Ulex Europaeus Lectin 10/14 3/14 0.01 .. . B. Inflammatory endothelial markers . ... CD123 (high endothelial venules) 1/12 12/16 0.0005 .... VCAM1 0/4 5/5 0.007 ... ICAM1 0/3 5/5 0.017 . ... CD62P 0/7 1/7 0.49 . ... Smooth muscle actin 0/15 12/12 0.000001 . .. C. Other inflammatory markers . ... PAR2 0/4 5/5 0.007 . ... PSGL1 0/7 8/8 0.0001 .... IL-1 a 0/6 7/7 0.0005 ... D. Other markers . ... RGS5 in situ 0/5 7/8 0.004 . ... Interferon a in situ (IFNA1 and IFNA2) 0/6 9/9 0.0001 ... CD123 (plasmacytoid dendrtic cells) 1/12 14/16 0.000005 . ... STAT1 0/7 7/10 0.006 . .. E. Endothelial cell death (apoptosis) . .... Cleaved Caspase 3 0/7 0/17 1 .. . F. Perivascular cell Turnover: . ... Ki67 Antigen 0/8 11/11 0.00001 . .. G. Endothelial cell turnover . ... Ki67 antigen 0/7 0/17 1 . . .. . {Data are numbers biopsies with positive immunohistochemical, lectin and in .. situ staining of skin biopsies over total number of biopsies available for . .. staining. ... {P values were calculated with Fisher’s exact test using 262 frequency tables. .. doi:10.1371/journal.pone.0001452.t001 Other markers of inflammation not limited to endothelial cells were significantly increased in scleroderma compared to normal tissue (quantitative results summarized in Table 1C). Immunohis- tochemical stains for VCAM1 and ICAM1 were elevated and statistically significant (p,0.05), consistent with published data for higher expression in scleroderma compared to normal[28], and cells positive for p selectin glycoprotein ligand 1(p = 0.0001), interleukin-1a (p = 0.0005) and protease activated receptor 2 (p = 0.007) were all increased in scleroderma compared to normal controls (quantitative results summarized in table 1 C). PAR2 appeared concentrated in smooth muscle and endothelial cells, with some positive cells scattered in the dermis biopsies (photomicrographs not shown). PAR2 is expressed in response to inflammatory stimuli and in turn appears to enhance endothelial proliferation, angiogenesis and immune mediator production resulting in a neurologically mediated itch [29–31], a clinical phenomenon seen often in early diffuse cutaneous scleroderma [30,32–34]. IL-1a and PSGL1 were similarly increased both in endothelial cells and in cells present in the dermal microvascular Vessel Recovery in Scleroderma unit (photomicrographs not shown). IL-1a activates complex pathways in endothelial cells, dramatically affecting function. PSGL1 is the high affinity counter receptor for selectins and is constitutively expressed on some kinds of inflamed endothelium [35]and PSGL1-selectin interactions play a role in homing and entry of inflammatory cells into sites of inflammation[36]. Scleroderma skin had an infiltrate of CD123+cells, an increase in interferon a mRNA expression and downstream signaling markers of interferon While looking at high endothelial venules in scleroderma skin (above), we found a population of strongly CD123+ cells infiltrating the dermis. Scleroderma dermis had many CD123+cells in the superficial horizontal plexus, significantly more than normal skin (p = 0.000005, Table 1D). CD123+ cells known as plasmacytoid dendritic cells are the primary producers of interferon a in the human body[37]. To look for the presence of interferon a mRNA expression we used IFNA1 and IFNA2 RNA in situ probes to look for expression of interferon a mRNA. 100% of Scleroderma patients were positive for interferon a and 0% of the normal had interferon a (p = 0.0001). Normal skin showed no detectable hybridization in the dermis and occasional faint hybridization in the epidermis whereas scleroderma tissue always expressed interferon a mRNA in the dermis and epidermis. The interferon a+cells, probably plasmacy- toid dendritic cells, were present next to small vessels in the superficial horizontal plexus in the upper dermis (Figure 2 B). In situ results were repeated 3 times and results were consistent. To further confirm the presence of interferon a, we used staining for downstream markers of type 1 interferon activity. Canonical type 1 interferon signaling involves the binding of type 1 interferon (interferon a b or v) to the type 1 interferon receptor, activation of the JAK/STAT pathway, and phosphorylation of STAT1[38]. Presence of phosphorylated STAT1 in the nucleus of cells STAT1 dimerization with another phosphorylated STAT and translocation into the nucleus. STAT1 phosphorylation was present in cellular nuclei in 7 of 10 scleroderma biopsies (depicted in Figure 2 C), but negative in all 7 normal controls tested (p = 0.006, Table 1 D). Scleroderma skin had increased mRNA expression of RGS5 and no sign of endothelial death or proliferation The apparent loss of capillaries in scleroderma led us to look for increased expression of RGS5 mRNA. We found that both RGS5 mRNA expression was significantly increased in scleroderma (Table 1 D). RGS5 is a regulator of G protein coupled receptor signaling with cardiovascular properties[39] and specific associa- tion with arteries[40]. For as yet unknown reasons, RGS5 is expressed in pericytes that coat angiogenic vessels and appearance of this gene correlates with loss of the ability to branch[41]. RGS5 in situ probes showed ,1RGS5+cells in normal skin. The same probes hybridized with .10 cells per hpf cells in the 7 of 8 biopsies of scleroderma skin, both in a perivascular distribution, and scattered throughout the dermal matrix in cells that seem to be myofibroblasts. There was a strong signal in the epidermis of the scleroderma patient as well (Figure 2 D). For the same reasons we looked at RGS5 we looked for direct evidence of endothelial cell death and/or signs that vessels were growing to replace the lost capillaries. To assess for signs of apoptosis, the tissue was examined for nuclear changes character- istic of apoptosis and stained for the apoptotic marker cleaved caspase 3 [42] (Table 1 E). Cleaved Caspase 3 staining is recorded PLoS ONE | www.plosone.org 3 January 2008 | Issue 1 | e1452