Figure 2. Non-Angiogenic Phenotype in Scleroderma Compared to Normal Controls. A) There was increased staining in CD123 in perivascular cells in the superficial horizontal plexus in skin biopsies from scleroderma patients compared with normal controls. B) Interferon a (IFNa) in situ hybridization exhibited a robust signal in scleroderma but was absent from controls. C.) Nuclear staining of phosphorylated STAT1 in scleroderma provided evidence of active interferon signaling, with STAT1 phosphorylation, dimerization and translocation to nucleus in response to interferon a. D.) RGS5, a marker of pericytes and arterial smooth muscle had increased mRNA expression in scleroderma in a perivascular distribution. E.) Ki67 a marker of cell cycle, showed no sign of Ki67 labeling of the endothelial cells in either normal controls or scleroderma. There was an increase in Ki67 positive perivascular cells in scleroderma. doi:10.1371/journal.pone.0001452.g002 as positive biopsies per total n. Numbers of caspase 3+cells per high power field in the scleroderma biopsies fell within the normal range. The only positive biopsy was one of the normal controls (photomicrograph not shown) and the quantitative results were not significantly different (p = 1). P values were calculated with Fisher’s exact test using 262 frequency tables. To assess endothelial replication, positive Ki67 antigen staining was used to indicate the presence of cells in the biopsy that were in the cell cycle, providing an estimate of proliferation. Ki67 staining is expressed as number of positive biopsies per total n. Cells in the follicles and epidermal layer were not counted. All biopsies were scanned for cells lining the lumen that labeled positive with Ki67 and none were found. We scored the presence of Ki67+cells in normal and scleroderma biopsies and carefully assessed cellular morphology to establish whether or not endothelial cells were proliferating (Table 1 F and G.). Comparison of Ki67 stain between scleroderma and normal skin biopsies demonstrated no endothelial cells in cell cycle in the superficial horizontal plexus or anywhere else in any of the biopsies of normal or scleroderma skin (Figure 2 E). Ki67+cells were found only in a perivascular Vessel Recovery in Scleroderma distribution. With no evidence that endothelial cells are prolifer- ating in the scleroderma biopsies, we suggest that there is no ongoing endothelial cell replacement. Scleroderma had a decreased numbers of capillaries Finally, to confirm that rarefaction exists in scleroderma, we quantified the number of vessels present in normal and scleroderma skin biopsies. Vessel counts based on finding red blood cells can be misleading when vessels are collapsed; therefore we used the presence of CD31 to define vascular profiles. We compared average numbers of vessels per high power field between forearm skin sections from 21 scleroderma patients and 13 site-matched normal controls. To determine if blood vessels decrease during the course of disease, we counted biopsies from our patient group whose disease duration was 2 years or less, and compared vessel density to biopsies whose disease duration was five years or greater and compared both disease groups to normal vessel counts (Figure 3A). The numbers of vessels per high power field in scleroderma was virtually identical both early (2 years or less) and late in disease (5 or more years) and there was no statistically significant difference (p = 0.48). Vessel counts of normal controls differed significantly compared with both early (p = 0.02) and late (p = 0.04) scleroderma and showed that the number of total vessels was decreased overall in scleroderma (Figure 3 A). To approximate capillary numbers, profiles of vessels were counted in skin sections stained with an antibody for CD31. Profiles were defined as clusters of positive CD31 stained cells within the dermis with no central lumen visible (Figure 3B). To approximate the numbers of larger vessels and the remainder of capillaries we counted lumens in the same sections. Lumens were defined as clusters of CD31 stained cells with a central lumen clearly visible (Figure 3B). Comparison of lumens and profiles between controls and scleroderma (Figure 3C) show that profiles were significantly decreased in scleroderma (p = 0.009) whereas lumens were not. (p = 0.18). This suggests that vascular loss in early diffuse scleroderma is in smaller vessels, likely representing capillaries. Scleroderma endothelia regained a normal endothelial marker and lost inflammatory markers after HDIT/HCT treatment Next we evaluated biopsies from patients with scleroderma who underwent HDIT/HCT. Our study of capillary counts, IHC and in situ study included biopsies from 7 patients at baseline and at a median of 5 (range 4–6) years after treatment. As noted in table 2, the patients from the clinical trial whose skin biopsies were evaluated for the effects of HDIT/HCT on the microvasculature, all experienced improvement in clinical assess- ment of scleroderma (results of clinical trial have been previously published)[19], including activities of daily life, stamina, energy, and shortness of breath. Each of the 7 patients also reported improvement in range of motion, hand flexion and reduction in skin tightening, and pathologically the degree of the dermal fibrosis improved [19]. We looked at endothelial phenotype, comparing endothelial marker expression by IHC, and found that alkaline phosphatase, Ulex europaeus lectin, and von Willebrand factor did not return to normal after HDIT/HCT. CD31 was consistently positive both before and after HDIT/HCT. VE cadherin stain was absent in a population of vessels in all baseline biopsies. No VE cadherin- negative vessels were present in 5 of 7 biopsies after HDIT/HCT (p = 0.01, Table 3A, see photomicrographs figure 4A and B for CD31 and VE cadherin). PLoS ONE | www.plosone.org 4 January 2008 | Issue 1 | e1452