Figure 3. Total Vessels Were Decreased and Capillaries in Scleroderma Skin Were Selectively Decreased. A.) This data represents the total number of vessels per hpf in 15 normal and 21 scleroderma skin biopsies stained with CD31. The average vessels/hpf is significantly decreased in scleroderma compared with normal controls (p = 0.02). B.) Normal skin IHC for CD31 demonstrates microvasculature. ‘‘Profiles’’ or capillaries are defined as linear CD31 staining with no central lumen (single arrows) and represent a robust estimate of capillary density. ‘Lumens’ represent larger vessels, and are defined as structures positive for CD31 that have a central lumen (circle). C.) Data are average numbers of ‘‘lumens’’ versus ‘‘profiles’’. Average ‘‘lumens’’, representing larger vessels, in scleroderma skin biopsies are not significantly different from normal skin (p = 0.18). Average ‘‘profiles’’, representing capillaries, are significantly decreased in scleroderma compared with controls P = 0.009. These results indicate that capillaries are decreased in scleroderma. doi:10.1371/journal.pone.0001452.g003 CD123+(high endothelial venules) endothelial cells significantly decreased (p = 0.01) after HDIT/HCT in scleroderma patients, although smooth muscle actin expression was not (p = 0.08). Vessel Recovery in Scleroderma Interferon a mRNA expression in scleroderma decreased after HDIT/HCT treatment Interferon a mRNA in situ hybridization, CD123 (plasmacytoid dendritic cells) and STAT1 were all significantly decreased after HDIT/HCT (Table 3C). At baseline, all 7 patients had markedly high transcript levels of interferon a RNA both in the dermis and epidermis whereas five of 7 patients after HDIT/HCT showed loss of RNA hybridization for interferon a in the dermis and epidermis (p = 0.01, see photomicrographs Figure 4C). The marker for plasmacytoid dendritic cells, CD123, present in all baseline biopsies preceding HDIT/HCT, was lost in 6 of 7 patients after HDIT/HCT (p = 0.01) To further confirm the loss of interferon a expression, presence of nuclear phosphorylated STAT1 was decreased (p = 0.001) after HDIT/HCT (Table 3B). The decrease in signs of interferon a, CD123+plasmacytoid dendritic cells and STAT1 may indicate that the interferon a producing cells are destroyed or deactivated by treatment, and in the context of the capillary counts below have interesting vascular implications. After HDIT/HCT scleroderma skin had decreased mRNA expression of RGS5 and no sign of endothelial death or proliferation RNA in situ hybridization for RGS5 at baseline shows that 6 of 7 scleroderma patients had increased positive cells (p = 0.0023, Table 3C). After HDIT/HCT, all 7 biopsies had less than one per high power field (depicted in Figure 4D). Biopsies before and after had numbers of perivascular cells in cell cycle greater than normal (p = 0.09, Table 3D). Ki67 was not seen in endothelial cells before and after HDIT/HCT (p = 1, Table 3E). IHC for cleavage of caspase 3 and histological analysis of the endothelial cells before and after HDIT/HCT showed very little cell death (p = 1, Table 3F). After HDIT/HCT, capillary numbers increased in those patients who lost all interferon a expression, and regained VE cadherin To demonstrate whether capillary counts increase after treatment, we quantified biopsies from the scleroderma patients who grew back capillaries at baseline and after HDIT/HCT. Patients after HDIT/HCT show significantly increasing capillary numbers (p = 0.015, Figure 5). Most interesting, because of the mechanistic implications, is the correlation of the capillary numbers with VE cadherin and interferon a expression. All 7 patients showed changes in interferon a and VE cadherin in baseline biopsies. In the 5 patients where capillary counts increased, IHC for VE cadherin was positive in all vessels (Table 4). In the same patients where VE cadherin expression returned to normal, IFNA1 and IFNA2 in situ hybridization (interferon a) was undetectable (Table 4, see Figure 4 C for photomicrographs). Late stage scleroderma did not resemble scleroderma after treatment Skin changes in scleroderma at a late stage of disease can include some softening and reduction of symptoms[43,44]. These late stage skin changes could be a potentially confounding factor for the scleroderma patients after HDIT/HCT since their disease duration spans 5 to 7 years after diagnosis. We therefore included as a final control, 4 skin biopsies from patients with a similar duration of disease (5–6 years from diagnosis). These biopsies were compared to the biopsies of the 7 patients after HDIT/HCT. PLoS ONE | www.plosone.org 5 January 2008 | Issue 1 | e1452