[8,9], requires histological studies using endothelial specific markers[12]. Capillary rarefaction has been identified, to our knowledge, in only three other pathologic entities: congestive heart failure, granulation tissue and hypertension[13,14]. Some researchers have suggested that rarefaction may contribute to the character- istic elevation in peripheral re´sistance in hypertension[14]. Scleroderma patients can also have cardiac failure associated with pulmonary fibrosis and pulmonary hypertension[15,16] but a link has not been found between the rarefaction in scleroderma and that in cardiac failure, and scleroderma patients are, as a group, hypotensive[17] rather than hypertensive. To investigate the vasculopathy associated with scleroderma we examined protein and mRNA molecules in endothelial cells and capillaries of skin biopsies from patients with diffuse cutaneous scleroderma and normal controls. In addition, we retrospectively studied skin biopsies from diffuse cutaneous scleroderma patients enrolled in a pilot study of the effect of high-dose immunosup- pressive therapy and autologous hematopoietic cell transplantation (HDIT/HCT)[18]. After HDIT/HCT, these patients all experi- enced significant clinical improvement[19] including decrease in the dermal fibrosis[19,20] and the modified Rodnan skin score (MRSS) and an increase in overall function as measured by the modified Health Assessment Questionnaire Disability Index (MHAQ) . We analyzed biopsies from these patients, both before and after treatment, and have demonstrated a correlation between changes in tissue expression of angiogenesis related molecules and capillary numbers. RESULTS Scleroderma vessels lost normal endothelial markers and gained inflammatory markers Normal dermal endothelium is characterized by several charac- teristic immunohistochemical markers including: CD31, a com- mon blood group antigen (Ulex europaeus lectin), von Willebrand factor, and VE cadherin[21]. The most widely used of these is CD31, which also stains leukocytes[22]. We found the expected vascular labeling of vessels in normal and scleroderma skin, with some positive leukocytes. Endothelium in capillary loops and precapillary arterioles in normal skin are also positive for enzymatic activity of endogenous alkaline phosphatase[23]. Figure 1 (A and B) depicts normal and scleroderma biopsies where serial sections have been stained with CD31 and Ulex europaeus lectin. In scleroderma skin a population of vessels have no luminal staining for Ulex europaeus lectin whereas normal skin has robust staining in all vessels. Von Willebrand factor, VE cadherin (Figure 1 C), and alkaline phosphatase all showed similar marked loss of luminal stain. Immunohistochemical and histochemical stain quantification showed significant loss in vessels in scleroderma biopsies for Ulex europaeus lectin (p = 0.01), alkaline phosphatase (p = 0.002), VE cadherin (p = 0.008), and von Willebrand factor (p = 0.033) (Table 1 Section A). The difference in sample numbers tested for each marker is a function of scarcity of tissue and when the IHC was done. Some samples were very small and IHC was done for only a few markers. Endothelial markers were scored according to presence of positive and negative vessels. Numbers of biopsies with vessels that all had stain on the endothelial cells are expressed as number of positive biopsies per total n. All endothelial markers showed a decrease in expression in scleroderma except CD31. We assessed the vasculature for signs of endothelial inflamma- tion and summarized the quantitative results in Table 1 B. High endothelial venules have been described in dermal vessels of Vessel Recovery in Scleroderma Figure 1. Endothelial Phenotype of Capillaries in Scleroderma Compared to Normal Controls. Markers of normal endothelium: A.) CD31 staining highlights capillaries in both scleroderma and normal controls. B.) Black arrows show Ulex europaeus lectin (Ulex) is lost from some vessels in scleroderma (compare with CD31 above). C.) VE Cadherin stain is lost from some vessels in scleroderma (arrows) whereas all vessels in normal skin are positive for VE cadherin. Both von Willebrand factor and alkaline phosphatase were similarly lost from scleroderma were similarly lost from scleroderma (data not shown) Markers of inflamed endothelium: D.) CD123, a marker for high endothelial venules, is increased in scleroderma as is E.) smooth muscle actin (SMA). In the capillaries (arrows) smooth muscle actin stain extends to the very top of the dermal papilla, unlike normal skin where smooth muscle actin stain ends at the superficial horizontal plexus (doubleheaded arrow). SMA staining is also increased in the media of vessels in the reticular dermis, (data not shown) doi:10.1371/journal.pone.0001452.g001 chronic dermopathies and are considered a sign of chronic inflammation[24]. The high endothelial venule phenotype has endothelial nuclei projecting into the lumen with characteristic proteins on the endothelial surface that provide a specialized site for lymphocyte migration [25]. CD123, CD62P, and smooth muscle actin are among the proteins expressed on inflamed vascular lumens[26,27]. Scleroderma patients clearly had in- creased inflammatory markers of endothelial cells compared to normal controls. The endothelial cells in scleroderma were found to have significantly increased expression of CD123 (high endothelial venule, p = 0.0005) and smooth muscle actin (p = 0.000001) although CD62P did not show a significant difference (p = 0.49). CD123+ (high endothelial venules), and smooth muscle actin staining patterns are depicted in Figure 1 D and E respectively. PLoS ONE | www.plosone.org 2 January 2008 | Issue 1 | e1452