Vessel Recovery in Scleroderma . ... Table 2. Clinical and pathological findings after HDIT and HCT correlated with vessel counts. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... Patient # SCpoedceim#en THiDmIeT/HafCteTr(years) vBeesfoserels HcoDuITn/tHCT VAeftsesrelHsDcIoTu/HntCT aDfrtoepr HinDMITR/HSCST ADfrtoepr iHnDdITe/rHmCaTl fibrosis CAhftaenrgHeDiInT/MHHCTAQ score . ... 9 0942 6 6.8 7.7 16 3 1 to 0 . ... 11{ 1105 5 6.14 7.9 30 5 2.5 to 0 ... . 12{,D 1226 1 8.2 8 1 0.75 to 0 .... 18 1888 5 5.2 12 18 3 2.125 to 1.5 ... 22J,£ 2218 4 6.59 3.43 22 4 . ... 24 2435 4 5.09 8.78 6 4 2.875 to 2.375 .... 26£ 2622 4 5.7 4.4 34 4 2.875 to 0.125 . . .. For each of the clinical parameters assessed, post-HDIT/HCT values were compared to pre-HDIT/HCT (baseline) values at the last time point in each of several post-HDIT/ . . HCT time windows . .. {Reported finger contractures as the only indication of evidence of scleroderma post HDIT/HCT. . .. {No clinical or pathologic data is available for the first 4 years post HDIT/HCT. Patient subsequently underwent right lower lobectomy 4 years 9 months post HDIT for .. squamous cell carcinoma. .. Jdeveloped pulmonary hypertension. . .. £did not have increased capillary counts in post HDIT/HCT biopsies. ... Ddid not have a count done on the one year biopsy and clinical data was not available for four year biopsy . . Abbreviations: mRSS-Modified Rodnan Skin Score for dermal fibrosis; MHAQ–modified Health Assessment Questionnaire Disability Index . .. doi:10.1371/journal.pone.0001452.t002 We compared the IHC results after HDIT/HCT with four patients whose disease duration is 5 years or longer. VE cadherin was negative in scleroderma of long duration, 4 of 4 Non-HDIT/ HCT scleroderma patients of 5 years or greater scleroderma duration still have vessels that do not express VE cadherin. 4 or . .. Table 3. Molecular markers of microvascular phenotype, . .. inflammation and cell cycle in skin biopsies from scleroderma . . before and after HDIT/HCT . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... BHeDfIoTr/eHCT AHfDteITr/HCT ... Type of molecular marker # pos/n{ # pos/n{ P value{ . . .. A) Endothelial markers . ... Alkaline Phosphatase 0/7 0/7 1 . ... VE Cadherin 0/7 5/7 0.010 .... CD31 6/7 7/7 0.49 ... Von Willebrand Factor 1/7 4/7 0.13 . ... Ulex Europaeus Lectin 2/7 3/7 0.49 . .. B Inflammatory . ... CD123 (high endothelial venules) 6/7 1/7 0.01 . ... Smooth muscle actin 5/5 2/5 0.08 . .. C. Other : . .... RGS5 in situ 6/7 7/7 0.0023 ... Interferon a in situ (IFNA1 and IFNA2) 7/7 2/7 0.01 . ... STAT1 6/6 0/6 0.001 .... CD123 (plasmacytoid dendritic cells) 6/7 1/7 0.01 ... D. Perivascular cell Turnover: . ... Ki67 Antigen 7/7 5/7 0.01 . .. E Endothelial cell turnover . ... Ki67 antigen 0/7 0/7 1 . ... Cleaved Caspase 3 0/7 0/7 1 . . . .. {Data are numbers of 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.t003 more years after HDIT/HCT however, 5 of 7 patients have regained all VE cadherin expression in biopsies (p = 0.007, Table 5 A). VE cadherin expression has therefore returned only after HDIT and is not an artifact of late stage disease. The CD123+endothelial cells (high endothelial venules) has resolved itself in 6 of 7 biopsies after HDIT/HCT, whereas Non- HDIT/HCT patients with similar (5 years or greater) disease duration all have CD123+ vessels with high endothelial morphol- ogy (p = 0.017, Table 5B). Similarly the expression of interferon a mRNA by in situ hybridization is undetectable in 5 of 7 biopsies, whereas 2of four late stage disease patients have interferon a+ cells in dermis (p = 0.047, Table 5C). The perivascular cells positive for CD123 are also increased in late stage patients but have disappeared in all but one of the post HDIT/HCT biopsies (p = 0.017, Table 5C). Loss of the expression of interferon a, absence of CD123+cells, as well as inflammation, appear also not to be an artifact of late stage disease but seem to be associated with HDIT/HCT. Limited amounts of available tissue prohibited us from obtaining results for RGS5. Profile counts showed a significant increase (p = 0.025) in the average number of capillaries per hpf in the five patients who grew back capillaries after HDIT/HCT, compared with Non-HDIT/ HCT scleroderma of 5–6 years duration (Figure 5). DISCUSSION In this study, the most impressive changes in the dermis of scleroderma patients were rarefaction of capillary loops, loss of expression of VE cadherin, and the appearance of mRNA for interferon a and RGS5. While we also observed changes in other molecules, the changes in these three molecules are particularly relevant because they have all been reported to play possible critical roles in angiogenesis. We also report that after HDIT/ HCT the above changes reverse, especially when increasing capillary counts are found in the tissue. We showed that these changes are unlikely to be the result of the natural history of scleroderma, and suggest that these findings may provide clues to the pathogenesis of this poorly understood disease. An endothelial phenotype that lacks normal markers is a puzzle and to the best of our knowledge this is previously unreported. One extreme possibility is that endothelial cells no longer line the vessels. We do not think this is true. On two samples of normal and PLoS ONE | www.plosone.org 6 January 2008 | Issue 1 | e1452