suggesting that there is no angiogenic response but we have not found a good explanation for this conundrum. A possible mechanism for the return of capillaries, consistent with the effects of HDIT/HCT is that endothelial precursor cells are required for angiogenesis[69–71]. An infusion of the CD34- selected hematopoietic cell graft may have therefore contributed to the re-capillarization. Unfortunately at the time of the HDIT/ HCT study, samples were not taken to analyze levels of circulating endothelial cells. In future studies it may be an excellent way to confirm that endothelial cells have returned to normal in scleroderma. Alternatively, and in our opinion more likely, HDIT/HCT is immunosuppressive and may be ablating some cell that inhibits endothelial regeneration or produces a factor or factors that inhibit endothelial cell regeneration. HDIT/HCT treatment has been investigated for other autoimmune diseases besides scleroderma including systemic lupus erythematosus and multiple sclerosis. It has been previously reported that there was recovery of CD4+ T cell counts after HDIT/HCT at 2 years after HDIT/HCT. However, sustained responses were observed in 63% of patients at a median of 4 years[72]. Immune recovery after HDIT/HCT is associated with increasing thymic-derived na¨ıve CD4+ T cells with a decrease in memory T cells, an increase in regulatory T cells, and broader clonal diversity than was present before HDIT/HCT[72–74] These late immune changes support the conclusion that sustained responses may have resulted from the immunomodulatory effects of HDIT/HCT in addition to the early immunosuppression and depletion of autoreactive T cells. After HDIT/HCT, we observed that those patients who grew back capillaries were different from scleroderma of long duration. The patients who received HDIT/HCT had more capillaries and also lost interferon a expression, and regained all VE cadherin expression, whereas the scleroderma in late stages did not. The interferon expression and CD123+cells are interesting in this context since one of the functions of the plasmacytoid dendritic cell is the breaking of tolerance[37], and it may be associated with some of the changes in T cell populations. The high levels of interferon a support the hypothesis that plasmacytoid dendritic cells may be part of the pathogenesis of scleroderma. Interferon a is antiproliferative in vivo[75]. The high local levels of interferon a in scleroderma skin may therefore be preventing the formation of new capillaries directly. In conclusion we propose that rarefaction is an early and possibly ‘‘pre-diagnosis’’ event in scleroderma. This capillary loss is associated with a change in endothelial phenotype of the remaining vessels that has not been previously described. The phenotype of these residual vascular structures appears to be mildly inflammatory but not apoptotic, with no sign of endothelial replication or replacement. We hypothesize that the presence of interferon a, the loss of VE cadherin and the presence of RGS5 are involved in the process which appears to keep the vessels static and produce fibrosis in this poorly understood disease. We report regeneration of capillaries and resolution of the non-angiogenic phenotype after HDIT/HCT. Our studies were somewhat limited by the retrospective nature, however, future planned studies of this autologous protocol and an allogeneic protocol will include capillaroscopic, ultrasound and MRI microvascular assessment in a subset of patients in the hope of defining these microvascular changes and assessing clinical importance. Regulation of VE cadherin expression, function of RGS5 producing smooth muscle, appearance of interferon a at other sites of vascular rarefaction, and the apparent ablation by HDIT/HCT of some cell involved in the inhibition of angiogenesis are obviously intriguing targets for continued study. Rarefaction itself is not well studied in the known examples of cardiac failure, hypertension and regression of Vessel Recovery in Scleroderma granulation tissue. Our studies may provide new clues to the more general problem of capillary loss. METHODS Patients in the study All tissues used in this study were biopsies supplied in collaboration with Stanford University, the University of California San Francisco, Boston University, Fred Hutchinson Cancer Research Center, and the University of Washington, collected and analyzed according to Institutional Review Board approved protocols for human study at each respective institution. Normal controls (Table 6) and scleroderma patient biopsies (Table 7) were supplied as sets of formalin fixed paraffin embedded blocks or slides. HDIT/HCT The clinical trial of HDIT/HCT for patients with scleroderma has been previously described[19]. Briefly, hematopoietic stem cells were mobilized with granulocyte colony stimulating factor, harvested from the peripheral blood by leukapheresis and then CD34-selected. HDIT/HCT consisted of total body irradiation (800 cGy) with partial lung shielding, high-dose cyclophosphamide (120 mg/kg) and antithymocyte globulin (Pfizer Inc., New York, NY). After HDIT/HCT, patients were transplanted with the autologous CD34-selected hematopoietic cell graft. Histochemistry Tissue was stained with hematoxylin & eosin and Movat’s pentachrome according to standard protocols. In addition to these two stains, tissue was stained for endogenous alkaline phosphatase. Briefly, the sections were cut, dewaxed with xylene and rehydrated with ethanol series prior to pretreatment with 100 mM glycine and 0.3% Triton X-100. They were then allowed to develop overnight in 5-Bromo-4-Chloro-39-Indolyphosphate p- Toluidine Salt/Nitro-Blue Tetrazolium Chloride (Roche). Coun- terstain is Nuclear Fast Red (Vector Labs). The slides were mounted with Aquamount (VWR Scientific). Immunohistochemistry (IHC) Antibodies are listed in Table 8 with dilution, manufacturer and clone number and secondary antibody. In-lab IHC Briefly, our protocol for IHC is as follows: paraffin sections were dewaxed and rehydrated prior to antigen retrieval using microwave method in sodium citrate pH 6.0. Slides were placed in citrate buffer and microwaved for 20 minutes on low power, allowed to cool and placed in blocking buffer for 30 minutes, incubated with primary antibody overnight at 4uC, then washed and incubated with biotinylated secondary antibody (Vector Labs Universal horse anti-mouse/rabbit/goat #BA-1300 or Vector Labs Universal horse anti-mouse/rabbit #BA-1400) for 30 minutes. After secondary antibody, the slides were washed and incubated for 30 minutes with avidin biotin complex reagent Blocking serum, and avidin biotin complex reagent were supplied in Vector Elite Universal Kit (Vector Labs #PK6100). The slides were washed again prior to detection with DAB with Nickel Chloride to create a grey-black color (Vector Labs # SK-4100), the slides were then counterstained with methyl green, dehydrated in ethanol series, cleared in xylene and mounted with Permount (Fisher Scientific). Phenopath IHC Immunohistochemistry done at Phenopath Labs (www.phenopath.com for their established protocols) is indicated in Table 8, along with protocols optimized for each antibody. PLoS ONE | www.plosone.org 9 January 2008 | Issue 1 | e1452