Figure 4. Immunohistochemistry and in situ results before and after HDIT/HCT show regain of VE cadherin and loss of RGS5 and Interferon a. A.) Immunohistochemistry for CD31 before and after HDIT and autologous HCT is positive in both biopsies, whereas B) when compared with CD31 stained biopsies above, VE cadherin is negative before but positive after HDIT and HCT. C.). Baseline biopsies of scleroderma patients prior to HDIT and autologous HCT all showed cells positive for interferon a (IFNa) in situ hybridization and were present both in the vasculature and scattered in the matrix. After HDIT and autologous HCT, interferon a mRNA was undetectable in 5 of 7 biopsies. Compared biopsies were run at the same time with the same conditions and probe concentration. D) In situ hybridization for RGS5 demonstrates positive mononuclear cells around vessels and some cells scattered in the matrix in the scleroderma biopsies prior to treatment with HDIT/ HCT. There was little or no detectable RGS5 mRNA after HDIT/HCT. doi:10.1371/journal.pone.0001452.g004 scleroderma skin, we performed electron microscopy (data not shown). The luminal cells of these samples lacked features of other cell types, which can, under some circumstances, form a lining. For example, we looked for the microplicated surface of macrophage or the altered polarity and microfilaments charac- teristic of smooth muscle cells and found none of these characteristics in the luminal cells in scleroderma vessels. One could speculate a variety of mechanisms to explain endothelial cells without normal markers: 1) differentiation of a cell type other than normal dermal microvascular endothelial cells into an endothelial-like morphology, which replaced the dead endothelial cells early in disease; 2) circulating precursors from other endothelial lineages (e.g. sinus, lymphatic) replace the normal dermal endothelial cell; 3) activating autoantibodies to endothelial cells or other circulating factors, which cause them to differentiate into an abnormal state and/or cause chronic inflammation; 4) Vessel Recovery in Scleroderma Figure 5. Capillary Counts Increase After HDIT and HCT Compared to Baseline and to Scleroderma of Long Duration. Data are average ‘‘profiles’’/hpf counted in scleroderma patients before (Baseline) and after (After HDIT/HCT) treatment with HDIT and HCT. ‘‘Late disease’’ are average ‘‘profiles’’/hpf in a separate group of patients with disease duration similar to patients after HDIT/HCT, included to provide an additional disease control. Baseline (biopsies for patients who grew back capillaries) had an average capillary number of 4.1/hpf. After HDIT/HCT, average capillary number (for patients who increased capillary number) rose to 5.8/hpf (p = 0.015). Patients with a disease duration of 5 or more years averaged 3.8 profiles/hpf and when compared with the patients who grew back capillaries(After HDIT/HCT) yielded a p value of 0.025. doi:10.1371/journal.pone.0001452.g005 effect of a pathological cell type on the endothelial cell expressome, or 5) an underlying mechanism or structural abnormality existent in scleroderma endothelial cells responsible for some part of the vascular abnormality similar to that found in the children of patients with malignant hypertension[45]. The bulk of capillaries in the skin are capillary loops branching from the superficial horizontal plexus into the rete pegs and consist of a single layer of endothelium with a few pericytes all above the border of the rete ridge[46]. This is a highly specific structure, optimized to improve thermal exchange. Our data showed that capillary loops, are lost from scleroderma skin at a very early stage. This loss was associated with a dramatic and unusual loss of expression of markers used to define the endothelial cell lineage including VE cadherin. We found significant loss of the smallest vessels in the baseline scleroderma skin biopsies (all 2 years or less from diagnosis). The low number of vessels appeared to remain constant at all stages of disease. After HDIT/HCT the endothelial marker which did return to normal in 5 of 7 patients, VE cadherin, is a calcium dependent cell adherens junction protein responsible during development for aggregation of cells and formation of endothelial tubes[47]. The loss of VE Cadherin in baseline scleroderma and the reappearance of VE cadherin after HDIT/HCT may be significant in terms of the angiogenic potential of the skin, since the same 5 patients whose VE cadherin expression returned to normal also increased the numbers of capillaries present in their skin. RGS5 mRNA expression was reversed in all 7 patients after treatment, and did not correlate specifically with regeneration of capillaries, since two of the seven did not increase average skin capillary numbers. This indicate that decreased RGS5 expression after HDIT/HCT is insufficient to regenerate capillaries by itself, but like the VE Cadherin data may suggest that some form of vasostatic process in scleroderma inhibited any angiogenic response prior to treatment. The hypothesis exists that part of pathogenesis of scleroderma is circulation of an endothelial toxic agent, loss of circulating endothelial precursors or some other less specified dysregulation of the angiogenic pathway[48,49]. How PLoS ONE | www.plosone.org 7 January 2008 | Issue 1 | e1452