Capillary Regeneration in Scleroderma: Stem Cell Therapy Reverses Phenotype? Jo N. Fleming1*, Richard A. Nash2, D. O. McLeod1, David F. Fiorentino3, Howard M. Shulman4, M. Kari Connolly5, Jerry A. Molitor6, Gretchen Henstorf7, Robert Lafyatis8, David K. Pritchard1, Lawrence D. Adams1, Daniel E. Furst9, Stephen M. Schwartz1 1 Department of Pathology, University of Washington, Seattle, Washington, United States of America, 2 Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America, 3 Department of Dermatology, Stanford University School of Medicine, Stanford, California, United States of America, 4 Clinical Research Division, Pathology Section, Fred Hutchinson Cancer Research Center and Seattle Cancer Care Alliance, Seattle, Washington, United States of America, 5 Department of Medicine, University of California at San Francisco, San Francisco, California, United States of America, 6 Department of Rheumatology, Virginia Mason Medical Center, Seattle, Washington, United States of America, 7 Program in Transplantation Biology, Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America, 8 Rheumatology Section, Boston University School of Medicine, Boston, Massachusetts, United States of America, 9 Department of Rheumatology, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, California, United States of America Background. Scleroderma is an autoimmune disease with a characteristic vascular pathology. The vasculopathy associated with scleroderma is one of the major contributors to the clinical manifestations of the disease. Methodology/Principal Findings. We used immunohistochemical and mRNA in situ hybridization techniques to characterize this vasculopathy and showed with morphometry that scleroderma has true capillary rarefaction. We compared skin biopsies from 23 scleroderma patients and 24 normal controls and 7 scleroderma patients who had undergone high dose immunosuppressive therapy followed by autologous hematopoietic cell transplant. Along with the loss of capillaries there was a dramatic change in endothelial phenotype in the residual vessels. The molecules defining this phenotype are: vascular endothelial cadherin, a supposedly universal endothelial marker required for tube formation (lost in the scleroderma tissue), antiangiogenic interferon a (overexpressed in the scleroderma dermis) and RGS5, a signaling molecule whose expression coincides with the end of branching morphogenesis during development and tumor angiogenesis (also overexpressed in scleroderma skin. Following high dose immunosuppressive therapy, patients experienced clinical improvement and 5 of the 7 patients with scleroderma had increased capillary counts. It was also observed in the same 5 patients, that the interferon a and vascular endothelial cadherin had returned to normal as other clinical signs in the skin regressed, and in all 7 patients, RGS5 had returned to normal. Conclusion/Significance. These data provide the first objective evidence for loss of vessels in scleroderma and show that this phenomenon is reversible. Coordinate changes in expression of three molecules already implicated in angiogenesis or anti-angiogenesis suggest that control of expression of these three molecules may be the underlying mechanism for at least the vascular component of this disease. Since rarefaction has been little studied, these data may have implications for other diseases characterized by loss of capillaries including hypertension, congestive heart failure and scar formation. Citation: Fleming JN, Nash RA, McLeod DO, Fiorentino DF, Shulman HM, et al (2008) Capillary Regeneration in Scleroderma: Stem Cell Therapy Reverses Phenotype?. PLoS ONE 3(1): e1452. doi:10.1371/journal.pone.0001452 INTRODUCTION Progressive systemic sclerosis, or scleroderma, is an uncommon connective tissue disease characterized by autoimmunity, diffuse fibrosis[1] in the skin and internal organs, and a vasculopathy with intimal hyperplasia of muscular and elastic arteries[2,3]. Ray- naud’s phenomenon, an excessive vasospastic reaction to cold or stress[4], precedes diagnosis of scleroderma by months or years[5], and is early evidence of the vasculopathy associated with scleroderma. The vasculopathy in scleroderma can result in non- healing ulcers, gangrene and digit loss[6], clinical features rarely encountered in primary Raynaud’s disease. Further evidence of endothelial damage in scleroderma are findings by nail fold capillaroscopy [7]. Typical findings include giant capillaries, hemorrhages, avascular areas and neovascular- ization[8]. This morphology resembles malformed capillary beds formed in response to vascular endothelial growth factor [9] and could be the result of increased levels of vascular endothelial growth factor produced in response to ischemia resulting from vasospasm and intimal hyperplasia[10]. Increased levels of vascular endothelial growth factor (VEGF) are known to be present in scleroderma[11] and high levels of VEGF are normally associated with increased angiogenesis. Instead there appears to be a loss of capillaries. Unfortunately, the prevailing belief that there is loss of capillaries in scleroderma is based on findings in nail fold capillaroscopy and is dependent on seeing columns of blood. The ischemia caused by Raynaud’s phenomenon, especially coupled with malformations caused by high levels of VEGF could cause capillary collapse and could be misinterpreted as loss of capillaries since nail fold microscopy would be unable to identify unperfused capillaries. Definitive evidence for rarefaction, as opposed to loss of capillary blood flow or malformation of newly formed vessels, Academic Editor: Pieter Reitsma, Leiden University Medical Center, Netherlands Received September 21, 2007; Accepted December 21, 2007; Published January 16, 2008 Copyright: 2008 Fleming et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by the scleroderma Research Foundation that had no role in the design and conduct of the study, in the collection, analysis, and interpretation of data, or in the preparation, review and approval of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * To whom correspondence should be addressed. E-mail: flemij@u.washington. edu PLoS ONE | www.plosone.org 1 January 2008 | Issue 1 | e1452