Vessel Recovery in Scleroderma . .. . Table 4. Increased vessel number following HDIT/HCT is correlated with return of normal immunohistochemical expression of VE ... cadherin and a interferon in skin biopsies in late stage scleroderma .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... Patient # vCehsasnegl/eh pinf Average THiCmTe(yaeftaerrs)HDIT/ BVeEfocaredhHeDriInT/HCT iBnetfeorrfeerHonDIaT/HCT VAEftecradHhDeIrTi/nHCT iAnftteerrfeHrDonIT/aHCT . .... 9 +0.9 6 neg pos pos neg ... 11 +1.8 5 neg pos pos neg . ... 12 +2.6 4 neg pos pos neg . ... 18 +7 5 neg pos pos neg ... 22{ 23.2 4 neg pos neg pos . ... 24 +3.7 4 neg pos pos neg . ... 26{ 21.3 4 neg pos neg pos . . .. {these 2 patient’s skin biopsies had decreased numbers of vessels . .. doi:10.1371/journal.pone.0001452.t004 this relates to the loss of VE cadherin and the appearance of RGS5 is not at all obvious. Interferon a is a cytokine known to be a potent inhibitor of angiogenesis[50,51]. Type 1 interferon has been found to be present in epithelial layers and epidermis in humans and identified as an endogenous inhibitor of angiogenesis in skin[52]. Interferon a treatment has been associated with new onset or exacerbation of scleroderma and Raynaud’s phenomena with digital gangrene[53– 55]. A type 1 interferon gene expression signature has been described in autoimmune connective tissues diseases, including scleroderma[56–58]. We found CD123+ interferon-producing cells[59] at the superficial horizontal plexus where the bulk of capillaries would be, and where angiogenesis should be taking place. Furthermore, increased STAT1 expression in the same areas confirms the presence of an interferon response. The evidence that expression of interferon a mRNA was decreased 5 of 7 patients undergoing HDIT/HCT combined with the unexpected observa- tion of vascular regeneration suggest that an antiangiogenic state may have been reversed by this treatment. We are unaware of any other study that has shown reversible rarefaction in any disease. Intriguingly, the two patients interferon a expression was still present were the same two patients that did not grow new capillaries and had vessels negative for VE Cadherin. . .. . Table 5. Scleroderma Patients after HDIT/HCT with Capillary . .. Regeneration, Compared with Patients of Similar Disease . .. Duration .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... AHfCtTer#HDIT/ Sdaumraetiodnise#ase ... Type of molecular marker pos/n{ pos/n{ P value{ . .. . A) Endothelial Markers . ... CD31 7/7 4/4 1 . ... VE Cadherin 5/7 0/4 0.007 . .. B Inflammatory markers . ... CD123 (high endothelial venules) 0/5 3/3 0.017 . ... C. Other Markers .... Interferon a in situ (IFNA1 and IFNA2) 0/5 2/2 0.047 ... CD123 (plasmacytoid dendritic cells) 0/5 3/3 0.017 . . .. . {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.t005 Existing observations suggest that vascular disease is paramount in the pathogenesis of scleroderma[60,61]. Certainly vasospasm, as in Raynaud’s, is a frequent, though not a clearly obligatory precursor to disease. It is also possible that rarefaction precedes diagnosis of scleroderma by many years and our data suggest this may be the case. There are no objective data of which we know showing elevated levels of cell death or apoptosis in any phase of this disease. Suggestions that apoptosis and rarefaction occur during active disease have been based on studies showing the presence of circulating factors able to kill endothelial cells in culture[62] but the relevance to endothelial death in vivo is not known. Similarly, endothelial autoantibodies in scleroderma have been classified as anti-microvascular and are cited by some as a cause of vessel loss[63,64]. Once again, these data are limited to in vitro studies that may not represent in vivo phenomena. Our studies were of skin biopsies only and, especially in the context of the HDIT/HCT trial, fresh tissue samples were limited. Given the number of possible circulating factors which may cause the death of endothelial cells or prevent their growth, it may be best in future studies to approach this problem with a discovery biology approach (such as proteomics or gene expression arrays) rather than a direct method. Also, since the natural history of the disease tends to decrease the levels of inflammatory factors as the disease moves from an inflammatory to a chronic sclerotic phase, it may not be possible to find significant differences without much larger numbers of patients than we have in this study. A larger study is underway and, given the clinical success of this one, and we may be able to assess antiendothelial antibodies and other circulating factors together with a more detailed assessment of cell death. We found no evidence of ongoing endothelial cell death in the scleroderma skin compared with normal controls, and must conclude that any endothelial cell death in scleroderma, implied by capillary rarefaction, must be an early or episodic event in scleroderma. To further support the suggestion that capillaries are lost very early, we found no evidence that capillary numbers continue to drop over time in scleroderma. If active capillary loss is occurring, there should be angiogenesis and vessel replacement in the superficial horizontal plexus at the post capillary venule[65] involving endothelial proliferation[66]. The pattern of malformed vessels seen in nail beds is, in fact, reminiscent of patterns of angiogenesis seen with localized over expression of VEGF[67,68]. Evidence published by others that circulating and local VEGF is increased in scleroderma has led to a dilemma. How can elevated VEGF be correlated with loss of the microvasculature? In the scleroderma dermis we see no evidence of endothelial replication in the superficial horizontal plexus PLoS ONE | www.plosone.org 8 January 2008 | Issue 1 | e1452