Evidence for reduced angiogenesis in bone marrow in SSc: immunohistochemistry and multiparametric computerized imaging analysis

Abstract
OBJECTIVE: Dysfunctional angiogenesis is a pathogenetic marker of SSc. Circulating levels of endothelial progenitor cells are reduced, and mesenchymal stromal cells show a reduced differentiation into endothelial cells and capacity to form capillaries. This suggests that pathophysiologically relevant changes may already exist in SSc bone marrow (BM) stromal cells that may affect downstream angiogenesis. The aim of this study is to evaluate, in SSc BM, angiogenesis, cellular immune system and fibrosis.

METHODS: Eight SSc patients affected by a severe dcSSc and screened for autologous haematopoietic stem cells transplantation (HSCT) underwent a BM biopsy. BM biopsies were compared with six healthy controls. To evaluate angiogenesis and cellular immunity, the following antibodies were used: vascular endothelial growth factor (VEGF), kinase insert domain-containing receptor/fetal liver kinase-1 (KDR/flk-1), MMP-9 and CD34. To evaluate fibrosis, silver impregnation for reticulum was used. The number of vessels, the mean area of vascularization, the perimeter and microvessel density (MVD) were measured with a multiparametric computerized imaging analysis.

RESULTS: A significant reduction in BM vascularity was found, while VEGF expression was much higher in SSc BM samples. Two patients had a Grade 2, whereas another two had a Grade 1 fibrosis.

CONCLUSION: In SSc, BM is characterized by a reduction of microvascular density and number of vessels and a significant increase of VEGF. This indicates that BM may be involved in the process of loss of angiogenesis, despite the presence of high local and systemic levels of VEGF.