Evidence for Reduced Angiogenesis in Bone Marrow of Systemic Sclerosis Patients Immunohistochemetry and Computerized Imagine Analysis.
Authors: Carrai Valentina , Alterini Renato , Saccardi Riccardo , Miniati Irene , Rigacci Luigi , Capaccioli Gloria , Donnini Irene , Rotunno Giada , Bosi Alberto , Cerinic Marco Matucci 
Published: October 2019

Abstract
Abstract 5126

Introdution
Systemic sclerosis (SSc) involves the microcirculation, the immune system, and the connective tissue eventually leading to fibrosis. Vascular dysfunction is one of the earliest events in SSc pathogenesis: endothelial damage leads to a dysregulation of angiogenesis and a loss of capillaries. The consequent chronic ischemia provokes a diffuse sufference of the tissues with formation of ulcers. We analyzed BM biopsies in a series of patients undergoing HSCT in our Centre for severe, progressive SSc in order to clarify the association between modification bone marrow angiogenesis and clinical features of this disease.

Materials and Methods
The main clinical feature of the patients are following: modified Rodnan Skin Score was more than 8 in four patients; nailfold videocapillaroscopy was active in three patients; pulmonary arterial hypertension was upper limit only in two patients; carbon monoxide diffusing capacity was more than 40% in all the patients; high resolution computed tomography showed fibrosis or ground glass lesions in all the patients; topoisomerase I and antinuclear antibody was positive in all the patients. Only one patient showed arrhythmia. Eight SSc bone marrow biopsies were studied, compared with five bone marrow biopsies of non malignant controls. To evaluate angiogenesis, following monoclonal antibodies (MoAb) were used: VEGF, KDR, MMP-9, CD34. Bone marrow fibrosis was evaluated by silver impregnation for reticulum. To identify BM microvessel, anti-CD34 was used. Sections were observed at 400x magnification by two different blinded observers. To calculate the number of vessels, vascular mean area expressed in mm2, vascular percent mean area, perimeter and MVD, a multiparametric, semi-automatic computerized imagine analysis was employed. For each section, we evaluated eight consecutive areas and each area was 16001,92 mm2 (total area was 128015,36 mm2). The VEGF, MMP-9, KDR expression was evaluated as percentage of positive cells in a total of eight consecutive areas at 400x magnification.

Results
All patients showed a mean cellularity of 40% (SD 5.24, range 30-45%). In four patients bone marrow fibrosis was detected: two patients were classified as I° grade and two as II°grade. The bone marrow biopsy specimens from patients with SSc show a substantial reduction in vascularity. A multiparametric computerized analysis demonstrated significantly reduction of MVD in SSc cases. The mean MVD in SSc bone marrow was 712,63 (SD 392.03, range 124,98-1312,34) whereas in control specimens it was 1364,58 (SD 44.20, range 1312,34-1402,14). The mean number of vessels (p0.004) and percent vascular mean area are lower in sclerosis than controls (p0.0009). A significant increased expression of VEGF was observed. The median VEGF rate was 48,75 (range 30-85%) with expression ≥ 50% in half patients and in two patients with advanced SSc this expression was more than 70%. KDR expression was significantly lower in SSc bone marrow than in controls (p=0,003). The median KDR rate was 13,25% (range 1-40%) and 42% (range 40-45%) in sclerosis and controls respectively. In all cases the expression of MMP-9 was significantly lower than controls (p=0,0009) with median rate of 13,06% (range 1-25%) while median expression in controls was 30% (range 20-40%).

Discussion
We demonstrated in patient with SSc a reduction of bone marrow vascularity despite the stricking increase of a number of angiogenic factors. VEGF expression in SSc bone marrow is high in all cases with a double median rate compared to controls (48,75% vs 4,25%) while MVD is lower in sclerosis than controls (712,63 vs 1364,58). In regard to these alterations in our study we have observed a reduction of KDR and MMP-9. The overproduction of VEGF can generate a negative feedback to KDR while the low levels of MMP9 found in SSc bone marrow may be due to a hyperproduction of MMP inhibitors contributing to the reduction of bone marrow vascularity. In conclusion, our data demonstrate that in SSc the angiogenic potential of bone marrow is reduced, mirroring the systemic microvascular condition characterised by loss of capillaries and desertification despite the increase of VEGF. The amount of reticular fibers, detected in bone marrow, suggests that the fibrotic process may affect also the bone marrow contributing further to the reduction of angiogenic potential.

Disclosures
No relevant conflicts of interest to declare.