Dados do Trabalho

Title

GRANULOMATOSIS WITH POLYANGIITIS: impairment of endothelial cell function and its role in vascular repair

Background

The circulating endothelial progenitor cells (EPC) have a fundamental role in angiogenesis, repair and vascular homeostasis. These cells isolated from peripheral blood generate in vitro endothelial colony-forming cells (ECFC) that have high proliferative capacity and stable phenotype throughout the culture. The aim of the study was to evaluate the functional capacity of ECFC in patients with GPA in vitro through migration and angiogenesis assay.

Materials and methods

Prospective study with adult patients diagnosed with GPA according to ACR criteria (1990) and Chappel Hill Consensus (2013). Patients without immunosuppressive drugs for at least 18 months and prednisone ≤10mg/day, with active and remission disease according to BVAS, were included. Controls were healthy volunteers and HUVEC (Human umbilical vein primary endothelial cells). ECFC were isolated from periferic blood after incubation in specific culture medium. The ECFC were characterized by flow cytometry (FACS). The MIGRATION or "scratch" wound-healing assay was performed with acquisition of images obtained hour by hour for 24 hours with Inverted Fluorescence Microscope and Color Camera. This assay was also performed after stimulation of these cells overnight with plasma of controls and active GPA patients. For the ANGIOGENESIS assay ECFC were plated in Matrigel™ for 24 hours and photographed after 15 and 24h (camera associated with an inverted microscope, with a 4X objective in phase contrast mode). All images were analyzed through Image J software.

Results

Peripheral blood was collected from 13 GPA patients. Success in the isolation and growth in ECFC culture was obtained in 7 patients (54%). Six were male and four were classified as active disease. ECFC from GPA patients had lower rates of migration compared to healthy controls and HUVEC. When submitted to plasma stimulation, the behavior was similar. Angiogenesis assay showed an impairment in the number of structures analysed (extremities, nodes and junctions) between the ECFC from GPA patients and health controls and HUVEC. When assessed separately according to disease activity no significant diferences were found between patients and controls.

Conclusions

The results suggest a functional alteration of the ECFC in GPA patients independent of external stimuli and consequent involvement in vascular repair.