Isolation and culture of human ECFC
Peripheral blood samples were collected from 13 GPA patients and 14 healthy controls. All GPA patients were anti-PR3 positive at the diagnosis. The culture was successful in 62% of GPA patients and 57% of controls. Nine patients presented disease activity at the time of blood collection. Culture success was observed in 55% of patients with disease activity and 75% of patients in remission.
On average, the first ECFC colonies of the GPA patients appeared on the 15th day, even as ECFC of the controls. Regarding the number of colonies, there was no significant difference at the end of the third passage between the controls and the active disease and remission patients. In one patient with active GPA, the colony count was 4 times higher than that of the others. The patients’ data and ECFC culture results are summarized in Table 1.
For longitudinal evaluation, samples were collected for the isolation and culture of ECFC at diagnosis and after remission induction with cyclophosphamide in three patients. Success was achieved in pre-treatment isolation in all three cases and in only one case after immunosuppression; that case had the highest number of pre-treatment colonies.
Characterization of GPA and control ECFC
The ECFC of GPA patients showed typical endothelial cobblestone morphology. FACS analysis confirmed that the ECFC were endothelial cells expressing specific markers (CD31 (PECAM), KDR (VEGFR), CD144 (VE-Kadhrin), CD146). Moreover, as expected, the ECFC were negative for CD133 and CD45 (monocyte markers) and presented a decreased expression of CD34 markers. The control ECFC demonstrated similar results (Figure 1).
Efficiency of the ECFC in forming capillary-like structures
The Matrigel assay showed a similar number of structures formed (extremities, junctions, nodes, meshes, and segments) by the ECFC in the GPA patients compared to the control group (p=0.18, p=0.57, p=0.49, p=0.76, and p=0.82, respectively). When classified according to the presence or absence of disease activity, it was observed that only remission patients showed a progressive decrease in the number of most structures after 15 h (Figure 2).
Migration assay
The migration assay was performed by the scratching method and the results were analysed comparing the values of each group every 4 hours, until the closing of gap area for a total of 24 hours. There was no significant difference in the proliferative capacity of the ECFC between GPA patients and controls (12th hour p=0.05), even when divided according to the presence or absence of disease activity (12th hour p=0.08) (Figure 3).
Otherwise, comparing the average of the lowest confluence percentage that each group (GPA, controls and HUVEC) reached in 24 hours with the mean time that each group took to close the gap area, no difference was found (p=0.20, p=0.91) .
Longitudinal analysis
Blood samples from three GPA patients were collected to analyse the ECFC behaviour before and after treatment. All three patients had glomerulonephritis and were PR3-ANCA positive. Successful ECFC isolation was observed in only one case after remission induction with endovenous cyclophosphamide.
ECFC isolated after cyclophosphamide showed significantly lower migration rates than those of ECFC isolated before treatment (p=0.0056) (Figure 4).
Plasma influence on migration in vitro
To analyse the influence of plasma on the migration capacity of ECFC, the authors performed the migration assay after ECFC overnight incubation with plasma from healthy controls and an active disease patient.
Comparing the lowest percentage of gap area reached in 24 hours between the groups (active GPA, remission GPA, control and HUVEC) in both conditions of plasma, there was less migration capacity in the group of remission GPA when incubated with control plasma (p=0.0020).
The data were also explored comparing the values of each group (active GPA, remission GPA, control and HUVEC) every 4 hours until the closing of gap area. When incubated with plasma from an active disease patient, although ECFC of GPA patients showed a decreased migration capacity compared to the ECFC of controls, no statistical significance was found (12th hour p=0.16, 16th hour p=0.36). In addition, it was observed a higher proliferative capacity was in the subgroup of ECFC from active disease patients, also without statistical significance (remission patients, p=0.31 and controls, p=0.74).
Considering the influence of control plasma, ECFC of remission GPA patients evidenced a significant lower migration capacity after the 4th hour (p=0.0001) (Figure 5).