Abstract
Background/Purpose: Vascular complications are a key pathological feature of systemic sclerosis (SSc) affecting the microcirculation and arterioles. Under normal circumstances the endothelium acts as a biological barrier supporting controlled permeability, immune surveillance and cellular trafficking. In SSc endothelial damage, contributes to barrier dysfunction and elevated immune cell infiltration and inflammation. The cause(s) of the initial endothelial dysfunction in SSc is unclear. Blood outgrowth endothelial cells (BOECs) are thought to home to sites of vascular injury and differentiate into endothelial cells, aiding the repair and restoration of normal endothelial functions. Circulating levels of BOECs have been shown to be reduced in SSc patients, and recent studies suggest that BOECs may be dysfunctional in vascular diseases. Here we sought to assess SSc-BOECs and their contribution to vascular dysfunction in SSc. Methods: BOECs were established from peripheral blood PBMCs from healthy donors (HD) and SSc patients. BOECs from HD (n=5) and SSc patients (n=6) were profiled using Illumina HT12 gene arrays and secreted inflammatory cytokines profiled by meso discovery scale (MSD) arrays. The capacity of SSc-BOECs (n=4) and HD-BOECs (n=4) to: 1) Establish biological barriers alone or in co-culture with mature endothelial cells, was assessed using electric cell-substrate impedance sensing (ECIS); 2) Support PBMC (n=4) trans-endothelial migration, by forming monolayers in 24-well transwell inserts stimulated with or without TNFα (10ng/ml). Results: Using gene set enrichment analysis, it was determined that SSc-BOECs exhibit a significantly altered gene expression profile including inflammatory chemokines and cytokines. In addition SSc-BOECs exhibited significantly elevated pro-inflammatory cytokine secretion compared to HD-BOECs, including IL-6 (P<0.05; 0.003ng/ml vs 0.73ng/ml) and IL-8 (P<0.01; 0.294ng/ml vs 1.7ng/ml). SSc and HD-BOECs exhibited a similar paracellular and transcellular barrier functions alone or in co-culture with mature endothelial cells as determined by ECIS. In contrast SSc-BOECs supported significantly elevated basal trans-endothelial PBMC migration (P<0.05) compared HD-BOECs. Whereas TNFα significantly induced trans-endothelial cell migration in both BOEC cell types (P<0.01 HD vs P<0.05 SSc). Conclusion: We have demonstrated that BOECs isolated from SSc patients exhibit a significantly altered gene profile compared to that of BOECs isolated from HC donors. Additionally, SSc-BOECs exhibit a pro-inflammatory profile, secreting high levels of cytokines including IL-6 and IL-8.BOECs isolated from SSc patients exhibited a similar capacity to form biological barriers compared to those from HDs. Conversely, SSc-BOEC monolayers supported a higher level of immune cell trans-endothelial migration. Our data suggests that BOECs integrated into the endothelium in SSc patients are dysfunctional and may promote immune cell infiltration and exacerbate the pro-inflammatory environment in vascular fibrotic lesions in SSc patients.