The Cytoprotective Effects of Human Endothelial Progenitor Cell-Conditioned Medium against an Ischemic Insult Are Not Dependent on VEGF and IL-8

Abstract

Endothelial progenitor cells (EPCs) promote revascularization and tissue repair mainly by paracrine actions. In the present study, we investigated whether EPC-secreted factors in the form of conditioned medium (EPC-CM) can protect cultured brain microvascular endothelial cells against an ischemic insult. Furthermore, we addressed the type of factors that are involved in the EPC-CM-mediated functions. For that purpose, rat brain-derived endothelial cells (rBCEC4 cell line) were exposed to EPC-CM pretreated with proteolytic digestion, heat inactivation, and lipid extraction. Moreover, the involvement of VEGF and IL-8, as canonical angiogenic factors, was investigated by means of neutralizing antibodies. We demonstrated that EPC-CM significantly protected the rBCEC4 cells against an ischemic insult mimicked by induced oxygen-glucose deprivation followed by reoxygenation. The cytoprotective effect was displayed by higher viable cell numbers and reduced caspase 3/7 activity. Heat inactivation, proteolytic digestion, and lipid extraction resulted in a significantly reduced EPC-CM-dependent increase in rBCEC4 viability, tube formation, and survival following the ischemic challenge. Notably, VEGF and IL-8 neutralization did not affect the actions of EPC-CM on rBCEC4 under both standard and ischemic conditions. In summary, our findings show that paracrine factors released by EPCs activate an angiogenic and cytoprotective response on brain microvascular cells and that the activity of EPC-CM relies on the concerted action of nonproteinaceous and proteinaceous factors but do not directly involve VEGF and IL-8.

Endothelial Progenitor Cells Conditioned Medium Supports Number of GABAergic Neurons and Exerts Neuroprotection in Cultured Striatal Neuronal Progenitor Cells

“…To eliminate the proteinaceous content in the CM, the CM was treated with proteinase K as described earlier 5,12 . In brief, aliquots of EBM-2 or CM medium were incubated with agarose beads-proteinase K (Sigma, Buchs, Switzerland; 5 U) in 15 ml polypropylene test tubes (BD Falcon, Allschwil, Switzerland) for 4 h at 37°C in a rotating roller drum.…”

“…Thereafter, the tubes were centrifuged 5 min at 2000 rpm to pellet the agarose beads-proteinase K, and the proteinase K-free supernatant was carefully collected and used for subsequent experiments. The treatment resulted in degradation of proteins as assessed in Coomassie Blue (Sigma, Buchs, Switzerland)-stained gels 5,12 (Fig. 1).…”

Endothelial progenitors increase vascularization and improve fibroblasts function that prevent medication‐related osteonecrosis of the jaw

“…*p < .05, **p < .01, ***p < .001 [Colour figure can be viewed at wileyonlinelibrary.com] function. Various studies have shown that the cytoprotective and angiogenic properties of EPCs substantially rely on paracrine mechanisms (Di Santo, Fuchs, Periasamy, Seiler, & Widmer, 2016;Odent Grigorescu et al, 2017;Rosell et al, 2013;Yang et al, 2010;Zhang et al, 2014). Burchfield and Dimmeler showed that the beneficial effect of EPC transplantation on diabetic wounds was mainly achieved by their paracrine action on keratinocytes, fibroblasts, and endothelial cells (Burchfield & Dimmeler, 2008).…”

Endothelial Progenitor Cell-Derived Factors Exert Neuroprotection in Cultured Cortical Neuronal Progenitor Cells

“…However, our earlier study identified lipidic factors as important mediators of the actions of EPC-CM 7 . Moreover, we have previously shown that EPC-CM contains a number of neurotrophic factors including brain-derived neurotrophic factor glial cell line-derived neurotrophic factor, neuritin, and vascular endothelial growth factor, which are critical for the developing cerebral cortex 8 , 30 . In addition, it has been reported that EPC secretome has the capacity to promote oligodendrocyte expansion and maturation in mice in a model of cerebral hypoperfusion 6 .…”