Abstract
BackgroundCell-based therapeutic strategies have been proposed as an alternative for brain repair after stroke, but their clinical application has been hampered by potential adverse effects in the long term. The present study was designed to test the effect of the secretome of endothelial progenitor cells (EPCs) from stroke patients (scCM) on in vitro human models of angiogenesis and vascular barrier.MethodsTwo different scCM batches were analysed by mass spectrometry and a proteome profiler. Human primary CD34+-derived endothelial cells (CD34+-ECs) were used for designing angiogenesis studies (proliferation, migration, and tubulogenesis) or in vitro models of EC monolayer (confluent monolayer ECs—CMECs) and blood–brain barrier (BBB; brain-like ECs—BLECs). Cells were treated with scCM (5 μg/mL) or protein-free endothelial basal medium (scEBM—control). CMECs or BLECs were exposed (6 h) to oxygen–glucose deprivation (OGD) conditions (1% oxygen and glucose-free medium) or normoxia (control—5% oxygen, 1 g/L of glucose) and treated with scCM or scEBM during reoxygenation (24 h).ResultsThe analysis of different scCM batches showed a good reproducibility in terms of protein yield and composition. scCM increased CD34+-EC proliferation, tubulogenesis, and migration compared to the control (scEBM). The proteomic analysis of scCM revealed the presence of growth factors and molecules modulating cell metabolism and inflammatory pathways. Further, scCM decreased the permeability of CMECs and upregulated the expression of the junctional proteins such as occludin, VE-cadherin, and ZO-1. Such effects were possibly mediated through the activation of the interferon pathway and a moderate downregulation of Wnt signalling. Furthermore, OGD increased the permeability of both CMECs and BLECs, while scCM prevented the OGD-induced vascular leakage in both models. These effects were possibly mediated through the upregulation of junctional proteins and the regulation of MAPK/VEGFR2 activity.ConclusionOur results suggest that scCM promotes angiogenesis and the maturation of newly formed vessels while restoring the BBB function in ischemic conditions. In conclusion, our results highlight the possibility of using EPC-secretome as a therapeutic alternative to promote brain angiogenesis and protect from ischemia-induced vascular leakage.
Highlights
Cell-based therapeutic strategies have been proposed as an alternative for brain repair after stroke, but their clinical application has been hampered by potential adverse effects in the long term
We initially compared the angiogenic properties of Endothelial progenitor cells (EPC)-secretome from three stroke patients and three healthy donors using Low‐scale production (LSP) conditions in CD34+-CD34 +-derived cord-blood hematopoietic endothelial cells (EC)
Our results suggest that scSP-Conditioned medium (CM) preserves their angiogenic properties, as they showed a similar effect on C D34+-ECs proliferation (Fig. 1a) and migration (Fig. 1b) compared to scHD-CM, whereas neither scHD-CM nor scSP-CM had any effect on tubulogenesis (Fig. 1c)
Summary
Cell-based therapeutic strategies have been proposed as an alternative for brain repair after stroke, but their clinical application has been hampered by potential adverse effects in the long term. BBB impairment and vascular disruption are early events following an ischemic stroke, exacerbating the brain injury and contributing to cognitive impairment [2, 5]. In this context, developing new therapies that combine the protection of the BBB integrity and the promotion of angiogenesis could be a potential strategy to improve the functional outcome after stroke. This hypothesis is supported by a body of evidence, suggesting that therapies promoting angiogenesis can stimulate neurogenesis and improve brain repair after stroke [6]
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