This study reveals for the first time the angiogenic gene-expression profile of OECs from ischemic stroke patients. Our results show that early colony-OECs from ischemic stroke patients display higher expression of proangiogenic-related genes, while expression of these set of genes in more mature populations (mature-OECs) is more similar to human cerebral microvascular endothelial cells.
A recent study has revealed changes in gene expression of late outgrowth EPC-derived endothelial cells from systemic sclerosis patients that could contribute to the endothelial dysfunction and may be relevant to the development of the vasculopathy . However, it is completely unknown the gene expression profile of EPCs from ischemic stroke patients.
Other authors have studied the differentiation process of cord blood-derived EPCs showing that the first stage involves the expression of genes related to cell adhesion to extracellular matrix; during the second stage, gene-expression profile reveals transcription of cell cycle and antiapoptotic genes; finally, after the proliferative stage, adherent EPCs acquire additional endothelial-specific characteristics through the expression of endothelial markers . Our results in the present study show that some of the genes previously described as expressed in EPCs from cord blood or peripheral blood of healthy volunteers such as IGF-1, MMP9, MMP2 and ID3[8, 9] are also expressed in EPCs from ischemic stroke patients. Additionally, it is known that IGF-1 is highly expressed in EPCs compared to mature endothelial cells or monocytes enhancing EPC differentiation involving PI3-kinase/phosphorylated Akt pathways of cell survival and proliferation [10, 11]. Our data also confirms that IGF-1 mRNA level is also increased in colony-OECs derived from stroke patients, suggesting a role for IGF-1 during the differentiation process of these cells.
Furthermore, it is well established that secreted matrix metalloproteinases (MMPs) from EPCs have a pivotal role in the ischemia-induced neovascularization since they actively participate in matrix degradation . In this context, we show that MMP9 and MMP2 are highly expressed in colony-OECs, further reinforcing the functional properties of this population. We also successfully validated the MMP2, MMP9, VEGF-C, THBS1 and NRP-2 results by qRT-PCR showing that the RT2 profiler PCR Array is a good method to assess gene expression levels even when differences in expression are small.
In addition, our findings also prove the overexpression of ID3 and TGFβR1 genes in colony-OECs, suggesting a role in the proliferation and differentiation of colony-OECs to endothelial cells. In fact, previous reports have demonstrated that ID3 −/− mutant mice show a markedly impaired EPC mobilization  and TGFβR1 has also been involved in the mobilization and differentiation of cord blood-derived EPCs . Recently, the capacity of EPCs to support the activity and function of resident differentiated cells by paracrine mechanisms has focused increased attention . Our study also demonstrates that colony-OECs show a higher proangiogenic gene expression pattern, suggesting that colony-OECs could potentially present a higher secretion of angiogenic growth factors.
The current study has revealed that the gene expression profile of mature-OECs from ischemic stroke patients partially resembles that of hCMEC/D3 cells. Recently, it has been reported that OECs acquire additional endothelial-specific characteristics based on transcriptomic-, proteomic-, and ultrastructural- analysis . Consistent with that study, our results also demonstrate that mature-OECs show an increase in MMP2, THBS1 and VEGF-C, reinforcing the endothelial nature of mature-OECs. Interestingly, we also observe an increase in mature-OECs expression of BAI-1, a member of the secretin receptor family involved in the inhibition of angiogenesis, supporting the hypothesis that angiogenesis is controlled by a local balance between stimulators and inhibitors.
In the context of cerebral ischemia, the first pre-clinical studies testing the therapeutic potential of EPCs administrated early EPCs [16, 17] but lately, new studies are exploring the role of OECs . It is important to define the nature of these cells in culture to identify the best population for transplantation.
A recent publication demonstrated that time in culture conditions did not alter phenotype of OECs with no significant change in antigen expression between early (passage 0–2), mid (passage 4–6) and late passage (passage 6–9) cells. Furthermore, they confirmed that late outgrowth endotelial progenitor cells resembled mature human umbilical vein endothelial cells (HUVEC) .
In conclusion, the present study shows that the expression pattern of the angiogenic-related genes from OECs of ischemic patents changes during their expansion but preserves the pro-angiogenic potential despite the ischemic insult. Therefore, it would be of great value to determine in which differentiation stage (i.e., whether colony or mature) would OECs be more effective in cell-based therapies, making them an attractive tool to be tested for autologous transplantation after ischemic stroke.
This study certainly includes some limitations. We included stroke patients between 24 h and 7 days after onset of symptoms because we demonstrated in a previous study that OECs from subacute strokes (obtained after 24 h of the symptoms onset) showed a higher expression and secretion of angiogenic factors than OECs obtained in the first hours following stroke . However, the temporal profile of OECs maturation in terms of angiogenic factors gene expression needs to be confirmed at different time points in a larger cohort of stroke patients. We focused our study in ischemic stroke patients because cell cultures from sex- and age-matched controls did not yield OECs under the same cell culture conditions. In this regard, we have already demonstrated that circulating EPCs are reduced in control subjects compared to stroke patients . Moreover, other authors have shown that hormonal status influences circulating EPC levels being significantly reduced in postmenopausal women related to reduce estradiol levels . Since our control group was primarily composed by women in menopausal age (76.5%, 70.6±7.4 years old) this could also partially explain our difficulty. Finally, it would be interesting to compare gene expression patterns of OECs with those of primary endothelial cells. However, the advantage of using hCMEC/D3 cells is that they model the human blood brain barrier (BBB) and avoid the inherent variability as well as the difficulty of sourcing that apply to primary endothelial cultures. Further studies, must confirmed our results in a larger number of patients and further explored at functional level to define its biological implications.