Atherosclerotic Pre-Conditioning Affects the Paracrine Role of Circulating Angiogenic Cells Ex-Vivo

Abstract

In atherosclerosis, circulating angiogenic cells (CAC), also known as early endothelial progenitor cells (eEPC), are thought to participate mainly in a paracrine fashion by promoting the recruitment of other cell populations such as late EPC, or endothelial colony-forming cells (ECFC), to the injured areas. There, ECFC replace the damaged endothelium, promoting neovascularization. However, despite their regenerative role, the number and function of EPC are severely affected under pathological conditions, being essential to further understand how these cells react to such environments in order to implement their use in regenerative cell therapies. Herein, we evaluated the effect of direct incubation ex vivo of healthy CAC with the secretome of atherosclerotic arteries. By using a quantitative proteomics approach, 194 altered proteins were identified in the secretome of pre-conditioned CAC, many of them related to inhibition of angiogenesis (e.g., endostatin, thrombospondin-1, fibulins) and cell migration. Functional assays corroborated that healthy CAC released factors enhanced ECFC angiogenesis, but, after atherosclerotic pre-conditioning, the secretome of pre-stimulated CAC negatively affected ECFC migration, as well as their ability to form tubules on a basement membrane matrix assay. Overall, we have shown here, for the first time, the effect of atherosclerotic factors over the paracrine role of CAC ex vivo. The increased release of angiogenic inhibitors by CAC in response to atherosclerotic factors induced an angiogenic switch, by blocking ECFC ability to form tubules in response to pre-conditioned CAC. Thus, we confirmed here that the angiogenic role of CAC is highly affected by the atherosclerotic environment.

Long Term Response to Circulating Angiogenic Cells, Unstimulated or Atherosclerotic Pre-Conditioned, in Critical Limb Ischemic Mice

“…Overall, these results reinforce the hypothesis that CACs-mediated revascularization is mainly achieved in a paracrine fashion. Thus, CAC's role is fundamental on the first days, when they participate in recruiting autologous progenitor cells [39][40][41] and promoting a mature and functional vascular network, in agreement with in vitro results where CACs secreted factors enhance ECFCs tubule formation [13,26,45].…”

“…As an alternative for those patients that are not suitable candidates for surgery, the use of pro-angiogenic cells such as endothelial progenitor cells (EPCs) has become a promising option, due to their potential to promote neovascularization in ischemic tissues. To date, two different subpopulations of EPCs have been described [ 9 , 10 , 11 , 12 , 13 ]: early EPCs, also called circulating angiogenic cells (CACs) or myeloid angiogenic cells, and late outgrowth EPCs or endothelial colony-forming cells (ECFCs). Both sets of cells have been tested to promote revascularization in ischemic diseases such as myocardial infarction or CLI [ 14 , 15 , 16 , 17 , 18 , 19 , 20 ].…”

“…Different studies suggest that CACs participate in vascular repair in a paracrine fashion, while ECFCs, recruited by CACs, might replace damaged endothelium and are probably responsible for new-vessel formation [ 9 , 24 , 25 ]. For instance, CACs release factors that appear to promote ECFCs tubule formation in vitro [ 13 , 24 ].…”

Current Status of Angiogenic Cell Therapy and Related Strategies Applied in Critical Limb Ischemia

“…Although MSCs secretomes are the most studied ones, secretomes of other stem/progenitor cells are also showing promising results. Very recently, we analyzed by a proteomic approach the secretome of CACs, identifying a significant number of angiogenic factors, and moreover, we demonstrated that incubation ex vivo of ECFCs with this secretome enhances ECFCs angiogenesis, in agreement with previous studies [ 77 , 110 ]. Moreover, ASCs secretome contains multiple angiogenic factors, which appear to promote, among others, survival, proliferation, and migration of ECs, as well as vasculogenesis [ 187 , 188 ].…”

“…Yoon et al evaluated this combined cell therapy, demonstrating a synergistic neovascularization involving several cytokines and matrix metalloproteinases (MMPs) [ 77 ]. Very recently, our group has also corroborated the potential of CACs to promote angiogenesis of ECFCs in vitro, and such effect was impaired under an atherosclerotic environment [ 110 ]. In the same way, different cell combinations have been tested.…”