Identification of the initial molecular changes in response to circulating angiogenic cells-mediated therapy in critical limb ischemia

Abstract

Background: Critical limb ischemia (CLI) constitutes the most aggressive form of peripheral arterial occlusive disease, characterized by the blockade of arteries supplying blood to the lower extremities, significantly diminishing oxygen and nutrient supply. CLI patients usually undergo amputation of fingers, feet, or extremities, with a high risk of mortality due to associated comorbidities. Circulating angiogenic cells (CACs), also known as early endothelial progenitor cells, constitute promising candidates for cell therapy in CLI due to their assigned vascular regenerative properties. Preclinical and clinical assays with CACs have shown promising results. A better understanding of how these cells participate in vascular regeneration would significantly help to potentiate their role in revascularization. Herein, we analyzed the initial molecular mechanisms triggered by human CACs after being administered to a murine model of CLI, in order to understand how these cells promote angiogenesis within the ischemic tissues.

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

“…As a first step, the identity of CAC was confirmed by flow cytometry as previously described [ 12 ] ( Supplementary Figure S1 ). In order to identify a differential secretion pattern of CAC in response to an atherosclerotic environment, a label-free quantitative proteomic analysis was carried out comparing the secretome of CAC in basal medium (C) with the secretome of CAC pre-treated ex-vivo with atherosclerotic factors (CAC+AP or AP).…”

“…In the last years, there has been a significant increase in the number of pre-clinical and clinical studies focused on the application of EPC pursuing neovascularization in response to ischemia or CVD risk factors [ 12 , 14 , 15 , 28 , 29 , 30 ]. Moreover, EPC are also under consideration as a preventative and/or treatment strategy to provide vascular repair in atherosclerosis [ 4 ].…”

“…They both differ not only in their differentiation status, surface markers or their capability to form colonies; they also seem to contribute to neovascularization in different ways. Thus, while CAC are associated to a more paracrine role, by trophic support and enhancement of endogenous repair process [ 4 , 12 ], ECFC participate more directly by replacing damaged endothelial cells (EC) [ 10 , 31 ]. The “paracrine effect” of CAC has been assigned based on in vivo studies in which the cells themselves or their conditioned media administrated to murine models of critical limb ischemia or similar ones [ 12 , 13 , 32 ], induced an increase of vascular density or wound healing, without being able to detect these cells at the short term within the system [ 12 ].…”

“…Thus, while CAC are associated to a more paracrine role, by trophic support and enhancement of endogenous repair process [ 4 , 12 ], ECFC participate more directly by replacing damaged endothelial cells (EC) [ 10 , 31 ]. The “paracrine effect” of CAC has been assigned based on in vivo studies in which the cells themselves or their conditioned media administrated to murine models of critical limb ischemia or similar ones [ 12 , 13 , 32 ], induced an increase of vascular density or wound healing, without being able to detect these cells at the short term within the system [ 12 ]. Additionally, other studies have confirmed the presence of several pro-angiogenic factors in the conditioned media of EPC such as VEGF and GC-SF [ 33 ], PDGF-α, PDGF-β or KGF [ 32 ], CD163 and platelet factors such as CXCL4 and CXCL7 [ 34 ], among others.…”

“…Since their discovery in 1997 [ 7 ], many researchers have focused on clarifying the identity of these cells, their nature, and characteristics. To date, two main sub-populations have been formally defined from the initially named EPC [ 8 , 9 , 10 ]: early EPC (eEPC), also called circulating angiogenic cells (CAC) or myeloid angiogenic cells (MAC), with limited proliferation capacity (lasting 7–10 days after isolation) and angiogenic potential through paracrine mechanisms [ 11 , 12 ]; and late outgrowth cells or endothelial colony-forming cells (ECFC), highly proliferative and with enormous vasculogenic properties, probably by direct engraftment and/or replacement of damaged cells [ 8 , 9 ].…”