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

Abstract

Critical limb ischemia (CLI), the most severe form of peripheral artery disease, results from the blockade of peripheral vessels, usually correlated to atherosclerosis. Currently, endovascular and surgical revascularization strategies cannot be applied to all patients due to related comorbidities, and even so, most patients require re-intervention or amputation within a year. Circulating angiogenic cells (CACs) constitute a good alternative as CLI cell therapy due to their vascular regenerative potential, although the mechanisms of action of these cells, as well as their response to pathological conditions, remain unclear. Previously, we have shown that CACs enhance angiogenesis/arteriogenesis from the first days of administration in CLI mice. Also, the incubation ex vivo of these cells with factors secreted by atherosclerotic plaques promotes their activation and mobilization. Herein, we have evaluated the long-term effect of CACs administration in CLI mice, whether pre-stimulated or not with atherosclerotic factors. Remarkably, mice receiving CACs and moreover, pre-stimulated CACs, presented the highest blood flow recovery, lower progression of ischemic symptoms, and decrease of immune cells recruitment. In addition, many proteins potentially involved, like CD44 or matrix metalloproteinase 9 (MMP9), up-regulated in response to ischemia and decreased after CACs administration, were identified by a quantitative proteomics approach. Overall, our data suggest that pre-stimulation of CACs with atherosclerotic factors might potentiate the regenerative properties of these cells in vivo.

The serum of COVID-19 asymptomatic patients up-regulates proteins related to endothelial dysfunction and viral response in circulating angiogenic cells ex-vivo

“…Remarkably, many of the proteins altered in CACs incubated ex vivo with the serums of asymptomatic donors have been previously associated with viral infection, infection by RNA virus and SARS, but also to ECs movement or proliferation, and endothelial dysfunction. Besides, the alteration of proteins related to leukocyte extravasation and movement in CACs exposed to the serum of PCR + people, corroborates the activation of the immune process in these cells (Eslava-Alcon et al 2020 ; Beltrán-Camacho et al 2021 ; Medina et al 2011 ). In response to pro-inflammatory stimuli such as viral infection, circulating EPCs initiate weak cell–cell interactions with the endothelium, promoting the expression of adhesion molecules such as E-selectin or ICAM-1 by these cells, which also promotes vascular permeability, EPCs adhesion and trans-endothelial migration (Krenning et al 2009 ), as well as leukocyte recruitment (Othumpangat et al 2016 ; Yu et al 2020 ; Dai et al 2008 ).…”

Unraveling the differential mechanisms of revascularization promoted by MSCs & ECFCs from adipose tissue or umbilical cord in a murine model of critical limb-threatening ischemia

“…Despite this, a recovery in BF could be seen by day 7, even in IC untreated mice, which might be explained by repairing mechanisms triggered in response to hypoxia an ischemic events, such as the formation of new capillaries and new muscle fibers [ 53 ], in an attempt to recover from the absence of oxygen and lack of nutrients, as previously reported [ 54 ]. Such vessel sprouting takes place during the first days post-ischemia, even in non-treated tissues [ 30 ]. Afterwards, the newly formed vessels either grow and become functional or, on the contrary, they are immature and fragile, and they might contribute to the tissue instability [ 55 , 56 ].…”

“…Perfusion was expressed as the ratio of left (ischemic) vs right (non-ischemic) limb, and blood flow values registered within anatomically defined regions of interest (ROIs). In parallel, motility impairment, inflammation, ulceration, and necrosis were also registered for all mice during the entire assay according to Tarlov [ 32 ] and ischemia scores [ 33 ], as described [ 5 , 29 , 30 ] (Supplementary Table S2). In order to get closer to the necrotic progression seen in our CLTI animal model, we defined a necrotic score based on the number of necrotic nails, fingers or paw.…”

“…OCT embedded tissues were cut transversally in 10 μm sections and placed in poly-L-lysine slides. In total, 3 tissue sections separated by 150 μm each were employed for vessel and cellular detection from all mice (n:8 per group), as described [ 5 , 29 , 30 ]. Primary antibodies employed were anti-α-actin smooth muscle (α-SMA) for vessel detection, anti-MOMA-2 for macrophages/monocytes detection, and anti-Ly-6G for neutrophils detection.…”

Assessment of endothelial colony forming cells delivery routes in a murine model of critical limb threatening ischemia using an optimized cell tracking approach

“…We and other researchers have already described the beneficial effects of cell therapy in murine models of CLTI, mainly by promoting blood flow perfusion recovery after enhancement of collateral vessel formation [ 33 , 42 , 43 ]. Similarly, the therapeutic effects of endothelial progenitor cells (EPCs) transplantation in different models of ischemia have already been described [ 11 , 12 , 44 – 46 ], and the potential use of ECFCs to promote revascularization in CLTI patients seems undeniable [ 47 – 49 ].…”