Angiogenic Mechanisms of Human CD34 ⁺ Stem Cell Exosomes in the Repair of Ischemic Hindlimb

Abstract

Rationale Paracrine secretions appear to mediate therapeutic effects of human CD34+ stem cells locally transplanted in patients with myocardial and critical limb ischemia as well as in animal models. Earlier, we had discovered that paracrine secretion from human CD34+ cells contains pro-angiogenic, membrane-bound nano-vesicles called exosomes (CD34Exo). Objective Here, we investigated the mechanisms of CD34Exo-mediated ischemic tissue repair and therapeutic angiogenesis by studying their miRNA content and uptake. Methods and Results When injected into mouse ischemic hindlimb tissue, CD34Exo, but not the CD34exo-depleted conditioned media, mimicked the beneficial activity of their parent cells by improving ischemic limb perfusion, capillary density, motor function and their amputation. CD34Exo were found to be enriched with pro-angiogenic miRNAs such as miR-126-3p. Knocking down miR-126-3p from CD34exo abolished their angiogenic activity and beneficial function both in vitro and in vivo. Interestingly, injection of CD34Exo increased miR-126-3p levels in mouse ischemic limb, but did not affect the endogenous synthesis of miR-126-3p suggesting a direct transfer of stable and functional exosomal miR-126-3p. miR-126-3p enhanced angiogenesis by suppressing the expression of its known target, SPRED1; simultaneously modulating the expression of genes involved in angiogenic pathways such as VEGF, ANG1, ANG2, MMP9, TSP1 etc. Interestingly, CD34Exo, when treated to ischemic hindlimbs, were most efficiently internalized by endothelial cells relative to smooth muscle cells and fibroblasts demonstrating a direct role of stem cell-derived exosomes on mouse endothelium at the cellular level. Conclusions Collectively, our results have demonstrated a novel mechanism by which cell-free CD34Exo mediates ischemic tissue repair via beneficial angiogenesis. Exosome-shuttled angiomiRs may signify amplification of stem cell function and may explain the angiogenic and therapeutic benefits associated with CD34+ stem cell therapy.

Type 2 diabetes and older age contribute to elevated plasma microparticle concentrations independent of chronic stroke

“…Thus, elevations in CD34+ MPs observed with in the older groups may be indicative of chronic low-grade inflammation associated with older age. Alternatively, CD34+ exosomes have been found in vitro to contain angiogenesis-promoting miRNAs (Sahoo & Losordo, 2014; Mathiyalagan et al , 2017). Although the size and mechanisms of release differ between exosomes and MPs, potential similarities in cargo between the two extracellular vesicles suggest that perhaps the elevations in CD34+ MPs are serving in a compensatory capacity to offset the elevations in CD62E+ MPs also associated with older age.…”

Transplantation of Endothelial Progenitor Cells Overexpressing miR-126-3p Improves Heart Function in Ischemic Cardiomyopathy

“…17 Recent research also revealed that knockdown of miR-126-3p from CD34 Exo abolished angiogenic activity and beneficial function both in vitro and in vivo. 18 These findings prompted us to investigate the therapeutic effect of miR-126-3p overexpression on heart function in ICM models.…”

“…10, 11 Based on work conducted by Jiang, it was also reported that human endometrial stem cell transplantation can improve cardiac function by regulating cytokine secretion. 12 The altered cytokine levels in peripheral blood mononuclear cells (PBMCs) were also found to correlate with the progression and severity of heart disease.…”

Concise Review: Cell Therapy for Critical Limb Ischemia: An Integrated Review of Preclinical and Clinical Studies

“…Although EVs are produced by many cell types, EVs from multiple progenitor cell lineages are released into the peripheral circulation and target distant sites with potent pro‐angiogenic stimuli. Mathiyalagan et al have recently shown that human CD34+ cell‐derived exosomes injected into the ischemic hindlimb of mice improved limb perfusion via EC uptake of miRNA‐126‐3p that suppressed expression of the sprouty‐family gene, SPRED1, and simultaneously upregulated VEGF, ANGF1, ANG2, and MMP9 expression . ECFC‐derived EVs incorporate into ECs via interaction with α4 and β1 integrins, and stimulate angiogenesis via delivery of mRNA associated with eNOS production and PI3K/AKT pathway .…”