Alginate-Encapsulated Mesenchymal Stromal Cells Improve Hind Limb Ischemia in a Translational Swine Model.

BACKGROUND: Cellular therapies have been investigated to improve blood flow and prevent amputation in peripheral artery disease with limited efficacy in clinical trials. Alginate-encapsulated mesenchymal stromal cells (eMSCs) demonstrated improved retention and survival and promoted vascular generation in murine hind limb ischemia through their secretome, but large animal evaluation is necessary for human applicability. We sought to determine the efficacy of eMSCs for peripheral artery disease-induced limb ischemia through assessment in our durable swine hind limb ischemia model. METHODS AND RESULTS: Autologous bone marrow eMSCs or empty alginate capsules were intramuscularly injected 2 weeks post-hind limb ischemia establishment (N=4/group). Improvements were quantified for 4 weeks through walkway gait analysis, contrast angiography, blood pressures, fluorescent microsphere perfusion, and muscle morphology and histology. Capsules remained intact with mesenchymal stromal cells retained for 4 weeks. Adenosine-induced perfusion deficits and muscle atrophy in ischemic limbs were significantly improved by eMSCs versus empty capsules (mean±SD, 1.07±0.19 versus 0.41±0.16, P=0.002 for perfusion ratios and 2.79±0.12 versus 1.90±0.62 g/kg, P=0.029 for ischemic muscle mass). Force- and temporal-associated walkway parameters normalized (ratio, 0.63±0.35 at week 3 versus 1.02±0.19 preligation; P=0.17), and compensatory footfall patterning was diminished in eMSC-administered swine (12.58±8.46% versus 34.85±15.26%; P=0.043). Delivery of eMSCs was associated with trending benefits in collateralization, local neovascularization, and muscle fibrosis. Hypoxia-cultured porcine mesenchymal stromal cells secreted vascular endothelial growth factor and tissue inhibitor of metalloproteinase 2. CONCLUSIONS: This study demonstrates the promise of the mesenchymal stromal cell secretome at improving peripheral artery disease outcomes and the potential for this novel swine model to serve as a component of the preclinical pipeline for advanced therapies.

A Swine Hind Limb Ischemia Model Useful for Testing Peripheral Artery Disease Therapeutics.

Currently, there is no large animal model of sustained limb ischemia suitable for testing novel angiogenic therapeutics for peripheral artery disease (PAD) such as drugs, genes, materials, or cells. We created a large animal model suitable for efficacy assessment of these therapies by testing 3 swine hind limb ischemia (HLI) variations and quantifying vascular perfusion, muscle histology, and limb function. Ligation of the ipsilateral external and bilateral internal iliac arteries produced sustained gait dysfunction compared to isolated external iliac or unilateral external and internal iliac artery ligations. Hyperemia-dependent muscle perfusion deficits, depressed limb blood pressure, arteriogenesis, muscle atrophy, and microscopic myopathy were quantifiable in ischemic limbs 6 weeks post-ligation. Porcine mesenchymal stromal cells (MSCs) engineered to express a reporter gene were visualized post-administration via positron emission tomography (PET) in vivo. These results establish a preclinical platform enabling better optimization of PAD therapies, including cellular therapeutics, increasing bench-to-bedside translational success. A preclinical platform for porcine studies of peripheral artery disease therapies including (1) a hind limb ischemia model and (2) non-invasive MSC viability and retention assessment via PET.

Adenosine from a biologic source regulates neutrophil extracellular traps (NETs).

Neutrophil extracellular traps (NETs) are implicated in autoimmune, thrombotic, malignant, and inflammatory diseases; however, little is known of their endogenous regulation under basal conditions. Inflammatory effects of neutrophils are modulated by extracellular purines such as adenosine (ADO) that is inhibitory or ATP that generally up-regulates effector functions. In order to evaluate the effects of ADO on NETs, human neutrophils were isolated from peripheral venous blood from healthy donors and stimulated to make NETs. Treatment with ADO inhibited NET production as quantified by 2 methods: SYTOX green fluorescence and human neutrophil elastase (HNE)-DNA ELISA assay. Specific ADO receptor agonist and antagonist were tested for their effects on NET production. The ADO 2A receptor (A2A R) agonist CSG21680 inhibited NETs to a similar degree as ADO, whereas the A2A R antagonist ZM241385 prevented ADO's NET-inhibitory effects. Additionally, CD73 is a membrane bound ectonucleotidase expressed on mesenchymal stromal cells (MSCs) that allows manipulation of extracellular purines in tissues such as bone marrow. The effects of MSCs on NET formation were evaluated in coculture. MSCs reduced NET formation in a CD73-dependent manner. These results imply that extracellular purine balance may locally regulate NETosis and may be actively modulated by stromal cells to maintain tissue homeostasis.