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.

VEGF-delivering PEG hydrogels promote vascularization in the porcine subcutaneous space.

For cell therapies, the subcutaneous space is an attractive transplant site due to its large surface area and accessibility for implantation, monitoring, biopsy, and retrieval. However, its poor vascularization has catalyzed research to induce blood vessel formation within the site to enhance cell revascularization and survival. Most studies focus on the subcutaneous space of rodents, which does not recapitulate important anatomical features and vascularization responses of humans. Herein, we evaluate biomaterial-driven vascularization in the porcine subcutaneous space. Additionally, we report the first use of cost-effective fluorescent microspheres to quantify perfusion in the porcine subcutaneous space. We investigate the vascularization-inducing efficacy of vascular endothelial growth factor (VEGF)-delivering synthetic hydrogels based on 4-arm poly(ethylene) glycol macromers with terminal maleimides (PEG-4MAL). We compare three groups: a non-degradable hydrogel with a VEGF-releasing PEG-4MAL gel coating (Core+VEGF gel); an uncoated, non-degradable hydrogel (Core-only); and naïve tissue. After 2 weeks, Core+VEGF gel has significantly higher tissue perfusion, blood vessel area, blood vessel density, and number of vessels compared to both Core-only and naïve tissue. Furthermore, healthy vital signs during surgery and post-procedure metrics demonstrate the safety of hydrogel delivery. We demonstrate that VEGF-delivering synthetic hydrogels induce robust vascularization and perfusion in the porcine subcutaneous space.

TREM2+ and interstitial-like macrophages orchestrate airway inflammation in SARS-CoV-2 infection in rhesus macaques.

The immunopathological mechanisms driving the development of severe COVID-19 remain poorly defined. Here, we utilize a rhesus macaque model of acute SARS-CoV-2 infection to delineate perturbations in the innate immune system. SARS-CoV-2 initiates a rapid infiltration of plasmacytoid dendritic cells into the lower airway, commensurate with IFNA production, natural killer cell activation, and a significant increase of blood CD14-CD16+ monocytes. To dissect the contribution of lung myeloid subsets to airway inflammation, we generate a longitudinal scRNA-Seq dataset of airway cells, and map these subsets to corresponding populations in the human lung. SARS-CoV-2 infection elicits a rapid recruitment of two macrophage subsets: CD163+MRC1-, and TREM2+ populations that are the predominant source of inflammatory cytokines. Treatment with baricitinib (Olumiant®), a JAK1/2 inhibitor is effective in eliminating the influx of non-alveolar macrophages, with a reduction of inflammatory cytokines. This study delineates the major lung macrophage subsets driving airway inflammation during SARS-CoV-2 infection.

Hydrogel delivery of purinergic enzymes improves cardiac ischemia/reperfusion injury.

RATIONALE: The innate immune response contributes to cardiac injury in myocardial ischemia/reperfusion (MI/R). Neutrophils are an important early part of the innate immune response to MI/R. Adenosine, an endogenous purine, is a known innate immune modulator and inhibitor of neutrophil activation. However, its delivery to the heart is limited by its short half-life (<30 s) and off-target side effects. CD39 and CD73 are anti-inflammatory homeostatic enzymes that can generate adenosine from phosphorylated adenosine substrate such as ATP released from injured tissue. OBJECTIVE: We hypothesize that hydrogel-delivered CD39 and CD73 target the local early innate immune response, reduce neutrophil activation, and preserve cardiac function in MI/R injury. METHODS AND RESULTS: We engineered a poly(ethylene) glycol (PEG) hydrogel loaded with the adenosine-generating enzymes CD39 and CD73. We incubated the hydrogels with neutrophils in vitro and showed a reduction in hydrogen peroxide production using Amplex Red. We demonstrated availability of substrate for the enzymes in the myocardium in MI/R by LC/MS, and tested release kinetics from the hydrogel. On echocardiography, global longitudinal strain (GLS) was preserved in MI/R hearts treated with the loaded hydrogel. Delivery of purinergic enzymes via this synthetic hydrogel resulted in lower innate immune infiltration into the myocardium post-MI/R, decreased markers of macrophage and neutrophil activation (NETosis), and decreased leukocyte-platelet complexes in circulation. CONCLUSIONS: In a rat model of MI/R injury, CD39 and CD73 delivered via a hydrogel preserve cardiac function by modulating the innate immune response.

Sodium alginate microencapsulation of human mesenchymal stromal cells modulates paracrine signaling response and enhances efficacy for treatment of established osteoarthritis.

Mesenchymal stromal cells (MSCs) have shown promise as osteoarthritis (OA) treatments; however, effective translation has been limited by high variability and heterogeneity of MSCs, suboptimal delivery strategies, and poor understanding of critical quality and potency attributes. Furthermore, most pre-clinical studies of MSC therapeutics for OA have focused on delaying OA development and not on treating established OA, which brings added clinical relevance. Thus, the objective of the current study was to assess the effects of sodium alginate microencapsulation on human MSC (hMSC) secretion of immunomodulatory cytokines in an OA microenvironment and therapeutic efficacy in treating established OA. A Medial Meniscal Transection (MMT) pre-clinical model of OA was implemented. Three weeks post-surgery, after OA was established, intra-articular injections of encapsulated hMSCs or nonencapsulated hMSCs were administered. Six weeks post-surgery, microstructural changes in the knee joint were quantified using microCT. Encapsulated hMSCs reduced articular cartilage degeneration and subchondral bone remodeling. A multiplexed immunoassay panel was used to profile the in vitro secretome of hMSCs in response to IL-1ß. Nonencapsulated hMSCs showed an indiscriminate increase in all cytokines in response to IL-1ß while encapsulated hMSCs showed a targeted secretory response with increased expression of pro-inflammatory (IL-1ß, IL-6, IL-7, IL-8), anti-inflammatory (IL-1RA), and chemotactic (G-CSF, MDC, IP10) cytokines. These data show that sodium alginate microencapsulation can modulate hMSC paracrine signaling and enhance the therapeutic efficacy of the hMSCs in treating established OA. This cytokine profile provides a foundation for the identification of key factors affecting the overall potency of hMSC therapeutics for OA. STATEMENT OF SIGNIFICANCE: While there has been considerable interest in material based MSC encapsulation for treatment of OA, there are critical gaps in our translational understanding of these biomaterial-based technologies for OA. More specifically, previous studies have several important limitations: (1) they have been largely focused on preventing OA development, which limits their translational utility and (2) little prior work has been done to delineate potential routes/mechanisms by which material encapsulation alters MSC therapeutic action. In our manuscript, we aimed to fill these gaps in knowledge by testing the hypotheses that: (1) hMSC encapsulation can attenuate established disease progression, which is a more clinically relevant scenario and (2) hMSC encapsulation significantly changes the secreted paracrine factors from hMSCs.

A Hydrogel Strategy to Augment Tissue Adenosine to Improve Hindlimb Perfusion.

[Figure: see text].

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.

Adenosine Production by Biomaterial-Supported Mesenchymal Stromal Cells Reduces the Innate Inflammatory Response in Myocardial Ischemia/Reperfusion Injury.

BACKGROUND: During myocardial ischemia/reperfusion (MI/R) injury, there is extensive release of immunogenic metabolites that activate cells of the innate immune system. These include ATP and AMP, which upregulate chemotaxis, migration, and effector function of early infiltrating inflammatory cells. These cells subsequently drive further tissue devitalization. Mesenchymal stromal cells (MSCs) are a potential treatment modality for MI/R because of their powerful anti-inflammatory capabilities; however, the manner in which they regulate the acute inflammatory milieu requires further elucidation. CD73, an ecto-5'-nucleotidase, may be critical in regulating inflammation by converting pro-inflammatory AMP to anti-inflammatory adenosine. We hypothesized that MSC-mediated conversion of AMP into adenosine reduces inflammation in early MI/R, favoring a micro-environment that attenuates excessive innate immune cell activation and facilitates earlier cardiac recovery. METHODS AND RESULTS: Adult rats were subjected to 30 minutes of MI/R injury. MSCs were encapsulated within a hydrogel vehicle and implanted onto the myocardium. A subset of MSCs were pretreated with the CD73 inhibitor, α,ß-methylene adenosine diphosphate, before implantation. Using liquid chromatography/mass spectrometry, we found that MSCs increase myocardial adenosine availability following injury via CD73 activity. MSCs also reduce innate immune cell infiltration as measured by flow cytometry, and hydrogen peroxide formation as measured by Amplex Red assay. These effects were dependent on MSC-mediated CD73 activity. Finally, through echocardiography we found that CD73 activity on MSCs was critical to optimal protection of cardiac function following MI/R injury. CONCLUSIONS: MSC-mediated conversion of AMP to adenosine by CD73 exerts a powerful anti-inflammatory effect critical for cardiac recovery following MI/R injury.

"Frontiers in Fontan failure: A summary of conference proceedings".

"Frontiers in Fontan Failure" was the title of a 2015 conference sponsored by Children's Healthcare of Atlanta and Emory University School of Medicine. In what is hoped to be the first of many such gatherings, speakers and attendees gathered to discuss the problem of long-term clinical deterioration in these patients. Specific focuses included properly defining the problem and then discussing different treatment strategies, both medical and surgical. The health of the liver after Fontan palliation was a particular point of emphasis, as were quality of life and future directions.

Pheochromocytoma Masquerading as "Diabetic Ketoacidosis".

Diabetic ketoacidosis is a routinely encountered diagnosis in medicine. Physicians are trained early on to look for precipitants. Most clinicians assess for medication compliance, infection, ischemia, and the like. We present a case of pheochromocytoma presenting as "diabetic ketoacidosis." The case serves as an example for broadening the differential diagnosis for patients with similar presentations. Additionally, the case helps inform our understanding of the so-called "stress reactions" that are commonly invoked in clinical rationale.

Alginate microencapsulation of human mesenchymal stem cells as a strategy to enhance paracrine-mediated vascular recovery after hindlimb ischaemia.

Stem cell-based therapies hold great promise as a clinically viable approach for vascular regeneration. Preclinical studies have been very encouraging and early clinical trials have suggested favourable outcomes. However, significant challenges remain in terms of optimizing cell retention and maintenance of the paracrine effects of implanted cells. To address these issues, we have proposed the use of a cellular encapsulation approach to enhance vascular regeneration. We contained human mesenchymal stem cells (hMSCs) in biocompatible alginate microcapsules for therapeutic treatment in the setting of murine hindlimb ischaemia. This approach supported the paracrine pro-angiogenic activity of hMSCs, prevented incorporation of hMSCs into the host tissue and markedly enhanced their therapeutic effect. While injection of non-encapsulated hMSCs resulted in a 22 ± 10% increase in vascular density and no increase in perfusion, treatment with encapsulated hMSCs resulted in a 70 ± 8% increase in vascular density and 21 ± 7% increase in perfusion. The described cellular encapsulation strategy may help to better define the mechanisms responsible for the beneficial effects of cell-based therapies and provide a therapeutic strategy for inducing vascular growth in the adult. As hMSCs are relatively easy to isolate from patients, and alginate is biocompatible and already used in clinical applications, therapeutic cell encapsulation for vascular repair represents a highly translatable platform for cell-based therapy in humans.

Cellular encapsulation enhances cardiac repair.

BACKGROUND: Stem cells for cardiac repair have shown promise in preclinical trials, but lower than expected retention, viability, and efficacy. Encapsulation is one potential strategy to increase viable cell retention while facilitating paracrine effects. METHODS AND RESULTS: Human mesenchymal stem cells (hMSC) were encapsulated in alginate and attached to the heart with a hydrogel patch in a rat myocardial infarction (MI) model. Cells were tracked using bioluminescence (BLI) and cardiac function measured by transthoracic echocardiography (TTE) and cardiac magnetic resonance imaging (CMR). Microvasculature was quantified using von Willebrand factor staining and scar measured by Masson's Trichrome. Post-MI ejection fraction by CMR was greatly improved in encapsulated hMSC-treated animals (MI: 34 ± 3%, MI + Gel: 35 ± 3%, MI + Gel + hMSC: 39 ± 2%, MI + Gel + encapsulated hMSC: 56 ± 1%; n = 4 per group; P < 0.01). Data represent mean ± SEM. By TTE, encapsulated hMSC-treated animals had improved fractional shortening. Longitudinal BLI showed greatest hMSC retention when the cells were encapsulated (P < 0.05). Scar size at 28 days was significantly reduced in encapsulated hMSC-treated animals (MI: 12 ± 1%, n = 8; MI + Gel: 14 ± 2%, n = 7; MI + Gel + hMSC: 14 ± 1%, n = 7; MI+Gel+encapsulated hMSC: 7 ± 1%, n = 6; P < 0.05). There was a large increase in microvascular density in the peri-infarct area (MI: 121 ± 10, n = 7; MI + Gel: 153 ± 26, n = 5; MI + Gel + hMSC: 198 ± 18, n = 7; MI + Gel + encapsulated hMSC: 828 ± 56 vessels/mm2, n = 6; P < 0.01). CONCLUSIONS: Alginate encapsulation improved retention of hMSCs and facilitated paracrine effects such as increased peri-infarct microvasculature and decreased scar. Encapsulation of MSCs improved cardiac function post-MI and represents a new, translatable strategy for optimization of regenerative therapies for cardiovascular diseases.

Computational protein design to reengineer stromal cell-derived factor-1α generates an effective and translatable angiogenic polypeptide analog.

BACKGROUND: Experimentally, exogenous administration of recombinant stromal cell-derived factor-1α (SDF) enhances neovasculogenesis and cardiac function after myocardial infarction. Smaller analogs of SDF may provide translational advantages including enhanced stability and function, ease of synthesis, lower cost, and potential modulated delivery via engineered biomaterials. In this study, computational protein design was used to create a more efficient evolution of the native SDF protein. METHODS AND RESULTS: Protein structure modeling was used to engineer an SDF polypeptide analog (engineered SDF analog [ESA]) that splices the N-terminus (activation and binding) and C-terminus (extracellular stabilization) with a diproline segment designed to limit the conformational flexibility of the peptide backbone and retain the relative orientation of these segments observed in the native structure of SDF. Endothelial progenitor cells (EPCs) in ESA gradient, assayed by Boyden chamber, showed significantly increased migration compared with both SDF and control gradients. EPC receptor activation was evaluated by quantification of phosphorylated AKT, and cells treated with ESA yielded significantly greater phosphorylated AKT levels than SDF and control cells. Angiogenic growth factor assays revealed a distinct increase in angiopoietin-1 expression in the ESA- and SDF-treated hearts. In addition, CD-1 mice (n=30) underwent ligation of the left anterior descending coronary artery and peri-infarct intramyocardial injection of ESA, SDF-1α, or saline. At 2 weeks, echocardiography demonstrated a significant gain in ejection fraction, cardiac output, stroke volume, and fractional area change in mice treated with ESA compared with controls. CONCLUSIONS: Compared with native SDF, a novel engineered SDF polypeptide analog (ESA) more efficiently induces EPC migration and improves post-myocardial infarction cardiac function and thus offers a more clinically translatable neovasculogenic therapy.

Oxygen-dependent quenching of phosphorescence used to characterize improved myocardial oxygenation resulting from vasculogenic cytokine therapy.

This study evaluates a therapy for infarct modulation and acute myocardial rescue and utilizes a novel technique to measure local myocardial oxygenation in vivo. Bone marrow-derived endothelial progenitor cells (EPCs) were targeted to the heart with peri-infarct intramyocardial injection of the potent EPC chemokine stromal cell-derived factor 1α (SDF). Myocardial oxygen pressure was assessed using a noninvasive, real-time optical technique for measuring oxygen pressures within microvasculature based on the oxygen-dependent quenching of the phosphorescence of Oxyphor G3. Myocardial infarction was induced in male Wistar rats (n = 15) through left anterior descending coronary artery ligation. At the time of infarction, animals were randomized into two groups: saline control (n = 8) and treatment with SDF (n = 7). After 48 h, the animals underwent repeat thoracotomy and 20 µl of the phosphor Oxyphor G3 was injected into three areas (peri-infarct myocardium, myocardial scar, and remote left hindlimb muscle). Measurements of the oxygen distribution within the tissue were then made in vivo by applying the end of a light guide to the beating heart. Compared with controls, animals in the SDF group exhibited a significantly decreased percentage of hypoxic (defined as oxygen pressure ≤ 15.0 Torr) peri-infarct myocardium (9.7 ± 6.7% vs. 21.8 ± 11.9%, P = 0.017). The peak oxygen pressures in the peri-infarct region of the animals in the SDF group were significantly higher than the saline controls (39.5 ± 36.7 vs. 9.2 ± 8.6 Torr, P = 0.02). This strategy for targeting EPCs to vulnerable peri-infarct myocardium via the potent chemokine SDF-1α significantly decreased the degree of hypoxia in peri-infarct myocardium as measured in vivo by phosphorescence quenching. This effect could potentially mitigate the vicious cycle of myocyte death, myocardial fibrosis, progressive ventricular dilatation, and eventual heart failure seen after acute myocardial infarction.

Cardiovascular disease in young women: a population at risk.

Ischemic heart disease (IHD) is a leading cause of morbidity in the United States and worldwide. In women, it is the leading cause of death in all age groups except young women who rarely have clinically evident disease. However, when young women less than age 50 develop IHD, they are at high risk for mortality. This may be due in part to delay in diagnosis or less aggressive treatment. Young women may be less aggressively treated with medical therapies and percutaneous or surgical interventions despite studies that have shown benefit in women as well as men. Young women are an especially important population to target for treatment and study since prevention of IHD during this stage of life can have great personal and societal health consequences. Epidemiological studies, including the INTERHEART study, have identified risk factors including hypertension, diabetes, metabolic syndrome, smoking, and sedentary lifestyle that explain much of IHD in women. Several factors, including diabetes, metabolic syndrome, and tobacco use, are stronger predictors of IHD in young women as compared with older women. Healthcare practitioners who encounter young women should aggressively treat risk factors, maintain an appropriate index of suspicion for IHD, and treat acute coronary syndromes promptly and intensively to reduce the burden of IHD in young women.

Acute myocardial rescue with endogenous endothelial progenitor cell therapy.

PURPOSE: Post-myocardial infarction heart failure is a major health concern with limited therapy. Molecular revascularisation utilising granulocyte-macrophage colony stimulating factor (GMCSF) mediated endothelial progenitor cell (EPC) upregulation and stromal cell derived factor-1α (SDF) mediated myocardial EPC chemokinesis, may prevent myocardial loss and adverse remodelling. Vasculogenesis, viability, and haemodynamic improvements following therapy were investigated. PROCEDURES: Lewis rats (n=91) underwent LAD ligation and received either intramyocardial SDF and subcutaneous GMCSF or saline injections at the time of infarction. Molecular and haemodynamic assessments were performed at pre-determined time points following ligation. FINDINGS: SDF/GMCSF therapy upregulated EPC density as shown by flow cytometry (0.12±0.02% vs. 0.06±0.01% circulating lymphocytes, p=0.005), 48hours following infarction. A marked increase in perfusion was evident eight weeks after therapy, utilising confocal angiography (5.02±1.7×10(-2)µm(3)blood/µm(3)myocardial tissue vs. 2.03±0.710(-2)µm(3)blood/µm(3)myocardial tissue, p=0.00004). Planimetric analysis demonstrated preservation of wall thickness (0.98±0.09mm vs. 0.67±0.06mm, p=0.003) and ventricular diameter (7.81±0.99mm vs. 9.41±1.1mm, p=0.03). Improved haemodynamic function was evidenced by echocardiography and PV analysis (ejection fraction: 56.4±18.1% vs. 25.3±15.6%, p=0.001; pre-load adjusted maximal power: 6.6±2.6mW/µl(2) vs. 2.7±1.4mW/µl(2), p=0.01). CONCLUSION: Neovasculogenic therapy with GMCSF-mediated EPC upregulation and SDF-mediated EPC chemokinesis maybe an effective therapy for infarct modulation and preservation of myocardial function following acute myocardial infarction.