Intramyocardial injection of hypoxia-conditioned extracellular vesicles increases myocardial perfusion in a swine model of chronic coronary disease.

Objective: Coronary artery disease remains a leading cause of morbidity and mortality worldwide. Patients with advanced coronary artery disease who are not eligible for endovascular or surgical revascularization have limited options. Extracellular vesicles have shown potential to improve myocardial function in preclinical models. Extracellular vesicles can be conditioned to modify their components. Hypoxia-conditioned extracellular vesicles have demonstrated the ability to reduce infarct size and apoptosis in small animals. Our objective is to assess the potential benefits of hypoxia-conditioned extracellular vesicles in a large animal model of coronary artery disease. Methods: Coronary artery disease was induced in 14 Yorkshire swine by ameroid constriction of the left circumflex coronary artery. Two weeks postsurgery, swine underwent a repeat left thoracotomy for injections of hypoxia-conditioned extracellular vesicles (n = 7) or saline (control, n = 7). Five weeks later, all animals underwent terminal harvest for perfusion measurements and myocardial sectioning. Results: Myocardial perfusion analysis demonstrated a trend toward increase at rest and a significant increase during rapid pacing (P = .09, P < .001). There were significant increases in activated phosphorylated endothelial nitric oxide synthase, endothelial nitric oxide synthase, phosphatidylinositol 3-kinase, phosphorylated protein kinase B, and the phosphorylated protein kinase B/protein kinase B ratio in the hypoxia-conditioned extracellular vesicles group compared with the control group (all P < .05). Additionally, there was a significant decrease in the antiangiogenic proteins collagen 18 and angiostatin (P = .01, P = .01) in the hypoxia-conditioned extracellular vesicles group. Conclusions: Intramyocardial injection of hypoxia-conditioned extracellular vesicles results in increased myocardial perfusion without a corresponding change in vessel density. Therefore, this improvement in perfusion is possibly due to changes in nitric oxide signaling. Hypoxia-conditioned extracellular vesicles represent a potential therapeutic strategy to increase myocardial perfusion in patients with advanced coronary artery disease.

Semaglutide Improves Myocardial Perfusion and Performance in a Large Animal Model of Coronary Artery Disease.

Objective: Coronary artery disease (CAD) is the leading cause of death worldwide. It imposes an enormous symptomatic burden on patients, leaving many with residual disease despite optimal procedural therapy, and up to 1/3 with debilitating angina amenable neither to procedures, nor to current pharmacologic options. Semaglutide, a glucagon-like peptide 1 agonist originally approved for management of diabetes, has garnered substantial attention for its capacity to attenuate cardiovascular risk. Although subgroup analyses in patients indicate promise, studies explicitly designed to isolate the impact of semaglutide on the sequelae of CAD, independently of comorbid diabetes or obesity, are lacking. Approach and Results: Yorkshire swine (n=17) underwent placement of an ameroid constrictor around the left circumflex coronary artery to induce CAD. Oral semaglutide was initiated postoperatively at 1.5 mg and scaled up in 2 weeks to 3 mg in treatment animals (SEM, n=8) for a total of 5 weeks, while control animals (CON, n=9) received no drug. All then underwent myocardial harvest with acquisition of perfusion and functional data using microsphere injection and pressure-volume loop catheterization. Immunoblotting, immunohistochemistry, and immunofluorescence were performed on the most ischemic myocardial segments for mechanistic elucidation. SEM animals exhibited improved left ventricular ejection fraction, both at rest and during rapid myocardial pacing (both p<0.03), accompanied by increased perfusion to the most ischemic myocardial region at rest and during rapid pacing (both p<0.03); reduced perivascular and interstitial fibrosis (both p <0.03); and apoptosis (p=0.008). These changes were associated with increased activation of the endothelial-protective AMPK pathway (p=0.005), coupled with downstream increases in endothelial nitric oxide synthase (p=0.014). Conclusion: This study is the first to reveal the capacity of oral semaglutide to augment cardiac function in the chronically ischemic heart in a highly translational large animal model, likely through AMPK-mediated improvement in endothelial function and perfusion to the ischemic myocardium.

Metformin Preconditioning Augments Cardiac Perfusion and Performance in a Large Animal Model of Chronic Coronary Artery Disease.

OBJECTIVE: To test the efficacy of metformin (MET) during the induction of coronary ischemia on myocardial performance in a large animal model of coronary artery disease (CAD) and metabolic syndrome (MS), with or without concomitant extracellular vesicular (EV) therapy. BACKGROUND: Although surgical and endovascular revascularization are durably efficacious for many patients with CAD, up to one-third are poor candidates for standard therapies. For these patients, many of whom have comorbid MS, adjunctive strategies are needed. EV therapy has shown promise in this context, but its efficacy is attenuated by MS. We investigated whether MET pretreatment could ameliorate therapeutic decrements associated with MS. METHODS: Yorkshire swine (n = 29) were provided a high-fat diet to induce MS, whereupon an ameroid constrictor was placed to induce CAD. Animals were initiated on 1000 mg oral MET or placebo; all then underwent repeat thoracotomy for intramyocardial injection of EVs or saline. Swine were maintained for 5 weeks before the acquisition of functional and perfusion data immediately before terminal myocardial harvest. Immunoblotting and immunofluorescence were performed on the most ischemic tissue from all groups. RESULTS: Regardless of EV administration, animals that received MET exhibited significantly improved ejection fraction, cardiac index, and contractility at rest and during rapid myocardial pacing, improved perfusion to the most ischemic myocardial region at rest and during pacing, and markedly reduced apoptosis. CONCLUSIONS: MET administration reduced apoptotic cell death, improved perfusion, and augmented both intrinsic and load-dependent myocardial performance in a highly translatable large animal model of chronic myocardial ischemia and MS.

Effects of diet-induced metabolic syndrome on cardiac function and angiogenesis in response to the sodium-glucose cotransporter-2 inhibitor canagliflozin.

INTRODUCTION: Sodium-glucose cotransporter-2 inhibitors are antidiabetic medications that have been shown to decrease cardiovascular events and heart failure-related mortality in clinical studies. We attempt to examine the complex interplay between metabolic syndrome and the sodium-glucose cotransporter-2 inhibitor canagliflozin (CAN) in a clinically relevant model of chronic myocardial ischemia. METHODS: Twenty-one Yorkshire swine were fed a high-fat diet starting at 6 weeks of age to induce metabolic syndrome. At 11 weeks, all underwent placement of an ameroid constrictor around the left circumflex coronary artery to induce chronic myocardial ischemia. After 2 weeks, swine received either control (CON) (n = 11) or CAN 300 mg by mouth daily (n = 10) for 5 weeks, whereupon all underwent terminal harvest. RESULTS: There was a significant increase in cardiac output and heart rate with a decrease in pulse pressure in the CAN group compared with CON (all P values < .05). The CAN group had a significant increase in capillary density (P = .02). There was no change in myocardial perfusion or arteriolar density. CAN induced a significant increase in markers of angiogenesis, including Phospho-endothelial nitric oxide synthase, Endothelial nitric oxide synthase, vascular endothelial growth factor receptor-1, heat shock protein 70, and extracellular signal-regulated kinases (all P values < .05), plausibly resulting in capillary angiogenesis. CONCLUSIONS: CAN treatment leads to a significant increase in capillary density and augmented cardiac function in a swine model of chronic myocardial ischemia in the setting of metabolic syndrome. This work further elucidates the mechanism of sodium-glucose cotransporter-2 inhibitors in patients with cardiac disease; however, more studies are needed to determine if this increase in capillary density plays a role in the improvements seen in clinical studies.

Proteomic Profiling of SGLT-2 Inhibitor Canagliflozin in a Swine Model of Chronic Myocardial Ischemia.

BACKGROUND: Sodium-glucose cotransporter-2 (SGLT2) inhibitors are known to be cardioprotective independent of glucose control, but the mechanisms of these benefits are unclear. We previously demonstrated improved cardiac function and decreased fibrosis in a swine model of chronic myocardial ischemia. The goal of this study is to use high-sensitivity proteomic analyses to characterize specific molecular pathways affected by SGLT-2 inhibitor canagliflozin (CAN) therapy in a swine model of chronic myocardial ischemia. METHODS: Chronic myocardial ischemia was induced in sixteen Yorkshire swine via the placement of an ameroid constrictor to the left circumflex coronary artery. After two weeks of recovery, swine received either 300 mg of CAN daily (n = 8) or a control (n = 8). After five weeks of therapy, the group of swine were euthanized, and left ventricular tissue was harvested and sent for proteomic analysis. RESULTS: Total proteomic analysis identified a total of 3256 proteins between the CAN and control groups. Three hundred and five proteins were statistically different. This included 55 proteins that were downregulated (p < 0.05, fold change <0.5) and 250 that were upregulated (p < 0.05, fold change >2) with CAN treatment. Pathway analysis demonstrated the upregulation of several proteins involved in metabolism and redox activity in the CAN-treated group. The CAN group also exhibited a downregulation of proteins involved in motor activity and cytoskeletal structure. CONCLUSIONS: In our swine model of chronic myocardial ischemia, CAN therapy alters several proteins involved in critical molecular pathways, including redox regulation and metabolism. These findings provide additional mechanistic insights into the cardioprotective effects of canagliflozin.

Intramyocardial Injection of Hypoxia-Conditioned Extracellular Vesicles Modulates Response to Oxidative Stress in the Chronically Ischemic Myocardium.

INTRODUCTION: Patients with advanced coronary artery disease (CAD) who are not eligible for stenting or surgical bypass procedures have limited treatment options. Extracellular vesicles (EVs) have emerged as a potential therapeutic target for the treatment of advanced CAD. These EVs can be conditioned to modify their contents. In our previous research, we demonstrated increased perfusion, decreased inflammation, and reduced apoptosis with intramyocardial injection of hypoxia-conditioned EVs (HEVs). The goal of this study is to further understand the function of HEVs by examining their impact on oxidative stress using our clinically relevant and extensively validated swine model of chronic myocardial ischemia. METHODS: Fourteen Yorkshire swine underwent a left thoracotomy for the placement of an ameroid constrictor on the left circumflex coronary artery to model chronic myocardial ischemia. After two weeks of recovery, the swine underwent a redo thoracotomy with injection of either HEVs (n = 7) or a saline control (CON, n = 7) into the ischemic myocardium. Five weeks after injection, the swine were subjected to terminal harvest. Protein expression was measured using immunoblotting. OxyBlot analysis and 3-nitrotyrosine staining were used to quantify total oxidative stress. RESULTS: There was a significant increase in myocardial expression of the antioxidants SOD 2, GPX-1, HSF-1, UCP-2, catalase, and HO-1 (all p ≤ 0.05) in the HEV group when compared to control animals. The HEVs also exhibited a significant increase in pro-oxidant NADPH oxidase (NOX) 1, NOX 3, p47phox, and p67phox (all p ≤ 0.05). However, no change was observed in the expression of NFkB, KEAP 1, and PRDX1 (all p > 0.05) between the HEV and CON groups. There were no significant differences in total oxidative stress as determined by OxyBlot and 3-nitrotyrosine staining (p = 0.64, p = 0.32) between the groups. CONCLUSIONS: Administration of HEVs in ischemic myocardium induces a significant increase in pro- and antioxidant proteins without a net change in total oxidative stress. These findings suggest that HEV-induced changes in redox signaling pathways may play a role in increased perfusion, decreased inflammation, and reduced apoptosis in ischemic myocardium. Further studies are required to determine if HEVs alter the net oxidative stress in ischemic myocardium at an earlier time point of HEV administration.

Crafting a Rigorous, Clinically Relevant Large Animal Model of Chronic Myocardial Ischemia: What Have We Learned in 20 Years?

The past several decades have borne witness to several breakthroughs and paradigm shifts within the field of cardiovascular medicine, but one component that has remained constant throughout this time is the need for accurate animal models for the refinement and elaboration of the hypotheses and therapies crucial to our capacity to combat human disease. Numerous sophisticated and high-throughput molecular strategies have emerged, including rational drug design and the multi-omics approaches that allow extensive characterization of the host response to disease states and their prospective resolutions, but these technologies all require grounding within a faithful representation of their clinical context. Over this period, our lab has exhaustively tested, progressively refined, and extensively contributed to cardiovascular discovery on the basis of one such faithful representation. It is the purpose of this paper to review our porcine model of chronic myocardial ischemia using ameroid constriction and the subsequent myriad of physiological and molecular-biological insights it has allowed our lab to attain and describe. We hope that, by depicting our methods and the insight they have yielded clearly and completely-drawing for this purpose on comprehensive videographic illustration-other research teams will be empowered to carry our work forward, drawing on our experience to refine their own investigations into the pathogenesis and eradication of cardiovascular disease.