Cynthia Marie Xu

Our novel lab-grown, human 3-D extracellular matrix (ECM) represents a novel therapeutic approach to prevent pathological remodeling and heart failure in an animal model of heart attack. This novel finding may help develop nonsurgical therapeutic modalities aimed at reducing the global burden of cardiovascular disease.

Small animal models have shown improved cardiac function with DPP‐4 inhibition, but many human studies have shown worse outcomes or no benefit. We seek to bridge the gap by studying the DPP‐4 inhibitor sitagliptin in a swine model of chronic myocardial ischemia using proteomic analysis. Thirteen Yorkshire swine underwent the placement of an ameroid constrictor on the left coronary circumflex artery to model chronic myocardial ischemia. Two weeks post‐op, swine received either sitagliptin 100 mg daily (SIT, n = 5) or no drug (CON, n = 8). After 5 weeks of treatment, swine underwent functional measurements and tissue harvest. In the SIT group compared to CON, there was a trend towards decreased cardiac index (p = 0.06). The non‐ischemic and ischemic myocardium had 396 and 166 significantly decreased proteins, respectively, in the SIT group compared to CON (all p < 0.01). This included proteins involved in fatty acid oxidation (FAO), myocardial contraction, and oxidative phosphorylation (OXPHOS). Sitagliptin treatment resulted in a trend towards decreased cardiac index and decreased expression of proteins involved in OXPHOS, FAO, and myocardial contraction in both ischemic and non‐ischemic swine myocardium. These metabolic and functional changes may provide some mechanistic evidence for outcomes seen in clinical studies.

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 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.

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.

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.

Human bone mesenchymal stem cell‐derived extracellular vesicles (HBMSC‐EV) have been used successfully in animal models of myocardial ischemia, yet have dampened effects in metabolic syndrome through unknown mechanisms. This study demonstrates the basal differences between non‐diabetic human coronary artery endothelial cells (HCAEC) and diabetic HCAEC (DM‐HCAEC), and how these cells respond to the treatment of HBMSC‐EV. HCAEC and DM‐HCAEC were treated with HBMSC‐EV for 6 h. Proteomics, western blot analysis, and tube formation assays were performed. Key metabolic, growth, and stress/starvation cellular responses were significantly altered in DM‐HCAEC in comparison to that of HCAEC at baseline. Proteomics demonstrated increased phosphorus metabolic process and immune pathways and decreased RNA processing and biosynthetic pathways in DM‐HCAEC. Similar to previous in vivo findings, HCAEC responded to the HBMSC‐EV with regenerative and anti‐inflammatory effects through the upregulation of multiple RNA pathways and downregulation of immune cell activation pathways. In contrast, DM‐HCAEC had a significantly diminished response to HBMSC‐EV, likely due to the baseline abnormalities in DM‐HCAEC. To achieve the full benefits of HBMSC‐EV and for a successful transition of this potential therapeutic agent to clinical studies, the abnormalities found in DM‐HCAEC will need to be further studied.

Dipeptidyl peptidase 4 inhibitors (DPP4i) may be cardioprotective based on several small animal and clinical studies, though randomized control trials have demonstrated limited benefit. Given these discrepant findings, the role of these agents in chronic myocardial disease, particularly in the absence of diabetes, is still poorly understood. The purpose of this study was to determine the effects of sitagliptin, a DPP4i, on myocardial perfusion and microvessel density in a clinically relevant large animal model of chronic myocardial ischemia. Normoglycemic Yorkshire swine underwent ameroid constrictor placement to the left circumflex artery to induce chronic myocardial ischemia. Two weeks later, pigs received either no drug (CON, n = 8) or 100 mg oral sitagliptin (SIT) daily (n = 5). Treatment continued for 5 weeks, followed by hemodynamic measurements, euthanasia, and tissue harvest of ischemic myocardium. There were no significant differences in myocardial function between CON and SIT as measured by stroke work (p > 0.5), cardiac output (p = 0.22), and end‐systolic elastance (p = 0.17). SIT was associated with increased absolute blood flow at rest (17% increase, IQR 12–62, p = 0.045) and during pacing (89% increase, IQR 83–105, p = 0.002). SIT was also associated with improved arteriolar density (p = 0.045) compared with CON, without changes in capillary density (p = 0.72). SIT was associated with increased expression of pro‐arteriogenic markers MCP‐1 (p = 0.003), TGFß (p = 0.03), FGFR1 (p = 0.002), and ICAM‐1 (p = 0.03), with a trend toward an increase in the ratio of phosphorylated/active PLCγ1 to total PLCγ1 (p = 0.11) compared with CON. In conclusion, in chronically ischemic myocardium, sitagliptin improves myocardial perfusion and arteriolar collateralization via the activation of pro‐arteriogenic signaling pathways.

Despite the overwhelming use of cellularized therapeutics in cardiac regenerative engineering, approaches to biomanufacture engineered cardiac tissues (ECTs) at clinical scale remain limited. This study aims to evaluate the impact of critical biomanufacturing decisions—namely cell dose, hydrogel composition, and size-on ECT formation and function—through the lens of clinical translation. ECTs were fabricated by mixing human induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs) and human cardiac fibroblasts into a collagen hydrogel to engineer meso-(3 × 9 mm), macro- (8 × 12 mm), and mega-ECTs (65 × 75 mm). Meso-ECTs exhibited a hiPSC-CM dose-dependent response in structure and mechanics, with high-density ECTs displaying reduced elastic modulus, collagen organization, prestrain development, and active stress generation. Scaling up, cell-dense macro-ECTs were able to follow point stimulation pacing without arrhythmogenesis. Finally, we successfully fabricated a mega-ECT at clinical scale containing 1 billion hiPSC-CMs for implantation in a swine model of chronic myocardial ischemia to demonstrate the technical feasibility of biomanufacturing, surgical implantation, and engraftment. Through this iterative process, we define the impact of manufacturing variables on ECT formation and function as well as identify challenges that must still be overcome to successfully accelerate ECT clinical translation.

In animal models, human bone marrow mesenchymal stem cell‐derived extracellular vesicles (MSC‐EV) have been found to have beneficial effects in cardiovascular disease, but only when administered via intramyocardial injection. The biodistribution of either intravenous or intramyocardial injection of MSC‐EV in the presence of myocardial injury is uncharacterized at this time. We hypothesized that intramyocardial injection will ensure delivery of MSC‐EV to the ischemic myocardium, while intravenous injection will not. Human bone marrow mesenchymal stem cells were cultured and the MSC‐EV were isolated and characterized. The MSC‐EVs were then labeled with DiD lipid dye. FVB mice with normal cardiac function underwent left coronary artery ligation followed by either peri‐infarct intramyocardial or tail vein injection of 3*10⁶ or 2*10⁹ particles of DiD‐labeled MSC‐EV or a DiD‐saline control. The heart, lungs, liver, spleen and kidneys were harvested 2 h post‐injection and were submitted for fluorescent molecular tomography imaging. Myocardial uptake of MSC‐EV was only visualized after intramyocardial injection of 2*10⁹ MSC‐EV particles (p = 0.01) compared to control, and there were no differences in cardiac fluorescence after tail vein injection of MSC‐EV (p = 0.5). There was no significantly detectable MSC‐EV uptake in other organs after intramyocardial injection. After tail vein injection of 2*10⁹ particles of MSC‐EV, the liver (p = 0.02) and spleen (p = 0.04) appeared to have diffuse MSC‐EV uptake compared to controls. Even in the presence of myocardial injury, only intramyocardial but not intravenous administration resulted in detectable levels of MSC‐EV in the ischemic myocardium. This study confirms the role for intramyocardial injection in maximal and effective delivery of MSC‐EV. Our ongoing studies aimed at developing bioengineered MSC‐EV for targeted delivery to the heart may render MSC‐EV clinically applicable for cardiovascular disease.