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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.
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Inibidores da Dipeptidil Peptidase IV , Isquemia Miocárdica , Suínos , Humanos , Animais , Inibidores da Dipeptidil Peptidase IV/uso terapêutico , Proteoma/metabolismo , Fosforilação Oxidativa , Fosfato de Sitagliptina/uso terapêutico , Proteômica/métodos , Miocárdio/metabolismo , Hipoglicemiantes/uso terapêutico , Modelos Animais de DoençasRESUMO
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.
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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.
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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.
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BACKGROUND: Although sodium-glucose cotransporter-2 inhibitors have been shown to improve cardiovascular outcomes in general, little is presently known about any sex-specific changes that may result from this therapy. We sought to investigate and quantify potential sex-specific changes seen with the sodium-glucose cotransporter-2 inhibitor canagliflozin (CAN) in a swine model of chronic myocardial ischemia. STUDY DESIGN: Eighteen Yorkshire swine underwent left thoracotomy with placement of an ameroid constrictor. Two weeks postop, swine were assigned to receive either control (F = 5 and M = 5) or CAN 300 mg daily (F = 4 and M = 4). After 5 weeks of therapy, swine underwent myocardial functional measurements, and myocardial tissue was sent for proteomic analysis. RESULTS: Functional measurements showed increased cardiac output, stroke volume, ejection fraction, and ischemic myocardial flow at rest in male swine treated with CAN compared with control male swine (all p < 0.05). The female swine treated with CAN had no change in cardiac function as compared with control female swine. Proteomic analysis demonstrated 6 upregulated and 97 downregulated proteins in the CAN female group compared with the control female group. Pathway analysis showed decreases in proteins in the tricarboxylic acidic cycle. The CAN male group had 639 upregulated and 172 downregulated proteins compared with control male group. Pathway analysis showed increases in pathways related to cellular metabolism and decreases in pathways relevant to the development of cardiomyopathy and to oxidative phosphorylation. CONCLUSIONS: Male swine treated with CAN had significant improvements in cardiac function that were not observed in female swine treated with CAN. Moreover, CAN treatment in male swine was associated with significantly more changes in protein expression than in female swine treated with CAN. The increased proteomic changes seen in the CAN male group likely contributed to the more robust changes in cardiac function seen in male swine treated with CAN.
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Canagliflozina , Isquemia Miocárdica , Proteômica , Inibidores do Transportador 2 de Sódio-Glicose , Animais , Feminino , Masculino , Canagliflozina/farmacologia , Canagliflozina/uso terapêutico , Doença Crônica , Modelos Animais de Doenças , Isquemia Miocárdica/metabolismo , Miocárdio/metabolismo , Fatores Sexuais , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêutico , SuínosRESUMO
BACKGROUND: Inflammation and disruption of cardiac metabolism are prevalent in the setting of myocardial ischemia. Canagliflozin, a sodium-glucose costransporter-2 inhibitor, has beneficial effects on the heart, though the precise mechanisms are unknown. This study investigated the effects of canagliflozin therapy on metabolic pathways and inflammation in ischemic myocardial tissue using a swine model of chronic myocardial ischemia. METHODS: Sixteen Yorkshire swine underwent placement of an ameroid constrictor to the left circumflex artery to induce chronic ischemia. Two weeks later, pigs received either no drug (n = 8) or 300 mg canagliflozin (n = 8) daily. Five weeks later, pigs underwent terminal harvest and tissue collection. RESULTS: Canagliflozin treatment was associated with a trend toward decreased expression of fatty acid oxidation inhibitor acetyl-CoA carboxylase and decreased phosphorylated/inactivated acetyl-CoA carboxylase, a promotor of fatty acid oxidation, compared with control ischemic myocardium (P = .08, P = .03). There was also a significant modulation in insulin resistance markers p-IRS1, p-PKCα, and phosphoinositide 3-kinase in ischemic myocardium of the canagliflozin group compared with the control group (all P < .05). Canagliflozin treatment was associated with a significant increase in inflammatory markers interleukin 6, interleukin 17, interferon-gamma, and inducible nitric oxide synthase (all P < .05). There was a trend toward decreased expression of the anti-inflammatory cytokines interleukin 10 (P = .16) and interleukin 4 (P = .31) with canagliflozin treatment. CONCLUSION: The beneficial effects of canagliflozin therapy appear to be associated with inhibition of fatty acid oxidation and enhancement of insulin signaling in ischemic myocardium. Interestingly, canagliflozin appears to increase the levels of several inflammatory markers, but further studies are required to better understand how canagliflozin modulates inflammatory signaling pathways.
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Isquemia Miocárdica , Inibidores do Transportador 2 de Sódio-Glicose , Simportadores , Suínos , Animais , Canagliflozina/farmacologia , Canagliflozina/uso terapêutico , Canagliflozina/metabolismo , Miocárdio/metabolismo , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêutico , Inibidores do Transportador 2 de Sódio-Glicose/metabolismo , Acetil-CoA Carboxilase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 3-Quinases/uso terapêutico , Isquemia Miocárdica/tratamento farmacológico , Isquemia Miocárdica/complicações , Isquemia Miocárdica/metabolismo , Inflamação/metabolismo , Glucose/metabolismo , Simportadores/metabolismo , Ácidos Graxos/metabolismo , Modelos Animais de DoençasRESUMO
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.
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Diabetes Mellitus , Vesículas Extracelulares , Células-Tronco Mesenquimais , Animais , Humanos , Vasos Coronários/metabolismo , Células Endoteliais/metabolismo , Células Cultivadas , Vesículas Extracelulares/metabolismo , Células-Tronco Mesenquimais/metabolismo , Diabetes Mellitus/metabolismoRESUMO
Objective: Limited treatments exist for nonoperative chronic coronary artery disease. Previously, our laboratory has investigated extracellular vesicle (EV) therapy as a potential treatment for chronic coronary artery disease using a swine model and demonstrated improved cardiac function in swine treated with intramyocardial EV injection. Here, we seek to investigate the potential cardiac benefits of EVs by using hypoxia-conditioned EVs (HEV). Specifically, this study aims to investigate the effect of HEV on apoptosis in chronically ischemic myocardium in swine. Methods: Fourteen Yorkshire swine underwent placement of an ameroid constrictor on the left circumflex artery. Two weeks later, swine underwent redo left thoracotomy with injection of either saline (control, n = 7) or HEVs (n = 7). After 5 weeks, swine were euthanized for tissue collection. Terminal deoxynucleotidyl transferase dUTP nick end labeling was used to quantify apoptosis. Immunoblotting was used for protein quantification. Results: Terminal deoxynucleotidyl transferase dUTP nick end labeling staining showed a decrease in apoptosis in the HEV group compared with the control (P = .049). The HEV group exhibited a significant increase in the anti-apoptotic signaling molecule phospho-BAD (P = .005), a significant decrease in B-cell lymphoma 2 (P = .006) and an increase in the phospho-B-cell lymphoma to B-cell lymphoma 2 ratio (P < .001). Furthermore, the HEV group exhibited increased levels of prosurvival signaling markers including phosphoinositide 3-kinase, phosphor-extracellular signal-regulated kinase 1/2, phospho-forkhead box protein O1, and phospho-protein kinase B to protein kinase B ratio (all P < .05). Conclusions: In chronic myocardial ischemia, treatment with HEV results in a decrease in overall apoptosis, possibly through the activation of both pro-survival and anti-apoptotic signaling pathways.
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OBJECTIVES: Sodium-glucose cotransporter-2 inhibitor, canagliflozin, improves myocardial perfusion to ischemic territory without accompanying changes in vascular density. We aimed to (1) characterize effects on angiogenic pathways, (2) use multiomics to identify gene expression and metabolite profiles relevant to regulation of myocardial blood flow, and (3) investigate drug effect on coronary microvascular reactivity. METHODS: A nondiabetic swine model of chronic myocardial ischemia and nondiabetic rat model were used to study functional and molecular effects of canagliflozin on myocardium and in vitro microvascular reactivity. RESULTS: Canagliflozin resulted in increased coronary microvascular vasodilation and decreased vasoconstriction (P < .05) without changes in microvascular density (P > .3). Expression of the angiogenic modulator, endostatin, increased (P = .008), along with its precursor, collagen 18 (P < .001), and factors that increase its production, including cathepsin L (P = .004). Endostatin and collagen 18 levels trended toward an inverse correlation with blood flow to ischemic territory at rest. Proangiogenic fibroblast growth factor receptor was increased (P = .03) and matrix metalloproteinase-9 was decreased (P < .001) with canagliflozin treatment. Proangiogenic vascular endothelial growth factor A (P = .13), Tie-2 (P = .10), and Ras (P = .18) were not significantly altered. Gene expression related to the cardiac renin-angiotensin system was significantly decreased. CONCLUSIONS: In chronic myocardial ischemia, canagliflozin increased absolute blood flow to the myocardium without robustly increasing vascular density or proangiogenic signaling. Canagliflozin resulted in altered coronary microvascular reactivity to favor vasodilation, likely through direct effect on vascular smooth muscle. Downregulation of cardiac renin-angiotensin system demonstrated local regulation of perfusion. VIDEO ABSTRACT.
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Isquemia Miocárdica , Inibidores do Transportador 2 de Sódio-Glicose , Suínos , Animais , Ratos , Vasodilatação , Canagliflozina/farmacologia , Canagliflozina/metabolismo , Canagliflozina/uso terapêutico , Fator A de Crescimento do Endotélio Vascular/farmacologia , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêutico , Endostatinas/metabolismo , Endostatinas/farmacologia , Endostatinas/uso terapêutico , Miocárdio/metabolismoRESUMO
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.
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Inibidores da Dipeptidil Peptidase IV , Isquemia Miocárdica , Suínos , Animais , Fosfato de Sitagliptina/farmacologia , Fosfato de Sitagliptina/uso terapêutico , Projetos Piloto , Circulação Coronária/fisiologia , Neovascularização Fisiológica , Isquemia Miocárdica/complicações , Miocárdio/metabolismo , Inibidores da Dipeptidil Peptidase IV/farmacologia , Inibidores da Dipeptidil Peptidase IV/uso terapêutico , Perfusão , Modelos Animais de DoençasRESUMO
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.
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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.
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Vesículas Extracelulares , Células-Tronco Mesenquimais , Infarto do Miocárdio , Camundongos , Animais , Humanos , Injeções Intravenosas , Distribuição Tecidual , Vesículas Extracelulares/metabolismo , Modelos Animais de DoençasRESUMO
We have previously shown that normoxia serum-starved extracellular vesicle (EV) therapy improves myocardial function, perfusion, and angiogenesis in a swine model of chronic myocardial ischemia. Hypoxia-modified EVs have increased abundance of anti-oxidant, pro-angiogenic, and pro-survival proteins. The purpose of this study is to investigate the differential effects of normoxia serum-starved EVs and hypoxia-modified EVs on myocardial function, perfusion, and microvascular density in chronically ischemic myocardium. Yorkshire swine underwent placement of an ameroid constrictor to the left circumflex artery to induce chronic myocardial ischemia. Two weeks later, the pigs underwent intramyocardial injection of either normoxia serum-starved EVs (NOR, n = 10) or hypoxia-modified EVs (HYP, n = 7). Five weeks later, pigs were euthanized, and ischemic myocardium was harvested. Hypoxia EV treatment was associated with improved contractility compared to NOR, as well as improved capillary density, without changes in arteriolar density. There were trends towards improved perfusion at rest and during pacing in the HYP group compared to NOR. Ischemic myocardium in the HYP group had increased pro-angiogenic Akt and ERK signaling and decreased expression of anti-angiogenic markers compared to the NOR group. In the setting of chronic myocardial ischemia, hypoxia-modified EVs may enhance contractility, capillary density, and angiogenic signaling pathways compared to normoxia serum-starved EVs.
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Vesículas Extracelulares , Isquemia Miocárdica , Suínos , Animais , Neovascularização Fisiológica , Circulação Coronária , Isquemia Miocárdica/metabolismo , Miocárdio/metabolismo , Hipóxia/metabolismo , Perfusão , Vesículas Extracelulares/metabolismo , Modelos Animais de DoençasRESUMO
Human bone marrow mesenchymal stem cell derived-extracellular vesicles (HBMSC-EV) are known for their regenerative and anti-inflammatory effects in animal models of myocardial ischemia. However, it is not known whether the efficacy of the EVs can be modulated by pre-conditioning of HBMSC by exposing them to either starvation or hypoxia prior to EV collection. HBMSC-EVs were isolated following normoxia starvation (NS), normoxia non-starvation (NNS), hypoxia starvation (HS), or hypoxia non-starvation (HNS) pre-conditioning. The HBMSC-EVs were characterized by nanoparticle tracking analysis, electron microscopy, Western blot, and proteomic analysis. Comparative proteomic profiling revealed that starvation pre-conditioning led to a smaller variety of proteins expressed, with the associated lesser effect of normoxia versus hypoxia pre-conditioning. In the absence of starvation, normoxia and hypoxia pre-conditioning led to disparate HBMSC-EV proteomic profiles. HNS HBMSC-EV was found to have the greatest variety of proteins overall, with 74 unique proteins, the greatest number of redox proteins, and pathway analysis suggestive of improved angiogenic properties. Future HBMSC-EV studies in the treatment of cardiovascular disease may achieve the most therapeutic benefits from hypoxia non-starved pre-conditioned HBMSC. This study was limited by the lack of functional and animal models of cardiovascular disease and transcriptomic studies.
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Doenças Cardiovasculares , Vesículas Extracelulares , Células-Tronco Mesenquimais , Animais , Humanos , Doenças Cardiovasculares/metabolismo , Proteômica , Vesículas Extracelulares/metabolismo , Hipóxia/metabolismo , Células-Tronco Mesenquimais/metabolismoRESUMO
Background Sodium-glucose cotransporter-2 inhibitors are cardioprotective independent of glucose control, as demonstrated in animal models of acute myocardial ischemia and clinical trials. The functional and molecular mechanisms of these benefits in the setting of chronic myocardial ischemia are poorly defined. The purpose of this study is to determine the effects of canagliflozin therapy on myocardial perfusion, fibrosis, and function in a large animal model of chronic myocardial ischemia. Methods and Results Yorkshire swine underwent placement of an ameroid constrictor to the left circumflex artery to induce chronic myocardial ischemia. Two weeks later, pigs received either no drug (n=8) or 300 mg sodium-glucose cotransporter-2 inhibitor canagliflozin orally, daily (n=8). Treatment continued for 5 weeks, followed by hemodynamic measurements, harvest, and tissue analysis. Canagliflozin therapy was associated with increased stroke volume and stroke work and decreased left ventricular stiffness compared with controls. The canagliflozin group had improved perfusion to ischemic myocardium compared with controls, without differences in arteriolar or capillary density. Canagliflozin was associated with decreased interstitial and perivascular fibrosis in chronically ischemic tissue, with reduced Jak/STAT (Janus kinase/signal transducer and activator of transcription) signaling compared with controls. In ischemic myocardium of the canagliflozin group, there was increased expression and activation of adenosine monophosphate-activated protein kinase, decreased activation of endothelial nitric oxide synthase, and unchanged total endothelial nitric oxide synthase. Canagliflozin therapy reduced total protein oxidation and increased expression of mitochondrial antioxidant superoxide dismutase 2 compared with controls. Conclusions In the setting of chronic myocardial ischemia, canagliflozin therapy improves myocardial function and perfusion to ischemic territory, without changes in collateralization. Attenuation of fibrosis via reduced Jak/STAT signaling, activation of adenosine monophosphate-activated protein kinase, and antioxidant signaling may contribute to these effects.
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Isquemia Miocárdica , Inibidores do Transportador 2 de Sódio-Glicose , Animais , Antioxidantes/farmacologia , Canagliflozina/farmacologia , Canagliflozina/uso terapêutico , Circulação Coronária , Modelos Animais de Doenças , Fibrose , Isquemia Miocárdica/complicações , Óxido Nítrico Sintase Tipo III , Perfusão , Proteínas Quinases , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêutico , SuínosRESUMO
OBJECTIVE: Extracellular vesicle (EV) therapy has been shown to mitigate inflammation in animal models of acute myocardial ischemia/reperfusion. This study evaluates the effect of EV therapy on inflammatory signaling in a porcine model of chronic myocardial ischemia and metabolic syndrome. METHODS: Yorkshire swine were fed a high-cholesterol diet for 4 weeks to induce metabolic syndrome, then underwent placement of an ameroid constrictor to the left circumflex artery to induce chronic myocardial ischemia. Two weeks later, pigs received intramyocardial injection of either saline (control) (n = 6) or EVs (n = 8). Five weeks later, pigs were put to death and left ventricular myocardial tissue in ischemic and nonischemic territories were harvested. Protein expression was measured with immunoblotting, and macrophage count was determined by immunofluorescent staining of cluster of differentiation 68. Data were statistically analyzed via Wilcoxon rank-sum test. RESULTS: EV treatment was associated with decreased expression of proinflammatory markers nuclear factor kappa B (P = .002), pro-interleukin (IL) 1ß (P = .020), and cluster of differentiation 11c (P = .001) in ischemic myocardium, and decreased expression of nuclear factor kappa B in nonischemic myocardium (P = .03) compared with control. EV treatment was associated with increased expression of anti-inflammatory markers IL-10 (P = .020) and cluster of differentiation 163 (P = .043) in ischemic myocardium compared with control. There were no significant differences in expression of IL-6, tumor necrosis factor alpha, arginase, HLA class II histocompatibility antigen DR alpha chain, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha, or phosphorylated nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha in ischemic myocardium or pro-IL1ß, IL-6, tumor necrosis factor alpha, IL-10, or nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha in nonischemic myocardium of EV-treated pigs compared with control. There were no differences in macrophage count in ischemic myocardium between EV-treated pigs and control. CONCLUSIONS: In the setting of metabolic syndrome and chronic myocardial ischemia, intramyocardial EV therapy attenuates proinflammatory signaling.
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Vesículas Extracelulares , Síndrome Metabólica , Isquemia Miocárdica , Suínos , Animais , Interleucina-10 , NF-kappa B/metabolismo , Síndrome Metabólica/terapia , Síndrome Metabólica/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Interleucina-6/metabolismo , Isquemia Miocárdica/metabolismo , Miocárdio/patologia , Vesículas Extracelulares/metabolismo , Modelos Animais de DoençasRESUMO
OBJECTIVE: Our recent studies using a porcine model of metabolic syndrome (MS) and chronic myocardial ischemia show that extracellular vesicle (EV) therapy improves blood flow and arteriogenesis in ischemic myocardium, although mechanisms of these changes are unclear. We hypothesized that in the setting of MS, EV therapy would decrease antiangiogenic signaling to mediate increased blood flow to chronically ischemic myocardium. METHODS: Yorkshire swine were fed a high-fat diet for 4 weeks to induce MS, then underwent placement of an ameroid constrictor to the left circumflex artery to induce chronic myocardial ischemia. Two weeks later, pigs underwent intramyocardial injection of vehicle (control, n = 6) or human bone marrow-derived EVs (n = 8). Five weeks later, left ventricular myocardium in ischemic territory was harvested. Protein expression was measured using immunoblot analysis, and data were analyzed using Wilcoxon rank sum test. Myocardial perfusion was measured with isotope-labeled microspheres, and correlation data were analyzed using Spearman rank correlation coefficient. RESULTS: EV treatment was associated with decreased expression of antiangiogenic proteins, angiostatin (P < .001) and endostatin (P = .043) in ischemic myocardium compared with control. In EV-treated pigs, there was a negative correlation between blood flow to ischemic myocardium and angiostatin (rs = -0.76; P = .037), but not endostatin expression (rs = .02; P = .98). EV treatment was also associated with decreased cathepsin D, which cleaves precursors to produce angiostatin and endostatin, in ischemic myocardium (P = .020). CONCLUSIONS: In the setting of MS and chronic myocardial ischemia, EV therapy is associated with decreased expression of antiangiogenic proteins, which might contribute to increased blood flow to chronically ischemic myocardium.
Assuntos
Vesículas Extracelulares , Síndrome Metabólica , Isquemia Miocárdica , Suínos , Humanos , Animais , Síndrome Metabólica/metabolismo , Angiostatinas/metabolismo , Modelos Animais de Doenças , Isquemia Miocárdica/complicações , Miocárdio/metabolismo , Vesículas Extracelulares/metabolismo , Circulação CoronáriaRESUMO
Introduction: Sodium-glucose cotransporter-2 inhibitors (SGLT2i) are cardioprotective, and canagliflozin (CANA), an SGLT2i, has been shown to improve perfusion, AMPK signaling, and oxidative stress in chronically ischemic myocardium. The aim of this study is to determine the effects of CANA in nonischemic myocardium on coronary collateralization, oxidative stress, and other molecular pathways determined by proteomic profiling. Methods: Yorkshire swine underwent placement of an ameroid constrictor to the left circumflex artery. Two weeks later, pigs received no drug (CON, n = 8) or 300 mg CANA daily (n = 8). Treatment continued for five weeks, followed by tissue harvest of nonischemic myocardium. Results: CANA was associated with decreased capillary density (p = 0.05) compared to CON, without changes in arteriolar density. Reduced capillary density did not correlate with reduced perfusion. Oxidative stress was reduced with CANA (22 % decrease). In the CANA group, there was a trend towards increased p-eNOS and eNOS, without a change in p-eNOS/eNOS ratio, p-Akt, Akt, and p-Akt/Akt ratio. There was no change in p-ERK1/2, but a decrease in total ERK1/2 and increase in p-ERK1/2/ERK1/2 ratio. There were no changes in expression of p-AMPK, AMPK, with a trend towards increased ratio of p-AMPK/AMPK. Proteomics analysis identified 2819 common proteins, of which 120 were upregulated and 425 were downregulated with CANA. Pathway analysis demonstrated wide regulation of metabolic proteins. Conclusions: The effects of CANA on myocardial perfusion and AMPK signaling in chronically ischemic myocardium are not found in nonischemic territory, despite attenuation of oxidative stress. Metabolic proteins are widely regulated in nonischemic myocardium with CANA.
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Background: Coronary artery disease remains a leading cause of death worldwide. Bone mesenchymal stem cell-derived extracellular vesicles (EVs) have shown promise in the setting of myocardial ischemia. Furthermore, the properties of the EVs can be modified via preconditioning of progenitor cells. Previous research from our lab demonstrated a significant decrease in proinflammatory signaling following treatment with EVs derived from starvation preconditioning of human bone mesenchymal stem cells (MVM EVs) in a porcine model of chronic myocardial ischemia. However, rodent models have demonstrated that the use of EVs derived from hypoxia preconditioning of bone mesenchymal stem cells (HYP EVs) may have extended benefits compared to MVM EVs. This study evaluated the effect of HYP EVs on inflammation in a swine model of chronic myocardial ischemia. We hypothesized that HYP EVs would have a greater anti-inflammatory effect than MVM EVs or saline (CON). Methods: Yorkshire swine fed a standard diet underwent placement of an ameroid constrictor to the left circumflex artery. Two weeks later, the animals received intramyocardial injection of saline (CON; n = 6), starvation-derived EVs (MVM; n = 10), or hypoxia-derived EVs (HYP; n = 7). After 5 weeks, myocardial perfusion was assessed, and left ventricular myocardial tissue was harvested. Protein expression was measured using immunoblotting. Data were analyzed via the Kruskal-Wallis test or one-way analysis of variance based on the results of a Shapiro-Wilk test. Coronary perfusion was plotted against relative cytokine concentration and analyzed with the Spearman rank-sum test. Results: HYP EV treatment was associated with decreased expression of proinflammatory markers interleukin (IL)-6 (P = .03), Pro-IL-1ß (P = .01), IL-17 (P < .01), and NOD-like receptor protein 3 (NLRP3; P < .01) compared to CON. Ischemic tissue from the MVM group showed significantly decreased expression of pro-inflammatory markers NLRP3 (P < .01), IL-17 (P < .01), and HLA class II histocompatibility antigen (P < .01) compared to CON. The MVM group also had decreased expression of anti-inflammatory IL-10 (P = .01) compared to CON counterparts. There were no significant differences in expression of tumor necrosis factor-α, interferon-γ, IL-12, Toll-like receptor-2, and nuclear factor kappa-light-chain-enhancer of activated B cells in either group . There was no correlation between coronary perfusion and cytokine concentration in the MVM or HYP groups, either at rest or with pacing. Conclusions: HYP EVs and MVM EVs appear to result in relative decreases in the degree of inflammation in chronically ischemic swine myocardium, independent of coronary perfusion. It is possible that this observed decrease may partially explain the myocardial benefits seen with both HYP and MVM EV treatment.
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Aim: To determine the differences in improvement in cardiac function by intramyocardial (IM) vs. intravenous (IV) injection of human bone mesenchymal stem cell-derived extracellular vesicles (HBMSC-EV) after acute MI. Methods: FVB mice underwent acute MI via left anterior descending coronary artery ligation and subsequent injection of: (1) IM saline control; (2) IM HBMSC-EV; (3) IV saline control; and (4) IV HBMSC-EV. Cardiac function was evaluated with weekly postoperative echocardiography. On postoperative day 28, the mice were euthanized, and the heart, lungs, liver, spleen, and kidneys were harvested. Given previous studies showing HBMSC-EV hepatic uptake after IV injection, the liver was evaluated for changes in inflammation, fibrosis, and proliferation. Results: On postoperative day 28, there were no significant differences in left ventricular ejection fraction (P = 0.6151), fractional shortening (P = 0.1135), or anterior border zone fibrosis (P = 0.6333) in any of the experimental groups. Interestingly, there was a strong trend demonstrating improvement in infarct size on fibrosis staining, which did not reach significance (P = 0.05620). There were no differences in hepatic inflammation, fibrosis, and proliferation. Conclusions: Although there was a trend in the improvement in infarct size, a single-dose administration of neither IM nor IV injection of HBMSC-EV resulted in significant improvement in post-MI cardiac function. A major limitation of this study is the lack of trials determining the optimal dose of HBMSC-EV needed in this model. However, the current study demonstrates that future studies are required to either optimize administration or bioengineer HBMSC-EV with cardiac-homing properties.