Comparative Analysis of Normoxia- and Hypoxia-Modified Extracellular Vesicle Therapy in Function, Perfusion, and Collateralization in Chronically Ischemic Myocardium

Abstract

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.

One Billion hiPSC-Cardiomyocytes: Upscaling Engineered Cardiac Tissues to Create High Cell Density Therapies for Clinical Translation in Heart Regeneration

“…Two Yorkshire swine (male and female; Tufts University, Medford, MA, USA), aged 8 to 10 weeks and weighing between 22 and 25 kg at the time of the initial procedure, were used. This pilot study was designed to assess technical feasibility using an established model of chronic myocardial ischemia developed by Dr. Sellke as previously described [30][31][32][33][34][35][36][37][38][39][40][41][42][43][44]. To induce chronic ischemia, a small left thoracotomy was performed, the pericardium was opened, and an ameroid constrictor composed of hygroscopic casein material cased in titanium (Research Instruments SW, Escondido, CA, USA) was sized and placed around the proximal left circumflex (LCX) coronary artery (Figure 2B).…”

Ischemic myocardial inflammatory signaling in starvation versus hypoxia-derived extracellular vesicles: A comparative analysis

“…HBMSCs (Lonza) were cultured in accordance with the manufacturer's recommendations using growth medium (MSCGM Bulletkit PT-3001; Lonza), as described previously. 13 HYP EVs were grown until they reached 80% confluence and then passaged to passage 7. At this stage, the medium was replaced with MSCGM medium, and the cells were placed in a humidified hypoxia chamber (MIC-101; Billups-Rothenberg) containing 5% carbon dioxide and 95% nitrogen.…”

“…At this stage, the medium was replaced with MSCGM medium, and the cells were placed in a humidified hypoxia chamber (MIC-101; Billups-Rothenberg) containing 5% carbon dioxide and 95% nitrogen. 13 The cells were incubated at 37 °C for 24 hours, after which the medium and the EVs were collected via ultracentrifugation as described previously. 13 Protein quantification was performed using a radioimmunoprecipitation assay (Kit 23225; Thermo Fisher Scientific).…”

“…In our previous investigation using a large animal model, our group successfully demonstrated that HYP EVs exhibit enhanced contractility, capillary density, and angiogenic signaling pathways compared to MVM EVs. 13 These findings suggest significant potential for HYP EVs in improving cardiac function compared to MVM EVs 13 ; however, the underlying mechanism remains unclear. To further understand the cardiac benefits of EVs, we plan to compare the anti-inflammatory effects of MVM EVs and HYP EVs in a swine model of chronic myocardial ischemia.…”

Extracellular Vesicles’ Role in Angiogenesis and Altering Angiogenic Signaling

“…Furthermore, two studies that investigated the impact of EVs derived from human bone mesenchymal stem cells on angiogenesis demonstrated that the application of these EVs to ischemic heart tissue resulted in improved angiogenic signaling via the Akt and ERK pathways. Moreover, these EV-treated tissues exhibited decreased levels of anti-angiogenic markers, suggesting their potential suitability for treating conditions characterized by a reduced blood flow or capillary density, such as heart failure or myocardial ischemia [ 87 , 88 , 89 ]. Another paper on glioblastoma demonstrated how vesicles play a crucial role in information exchange between malignant and vascular cells, the release of growth factors and cytokines by endothelial cells, activation of the PI3K.AKT signaling pathway, and the movement of pericytes.…”