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

Figure 1. Intramyocardial injection of hypoxia-modified EVs increases left ventricular contractility compared to the injection of normoxia serum-stressed EVs. Swine treated with 50 µg hypoxia-modified (HYP, n = 7) EVs had improved contractility as measured by dP/dtmax and the x-intercept (Lw) of the pre-load recruitable stroke work relationship (PRSW) compared to swine treated with 50 µg normoxia serum-starved EVs (NOR, n = 10). There was a trend towards improved stroke work and end-systolic elastance in the HYP group compared to NOR, without changes in cardiac output or stroke volume. Upper and lower borders of box represent upper and lower quartiles; middle horizontal line represents median; upper and lower whiskers represent maximum and minimum values. CO, cardiac output; SW, stroke work; SV, stroke volume; dP/dT, change in pressure over change in time; Ees, end-systolic elastance. ** p < 0.01, **** p < 0.0001.

Figure 2. Perfusion to chronically ischemic myocardium was not significantly changed with hypoxia-modified EVs compared to normoxia EVs. Swine treated with hypoxia-modified (HYP, n = 7) EVs had nonsignificant trends towards increased perfusion to chronically ischemic myocardial territory compared to swine treated with normoxia serum-starved EVs (NOR, n = 10). Upper and lower borders of box represent upper and lower quartiles; middle horizontal line represents median; upper and lower whiskers represent maximum and minimum values.

Figure 3. Hypoxia-modified EVs increased capillary density to chronically ischemic myocardium compared to normoxia EVs. (A) Swine treated with hypoxia-modified (HYP, n = 7) EVs had improved capillary density as measured by isolectin B4 immunofluorescent staining compared to treatment with normoxia serum-starved EVs, without changes in arteriolar density as measured by α-smooth muscle actin staining. The upper and lower borders of the box represent upper and lower quartiles; the middle horizontal line represents the median; the upper and lower whiskers represent maximum and minimum values. ** p < 0.01. (B) Representative images (20×) of ischemic myocardial tissue stained with α-smooth muscle actin (α-SMA) for arteriolar density (red), isolectin B4 for capillary density (green), and 4′,6-diamidino-2-phenylindole (DAPI) for nuclei staining (blue).

Figure 4. Hypoxia-modified EV treatment increased pro-angiogenic signaling and decreased anti-angiogenic signaling in chronically ischemic myocardium compared to normoxia EV treatment. Immunoblot results from total protein lysates of chronically ischemic myocardium in swine treated with hypoxia-modified EVs (HYP, n = 7) compared to normoxia serum-starved EVs (NOR, n = 10) are shown for pro-angiogenic markers Akt, phosphorylated (p-) Akt, and the ratio of p-Akt to Akt; extracellular signal-regulated kinase 1/2 (ERK1/2), p-ERK1/2, and the ratio of p-ERK 1/2 to ERK1/2; endothelial nitric oxide synthase (eNOS), p-eNOS, and the ratio of p-eNOS and eNOS; and vascular endothelial cadherin (VE-Cadherin). Immunoblot results are also shown for anti-angiogenic markers angiostatin and endostatin. Upper and lower borders of box represent upper and lower quartiles; middle horizontal line represents median; upper and lower whiskers represent maximum and minimum values. ** p < 0.01, *** p < 0.001, **** p < 0.0001.