Delayed delivery of endothelial progenitor cell-derived extracellular vesicles via shear thinning gel improves postinfarct hemodynamics

Abstract

Background: Extracellular vesicles (EVs) are promising therapeutics for cardiovascular disease, but poorly-timed delivery might hinder efficacy. We characterized the time-dependent response to endothelial progenitor cell (EPC)-EVs within an injectable shear-thinning hydrogel (STG+EV) post-myocardial infarction (MI) to identify when an optimal response is achieved.

Methods: The angiogenic effects of prolonged hypoxia on cell response to EPC-EV therapy and EV uptake affinity were tested in vitro. A rat model of acute MI via left anterior descending artery ligation was created and STG+EV was delivered via intramyocardial injections into the infarct border zone at time points corresponding to phases of post-MI inflammation: 0 hours (immediate), 3 hours (acute inflammation), 4 days (proliferative), and 2 weeks (fibrosis). Hemodynamics 4 weeks post-treatment were compared across treatment and control groups (phosphate buffered saline [PBS], shear-thinning gel). Scar thickness and ventricular diameter were assessed histologically. The primary hemodynamic end point was end systolic elastance. The secondary end point was scar thickness.

Results: EPC-EVs incubated with chronically versus acutely hypoxic human umbilical vein endothelial cells resulted in a 2.56 ± 0.53 versus 1.65 ± 0.15-fold increase (P = .05) in a number of vascular meshes and higher uptake of EVs over 14 hours. End systolic elastance improved with STG+EV therapy at 4 days (0.54 ± 0.08) versus PBS or shear-thinning gel (0.26 ± 0.03 [P = .02]; 0.23 ± 0.02 [P = .01]). Preservation of ventricular diameter (6.20 ± 0.73 mm vs 8.58 ± 0.38 mm [P = .04]; 9.13 ± 0.25 mm [P = .01]) and scar thickness (0.89 ± 0.05 mm vs 0.62 ± 0.03 mm [P < .0001] and 0.58 ± 0.05 mm [P < .0001]) was significantly greater at 4 days, compared wit PBS and shear-thinning gel controls.

Conclusions: Delivery of STG+EV 4 days post-MI improved left ventricular contractility and preserved global ventricular geometry, compared with controls and immediate therapy post-MI. These findings suggest other cell-derived therapies can be optimized by strategic timing of therapeutic intervention.

Keywords: delayed therapy; extracellular vesicles; myocardial infarction; shear thinning gel.

Figure 1.. EPCs are harvested from the long bones of Wistar rats and plated.

EVs released from cells are isolated from CM. STG+EV is prepared by suspending EVs within STG. LAD ligation creates an LV infarct. At the designated post-MI intervention time, STG+EV is injected intramyocardially in the infarct borderzone. Post-treatment hemodynamics were assessed at 4 and 6 weeks post-MI.

Figure 2.. EPC-EVs promote a robust angiogenic response from chronically hypoxic HUVECs.

HUVECS were exposed to chronic hypoxia (7 days, 5% O2) to mimic an ischemic environment. Naive and preconditioned HUVECs were incubated with EVs in 5% O2 for 14 hours. Chronically hypoxic HUVECs demonstrated increased vascular tubule mesh formation compared to the naive group (p=0.049).

Figure 3.. Hypoxic HUVECs demonstrate robust EPC-EV uptake ability.

EPC-EV uptake by preconditioned HUVECS (4 days, 5% O2) was compared to that of naive HUVECs at 3 and 14 hours after EPC-EV incubation in a hypoxic environment. After 14 hours, preconditioned HUVECs exhibited greater EV uptake than both naive (p<0.05) and preconditioned (p<0.01) cells in 3 hours of hypoxia.

Figure 4.. Representative H&E of infarct over time.

Inflammatory cell infiltrate was evident at all timepoints. At 4 days there was delineation of scar and borderzone regions. Thinning of the LV wall from necrosis progressed in the 2 week group. Black arrows = borderzone. Blue arrows = infarct. Imaged at 4x and 10x magnification under brightfield. Scale 500um.

Figure 5.. Hemodynamics panel.

Data plotted as mean +/− SEM. Treatment groups indicate timepoints post-MI at which therapy was administered. Treatment at 4d post-MI shows greatest improvement in measures of Ees, EF, cardiac output, stroke work, and dP/dt max.

Figure 6.. Delayed delivery of STG+EV limits infarct thinning.

A) Representative heart sections at 4 weeks post-MI were stained with Masson’s Trichrome. B) Scar thickness was calculated. The 4d group (p<0.0001) had the greatest scar thickness compared to controls.