HIF-1α overexpression in mesenchymal stem cell-derived exosome-encapsulated arginine-glycine-aspartate (RGD) hydrogels boost therapeutic efficacy of cardiac repair after myocardial infarction

Fig. 1. Lentiviral transduction, isolation and characterization of MSCs derived EVs. (A) Successful lentiviral transduction was confirmed by positive fluorescence signal under microscope. Scale bar ​= ​100 ​μm n= 3 for each group. (B) Western blot images showed HIF-1α protein levels in HIF-1α-MSCs and NC-MSCs groups. n= 3 for each group. (C) EVs were isolated from HIF-1α-MSCs and NC-MSCs groups using a standard protocol of serial, differential centrifugation and ultracentrifugation methods. (D) TEM images of EVs derived from HIF-1α-MSCs and NC-MSCs. n= 3 for each group. Scale bar ​= ​100 ​nm ​(E) The exosomal protein markers of TSG101, CD63, and CD81 in EVs isolated from HIF-1α-MSCs and NC-MSCs. n= 3 for each group. (F) The particle size distribution and particle concentration were analyzed by nanoparticle tracking analysis. n= 3 for each group. Continuous variables were described by means ​± ​SEM. ∗∗P ​< ​0.01.

Fig. 2. EVs isolated from MSCs could be absorbed by cardiomyocytes and endothelial cells in vitro. (A and B) DiR-labeled EVs with various concentrations was co-cultured with cardiomyocytes and internalization of EVs was detected. (C) Confocal images showed that red fluorescence of dye Dil labeled EVs were endocytosed by HUVECs and cardiomyocytes 6 ​h and 24 ​h after incubation. Scale bar ​= ​50 ​μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3. HIF-1α-EVs exhibited an anti-apoptosis effect in cardiomyocytes and a pro-angiogenesis effect in HUVECs in vitro. (A) Hoechest33342/PI double staining was used to observe the survival of cardiomyocytes treated with PBS, MSCs-EVs or HIF-1α-EVs under H/SD condition. Scale bar ​= ​100 ​μm. (B) Caspase3/caspase7 activity of cardiomyocytes treated with PBS, MSCs-EVs or HIF-1α-EVs under H/SD condition. Scale bar ​= ​100 ​μm. (C) HIF-1α EVs improved cell viability of cardiomyocytes under H/SD condition. (D) Quantitative analysis of viable cells treated with PBS or EVs. (E) Quantitative analysis of caspase3/caspase7 positive cells between the three groups. (F) Quantitative analysis of TUNEL-positive cells between the three groups. (G) Representative photographs showing the TUNEL-positive cells in cardiomyocytes among the different groups. Green, TUNEL-positive nuclei; red, α-actin; blue, DAPI-stained nuclei. Scale bar ​= ​100 ​μm. (H) Representative images showing tube formation in HUVECs treated with PBS, MSCs-EVs or HIF-1α-EVs. Scale bar ​= ​100 ​μm. (I) Quantification of tube length in each group. Continuous variables were described by means ​± ​SEM. ∗P< 0.05, ∗∗P< 0.01, ∗∗∗∗P< 0.0001…

Fig. 4. HIF-1α-EVs effectively preserved cardiac function in a rat MI model. (A) Dil-labeled exosomes were injected into the infarcted heart of rats for 6 ​h. Representative images of post-MI heart sections stained with Dil-labeled Exo (red), α-actin (green), and DAPI (blue). Scale bar ​= ​50 ​μm (B) Representative echocardiogram of rat heart in different groups at 28 days post-MI. (C and D) Significantly enhanced LVEF and LVFS in rats transplanted with MSCs-EVs and HIF-1α-EVs compared with control groups. (E) HE staining at the border zone on Day 28 after MI. (F) Masson Trichrome staining at the border zone on Day 28 after MI. (G) Quantitative data for the fibrotic area. (H) Quantification of IL-6 expression level in the infarct border zone of rat heats. Continuous variables were described by means ​± ​SEM. n=5–6 for each group. ∗P< 0.05, ∗∗P< 0.01, ∗∗∗P< 0.001, ∗∗∗∗P< 0.0001 vs. PBS group; #P< 0.05, ##P< 0.01 vs. NC-EVs group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5. HIF-1α-EVs inhibited apoptosis and promoted angiogenesis in a rat MI model. (A)TUNEL staining at the border zone on Day 28 post-MI with TUNEL-positive cells. Red, TUNEL-positive nuclei; Green, α-actin; blue, DAPI-stained nuclei. Scale bar ​= ​50 ​μm (B) Quantification of TUNEL-positive cells in each group. (C) Neovascularization at the border zone on Day 28 post-MI was identified by staining with a-SMA (red) and CD31 (green) and nuclei (blue). Scale bar ​= ​50 ​μm (D and E) Quantification of a-SMA-positive cells, CD31-positive cells among different groups. (F) Border zone sections obtained 4 weeks after MI were immunofluorescent stained for Con43 expression. Red, Conx43; Green, α-actin; blue, DAPI-stained nuclei. Continuous variables were described by means ​± ​SEM. n=5–6 for each group. ∗P< 0.05, ∗∗P< 0.01, ∗∗∗P< 0.001, ∗∗∗∗P< 0.0001 vs. PBS group; #P< 0.05, ##P< 0.01 vs. NC-EVs group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6. EVs/RGD-biotin hydrogels enhanced the retention and stability of HIF-1α-EVs. (A) Chemical structure of the RGD-biotin molecule. (B) SEM image of RGD-biotin hydrogel revealed the formation of entangled nanofibers formed by the RGD-biotin molecule. (C) Biotin-GFFYGRGD was dissolved in PBS. After that, the suspension was heated to about 95 ​°C in order to dissolve the compound completely, and hydrogels formed after cooling back to room temperature. (D) EVs were incorporated in Biotin-GFFYGRGD and then injected into infarcted rat hearts. (E) The fate of EVs in vivo was tracked by bioluminescence imaging. Representative images are from rats receiving EVs incorporated in RGD hydrogels (EV/RGD-biotin) and naked EVs. (F) Quantification of bioluminescence imaging after injection. Continuous variables were described by means ​± ​SEM. n=3 for each group.

Fig. 7. EVs/RGD-biotin hydrogels improved Cardiac function after AMI. (A) Representative echocardiogram of rat heart between HIF-1α-EVs group and HIF-1α-EVs RGD-Biotin group at 28 days post-MI. (B and C) Significantly enhanced LVEF and LVFS in rats transplanted with MSCs-EVs and HIF-1α-EVs compared with control groups. (D) HE staining and Masson Trichrome staining at the border zone on Day 28 after MI. (E) Quantitative data for the fibrotic area. Continuous variables were described by means ​± ​SEM. n=4–6 for each group. ∗P< 0.05, ∗∗P< 0.01.

Fig. 8. Inhibition of miR-221-3p Reversed the Protective Effects of EVs on Apoptosis and Angiogenesis. (A) Expression values for miR-221–3p in MSCs-EVs and HIF-1α-EVs by qRT-PCR. (B) Experimental design. To inhibit miR-221–3p in HIF-1α-EVs. HIF-1α-EVs-inhibitormiR−221 and HIF-1α-EVs-inhibitorNC were incubated with cardiomyocytes or HUVECs under H/SD condition. (C and D) TUNEL staining at the border zone 4 weeks after MI, and quantification analysis of percentage of apoptotic cardiomyocytes between two groups. Scale bar ​= ​100 ​μm (E–F) Caspase3/caspase7 activity and quantification analysis in cardiomyocytes treated with HIF-1α-EVs-inhibitormiR−221 and HIF-1α-EVs-inhibitorNC under H/SD condition. Scale bar ​= ​100 ​μm (G) Representative images showing tube formation in HUVECs treated with HIF-1α-EVs-inhibitormiR−221 and HIF-1α-EVs-inhibitorNC. Scale bar ​= ​100 ​μm (H) Quantification of tube length in each group. Continuous variables and categorical variables were described by means ​± ​SEM and percentages. n=6 for each group. ∗P<0.05; ∗∗P ​< ​0.01; ∗∗∗P ​< ​0.001.

Fig. 9. Schematic of the working hypothesis. EVs released by MSCs with stabilized overexpression of HIF-1α contribute to angiogenesis and anti-apoptosis in part via enhanced expression of miR-221–3p. RGD hydrogels increased the therapeutic efficacy of HIF-1α engineered MSCs-derived EVs at a functional, histopathological and molecular level.