Intramyocardial injection of pre-cultured endothelial progenitor cells and mesenchymal stem cells inside alginate/gelatin microspheres induced angiogenesis in infarcted rabbits

Abstract

Background: Using several strategies, the stimulation of angiogenesis can alleviate the pathological complications of post-myocardial ischemia. Here, endothelial progenitor cells (EPCs) and mesenchymal stem cells (MSCs) were pre-cultured inside the alginate/gelatin (Alg/Gel) microspheres in the presence of SDF-1α for 7 days, and their angiogenesis potential was monitored in infarcted rabbits.

Methods: The decapsulated cells were monitored in terms of cell dynamic growth and angiogenesis potential in vitro and after injection into ischemic myocardium in rabbits.

Results: Based on the data, 7-day incubation inside the Alg/Gel microspheres led to the stimulation of angiogenesis profile (Ang-1↑, -2↑, Tie-2↑), migration (MMP-2↑, and − 9↑), and autophagic response (Beclin-1↑, LC3↑, and p-62↓) in EPCs containing groups in the presence of SDF-1α. PCR array analysis revealed the expression of angiogenesis-related genes in the presence of SDF-1α. These features coincided with the stimulation of in vitro tubulogenesis properties in EPCs and EPCs + SDF-1α groups. The injection of cells from different groups into the infarcted rabbits led to the reduction of fibrotic area in ischemic myocardium in MSCs-bearing groups, and these effects were intensified in the presence of SDF-1α. Pre-treatment of EPCs and MSCs increased the recruited immune cells into the ischemic area within the myocardium. It was suggested that SDF-1α stimulated the local vascular density (CD31 capillaries, and α-SMA arterioles) in EPCs-bearing groups compared to MSCs and MSCs + SDF-1α groups.

Conclusions: These data indicate that pre-culture of EPCs and MSCs inside the Alg-based hydrogels can increase the regenerative potential of these cells, especially when exposed to stimulatory cytokines such as SDF-1α for the alleviation of ischemic changes.

Supplementary Information: The online version contains supplementary material available at 10.1186/s12964-025-02301-0.

Keywords: Angiogenesis; Endothelial progenitor cells; Mesenchymal stem cells; Myocardial infarction; Rabbits.

Fig. 1

The schematic diagram of the experimental procedure. EPCs and MSCs were isolated from human bone marrow samples. The synthesis of Alg/Gel hydrogel was done using CaCl₂ as a cross-linking agent. EPCs, MSCs, and/or their combination were incorporated into Alg/Gel microspheres in the presence and/or absence of SDF-1α using CaCl₂ solution under high-voltage conditions. After a 7-day in vitro culture, physicochemical assay, angiogenesis properties, migration capacity, and autophagy response were measured. For the in vivo assay, cells were decapsulated using tri-sodium citrate buffer and injected into a rabbit model of myocardial infarction. The histological changes, fibrosis, and angiogenesis properties were analyzed after 30 days post-transplantation

Fig. 2

Characterization of EPCs at passage 3 using Dil-Ac-LDL uptake (A). The cultured EPCs efficiently internalized red-colored Dil-Ac-LDL, indicating lipoprotein lipase activity (Blue: DAPI). FTIR (B). Swelling ratio (C; n = 3). Degradation rate (D; n = 3). Mechanical properties (E;n = 3)

Fig. 3

SEM images (A). Upper panel: encapsulated cells can appropriately the polymeric network of Alg/Gel structure (yellow arrows). Lower panel: microsphere surface. Data show the bulging of cells from the external surface of Alg/Gel microspheres, indicating an appropriate cell localization and migration (yellow arrows). Bright-field images related to encapsulated EPCs and MSCs in the presence or absence of SDF-1α in side Alg/Gel microspheres after 7 days in in vitro conditions (B). Based on the data, cells were evenly distributed inside the microspheres. Non-significant differences were found in terms of microsphere diameter after 7 days (n = 50). Measuring survival rate using MTT assay after 7 days (n = 3). One-way ANOVA with Tukey post hoc analysis. **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 4

Monitoring protein levels of angiogenesis (Ang-1, -2, and Tie-2), migration (MMP-2, and − 9), and autophagy factors (Beclin-1, LC3, p62) in encapsulated EPCs, MSCs inside the Alg/Gel microspheres with/without SDF-1α after 7 days. Data were obtained from three pooled samples

Fig. 5

Monitoring the tubulogenesis properties of EPCs, MSCs pre-cultured inside Alg/Gel microspheres after 7 days (A-B; n = 3). The migration rate was monitored using the Transwell insert system (C; n = 11). PCR array analysis for the detection of angiogenesis profile (D; n = 3). Fold-changes greater than 2 are indicated in red; fold-changes less than − 2 are indicated in blue. One-way ANOVA with Tukey post hoc analysis. **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 6

Induction of MI in rabbits using LAD ligation (A). Cells were administered into the border zone of ischemic sites (yellow arrows and dashed circular area). Histological analysis revealed the existence of blue-colored collagen fibers in different groups (B; black arrows). Measuring the fibrotic area per cardiac tissue slide using Masson’s trichome staining (C; n = 6). H & E staining (D). Number of immune cells infiltrated into the periphery of the ischemic zone in MI rabbits (E; n = 6). One-way ANOVA with Tukey post hoc analysis. *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001

Fig. 7

Monitoring the arteriogenesis and angiogenesis properties of EPCs and MSCs pre-cultured inside the Alg/Gel microspheres for 7 days in the presence and absence of SDF-1α in MI rabbits after 30 days (A-B). Local density of α-SMA⁺ arterioles (A) and CD31⁺ capillaries (B). One-Way ANOVA with Tukey post hoc. (n = 6 rabbits per group, and data were obtained from 10 serial HPF). *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001