Factors secreted by mesenchymal stem cells and endothelial progenitor cells have complementary effects on angiogenesis in vitro

Abstract

Stem cell-based therapy for myocardial regeneration has reported several functional improvements that are attributed mostly to the paracrine effects stimulating angiogenesis and cell survival. This study was conducted to comparatively evaluate the potential of factors secreted by mesenchymal stem cells (MSCs) in normoxic and hypoxic conditions to promote tissue repair by sustaining endothelial cell (EC) adhesion and proliferation and conferring protection against apoptosis. To this aim, a conditioned medium (CM) was generated from MSCs after 24-h incubation in a serum-free normal or hypoxic environment. MSCs exhibited resistance to hypoxia, which induced increased secretion of vascular endothelial growth factor (VEGF) and decreased levels of other cytokines, including stromal-derived factor-1 (SDF). The CM derived from normal (nMSC-CM) and hypoxic cells (hypMSC-CM) induced similar protective effects on H9c2 cells in hypoxia. Minor differences were noticed in the potential of normal versus hypoxic CM to promote angiogenesis, which were likely connected to SDFα and VEGF levels: the nMSC-CM was more effective in stimulating EC migration, whereas the hypMSC-CM had an enhanced effect on EC adhesion. However, the factors secreted by MSCs in normoxic or hypoxic conditions supported adhesion, but not proliferation, of ECs in vitro, as revealed by impedance-based dynamic assessments. Surprisingly, factors secreted by other stem/progenitor cells, such as endothelial progenitor cells (EPCs), had complementary effects to the MSC-CM. Thus, the EPC-CM, in either a normal or hypoxic environment, supported EC proliferation, but did not sustain EC adhesion. Combined use of the MSC-CM and EPC-CM promoted both EC adhesion and proliferation, suggesting that the local angiogenesis at the site of ischemic injury might be better stimulated by simultaneous releasing of factors secreted by multiple stem/progenitor cell populations.

FIG. 1.

The effect of hypoxia on mesenchymal stem cells (MSCs). (a) Increase of heme oxygenase-1 (HMOX-1) protein level in MSCs after 24-h exposure to hypoxic conditions. lane 1: normal MSCs (before starvation); lane 2: normoxic MSCs (after 24-h incubation in serum-free DMEM and hypoxic conditions); lane 3: hypoxic MSCs. (b) Minimal increase in Caspase-3 activity (1.7-fold as compared to normal cells) was determined in MSCs after hypoxic treatment. The data represent the mean values from 5 independent experiments performed in duplicate (*P<0.05). (c) The bax/bcl-2 mRNA ratio in MSCs after exposure to hypoxia, as determined by RT–polymerase chain reaction (PCR) analysis. The data represent the mean values from 3 independent experiments performed in triplicate. (d) Fluorescence microscopy of MSC after hypoxic treatment illustrating the accumulation of JC-1 within mitochondria as red aggregate (demonstrating normal mitochondrial membrane potential [ΔΨm] and intact mitochondria) and no evidence of JC-1 green monomers into the cytosol. DMEM, Dulbecco's modified Eagle's medium; RT, real time.

FIG. 2.

Angiogenic factors secreted by MSCs in normal and hypoxic conditions. (a) Vascular endothelial growth factor (VEGF) level quantified in the culture supernatant after 24-h incubation of MSCs in normal and hypoxic conditions (ELISA assay). The data represent the mean values from 5 independent experiments (*P<0.05). (b) Stable expression (at both protein and mRNA levels) and constant secretion levels of transforming growth factor-β1 (TGF-β1) in MSCs after exposure to hypoxia. (c) ELISA quantification of SDF-1 in the MSC lysate and supernatant in normal and hypoxic conditions. The data represent the mean values from 3 (cell lysate) and 5 (supernatant) independent experiments (***P<0.005). (d) Real-time PCR quantification of SDF-1 expression in normal and hypoxic MSCs. The data represent the mean values from 3 independent experiments performed in triplicate. (e) The profile of MSC-secreted cytokines in normal conditions. The image is representative of 3 experiments. (f) Quantification of the cytokine expression level in MSC supernatant obtained in normal and hypoxic conditions. The data represent the mean values of 3 experiments performed in duplicates. (g) Qualitative ELISpot determination of the pro- and active forms of MMP-9 released from MSCs after exposure to hypoxic conditions.

FIG. 3.

Protective effects of MSC-conditioned medium (CM) on cardiac myocytes in hypoxic conditions. (a) (Top) Representative picture of myocytes stained with JC-1 after exposure to hypoxia. Note that polarized mitochondria appear as punctuate red fluorescence, whereas depolarized mitochondria exhibit the characteristic diffuse green monomer fluorescence. (Below) Assessment of ΔΨm loss expressed as the increase in 535 nm/610 nm fluorescence ratio and the effect of normal MSC-CM (nMSC-CM) and hypoxic MSC-CM (hypMSC-CM) in preventing ΔΨm loss (left). Data are mean±S.D. of results obtained in 1 experiment and representative of 3 (***P<0.005). (b) Quantitative determination of apoptotic H9c2 nuclei after exposure to hypoxia in the presence and absence of MSC-CM. The diagram represents the percentage of apoptotic cell nuclei determined by Hoechst staining. A minimum of 1,000 cells per assay was counted. The upper panel shows the fluorescence microscopy of Hoechst staining reaction illustrating apoptotic cells (right, arrowheads) and several dividing cells (left, arrows) that were excluded from the counting (*P<0.05). (c) The level of Caspase-3 activity determined in H9c2 cells after 24-h exposure to hypoxia and the protective role of nMSC-CM and hypMSC-CM on the Caspase-3 activation. The data represent the mean values from 5 independent experiments performed in duplicate. (d) Dynamic assessment of myocyte response to hypoxia and reoxygenation in the presence of MSC-CM (original recording). Cells incubated in culture medium with no serum define the baseline, and the plot shows data normalized to the time point before the beginning of starvation (1 representative experiment from 3). The right diagram illustrates the normalized cell index values at 24 h after reoxygenation. The average is resulted from 3 independent experiments performed in duplicates (**P<0.01). Color images available online at www.liebertpub.com/scd

FIG. 4.

The vasculogenic potential of MSC-CM. (a) Matrigel containing nMSC-CM, hypMSC-CM, or FGFb evaluated after 7 days from in vivo implantation. Arrowheads illustrate capillary-like structures. The diagram illustrates the quantification of the cells within the Matrigel (*P<0.05; **P<0.01). (b) Original recording illustrating the time-dependent chemotactic migration of EA.hy926 (in duplicates) in response to nMSC-CM and hypMSC-CM. The diagram on the right illustrates the cell migration index of endothelial cells (ECs) after 3 h. The average is resulted from 3 independent experiments performed in duplicates. (c) Scratch test assay on ECs. (left) Cells were scratched, photographed at time 0, and incubated in the presence of nMSC-CM and hypMSC-CM. Photographs were taken again after 8 h of culture. (right) Quantification of the covered area as percentage of the initial scratched area. The diagram illustrates the mean±S.D. of 3 independent experiments (***P<0.005). Color images available online at www.liebertpub.com/scd

FIG. 5.

The effect of nMSC-CM and hypMSC-CM on the adhesion and proliferation of ECs in vitro (original recordings). (a) Dynamic assessment illustrating the initial EC attachment to E-plate surface under the influence of nMSC-CM and hypMSC-CM as compared to serum or no-serum controls. The diagram below illustrates the mean values of cell indexes at 3 h after plating (resulted from 3 experiments) (*P<0.05; **P<0.01). (b) Real-time monitoring of the effect of MSC-CM on the EC adherence and proliferation. The plot shows the ability of nMSC-CM and hypMSC-CM to support EC adhesion and their failure to support EC proliferation in comparison to serum. The diagram below illustrates the cell index 48 h after cell seeding (resulted from 3 experiments) (***P<0.005). (c) Real-time monitoring of the effect of nMSC-CM and hypMSC-CM on EC proliferation after adherence. The plot shows data normalized to the last time point before replacing the serum-containing medium with MSC-CM. The diagram below illustrates the normalized cell index 12 h after cell plating (resulted from 3 experiments). All the recordings show the mean values obtained from duplicates and are representatives from 3 independent experiments. Color images available online at www.liebertpub.com/scd

FIG. 6.

Comparative effects of MSC-CM and endothelial progenitor cell (EPC)-CM on the adherence and proliferation of ECs. (a) Effect of MSC-CM and EPC-CM on EC adherence and proliferation (original recording). Note that MSC-CM, but not EPC-CM, sustains EC adherence; however, MSC-CM is not able to support cell proliferation. (b) Effect of MSC-CM and EPC-CM on EC proliferation after serum-induced adherence (original recording). In contrast to MSC-CM, EPC-CM supports EC proliferation, at a level comparable to serum. (c) Dynamic growth curves of ECs exposed to the culture medium containing both MSC-CM and EPC-CM (original recording). The 3 recordings illustrate representative experiments performed in duplicate, and similar patterns were obtained in 3 experiments with different conditioned media. (d) Diagram illustrating EC proliferation (MTT assay) in the presence of MSC-CM, EPC-CM, and the combination of MSC-CM and EPC-CM (**P<0.05; ***P<0.005). Color images available online at www.liebertpub.com/scd