Mesenchymal Stem Cell Secretion of SDF-1α Modulates Endothelial Function in Dilated Cardiomyopathy

Courtney Premer, Amarylis Wanschel, Valeria Porras, Wayne Balkan, Tatiana Legendre-Hyldig, Russell G Saltzman, Chunming Dong, Ivonne Hernandez Schulman, Joshua M Hare, Courtney Premer, Amarylis Wanschel, Valeria Porras, Wayne Balkan, Tatiana Legendre-Hyldig, Russell G Saltzman, Chunming Dong, Ivonne Hernandez Schulman, Joshua M Hare

Abstract

Background: Endothelial dysfunction contributes to the pathophysiology of dilated cardiomyopathy (DCM). Allogeneic but not autologous mesenchymal stem cells (MSCs) improve endothelial function in DCM patients. We hypothesized that these effects are modulated by release of stromal derived factor-1α (SDF-1α).

Methods: Plasma TNFα and endothelial progenitor cell-colony forming units (EPC-CFUs) were assessed at baseline and 3-months post-injection in a subset of POSEIDON-DCM patients that received autologous (n = 11) or allogeneic (n = 10) MSCs. SDF-1α secretion by MSCs, endothelial cell (EC) TNFα mRNA expression, and levels of reactive oxygen species (ROS) in response to SDF-1α were measured in vitro.

Results: As previously shown, DCM patients (n = 21) had reduced EPC-CFUs at baseline (3 ± 3), which were restored to normal by allogeneic MSCs 3-months post-treatment (Δ10 ± 4). DCM patients had elevated baseline plasma TNFα (n = 15, 22 ± 9.4 pg/mL). Allogeneic MSCs (n = 8) decreased, and autologous MSCs (n = 7) increased, plasma TNFα (-7.1 ± 3.1 vs. 22.2 ± 17.1 pg/mL, respectively; P = 0.0005). In culture, autologous MSCs (n = 11) secreted higher levels of SDF-1α than allogeneic MSCs (n = 6) [76.0 (63.7, 100.9) vs. 22.8 (7.2, 43.5) pg/mL, P = 0.0002]. SDF-1α and plasma TNFα negatively correlated with EPC-CFUs in both treatment groups (R = -0.7, P = 0.0004). ECs treated with 20 ng SDF-1α expressed lower levels of TNFα mRNA than cells treated with 100 ng (0.7 ± 0.2 vs. 2.1 ± 0.3, P = 0.0008). SDF-1α at low but not high concentration inhibited the generation of ROS.

Conclusion: MSC secretion of SDF-1α inversely correlates with EPC-CFU production in DCM patients and therefore may be a modulator of MSC therapeutic effect in this clinical setting.

Clinical trial registration: https://ichgcp.net/clinical-trials-registry/NCT01392625, identifier NCT01392625.

Keywords: SDF 1α; allogeneic; dilated cardiomyopathy; endothelial dysfunction; mesenchymal stem cells.

Copyright © 2019 Premer, Wanschel, Porras, Balkan, Legendre-Hyldig, Saltzman, Dong, Schulman and Hare.

Figures

FIGURE 1
FIGURE 1
The relationship between patient TNFα and endothelial colony formation at baseline and 3-months post autologous and allogeneic mesenchymal stem cell (MSC) treatment. (A) Patients who received allogeneic MSC treatment (n = 8) had a significant reduction in plasma TNFα from baseline to 3 months post treatment (24.5 ± 5.6 to 17.4 ± 3.7 pg/mL, P = 0.0005∗∗∗). (B) Patients who received autologous MSC treatment (n = 7) had no change in plasma TNFα from baseline to 3 months post injection (25.3 ± 13.3 to 42.4 ± 33.1 pg/mL, P = NS). (C) There is a significant difference comparing the effect of allogeneic vs. autologous on the reduction of plasma TNFα 3 months post treatment (Δ−7.1 ± −1.9 vs. Δ17.1 ± 19.8 pg/mL, P = 0.009∗∗). (D) Neither allogeneic nor autologous MSCs are secreting notable levels of TNFα (0.37 ± 0.7 vs. 0.1 ± 0.2 pg/mL, P = NS). (E) There was a significant correlation between the change in plasma TNFα and the change in EPC colony formation from baseline to 3-months post injection (n = 15) (R = −0.7, P = 0.0004∗∗∗).
FIGURE 2
FIGURE 2
Stromal derived factor-1α (SDF-1α) as a biomarker for endothelial function. (A) Schematic illustrating that mesenchymal stem cells (MSCs) secrete SDF-1α which stimulates the mobilization and function of endothelial progenitor cells (EPCs). (B) Allogeneic MSCs (n = 6) secrete significantly lower levels of SDF-1α compared to autologous MSCs (n = 11) 22.8 (7.2, 43.5) vs. 76.0 (63.7, 100.9) pg/mL, P = 0.0005∗). (C) There was a significant inverse correlation between MSC SDF-1α secretion and the change in EPC-CFUs from baseline to 3-months post injection (n = 21, R = −0.7, P = 0.0004∗). (D) Additionally, there was a correlation between the amount of SDF-1α secretion from cultured MSCs and the change in TNFα from baseline to 3-months post injection compared to (n = 15) (R = 0.7, P = 0.003∗∗).
FIGURE 3
FIGURE 3
Stromal derived factor-1α levels modulate the expression of TNFα expression in human coronary artery endothelial cells (HCAECs). (A) HCAECs exposed to low levels (20 ng) of SDF-1α express low levels of TNFα mRNA (0.7 ± 0.2) comparable to the PBS control, whereas those exposed to high levels (100 ng) express high levels of TNFα mRNA compared to control (2.1 ± 0.3, p = 0.005∗∗). Furthermore, there is a significant difference comparing low dose vs. high dose (p = 0.004∗) as well as comparing all three groups together (p = 0.0008). (B) HCAECs stimulated with lipopolysaccharide (LPS) significantly upregulate TNFα mRNA (Δ11.3 ± 3, p = 0.001∗∗), and low dose (20 ng) of SDF-1α attenuate this elevation (Δ−4.8 ± 1.6, p = 0.03∗). ∗p < 0.001.
FIGURE 4
FIGURE 4
Low dose SDF-1α is protective against both mitochondrial reactive oxygen species (ROS) AND nitrosative stress. (A) Representative immunofluorescence images of human coronary artery endothelial cells (HCAECs) incubated with the mitochondrial O2– – sensitive fluorescent dye MitoSOX Red (10 um), and DAPI (blue) after treatment with SDF-1α recombinant (20 ng; n = 4), allogeneic MSC conditioned medium (n = 4) or autologous MSC conditioned medium (n = 5) and stimulated with LPS (1 ug/mL). (B) Quantification of MitoSOX Red fluorescence showed that LPS significantly increased superoxide production compared to control (301676 ± 7843 vs. 112664 ± 22307, p < 0.0001∗), whereas SDF-1α, allogeneic MSC conditioned medium, and autologous MSC conditioned medium prevented superoxide production induced by LPS (58674 ± 8145, 52665 ± 33625, 39535 ± 13687 AU, p < 0.0001∗, respectively. (C) Representative immunofluorescence images via confocal microscopy of HCAECs incubated with H2-DCF-DA (Green) that detects intracellular peroxides including H2O2 and DAPI (blue). (D) Quantification of the average of DCF fluorescence showing LPS induced significantly higher rates of hydrogen peroxide production compared to control (22491 ± 16371 vs. 4464 ± 2047, p < 0.0001∗), while SDF-1α, allogeneic MSC conditioned medium, and autologous conditioned medium attenuated cell hydrogen peroxide production (6172 ± 3920 AU, p < 0.01∗∗∗, 9753 ± 4751 AU, p < 0.05#, 46941 ± 89798, p < 0.05#, respectively). (E) Representative pictures of nitrotyrosine (3NT) expression in HCAECs stimulated with LPS followed by SDF-1α, allogeneic MSC conditioned medium, and autologous conditioned medium. (F) Quantification of 3NT fluorescence demonstrating that LPS significantly increased nitrotyrosine expression (75894 ± 13874 vs. 43546 ± 10020 AU, p < 0.001∗∗, and only SDF-1α and allogeneic MSC conditioned medium reduced nitrotyrosine expression (52132 ± 4395 AU, p < 0.05#, 34761 ± 4898 AU, p < 0.0001∗∗∗). Autologous MSC conditioned medium did not block LPS induced nitrotyrosine expression (56949 ± 21648 vs. 75894 ± 13874, respectively, p = NS).
FIGURE 5
FIGURE 5
Injection of MSCs into patients with dilated cardiomyopathy reduces elevated TNFα levels and improves endothelial function as measured by an improvement in endothelial progenitor cell (EPC) colonies and flow-mediated vasodilation (FMD). These effects remain intact when SDF-1∝ levels are low. On the other hand, when SDF-1α levels are high, the beneficial effects of MSCs are inhibited.

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