Therapeutic Cell Protective Role of Histochrome under Oxidative Stress in Human Cardiac Progenitor Cells

Abstract

Cardiac progenitor cells (CPCs) are resident stem cells present in a small portion of ischemic hearts and function in repairing the damaged heart tissue. Intense oxidative stress impairs cell metabolism thereby decreasing cell viability. Protecting CPCs from undergoing cellular apoptosis during oxidative stress is crucial in optimizing CPC-based therapy. Histochrome (sodium salt of echinochrome A-a common sea urchin pigment) is an antioxidant drug that has been clinically used as a pharmacologic agent for ischemia/reperfusion injury in Russia. However, the mechanistic effect of histochrome on CPCs has never been reported. We investigated the protective effect of histochrome pretreatment on human CPCs (hCPCs) against hydrogen peroxide (H₂O₂)-induced oxidative stress. Annexin V/7-aminoactinomycin D (7-AAD) assay revealed that histochrome-treated CPCs showed significant protective effects against H₂O₂-induced cell death. The anti-apoptotic proteins B-cell lymphoma 2 (Bcl-2) and Bcl-xL were significantly upregulated, whereas the pro-apoptotic proteins BCL2-associated X (Bax), H₂O₂-induced cleaved caspase-3, and the DNA damage marker, phosphorylated histone (γH2A.X) foci, were significantly downregulated upon histochrome treatment of hCPCs in vitro. Further, prolonged incubation with histochrome alleviated the replicative cellular senescence of hCPCs. In conclusion, we report the protective effect of histochrome against oxidative stress and present the use of a potent and bio-safe cell priming agent as a potential therapeutic strategy in patient-derived hCPCs to treat heart disease.

Keywords: cardiac progenitor cells; cell therapy; echinochrome A; histochrome; oxidative stress.

Figure 1

Effects of histochrome treatment on human cardiac progenitor cells (hCPCs) characterization. (A) Chemical structure of echinochrome A—active substance of the histochrome drug. (B) hCPCs were treated with different concentrations of histochrome for 24 h and viability was measured using cell viability, Proliferation & Cytotoxicity assay (CCK assay). Data are presented as the mean ± standard deviation (SD). **, p < 0.01 versus 0 μM, ***, p < 0.001 versus 0 μM. n = 6 (C) Morphological analysis of hCPCs pretreated with histochrome. Scale bar = 100 μm, (D) Expression of stem cell marker by flow cytometric analysis, n = 3. Error bars indicate standard effort of the mean (S.E.M)

Figure 2

Intracellular reactive oxygen species (ROS) and mitochondrial ROS scavenging activity of histochrome in hCPCs. (A) hCPCs were pretreated with histochrome at 0 μM, 5 μM, 10 μM, and 20 μM for 24 h followed by the addition of 600 μM H₂O₂ for 1 h. Intracellular ROS scavenging activity was measured using CellRox staining. Representative image of increased intensity of CellRox produced by ROS and decreased intensity on pretreating with histochrome. Data are presented as the mean ± SD of three independent experiments. Scale bar = 100 μm, ### p < 0.01 versus -H₂O₂ -histochrome; * p < 0.05; ** p < 0.01 versus +H₂O₂ -histochrome, n = 3. Error bars indicate S.E.M. (B) 2’,7’–difluorofluorescin diacetate (H₂-DFFDA)assay was used to measure cellular ROS production. *** p < 0.001 versus -H₂O₂ -histochrome; ###, p < 0.001 versus +H₂O₂ -histochrome, n = 3. Error bars indicate S.E.M. (C) After pretreatment with histochrome for 24 h, hCPCs were exposed to H₂O₂ for 1 h and mitochondrial superoxide production was measured with MitoSOX staining. Representative image of the increased intensity of MitoSOX and decreased intensity on pretreatment with histochrome. Scale bar = 100 μm.

Figure 3

Anti-apoptotic effect of histochrome against H₂O₂-induced cell death. (A) hCPCs were pretreated with 10 µM of histochrome for 24 h and then exposed to 1 mM H₂O₂ for 4 h. Apoptotic cells were quantified by fluorescence-activated cell sorting (FACS) analysis with Annexin V / 7-AAD staining. ** p < 0.01; *** p < 0.001 versus -H₂O₂ -histochrome; ### p < 0.001 versus +H₂O₂ -histochrome. (B) Representative images showing the morphology of hCPCs pretreated with histochrome (0 µM, 5 µM, 10 µM, and 20 µM) in the presence of H₂O₂-induced oxidative stress. Morphology of hCPCs was observed by phase contrast microscope. Scale bar = 50 μm (C) Live cells were quantified by phalloidin (green fluorescence) intensity. *** p < 0.001 versus -H₂O₂ -histochrome; # p < 0.05 versus +H₂O₂ -histochrome. Scale bar = 100 μm, n = 3. Error bars indicate S.E.M.

Figure 4

hCPCs pretreated with histochrome show downregulation of pro-apoptotic signals and upregulation of anti-apoptotic signals under the oxidative stress condition. (A) hCPCs were pretreated with histochrome for 24 h, oxidative stress was induced in hCPCs by 1 mM H₂O₂. Expression of apoptosis signaling-related proteins was determined by western blotting. (B) Immunofluorescence was performed with the DNA damage marker γH2A.X to quantify DNA damage of hCPCs. Images were captured using a LionHeart FX automated microscope (Biotek, Winooski, VT, USA). *** p < 0.001 versus +H₂O₂, n = 5. Error bars indicate S.E.M.

Figure 5

Effect of prolonged treatment with histochrome on hCPCs senescence. (A) Representing images of senescence-β-galactosidase (SA- β-gal) stained hCPCs (Scalebar = 50 μm). (B) SA- β-gal positive cells were quantified and presented as a graph (*** p < 0.001, versus Young; ### p < 0.001, versus Sene) Error bars indicate S.E.M. Abbreviation: Sene, senescent hCPCs (passage 13); sene + histochrome, prolonged treatment with histochrome (till passage 13).

Figure 6

Schematic representation of cytoprotective effects of histochrome against H₂O₂-induced cell death via reduction of DNA damage and activation of survival signaling.