Role of hydrogen peroxide preconditioning in mesenchymal stem cell-mediated heart regeneration: Molecular insights

Abstract

Mesenchymal stem cells (MSCs) possess unique properties such as immunomodulation, paracrine actions, multilineage differentiation, and self-renewal. Therefore, MSC-based cell therapy is an innovative approach to treating various degenerative illnesses, including cardiovascular diseases. However, several challenges, including low transplant survival rates, low migration to the ischemic myocardium, and poor tissue retention, restrict the application of MSCs in clinical settings. These undesirable cell therapy outcomes mainly originated due to the overproduction of reactive oxygen species (ROS) in the injured heart. MSCs' stress-coping capacity can be enhanced by preconditioning them under conditions similar to the microenvironment of wounded tissues. Hydrogen peroxide (H₂O₂) is a ROS that has been shown to activate protective cellular mechanisms such as survival, proliferation, migration, paracrine effects, and differentiation at sublethal doses. These processes are induced via phosphatidylinositol 3-kinase/protein kinase B, p38 mitogen-activated protein kinases, c-Jun N-terminal kinase, Janus kinase/signal transducer and activator of the transcription, Notch1, and Wnt signaling pathways. H₂O₂ preconditioning could lead to many clinical benefits, including ischemic injury reduction, enhanced survival of cellular transplants, and tissue regeneration. In this review, we present an overview of stem cell preconditioning methods and the biological functions activated by H₂O₂ preconditioning. Furthermore, this review explores the molecular mechanisms underlying the protective cellular functions stimulated under H₂O₂ preconditioning.

Core Tip: Hydrogen peroxide (H₂O₂) preconditioning is reported to increase protective mechanisms in mesenchymal stem cells and enhance their ability to regenerate in damaged hearts. However, its molecular mechanism remains under-explored. This review highlights the studies on the protective biological functions and regulatory mechanisms associated with H₂O₂ preconditioning in heart regeneration. Moreover, the role of the H₂O₂-activated redox-sensitive transcription factors and their associated pathways in cell survival, proliferation, migration, paracrine effect, and cardiac differentiation is discussed. The molecular insights provided in this review will possibly help in the development of cardiovascular regenerative therapy.

INTRODUCTION

Redox imbalance, which is caused by a compromised antioxidant system or an overabundance of reactive oxygen species (ROS), is a major factor in cardiovascular disorders such as myocardial infarction (MI), ischemia-reperfusion damage, end-stage heart failure, and cardiomyopathy[1]. Heart transplantation remains the most effective treatment for heart failure because existing treatments have limited efficacy and cannot restore the heart's normal functions. However, this procedure is limited due to donor organ unavailability, immunological rejection, long-term complications, and higher expenses[2].

Mesenchymal stem cell (MSC) therapy has shown promise as an alternative method for restoring heart function in individuals with cardiovascular disorders[3]. MSCs are adult stem cells that can self-renew and differentiate into several lineages. These cells are found in bone marrow, adipose tissue, umbilical cord, and several other tissues and organs[4]. Studies have demonstrated that transplanted MSCs can differentiate into vascular cells and cardiomyocytes in heart models[5]. Since stem cell self-renewal and differentiation are dependent upon the redox balance of the tissue, stem cells cannot survive in inflamed tissue for long periods due to the hostile microenvironment[6].

Preconditioning is a technique that refers to the brief exposure of cells to mild stressors to get them ready for the harsh environment that they may experience in the diseased tissue[7]. Numerous preconditioning methods, such as chemical agents, hypoxia, gene modification, pharmacological agents, and physical factors have been investigated to increase the overall effectiveness of MSC transplantation[8]. It has been reported that pretreatment of MSCs with a controlled stressor can improve their proliferative, secretory, migratory, and differentiated properties[9]. Numerous studies have demonstrated that hydrogen peroxide (H₂O₂) acts as a signaling molecule at low concentrations, enabling cells to establish a cytoprotective response against stress[10,11]. In this review, we discuss MSC preconditioning methods with an emphasis on the stem cell functions modulated by H₂O₂ preconditioning. We also provide an overview of the molecular mechanisms underlying the protective cellular mechanism stimulated by H₂O₂ preconditioning.

MSCS IN HEART DISEASES

MSCs are increasingly being used in regenerative medicine to develop cell treatments. MSCs have demonstrated remarkable therapeutic results in several preclinical disease models, including cardiac diseases[12]. It has been demonstrated that MSCs, their genetic modifications, conditioned medium, or extracellular vesicles (EVs), promote regeneration after cardiac tissue injury[4]. Moreover, various combinations of cardiac transcription factors (TFs) such as GATA binding protein 4 (GATA-4), Mef2c, and NK2 homeobox 5 (Nkx2.5) play important roles in cardiac differentiation and heart regeneration[13]. Paracrine signals including insulin-like growth factor (IGF), hepatic growth factor, and vascular endothelial growth factor (VEGF) are reported to stimulate transdifferentiation of stem cells into cardiomyocytes and endothelial cells[14]. Studies reported that anti-inflammatory, anti-fibrotic, anti-apoptotic, and angiogenic properties are mediated through MSC-derived exosomal microRNAs (miRNAs)[4]. However, the long-term clinical translation of MSC therapy is hindered by the poor survival of MSCs in damaged hearts. Therefore, it is crucial to discover new methods to improve the therapeutic efficiency of MSCs. There is growing evidence that specific preconditioning methods can prime MSCs for cell therapy in vitro. Moreover, preconditioning techniques can maximize the reparative and regenerative capacity of MSCs by improving their paracrine, engraftment, and survival properties[8]. Presently, several in vitro preconditioning techniques have been used to increase MSCs' ability to regenerate in damaged hearts.

CELL PRECONDITIONING METHODS

Hypoxic preconditioning

Hypoxic preconditioning is a method where cells are exposed to moderate hypoxia for a shorter period of time which results in increased resistance to severe ischemic conditions[15]. Preconditioning techniques regulate a range of biological processes by either activating or suppressing chemical signals and signal transduction pathways[9]. Hypoxic preconditioned MSCs (HP-MSCs) release a wide range of substances with anti-apoptotic, pro-angiogenic, trophic, immunomodulatory, or immunosuppressive properties[16]. A number of studies have demonstrated the therapeutic potential of HP-MSCs in heart regeneration[9,17,18]. It has been reported that HP-MSCs following transplantation provide cardioprotective benefits in terms of increased neovascularization and reduced fibrosis[19]. According to research, controlled hypoxia is reported to improve cardiovascular health and oxygen supply by increasing blood vessel density, myocardial contractility, and erythropoietin (EPO) production[20]. Moreover, hypoxia is reported to enhance EVs loaded with miRNAs such as miR-210 to heal MI[21].

Preconditioning with mechanical/electrical /magnetic stimuli

A number of physical preconditioning methods such as shear stress, stretch, compression, and electrical or magnetic fields have shown promising results in the field of regenerative medicine[22]. These methods are reported to enhance MSCs’ ability to regenerate damaged tissues[7,23]. It has been reported that shear stress improves MSC alignment and stimulates the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/endothelial nitric oxide (NO) synthase pathway, which in turn promotes angiogenesis and healing of ischemic cardiac tissues[24]. Cyclic stretch is reported to enhance survival and cardiac differentiation of MSCs through the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinases (ERK) and YAP/TAZ pathways[25,26]. Electrical stimulation has been shown to increase calcium signaling and the Wnt/β-catenin pathway, which results in cardiac differentiation of MSCs[27]. MSCs preconditioned with magnetic fields are reported to boost the repair mechanism of the injured heart via increased migration and homing[28].

Preconditioning with biological and pharmacological agents

Biological and pharmacological preconditioning agents have been shown to enhance MSC survival, proliferation, differentiation, and paracrine activities in the injured myocardium[8]. MSCs enhance their survival and integration properties in infarcted heart tissue once preconditioned with growth factors and cytokines such as fibroblast growth factor (FGF)-2, VEGF, and stromal cell-derived factor-1 (SDF-1). These paracrine factors have been shown to activate pro-survival and anti-apoptotic pathways PI3K/Akt and ERK1/2 in preconditioned MSCs[29]. C-X-C motif chemokine receptor 4 (CXCR4)/stromal cell-derived factor-1 (SDF-1) signaling is reported to play an important role in MSC survival in the ischemic myocardium which results in infarct repair and enhanced vascular density[30]. Research indicates that EPO treated MSCs reduce apoptosis and fibrosis, and enhance survival through the PI3K/Akt pathway in MI animal models[31]. It has been reported that tumor necrosis factor-alpha preconditioned MSCs release anti-inflammatory cytokines, which in turn enhance the immunomodulatory properties of MSCs[32]. Additionally, MSCs preconditioned with growth differentiation factor 15 are reported to show anti-inflammatory properties in an MI model via the AMP-activated protein kinase (AMPK) pathway[33]. These preconditioning approaches primarily provide cytoprotection. Yet, we should examine preconditioning strategies which more accurately replicate the ischemic state of the infarcted heart while enabling positive tissue regeneration and differentiation, besides increasing cell resistance to stress. ROS, proinflammatory cytokines, and other stress-related molecules are present in high concentrations in the infarcted myocardial environment. However, H₂O₂ is a well-defined ROS, which at sublethal doses can act as a second messenger and also a mediator of oxidative stress. Recently, it has been shown that preconditioning MSCs with low doses of H₂O₂ for shorter periods makes them more resilient to oxidative stress. These H₂O₂ preconditioned MSCs[34-38] have been reported to activate various protective mechanisms to improve therapeutic potential of stem cells in cell therapy procedures[39-41]. It has been reported that H₂O₂ preconditioning enhance tissue engraftment and accelerate wound healing in mice via the activation of the SDF-1/CXCR4 and PI3K/Akt/mammalian target of rapamycin pathways and inhibition of glycogen synthase kinase-3β in MSCs[35]. Another study demonstrated that H₂O₂ pretreated human umbilical cord vein MSCs repaired ovary injured in mice, presumably by increasing the migration and survival of the stem cell[36]. Recent research provides shreds of evidence that the paracrine factors released from H₂O₂-preconditioned human adipose-derived stem cells efficiently reduce ultraviolet (UV) B-induced oxidative stress by a higher nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant system[37]. Mahmoudi et al[39] in 2020 reported that optimized concentration of H₂O₂ can enhance therapeutic potential of MSCs in idiopathic pulmonary fibrosis. A study carried out by Boopathy et al[42] has demonstrated cardiac differentiation of H₂O₂ preconditioned MSCs via Notch1 and Wnt11 signaling pathways. These studies provide evidence for the hypothesis that H₂O₂ preconditioning is a strategy for promoting tissue-regeneration, wound healing, cell survival, and differentiation. Hence, the mechanism of H₂O₂ preconditioning needs to be explored further.

POTENTIAL CELLULAR MECHANISMS STIMULATED BY H₂O₂ PRECONDITIONING IN MSCS

H₂O₂ preconditioned MSCs promote survival

For efficient utilization of MSCs in heart regeneration, their survival must be increased by triggering adaptive cellular responses. A number of research studies support the narration that H₂O₂ preconditioning induces cell survival in MSCs by upregulating various signaling pathways such as Janus kinase/signal transducer and activator of the transcription, Akt, Nrf1, Nrf2, p38 MAPK, PI3K/Akt, Notch1, and Wnt11[34,42-44]. In 2020, Garrido Pascual[45] reported that preconditioning with 0.25 mmol H₂O₂ for 1 hour regulates the effects of oxidative stress in transplanted MSCs by increasing the expression of the transcription factor Nrf2 and antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), heme oxygenase-1 (HO-1), and glutathione peroxidase (GPx)-1. Moreover, these preconditioned MSCs are reported to increase cell survival by reducing the secretion of pro-inflammatory molecules cyclooxygenase-2 and interleukin (IL)-1β and by increasing adenosine triphosphate production. Sadidi et al[46] in 2009 have also reported increased survival and growth of preconditioned MSCs via the PI3K/Akt signaling pathway. According to the research by Nouri et al[34] in 2019, MSCs pretreated with 20 μM H₂O₂ for 12 hours exhibited enhanced cell survival in the ischemic heart via the hypoxia-inducible factor-1α (HIF-1α) and PI3K/Akt pathways.

H₂O₂ preconditioned MSCs promote proliferation

Adequate cell count is a key factor that boosts MSCs’ capability to repair and regenerate damaged tissues[47]. MSCs’ functional characteristics such as proliferation, adhesion, and migration are reported to be enhanced by H₂O₂ preconditioning[41]. In 2012, Zhang et al[48] reported that preconditioning of MSCs with a low concentration of H₂O₂ stimulates cell migration and proliferation via IL-6 secretion. Moreover, H₂O₂ treated cells have been shown to modulate the production of phase II antioxidant enzymes via the PI3K/Akt and p38 MAPK pathways, thus promoting proliferation[44]. Similar proliferation results of preconditioned stem cells have been reported in a in vitro study of 2019 by Castro et al[49], who stated that the cryopreserved H₂O₂ preconditioned stem cells show improved bioactivity against oxidative stress through increased adhesion and cell cycle progression, suggesting a greater ability for proliferation.

H₂O₂ preconditioned MSCs promote migration

Cell migration plays a key role in various physiological processes including tissue regeneration and repair. A number of studies[34] have reported that H₂O₂ preconditioning enhances MSCs’ migration to ischemic myocardium following transplantation[41,48-51]. According to Zhang et al[48]’s research, H₂O₂ treated MSCs increase IL-6 secretion, which is found to be necessary for endothelial cell migration and proliferation, thus contributing to the remodeling of post-myocardial damage in the MI model. It has been reported that MSCs preconditioned with a non-toxic dose of H₂O₂ enhance cell migration and survival via modulation of Bax/Bcl-2 ratio and pro-survival proteins Bcl-2[34]. Notably, the destabilization of redox-sensitive phosphatase Dusp6 is a crucial step required for heart regeneration. H₂O₂ is reported to destabilize Dusp6 and activate the MAPK/ERK pathway, thus controlling several cellular activities, including migration and proliferation[50]. Moreover, it has been reported that pretreatment of MSCs with cytokines and H₂O₂ regulates their adhesion to cardiac endothelium, thus improving cell migration[43].

H₂O₂ preconditioned MSCs enhance release of paracrine factors

Preconditioning enhances MSCs' therapeutic potential by stimulating the release of paracrine chemicals through a variety of signaling pathways. A study has been carried out by Pankajakshan et al[52] has shown the H₂O₂ preconditioned MSCs release key paracrine factors involved in heart regeneration, such as VEGF, FGF-2, IGF-1, and IL-6. These soluble factors support heart regeneration via angiogenesis, cell survival, and immunomodulation[52]. A possible method to boost MSC-mediated tissue regeneration was suggested by a study, where MSCs were preconditioned with H₂O₂. These preconditioned MSCs showed higher proliferation and decreased inflammation rate through paracrine effects[53]. Furthermore, the research conducted by Pendergrass et al[54] described that H₂O₂ preconditioned cardiac progenitor cells stimulate neoangiogenesis in the peri-infarct region of an ischemia-reperfusion injury model. Another investigation revealed that H₂O₂ preconditioned MSCs stimulate the production of IL-6, which in turn enhances neovascularization and decreases cardiac fibrosis[48]. Moreover, H₂O₂ preconditioned cardiac progenitor cells show reduced fibrosis and enhanced angiogenesis and vascular density via the PI3K-dependent pathway[54].

H₂O₂ preconditioned MSCs enhance cardiac differentiation

H₂O₂ has been shown to act as signaling molecules that affect several biological pathways. Recently, it has been demonstrated that the key cardiovascular pathways, Notch1 and Wnt11, are activated in H₂O₂ preconditioned MSCs[55]. Notch1 signaling activation is reported to improve MSC based therapies for cardiac repair and regeneration[56]. A study conducted by Boopathy et al[42] reported that H₂O₂ mediated oxidative stress activates Notch1 signaling, which promotes cardiogenic gene expression in bone marrow derived MSCs. Exogenous H₂O₂ at low concentrations shows higher expression of cardiogenic TFs, cytokines, and a number of stem cells derived beating cardiomyocytes[57]. It is well-documented that H₂O₂ preconditioning enhances various cell protective mechanisms, survival, proliferation, migration, paracrine effects, and differentiation, by activating intracellular signaling pathways (Figure 1 and Table 1)[34,41-45,48-50,54,58-61]. However, there is still a large gap that needs to be filled with a thorough investigation of the underlying mechanisms of H₂O₂ preconditioning in heart regeneration.

Figure 1.

Hydrogen peroxide preconditioned mesenchymal stem cells promote heart regeneration. Redox sensitive transcription factors activated by hydrogen peroxide preconditioning regulate various biological functions such as cell survival, proliferation, migration, paracrine effects, and differentiation. This leads to an improvement in the regeneration of the heart. AP-1: Activator protein-1; HIF-1α: Hypoxia-inducible factor-1α; H₂O₂: Hydrogen peroxide; MSCs: Mesenchymal stem cells; Nrf2: Nuclear factor erythroid 2-related factor 2.

Table 1.

Hydrogen peroxide preconditioning promote heart regeneration

Cells	Cellular mechanism(s)	Redox sensitive transcription factor(s)	Pathway(s)	Major findings	Ref.
Adipose-derived stem cells (hASCs)	Survival	Nrf2	-	Increased: Antioxidant response; reduced: Inflammation; bioenergetic adaptation; nuclear factor-kappa B modulation	Garrido Pascual[45]
Cardiomyocytes	Survival	Nrf2	-	Increased: Mitochondrial membrane potential; reduced: Oxidative stress, apoptosis in cardiomyocytes	Dreger et al[58]
Cardiomyocytes	Proliferation and survival	Nrf1 and Nrf2	P38 MAPK and phosphatidylinositol 3-kinase/Akt	Increased: Phase II enzymes and Bcl2; reduced: Oxidative stress, apoptosis, and caspase-3; resistance to oxidative stress in ischemia-reperfusion injury	Angeloni et al[44]
Zebrafish cardiomyocytes	Proliferation and survival	P53	-	Increased: Proliferation and redox homeostasis; reduced: Reactive oxygen species levels and scar formation	Ye et al[59]
Human Wharton's jelly-derived MSCs	Proliferation, survival, and migration	Hypoxia-inducible factor-1α	Akt	Increased: Resistance to oxidative stress and cell viability; reduced: Apoptosis	Nouri et al[34]
Human MSCs	Survival	-	-	Increased: Adhesion	O'Leary[43]
Mice myocytes	Survival	Catalase	-	Regulate calcium handling and improve ventricular diastolic dysfunction; preserve sarcoplasmic reticulum calcium ATPase activity	Qin et al[60]
Wharton's Jelly-derived MSCs	Survival, migration, paracrine effect, and differentiation	-	-	Increased: Neovascularization and interleukin-6; reduced: Fibrosis	Zhang et al[48]
HASCs	Proliferation, paracrine effects, and migration	-	-	Increased: Insulin-like growth factor-1, PFKFB4, pyruvate dehydrogenase kinase isoenzyme 1, resistance to oxidative stress, glycolysis; reduced: Oxidative phosphorylation	Castro et al[49]
Human decidua basalis mesenchymal stem/stromal cells	Proliferation, migration, and survival	-	-	Increased: Colony formation, adhesion, migration, wound-healing, tube formation, and oxidative stress resilience	Khatlani et al[41]
Zebrafish heart lines	Migration, and differentiation	-	MAPK and extracellular signal-regulated kinases 1/2	Increased: Duox and NADPH oxidase 2; reduced: Dusp6	Han et al[50]
Cardiac progenitor cells	Paracrine effect, differentiation, survival	-	-	Increased: Angiogenesis, endothelial cell density, survival, and cardiac function post-I/R injury; reduced: Fibrosis, apoptosis	Pendergrass et al[54]
Mouse cardiac myocytes	Survival and paracrine effect	-	AMP-activated protein kinase and Akt	Increased: Myocyte contractility; selective activation of nitric oxide synthase isoforms	Sartoretto et al[61]
Bone marrow-derived MSCs	Differentiation	-	Notch1	Increased: Flt1, vWF, PECAM1, NK2 homeobox 5, αMHC, Notch1, Wnt11, cardiac repair and regeneration	Boopathy et al[42]

Akt: Protein kinase B; HASCs: Human adipose-derived stem cells; MAPK: Mitogen-activated protein kinase; MSCs: Mesenchymal stem cells; Nrf2: Nuclear factor erythroid 2-related factor 2.

Role of H₂O₂-activated redox sensitive TFs in heart regeneration

Mammalian cells continuously produce ROS as byproducts of electron transport activities. At physiological levels, ROS serves a crucial role in redox signaling through various post-translational modifications. However, increased ROS production causes molecular damage, a condition known as oxidative distress. Therefore, maintaining the redox balance is crucial for normal cell functioning[62]. H₂O₂, a ROS and an essential redox signaling agent, is produced by several metabolic activities in mammalian cells[63]. It is an intracellular messenger that regulates a number of physiological processes by reversible oxidation of the thiol side chain of cysteine residues of the effector proteins, thereby modulating the downstream gene expression and cell behaviors[57]. Heart development is regulated by a network of TFs such as Nkx2.5, GATA-4, Mef2c, and Tbx5[13]. Cardiac TFs might not be directly targeted by ROS, but their regulation can be controlled by redox sensitive TFs[57]. According to earlier research[64], oxidative stress caused by low-concentration H₂O₂ supplementation to culture media activates redox sensitive TFs, including Nrf2, activator protein-1 (AP-1), p53[65], and HIF-1α[34]. These TFs control the intracellular signaling pathways involved in the development/regeneration of the heart[58,59,66,67]. However, the precise role of redox sensitive TFs in heart repair is not fully understood yet. Therefore, it is now essential to uncover their molecular mechanism to target specific biological pathways to ensure improved survival, proliferation, and differentiation of cells in ischemic myocardium, thus increasing the outcome of cardiovascular regenerative therapy.

Nrf2

The basic leucine zipper transcription factor Nrf2 is encoded by the human NFE2 L2 gene. In normal conditions, Nrf2 resides in the cytoplasm because it assembles with kelch-like ECH-associated protein 1 (Keap1). Keap1 is the main negative regulator of Nrf2 that constantly ubiquitylates and degrades Nrf2. Deubiquitination, which is caused by the disruption of the Keap1-Cul3 E3 Ligase complex, releases and accumulates Nrf2 in the nucleus. In the nucleus, Nrf2 interacts with Maf proteins and binds to the antioxidant-response elements (ARE). This interaction promotes the transcription of Nrf2 target genes such as HO-1, GPx, CAT, and SOD. These enzymes are reported to promote cell viability, migration, and proliferation by balancing the redox status of cells[68].

Nrf2's complex transcriptional, translational, and post-translational network regulation allows it to coordinate adaptive responses to different types of stress. It has been proposed that Nrf2 acts as a mediator of protective responses since it is activated in response to moderate concentrations of various stressors and conditions[69]. In the process of tissue regeneration, stem and progenitor cell proliferation is reported to be promoted by Nrf2[70]. Moreover, Nrf2 regulates apoptosis through inhibition of pro-apoptotic and upregulation of anti-apoptotic proteins. In addition, Nrf2, directly or indirectly, controls the expression of cell cycle genes like cyclin D1[71] and c-Myc[72]. It has been documented in various studies that 10–500 μM H₂O₂ for 10 minutes to 6 hours can activate Nrf2 in a variety of cells[37,73,74]. In a study in 2022, Ishii et al[73] reported that H₂O₂-activated kinases liberate Nrf2 from caveolin-1, facilitating its nuclear translocation via p38/nSMase2/ceramide signaling. Furthermore, it has been demonstrated that the H₂O₂ activated Nrf2 enters the nucleus, attaches to AREs in target gene promoters, and upregulates the expression of various antioxidant enzymes, which guards cardiac cells from oxidative damage[58]. In 2022, a similar finding was reported by Yang et al[75] that Nrf2 induces the expression of HO-1 in the heart. This enzyme breaks down heme into biliverdin, carbon monoxide, and free iron, providing cytoprotective benefits against oxidative damage[75]. Moreover, it has been reported that Nrf2 activation promotes the pentose phosphate pathway and glucose metabolic reprogramming, which lessens cardiac dysfunction[76].

AP-1

The transcription factor AP-1 is produced by various dimeric combinations of Fos and Jun families. The synthesis of new Jun and Fos family proteins is triggered by ROS and is dependent on pre-existing TFs[77]. AP-1 is reported to control the signal transduction pathways that lead to cell growth and transformation[78]. Moreover, AP-1 activity is noticeably increased in the presence of antioxidants such as pyrrolidine dithiocarbamate and butylated hydroxyanisole[79]. In response to a variety of stimuli, such as growth hormones, cytokines, oxidative stress, and other environmental signals, AP-1 attaches to the TPA response element in target genes' promoter regions and regulates the transcription of stress responses, apoptosis, differentiation, and cell proliferation[80].

In 2013, a study conducted by Windak et al[81], reported that c-Jun and c-Fos play critical role in the induction of cardiac fetal genes. c-Jun has been shown to shield the heart against pathologic remodeling and maintain the correct arrangement of the cytoskeleton and sarcomeric structure in cardiomyocytes. Moreover, deletion of c-Jun in cardiomyocytes is linked to a higher incidence of cardiac apoptosis and fibrosis. ROS-dependent activation of c-Jun is reported to be controlled by the c-Jun N-terminal kinase (JNK)-dependent and MAPK family signaling pathway[81]. It has been reported that the coordinated activation of JNK, ERK, and p38 MAPKs increases the DNA binding and transactivation capacity of c-Jun in P19 cells[82]. According to the bioinformatics investigations, the cardiac transcription factor GATA-4 promoter region contains three putative binding sites for AP-1 that bind to activated c-Jun and promote the redox-dependent increase in GATA-4 transcription. This process possibly drives the differentiation of stem cells into cardiac cells during the process of embryonic development[83].

AP-1 production is stimulated by various stimuli such as UV light, ionizing radiation, or H₂O₂[84]. In 2006, Kikuta et al[85] reported that 100 μM H₂O₂ for 25 hours activates AP-1 by MAPKs, which phosphorylate AP-1 components and increase their transcriptional activity. In earlier studies, it was reported that H₂O₂ treatment increases AP-1 DNA binding, Jun nuclear kinase activity, and the levels of c-Fos and c-Jun mRNA[86]. AP-1 is reported to regulate the expression of certain antioxidant enzymes and proteins, as well as transforming growth factor-1 beta (TGF-1β)[87], cyclin D1[88], and many cytokines genes, which help in cell cycle progression. Moreover, AP-1 is reported to regulate cellular functions necessary for heart repair, thus playing a crucial role in cardiac regeneration. A recent study in 2020 by Beisaw et al[66], demonstrated that AP-1 regulates the expression of metabolism and cell cycle progression related genes during the process of heart regeneration as well as facilitates the expression of genes necessary for cardiomyocyte regeneration by regulating chromatin accessibility. According to Zuppo et al[89], cardiomyocyte proliferation is hampered when AP-1 expression is reduced, highlighting its crucial function in cell proliferation.

HIF-1α

The transcription factor HIF-1α is essential for coordinating cellular reactions to hypoxia or low oxygen supply[90]. Under normoxic conditions, prolyl hydroxylase domain (PHD) enzymes hydroxylate HIF-1α at particular proline residues. This post-translational alteration helps the von Hippel-Lindau E3 ubiquitin ligase complex to ubiquinate and degrade HIF-1α by proteasomal destruction[91]. Hypoxic preconditioning has been shown to increase the expression of HIF-1α through inhibition of PHDs. Similarly, ROS is also reported to stabilize HIF-1α by inhibiting PHD enzymes[92].

HIF-1α is reported to regulate angiogenesis, metabolism, and survival pathways, thus preserving cellular homeostasis in stressful situations[90]. Moeinabadi-Bidgoli et al[7] in 2021 reported that HIF-1α stimulates the production of SDF-1/CXCR4/CXCR7 signaling molecules, which play a crucial role in cell migration. Moreover, it enhances cell survival and proliferation through PI3K/Akt signaling[7]. In 2015, a study conducted by Zimna and Kurpisz[93], demonstrated that HIF-1α increases vessel formation by stimulating VEGF production. Moreover, HIF-1α at low oxygen levels, stimulates the expression of genes that produce glycolytic enzymes and pyruvate dehydrogenase, thus regulating metabolic reprogramming[94]. In 2014, a study conducted by Kuiper et al[95], demonstrated that H₂O₂ stabilizes HIF-1α by regulating intracellular ascorbate concentration. Similarly, another study reported that H₂O₂ enhances cell survival under stress conditions possibly through the stabilization of HIF-1α[96].

Preconditioning with a non-cytotoxic dose of H₂O₂ is reported to increase cell tolerance in stem cells. Chang et al[97] in 2008 reported that HIF-1α inhibition can reverse the H₂O₂ preconditioning mediated increased tolerance. Sun et al[98] in 2020 reported that HIF-1α overexpressing MSCs enhance cardiac functions and angiogenesis by increasing MSCs’ adhesion, migration, and paracrine effect. These results have suggested that HIF-1α is an important mediator in stem cell preconditioning for cardiac cell therapy. Similar results were reported by Cerrada et al[51]’s study which demonstrated that HIF-1α engineered MSCs enhance the expression of cell adhesion, migration, and paracrine factors genes. These genetically modified MSCs promote cardiac function in terms of angiogenesis, neovascularization, and heart regeneration. Moreover, expression of HIF-1α is reported to increase stem cell survival and cardiac differentiation[51].

P53

The tumor suppressor protein p53 plays a crucial role in the regulation of the cell cycle, DNA repair, apoptosis, and senescence. P53 is reported to activate NCF2/p67phox, a component of NADPH oxidase 2, which produces ROS[99]. However, it can also inhibit ROS accumulation in low-stress settings[100]. It has been established that p53 regulates several antioxidant enzymes, such as SOD2, GPx1[101], CAT[102], Grx[103], Prxs, and Sestrins[104]. Aubrey et al[105] in 2017 reported that p53 prevents apoptosis in mild stress situations by upregulation of anti-apoptotic genes like Bcl-2 and the downregulation of pro-apoptotic genes like PUMA and NOXA. This process enables cells to endure stress and bounce back[105]. In addition, p53 regulates DNA repair and antioxidant responses, which are essential for tissue regeneration and homeostasis[106]. Under stress conditions, p53 preserves energy balance by regulating metabolic processes including glycolysis and oxidative phosphorylation, which in turn improves cell survival and proliferation[107]. Moreover, p53 is reported to stimulate the production of growth factors like VEGF[108] and TGF-β[109] as well as cytokines (IL-6 and IL-8)[110] which in turn regulate inflammation, angiogenesis, and tissue regeneration. Furthermore, p53 controls the expression of cell adhesion and cytoskeletal dynamics proteins such as cadherins and integrins, Rho GTPases, and actin-binding proteins. These proteins play an important role in cell adhesion and migration during the process of tissue regeneration[111].

P53 signaling is reported to play an important role in heart development as well as essential to maintaining normal heart architecture and functions[112]. It has been reported that a low concentration of H₂O₂ induces the expression of p53, which in turn activates the oxidative stress combating genes such as NADPH, and glutathione (GSH) synthesis and nucleotide production genes[61]. In 2021, Goshovska et al[113] demonstrated that GSH deficiency is associated with myocardium damage during cardiac ischemia-reperfusion. Whereas, higher expression of GSH provides cardioprotection by regulating NO, AMPK, and peroxisome proliferator-activated receptor-α signaling[113]. Ye et al[59] in 2020 reported that H₂O₂ acts as an essential signaling molecule for zebrafish heart regeneration. Moreover, the expression of p53 isoform Δ133p53 I has been shown to increase by low-level ROS. It has been reported that Δ133p53 I, by interacting with full-length p53, promotes cell survival by enhancing the expression of antioxidant genes including GPx1a, Sestrin1, Sestrin2, aldh4, SOD1, and SOD2[59]. These p53 target genes are reported to maintain redox homeostasis within the regenerating tissue[114].

CONCLUSION

In this review, we present an overview of the protective cellular mechanisms stimulated in H₂O₂ preconditioned MSCs while covering the role of H₂O₂-activated redox sensitive TFs in heart regeneration. Cells continuously produce ROS to maintain normal physiological functions. However, increased ROS production causes molecular damage, a condition known as oxidative distress. H₂O₂, a ROS, acts as an intracellular messenger that regulates several physiological processes. H₂O₂ preconditioned MSCs have the potential to regulate biological processes related to heart regeneration such as survival, proliferation, migration, paracrine activities, and differentiation. As highlighted in this review, H₂O₂ might not directly regulate heart regeneration, but oxidative stress caused by a low concentration of H₂O₂ activates redox sensitive TFs such as Nrf2, AP-1, p53, and HIF-1α, which in turn control the intracellular signaling pathways involved in the development/regeneration of the heart. Combining H₂O₂ preconditioning with other induction factors may further promote stem cell-mediated heart regeneration. For this reason, it could be a major player in new therapies involving the use of stem cells for the heart diseases. In addition to heart regeneration, H₂O₂ preconditioned MSCs have also been found to be promising in increasing in vivo cell survival, wound healing, and regeneration of several tissues. These results indicate a broader translational perspective for H₂O₂ preconditioning in the field of regenerative medicine. However, in the future, it is critical to optimize H₂O₂ concentration and treatment time to enhance its regenerative effects and prevent oxidative damage.