Function of CXCR4-enriched exosomes:
The potential role of CXCR4-enriched exosomes was demonstrated for the repair of the infarcted heart. In vitro studies showed that CXCR4-enriched exosomes released by MSCCR4 were transferred to cardiomyocytes, leading to an increase in cardiomyocyte survival under hypoxic conditions. This was also associated PI3K/Akt signaling pathway activation. While in vivo studies demonstrated that cell sheets pretreated with CXCR4-enriched exosomes promoted angiogenesis and demonstrated subsequent cardiac function improvement. Evidence was also provided showing that MSCCR4 enhances the release of proangiogenic factors and promotes MSC endothelial differentiation under hypoxic conditions and that cardiac functions improved when MSCCR4 cell patches were implanted into myocardial ischemic hearts [1].
CXCR4 is a G-protein-coupled receptor and can activate several G-protein-mediated downstream signaling pathways after stimulation. One endpoint of CXCR4 signaling is the activation of transcription factors. SDF-1𝛼CXCR4 axis was reported to be critical for activation of PI3K in ischemic cardiomyocytes, thereby mediating acute cardioprotection. Furthermore, TUNEL assay revealed that apoptosis in cardiomyocytes treated with MSCCR4-derived exosomes was significantly decreased as compared to control group, whereas this protective effect was abolished by PI3K/Akt inhibitor, LY294002, which led us to hypothesize that MSCCR4-derived exosomes could promote cardioprotection through the Akt signaling pathway [1].
VEGF signaling pathway plays an essential role in the vascular homeostasis and the angiogenic cascade. The antiapoptosis activity of IGF-1α has been attributed to downregulation of cleaved caspase-3. It is well proved that MSCCR4-derived exosomes activate a series of downstream growth factors, such as IGF-1 and Akt. Cells were treated with LY294002, a PI3k/Akt inhibitor, to determine paracrine effcts of MSCCR4-derived exosomes which were mediated through Akt signaling [1].
It was shown previously that exosomes from MSCCR4 deleting CXCR4 genes by siRNA did not show cardiac protection effects in vitro as well as functional recovery of the infarcted heart in vivo, indicating that CXCR4 plays critical role in stem cell functions. Cardiovascular functions were improved by transplantation of exosome-treated MSC via increased angiogenesis in the infarcted heart. Moreover, exosomes from MSC overexpressing CXCR4 showed better efficiency for reducing left ventricular remodeling and promoting restoration of heart function after MI, confirming that CXCR4 is a key factor for angiogenesis and cell survival. These studies demonstrated that overexpression of CXCR4 in MSC would be an effective strategy to enhance the release of exosomes containing cardioprotective factors [1].
Paracrine role of cardiac stem cells CSC in Cardioprotection:
CSC-based therapy holds tremendous promise in the treatment of heart diseases for cardiac regeneration. However, little is known about implication of paracrine action by CSCs in protection of the ischemic heart. Evidence has demonstrated that paracrine action is one of major protective mechanisms provided by stem cells in the ischemic heart. Paracrine molecules secreted by stem cells can suppress apoptosis, promote local angiogenesis, reduce inflammation, and improve cardiac function after myocardial ischemia. It was also shown that CSCs are capable of producing a substantial amount of protective factors. Implantation of CSCs into isolated mouse hearts before ischemia improved myocardial function after 25-minute ischemia and 40-minutes reperfusion (I/R), indicating the acute protection of CSCs in the heart without their differentiation.
Protective effects after pretreatments of the stem cells:
Clinical trials proved that cell-based therapies are safe and can reduce the organic and functional lesions of the post-ischemic myocardium. The engraftment and survival of the therapeutic cells are poor because of the harsh post-ischemic environment with an inflammatory response and apoptotic signals. To counter the effects of the former possibility various pretreatment methods started to emerge. Preconditioning the cells might help them to bear with the post-infarct environment and ischemic preconditioning, heat shock and pharmacological pretreatments were demonstrated to help the survival and efficacy of the added cells. Using poly (ADP-ribose) polymerase inhibition also significantly lowered the sensitivity of the cells to the oxidative stress and improved their survival. A new possible candidate for a pretreatment agent might be hydrogen sulfide as this molecule was shown to be remarkably effective in preventing ischemia– reperfusion injury [5].
While for many years hydrogen sulfide (H2S) was known as a hazardous toxic gas today it is proven that H2S is an endogenously produced gasotransmitter involved in several reactions. In mammals, the major amount of H2S is produced from L-cysteine by cysthationine β-synthase (CBS, E.C. 4.2.1.22.) and cystathionine γ-lyase (CSE, E.C.4.4.1.1.1.). The effects of H2S on the human body are widespread. The first revealed effect was the inhibition of the cytochrome c oxidase, and the opening of KATP channels; today many molecular targets are known to be responsible for the various physiological and pathophysiological effects of H2S. Importantly, it was demonstrated in several in vitro and in vivo studies that the H2S-treatment is protective on cells undergoing ischemia–reperfusion injury, especially after myocardial ischemia. Adipose tissue-derived stem cells as therapeutic cells because these cells constitute an attractive pool for autologous adult stem cells due to their relatively easy harvest from patients via minimally invasive liposuction.
H2S can alter the proliferation rate of hASCs as long-term, repeated exposure to NaHS increased, while inhibition of endogenous H2S production decreased cellular proliferation. Rationale behind using hASCs was based on multiple reasons: survival and the efficacy of therapeutic hASCs and human bone marrow derived stem cells (hBMSCs) are similar in the used model. Harvesting ASCs from the human body is easier and more secure compared to hBMSCs their application in cell therapies is more feasible [5].
The infusion of CSC-conditioned media (CM) before ischemia or during the initial of reperfusion protected post-ischemic myocardial function, further confirming paracrine protective effect of CSCs. Also it has been documented that estrogen favorably modulates endothelial progenitor cell activities. It was also shown that treatment with estrogen up-regulated mesenchymal stem cells (MSCs) production of protective factors, and 17b-estradiol-pretreated MSCs exhibited greater cardiac protection compared with untreated MSCs in a rodent myocardial I/R model. The role of estrogen in regulation of CSC paracrine function, however, is unknown. E2-pretreated CSCs mediated greater cardioprotection after myocardial I/R injury compared with untreated CSCs. In addition, treatment with E2 increased production of VEGF and SDF-1 in CSCs and that CM from E2-treated CSCs protected cardiomyocytes from hypoxia induced cell death. Furthermore, implantation of E2-pretreated CSCs reduced active caspase-3 level and up-regulated STAT3 activation, in the isolated mouse hearts subjected to acute I/R injury when compared with untreated CSC group. It was previously indicated that MSCs from female animals conveyed greater cardiac protection compared with MSCs from male animals when infused into the ischemic heart. Female MSCs produced more protective factors and exhibited improved survival than did male MSCs in response to injury. Evidences indicated that MSCs and CSCs share some features in terms of cell morphology and expression of stem cell surface markers. Therefore, it is possible that E2 might play the similar role in modulating paracrine protection of CSCs as it did in MSCs. In addition, in a setting related to the clinical situation, infusion of E2-pretreated CSCs during the initiation of reperfusion protected post-ischemic myocardial functional recovery. Several growth factors and cytokines derived from stem cells have been reported to protect cardiac function, improve cell survival, correct ventricular remodeling, and reduce tissue damage in the ischemic heart. Among these, VEGF has been confirmed as a critical factor in mediating angiogenesis and cardiac protection in the ischemic myocardium. In addition, SDF-1, an important chemokine, has recently received much attention for cardiac repair. SDF- 1 has been reported to increase stem cell attraction to the sites of injury, thereby promoting angiogenesis, improving cardiac function, and advancing myocardial structure after myocardial ischemia. Delivery of SDF-1 into the myocardium improved post-ischemic cardiac function likely due to its directly protective effect on myocardial tissue. Previously indicated that SDF-1 was identified as one of the abundant paracrine factors in CSCs and played a critical role in CSC-mediated acute protection following myocardial I/ R. Paracrine action of E2- treated CSCs under hypoxic environment. We found that hypoxia induced more production of VEGF, SDF-1, and HGF in CSCs compared with normoxia. I/R injury cause cell death in the heart, leading to myocardial dysfunction and cardiac damage. Both VEGF and SDF-1 have been shown to improve cell survival. Pre-ischemic infusion of E2-treated CSCs decreased active caspase-3 levels and attenuated myocardial damage after I/ R. It is noteworthy that CM from E2-treated CSCs did provide greater protection in cardiomyocytes against hypoxia-induced cell death compared with CSC CM [6].
The activated inflammatory response and cytokine elaboration following myocardial infarction together contribute to cardiac remodeling and eventual host outcome. Cytokines are released immediately following ischemia in order to modulate tissue repair and adaptation. Previous studies revealed that MSCs treated with inflammatory mediators activate a series of pathophysiological processes, including cell survival, cell migration, cell adhesion, chemokine release, induction of angiogenesis and modulation of immune responses. Several studies have demonstrated that pretreatment of MSCs with cytokines, which were released by the myocardium following ischemic injury, increased MSC-mediated cardioprotection following acute myocardial infarction (AMI). Interleukin (IL)-1β and tumor necrosis factor (TNF)-α, are not constitutively expressed in normal myocardium; however, their levels markedly increase in the infarct and non-infarct areas following AMI. Since cell adhesion molecules and their ligands, extracellular matrix components, chemokines and specialized bone marrow niches all have roles in the precise regulation of MSC adhesion to endothelial cells, it was reported that VCAM-1 together with its ligand very late antigens-4 (VLA-4) was able to bind to stromal or endothelial cells, which subsequently facilitated stem cell homing. Furthermore, blockage of VCAM-1 or VLA-4 markedly reduced stem cell migration and adhesion ability [6]. It was identified that IL-1β and TNF-α stimulation significantly elevated the VCAM-1 secretion and adhesion ability of MSCs, and the combination of these two cytokines potentiated this effect. Furthermore, intra-myocardial injection with MSCs which were pretreated with IL-1β and TNF-α markedly improved the myocardial function and decreased the collagen deposition in infarcted myocardium in rats. Several investigations have demonstrated that VCAM-1 has a key role in MSC-mediated adhesion and immunosuppression. VCAM-1 is also important in the adhesion and migration of leukocytes through brain microvascular endothelial cells via binding to the α4β1 and α4β7 integrins. In several previous studies, the adhesion of MSCs to endothelial cells was significantly eliminated following incubation with monoclonal blocking antibodies against VCAM-1. It was reported that PI3K participated in the regulation of VCAM-1 expression and in intracellular signal transduction of the cell migration, which were induced by TNF-α in human endothelial cells. Other studies suggested that IL-1β induced MSC migration and adhesion through NF-κb. When MSCs were cultured in the presence of two typical inflammatory mediators, IL-1β and TNF-α, and administered to rats following experimentally-induced myocardial ischemic injury. The expression of VCAM-1 and the cardiac function of the left ventricular region were then assessed. It was identified that the expression of the adhesion molecule significantly increased following treatment with either of the cytokines, as did the cardiac function of the rats [7].
Remote gene therapy induced protection of the heart from an ischemic episode when DNA encoding for insulin-like growth factor I or vascular endothelial growth factor was injected into the skeletal muscle. DNA encoding for hypoxia-inducible factor 1-alpha (HIF-1a) or heme oxygenase-1 (HMOX-1), injected into the skeletal muscle, was beneficial for heart cells ex vivo, in vitro, and in vivo. cardioprotection provided by HIF-1a was similar to that of its direct target HMOX1. Nevertheless, HIF-1a overexpression in skeletal muscle caused a general angiogenesis, which might be procarcinogenic in vivo. HMOX-1 degrades heme into three end-metabolic products with individual cardioprotective effects: free iron, carbon monoxide, and biliverdin, which is rapidly converted to bilirubin. Besides the cardioprotective effect of HMOX-1 in remote gene therapy, several other studies have shown that transfection of MSC with HMOX-1 before local transplantation also protected the infarcted heart. The efficacy of MSC remote transplantation on cardioprotection was evaluated on hearts subjected to global ischemia followed by reperfusion, using a Langendorff perfusion system, as well as in vivo, on the infarcted hearts. Ex vivo analysis of the hearts showed that pretreatment of the animals with mASC 3 days before heart isolation resulted in smaller infarct areas and improved cardiac function after ischemiareperfusion injury when compared to sham-treated mice. Others have previously reported additional cardioprotective effects of HMOX-1-overexpressing MSC after intramyocardial transplantation. Animals transplanted with HMOX-1-mASC had no increased plasma bilirubin as compared to sham- or na€ıve mASC-treated animals, and in vivo downregulation of HMOX-1 gene expression occurred in both naiive or HMOX-1-mASC 3 days after transplantation. Furthermore, gene expression patterns of pro- and anti-inflammatory factors in HMOX-1-mASC were altered both before transplantation and afterward. Gene transcripts enriched in cells after transplantation included cytokines and chemokines generally involved in immune regulatory effects. In addition, PTX3 and TSG-6 were identified as factors possibly involved in the cardioprotective effects. PTX3 is a protein that has both atheroprotective and cardioprotective functions [23, 48], while TSG-6 (a molecule induced by TNF-α) has an important role in remodelling of the ischemic heart by reducing the inflammatory response and infarct area.
Stem cell delivery methods used in clinical studies are generally associated with poor engraftment, possibly caused by a local inflammatory environment. Furthermore, most clinical studies have delivered stem cells directly into the myocardium, which is a rather invasive strategy. The inefficiency of the existing approaches for cell transplant demands the development of other strategies for the treatment of acute and chronic myocardial ischemia [8].