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a Clarian Cardiovascular Surgery, Methodist Hospital, Indianapolis, Indiana
b Department of Surgery, Indiana University School of Medicine, Indianapolis, Indiana
c Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, Indiana
d Center for Immunobiology, Indiana University School of Medicine, Indianapolis, Indiana
= tumor necrosis factor-alpha; TNFR1 = tumor necrosis factor receptor 1; TNFR2 = tumor necrosis factor receptor 2; VEGF = vascular endothelial growth factor
* Address correspondence to Dr Meldrum, 2017 Van Nuys Medical Science Building, 635 Barnhill Dr, Indianapolis, IN 46202 (Email: dmeldrum{at}iupui.edu).
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Abstract |
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 Top Abstract IntroductionMethodsStem Cell History and...Mesenchymal Stem CellsCytokines Affect Mesenchymal...Ex Vivo Strategies Alter...CommentAcknowledgmentsReferences |
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Introduction |
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TopAbstractIntroductionMethodsStem Cell History and...Mesenchymal Stem CellsCytokines Affect Mesenchymal...Ex Vivo Strategies Alter...CommentAcknowledgmentsReferences |
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Stem cell therapy has the potential to be a key component in the field of regenerative cardiovascular medicine [2]. Mesenchymal stem cells, in particular, may be a leading candidate for cell-based therapy for the ischemic heart. These cells are a unique subset of stem cells that can be isolated from the bone marrow, adipose tissue, and even umbilical cord blood [3, 4]. The MSCs are multi-potent, and in response to ischemia–reperfusion injury (I-R) they secrete a variety of cytokines that are cardioprotective or angiogenic [5, 6]. In addition, MSCs engraft into injured myocardium and potentially differentiate into cardiac myocytes [5]. However, the engraftment and functional results are extremely variable in the literature and modest at best in a number of clinical trials [5, 7–12].
Cytokines likely affect the function of MSCs and represent one possible explanation for the variable results seen in the literature. Therefore, increasing our understanding of the cytokines that affect MSC function and the methods available to clinicians to modify MSCs ex vivo to capitalize on the positive or negative effects of cytokines may lead to future therapeutic gains.
This review provides: (1) a general overview of stem cells and MSCs in particular, (2) critically analyzes several cytokines known to alter MSC function, and (3) discusses methods to manipulate cytokine-activated MSCs to improve MSC function for potential clinical application.
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Methods |
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Stem Cell History and Terminology |
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TopAbstractIntroductionMethodsStem Cell History and...Mesenchymal Stem CellsCytokines Affect Mesenchymal...Ex Vivo Strategies Alter...CommentAcknowledgmentsReferences |
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By definition, stem cells must possess two characteristics: self-renewal and the ability to differentiate. In other words, a stem cell undergoing mitosis will either form clonogenic offspring (replicas that retain the parent stem cell properties) or offspring that eventually enter terminal differentiation into specific cell lineages [15, 16]. With respect to differentiation potential, stem cells are classified as totipotent, pluripotent, or multipotent. Totipotent zygotes (fertilized eggs) have the ability to differentiate into all cell lineages including extraembryonic tissue. Pluripotent embryonic stem cells from the inner cell mass of the blastocyst form tissue from all three germ layers but not extraembryonic tissue. Multipotent stem cells differentiate into a limited number of cell types usually of the same germ layer [15, 17]. Stem cells are also characterized by plasticity [18]. Plasticity implies that stem cells may differentiate into mature cell types outside their original lineage in response to micro-environmental cues.
In general, stem cells can be classified as either embryonic stem cells or adult stem cells. Adult stem cells can be further categorized as skeletal myoblasts, resident cardiac stem cells, and bone marrow-derived stem cells. Bone marrow-derived stem cells currently encompass hematopoietic stem cells, endothelial progenitor cells, and mesenchymal stem cells [19, 20].
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Mesenchymal Stem Cells |
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–, HLA-DR1–), and with stimulation differentiate in vitro into osteoblasts, adipocytes, or chondroblasts [22]. Unless the preceding criteria are met, the society recommends the term mesenchymal stromal cell be used. However, the position statement endorses the use of the acronym MSCs for both mesenchymal stromal cells and mesenchymal stem cells, which unfortunately may lead to continued ambiguity in the literature. It is important to note that these are the minimal requirements, and caution must be used when applying these standards across different species as interspecies variations are known to exist with respect to cell surface markers [23]. From the clinical perspective, MSCs possess at least four attributes that make them attractive for cell-mediated cardiac transplantation: (1) paracrine signaling, (2) immunomodulation, (3) ease of ex vivo expansion, and (4) potential for cardiomyoplasty.
Paracrine Signaling
Caplan, Haynesworth, and their associates [24, 25] were among the first to describe the ability of MSCs to express cytokines. These cytokines were believed to exert "trophic" effects that assisted in tissue repair outside of any potential MSC differentiation [24, 25]. Supporting this concept, our group has shown that activated MSCs secrete a wide array of cytokines (eg, vascular endothelial growth factor, hepatocyte growth factor) that are associated with functional recovery of the heart after I-R [26–29]. This functional recovery is seen with acute injury, and thus is more likely associated with nongenomic signaling rather than long-term differentiation or engraftment.
Immunomodulation
Mesenchymal stem cells are believed to be a uniquely immuno-privileged cell type as they do not express MHC class II molecules or co-stimulatory surface markers for T-cell activation (CD40, CD80, and CD86) [30–32]. The lack of immunogenic cell surface markers lends itself to allogenic and xenotransplantation where MSCs potentially evade or even change the native immune system at the site of injury by exerting immunosuppressive effects on T-cells [32]. In vivo animal research in myocardial infarct models supports the immune privileged status of MSCs. Successful allogenic transplantation of MSCs after myocardial infarction has been described in immunocompetent animal models (ie, rodents, swine) without immunosuppression [33]. These unique immune properties even extend across species, as several studies have demonstrated the presence of murine MSCs in the infarcted rat heart without evidence of immune system activation [34, 35].
Cell Culture
Despite the fact that MSCs represent less than 0.01% of nucleated cells found in the bone marrow, MSCs can be easily cultured to the high numbers needed for transplantation [33, 36, 37]. In brief, bone-marrow derived MSCs are harvested from the iliac crest in humans or from the femurs or tibias of animals. The MSCs are selected from the heterogeneous cell populations from the bone marrow based on plastic adherence to the cell culture container, as well as cell-surface marker selection (positive or negative) with antibodies. Although in-depth details of MSC harvesting and culture are beyond the scope of this review, the reader is referred to excellent reviews by Meirelles da Silva and Nardi [36] and Alhadlaq and Mao [38].
Cellular Cardiomyoplasty
Cell-based cardiomyoplasty for the ischemic heart involves the implantation of MSCs into the injured myocardium in an attempt to regenerate myocardium. However, engraftment may be limited, in part, due to the mechanical stresses of the beating heart [5, 9, 12, 39]. These observations led to the supposition that the extracellular matrix may be a key component to improving progenitor or stem cell engraftment after acute myocardial injury. In other words, replacement of the extracellular matrix in the infarcted area may lead to improved MSC engraftment. The Myocardial Assistance by Grafting a New Bioartificial Upgraded Myocardium (MAGNUM) Trial of 2008 tested this hypothesis. The trial demonstrated the safety and feasibility of cellular cardiomyoplasty using autologous mononuclear bone marrow cells injected into the heart tissue followed by fixation of a bone-marrow, cell-seeded matrix over the epicardium [40]. The trial showed an increase in viable tissue within the infarct scar, decreased wall stress, and attenuated ventricular remodeling. These results will hopefully lead to a larger randomized control trial in the near future.
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Cytokines Affect Mesenchymal Stem Cell Function |
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, high mobility group box 1, transforming growth factor-β1, hepatocyte growth factor, and insulin-like growth factor-1 are discussed.
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is a pleiotrophic inflammatory cytokine [41–46] known to alter MSC cytokine production, increase human MSC proliferation, and induce MSC basement membrane invasion by activating nuclear factor kappa-B, which potentially improves MSC engraftment [47]. Conversely, TNF- is known to inhibit chondrocyte and myocyte differentiation by activating nuclear factor kappa-B [48, 49]. In addition, TNF- alters MSC cytokine expression. These multiple effects may in part be explained by differential signaling by the TNF- associated receptors tumor necrosis factor receptor 1 (TNFR1 [55-kDa]) and tumor necrosis factor receptor 2 (TNFR2 [75-kDa]).
Tumor necrosis factor- is known to activate TNFR1, as well as the Fas receptor leading to increased apoptosis through cytosolic death domains in myocytes [41]. In addition to increased cell death, knockout of TNFR1 is associated with altered MSC cytokine production depending on the stimulus and gender of the stem cell. For example, TNF- stimulation of TNFR1 knockout MSCs increases vascular endothelial growth factor (VEGF) production, decreases production of TNF-, and increases production of proinflammatory interleukin (IL)-6. Similarly, lipopolysaccharide stimulation of TNFR1 knockout MSCs increases VEGF and decreases TNF-; however, IL-6 production is decreased [50]. The gender of the stem cell also plays a role in the effects of TNFR1 on MSCs, as cells derived from female mice appear to have innate resistance to TNFR1 signaling. We have shown that in female TNFR1 knockout MSCs, TNF-, IL-6, and VEGF levels are the same as wild-type mice, unlike male MSCs [27]. These data indicate that: (1) the TNFR1 is associated with induction of apoptosis in response to TNF-, and (2) the TNFR1 knockout MSCs alter cytokine production, depending on the stimulus and stem cell gender.
Tumor necrosis factor receptor 2 is associated with decreased MSC proinflammatory cytokine expression. In vitro, TNFR2 knockout MSCs after TNF- stimulation produce less VEGF [50]. VEGF is believed to play a pivotal role in MSC transplantation, inducing neovascularization, stimulating migration of stem cells, and inhibiting apoptosis [51, 52]. Indeed, we have recently demonstrated that VEGF is a crucial factor in mediating MSC cardioprotection after acute I-R [29]. Therefore, TNFR2 signaling may be a potential cause for improved function in vivo. One study corroborates this hypothesis as MSCs transfected with TNFR2 improved left ventricular function and decreased proinflammatory cytokine production (TNF-, IL-1β, IL-6) after permanent ligation of the left anterior descending coronary artery (LAD) in rodents [53]. This study must be viewed with caution as the authors do not mention verification of TNFR2 overexpression after transfection.
Taken together, TNF- alters MSC cytokine production by TNFR1 and TNFR2. There is some functional data indicating that TNFR2 overexpressing MSCs improve heart function after MI. However, further investigation is needed regarding the role of activated TNFR1 and TNFR2 on MSC function. This is an active area of research in our group.
High Mobility Group Box 1
High mobility group box 1 (HMGB1) is a nuclear protein released by inflammatory cells and necrotic cells that acts as a proinflammatory cytokine signaling injury and leading to tissue repair by recruiting stem cells. Germani and colleagues recently demonstrated that HMGB1 administration in a murine model of myocardial infarction-activated cardiac stem cells and promoted differentiation into cardiomyocytes [54]. With respect to MSC function, extracellular HMGB1 suppresses proliferation and induces differentiation into osteocytes. Interestingly, HMGB1 treatment of MSCs increases MSC migration and does not affect the anti-proliferative effects of MSCs on T-cells [55]. High mobility group box 1 is also particularly interesting from a receptor standpoint as Toll-like receptors 2 and 4, as well as the receptor for advanced glycation end products (RAGE) are associated with HMGB1 signaling. Although it is not clear how much each pathway mediates the effects of HMGB1, it is clear that HMGB1 increases proinflammatory cytokines release, such as TNF-, IL-1, and IL-6 from macrophages [56]. In short, although we know that HMGB1 improves MSC migration and does not appear to affect MSC immunomodulation, HMGB1 decreases MSC proliferation and induces MSC terminal differentiation into osteocytes. Counteracting the latter two effects and identifying the cytokines expressed by HMGB1-stimulated MSCs merits further investigation.
Transforming Growth Factor-β1
Transforming growth factor-β1 is a multipotent cytokine associated with angiogenesis and anti-inflammatory effects. In vitro, TGF-β1 in conjunction with bone morphogenetic protein-2 induces human MSC expression of cardiac surface markers [57] and increases MSC production of VEGF through the Akt pathway [58]. In vivo, TGF-β1 is significantly elevated after myocardial infarction [58] and is associated with decreased TNF- levels [59, 60]. Although these results are encouraging, TGF-β1 overexpressing MSCs may not be a viable solution. Transforming growth factor-β1 is also associated with cardiomyocyte hypertrophy and fibroblast proliferation related to pressure overload as seen in the development of heart failure [59, 61]. This is potentially explained at the molecular level where TGF-β1 has two binding receptors (ie, types I and II with multiple subtypes). Transforming growth factor-β1 binds a heteromeric combination of the two receptors. The type 2 receptors transfer the signal to the type 1 receptor for intracellular messaging [62]. Transforming growth factor-β1 binding initiates signaling by Smad, which may mediate these long-term remodeling effects [59, 61]. The Smads are intracellular proteins that act as nuclear transcription factors [63]. In transgenic hamsters designed to develop cardiomyopathy, normalization of Smad 2 decreased the accelerated collagen turnover believed to lead to cardiomyopathy [64].
Taken together, TGF-β1 is a cytokine with the potential to induce neoangiogenesis, decrease inflammatory signaling, and induce MSC differentiation. However, the long-term effects of TGF-β1 on cardiac remodeling may need to be addressed if MSCs overexpressing TGF-β1 are believed to be of therapeutic value. Alternatively, preconditioning MSCs with TGF-β1 prior to injection into the ischemic heart may avoid the sequelae of chronic TGF-β1 exposure.
Hepatocyte Growth Factor
Hepatocyte growth factor is a cytokine of mesenchymal origin involved in cell migration, proliferation and survival. Forte and colleagues [65] demonstrated that HGF exposure resulted in MSC migration but inhibited proliferation. In addition, prolonged exposure to HGF (48 hours) induced cardiac surface marker expression on MSCs [65]. Similar to the other cytokines, HGF signals by multiple intracellular pathways. Hepatocyte growth factor activates the mesenchymal-epithelial transition factor (MET) receptor on MSCs, which in turn signals through the ERK, p38, and P13K/Akt pathways. Although the full effects of each pathway are not known, it is known that the negative effect of HGF on MSC proliferation is reversed with a p38 inhibitor. It is also known that inhibition of P13K decreases MSC migration [65]. Nevertheless, despite the possible negative effects on proliferation, HGF overexpressing mesenchymal stem cells have been shown to engraft and improve myocardial function in a rodent LAD occlusion model [66].
Insulin-Like Growth Factor-1
Insulin-like growth factor-1 (formerly known as somatomedin C) is associated with cardiomyocyte cell survival in the setting of I-R by attenuating B-cell lymphoma 2 (Bcl-2)-associated X protein and caspase 3 activation [67]. This effect has not been studied in MSCs yet; however, IGF-1 is known to enhance the migration of MSCs. Li and colleagues [68] stimulated rat MSCs with IGF-1 and noted a marked increase in the chemokine receptor CXCR4 (the receptor for stromal cell derived factor-1). Upregulation of CXCR4 led to significantly improved response to stromal cell-derived factor-1 with resultant improvement in MSC migration. Li and colleagues [68] also demonstrated that this effect was mediated by PI3K. The role of IGF-1 on in vivo MSC function was recently explored by Haider and colleagues [69] who demonstrated that IGF-1 overexpressing MSCs improved survival and engraftment in the rodent LAD occlusion model. This increased MSC engraftment resulted in decreased infarct size and increased angiogenesis in the infracted heart. Finally, MSC pretreatment with IGF-1 prior to tail vein injection has been shown to improve stem cell engraftment after MI in a rodent model 4 weeks post-transplantation [70].
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Ex Vivo Strategies Alter the Impact of Cytokines On Stem Cell Function |
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and lipopolysaccharide stimulation [27, 50]. This data has led to ongoing functional studies to determine which receptor is associated with myocardial protection after I-R. One drawback to permanent receptor deletion is that it requires germ cell mutations, which are not feasible in harvested human donor MSCs. Therefore, targeted receptor ablation with siRNA may be a potential option to modify human stem cell receptors ex vivo on a temporary basis. Although we have not used the technique to modify receptors, we have used siRNA to disrupt VEGF production and demonstrate the critical role of VEGF in myocardial recovery from I-R [29]. Finally, with respect to receptor overexpression, viral vectors (or potentially siRNA targeting an inhibitory pathway for receptor expression) can be used to overexpress receptors in MSCs. Bao and colleagues [53] recently overexpressed TNFR2 in MSCs and demonstrated improved myocardial function after LAD ligation in rats. Potentially, these short-term modifications of receptors with either siRNA or vectors may lead to improved MSC function with potential application in clinical trials.
Intracellular Messenger Modification
Modifying the MSC intracellular signaling cascade activated by a cytokine of interest is another technique that may be applied to stem cell-mediated cardiac therapy. As mentioned earlier, PI3K/Akt is associated with improved migration of MSCs. Recently, investigators created MSCs that overexpress Akt. This modification enhanced MSC survival and function. In addition, MSCs that overexpressed phosphorylated-Akt (activated form of Akt) were associated with improved cardiac function after myocardial infarction [72], likely in association with extracellular secreted frizzled related protein 2 (Sfrp2) [73]. Interestingly, wild-type MSC survival was similarly improved after treatment with conditioned media from the Akt overexpressing MSCs implying a significant role for paracrine signaling in stem cell-mediated cardioprotection [72]. Another group similarly overexpressed Bcl-2 in MSCs, as Bcl-2 is associated with decreased apoptosis through regulation of mitochondrial function, which is believed to lead to improved cardioprotection during ischemia. Indeed, rat MSCs that are genetically modified to overexpress Bcl-2 had improved capillary density in the infarct border zone, improved left ventricular function, and increased MSC engraftment after LAD ligation [74]. Taken together, these studies imply that genetic ex vivo modification of intracellular cascades linked to a known cytokine may allow us to increase the function of the MSC in response to external stimulation. It may also be possible that intracellular modification may obviate the need for the external cytokine stimulation.
Cytokine Overexpression
Given the evidence that cytokines can activate MSCs potentially leading to cytoprotective cytokine production, it stands to reason that overexpression of cytokines may lead to improved MSC function. Viral transfer of the coding regions for the cytokines for HGF, IGF-1, and VEGF has been successfully performed [66, 69, 75]. As previously discussed, overexpression of HGF or IGF improved MSC function in the setting of ischemia. Vascular endothelial growth factor overexpressing MSCs were similarly associated with smaller infarct sizes and improved cardiac function after LAD ligation in rodents [75]. It is unclear if the overexpression of the cytokine affects the MSC or the injured heart, or both. However, it is clear that overexpression of cytokines is a potential tool for MSC modification.
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Comment |
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TopAbstractIntroductionMethodsStem Cell History and...Mesenchymal Stem CellsCytokines Affect Mesenchymal...Ex Vivo Strategies Alter...CommentAcknowledgmentsReferences |
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In summary, this review outlines some of the latest developments in MSC research for cell-based therapy for myocardial infarction and I-R. On the other hand, this review also highlights one of the major challenges that surgeon-scientists face, which is the inability to see the entire effects of one change (eg, knockout or genomic change) at the molecular level. Although the effects of modifying cytokine expression or receptors for myocardial therapy seem promising, the systematic consequences and the cross-talk among pathways must be further investigated prior to clinical application.
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Acknowledgments |
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B = nuclear factor kappa-B; RAGE = receptor for advanced glycation end products; TGF-β1 = transforming growth factor-beta 1; TLR = toll-like receptor; TNF = tumor necrosis factor; TNFR1 = tumor necrosis factor receptor 1; TNFR2 = tumor necrosis factor receptor 2; VEGF = vascular endothelial growth factor.)