2. BioTech - IJBTR - Mesenchymal - Fatemeh Pourrajaba - Iran

International Journal of Bio-Technology and Research (IJBTR) ISSN 2249-6858 Vol.2, Issue 2 Sep 2012 8-18 Š TJPRC Pvt. Ltd.,

MESENCHYMAL STEM CELLS AND MOLECULAR MECHANISMS, FEASIBLE CANDIDATES FOR NEUROPROTECTION AND REGENERATION FATEMEH POURRAJAB1,3, SEYED HOSAIN HEKMATIMOGHADAM1,2,4 & SEYED KHALIL FOROUZANNIA2, 1

2 3

4

Yazd Research Center of Sciences and Molecular Biothecnology, Shahid Sadoughi University of Medical Sciences, Yazd, Iran

Yazd Cardiovascular Research Center, Shahid Sadoughi University of Medical Sciences, Yazd, Iran

Department of Clinical Biochemistry and Molecular Biology, Faculty of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran

Department of Laboratory Sciences, School of Paramedicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran

ABSTRACT Cell therapy is an attractive strategy for protection or restoration of degenerative diseases.

Human bone marrow-derived mesenchymal stem cells (BMSCs) reservoirs of the reparative mechanisms, exhibit a beneficial population for treatment of neurodegenerative diseases. The key players involved in BMSC tropism are up-regulated signals after ischemia/reperfusion or inflammation.

Molecular signals also induced by injuries, trigger BMSC migration, homing and invasion. Cytokines and chemoattractant in particular VEGF, G-CSF, PDGF, FGF, CCRs and matrix proteases imply pivotal roles in reparative pathways. Under homing, BMSCs induce immunomodulatory and regenerative mechanisms as well as milieu-dependent differentiation to express phenotypes of the local microenvironment, great potentials for treatment of neurodegenerative and ischemic diseases.

KEYWORDS: Neuroprotective, Immunomodulator, Genetic Reprogramming, Modulating Hypoxia.

BENEFICIAL FEATURES OF BONE MARROW MESENCHYMAL STEM CELLS Human bone marrow-derived mesenchymal stem cells (BMSCs) are of therapeutic interest in a variety of neurological diseases, a population which can differentiate into multiple cellular lineages. The autologous origin of BMSCs avoids the risk of immune rejection and being adult cells weaken the possibility of tumor development. Furthermore, they also have anti-tumor, immunomodulatory and antiinflammatory effects (1). Although the mechanisms underlying the immunosuppressive effect of MSCs has not been clearly defined, but their immunosuppressive properties have already been exploited in the clinical settings. Beside, BMSCs in human body represent reservoirs of reparative cells and capable of homing to the site of diseases. They include a high quality for provoking the regeneration pathways in the injured tissue without fibrous tissue formation (2).

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Mesenchymal Stem Cells and Molecular Mechanisms, Feasible Candidates for Neuroprotection and Regeneration

When, BMSCs are co-cultured rescue dorsal root ganglia from dying and allow the long-lasting survival and maturation, otherwise neurons are committed to die. The BMSC rescue effects on the neurons is achieved only by direct contact (3, 4). In experimental models of ischemia, tumors, and neurodegenerative diseases, many studies have imply that BMSCs migrate to site of lesions in the brain to ameliorate functional deficits (5,6). BMSCs can also be used as vehicles to deliver therapeutic compounds or genes to site of prions (7).

Under adequate stimuli and contact stimulation, BMSCs express typical markers of specific cell lines including glial cells and neurons. The neuronal differentiation seems to be increased by the autorelease of specific molecules such as bone morphogenetic proteins or chemicals such as Dimethyl Sulfoxide and b-mercaptoethanol or by experimental conditions that increase the cyclic-AMP level.

BMSCs arrange in elongated cells to form a flattened layer among which the neuronal processes can be done (8, 9). Beside neurotrophic factors released by BMSCs, contribution of cell-cell interaction is also essential to prevent neuron dying and survival. Direct cellular interactions promote a proteomic change or a genetic switching in BMSCs, which enables them to mediate different functions (10).

TROPHIC FACTORS BY BMSCS TO MODULATE THE MICROENVIRONMENT Originally thought, BMSCs mediate their therapeutic action by stemness multi-potency now, it has become clear that the secretion of multiple growth factors and cytokines (trophic action) is primarily responsible for therapeutic benefits (11). In addition to replace lost cells, BMSCs provide a source of trophic factors to modulate the immune system and to prevent further neuron-degeneration (12). In experimental autoimmune encephalomyelitis (EAE), BMSCs improve clinical outcomes via immunomodulation and reduction in central nervous system inflammation (15). Particularly, secreted cytokines play pivotal roles in modulating the microenvironment and inducing morphological changes (11,13,14). The studies detected the expression of IL-1β, IL-6, IL-8, MCP-1, VEGF, G-CSF, SCF and IL-11 (14,16) . Of these cytokines, IL-6 plays an important role in the differentiation and regeneration of various stem cells, inhibits osteoblast development and promotes cell survival and proliferation. While IL-1β, a major intermediary of inflammation and immunological reactions, regulate the expression of IL6 (17). Additionally, TSG-6 is another anti-inflammatory factor secreted by BMSCs to mediate disruption (18,19). One of the most important cytokines, the vascular endothelial growth factor (VEGF) induces both BMSCs and endothelials for recovery of microvascular injury (16,18,20). VEGF is well known for its ability to mobilize bone marrow progenitor cells for participate in myogenesis and angiogenesis (21). VEGF can per se be secreted by BMSCs and boost the regenerative activity of progenitor cells to differentiate into myocytes and endothelial cells (18,22,23). VEGF well known for its participating in angiogenesis promotes also differentiation of stem cells into neuronal and endothelial cells (18, 34). VEGF contributing to progenitor cell mobilization also evokes pronounced trophic factors with angiogenic, cytoprotective and anti-inflammatory properties (32). In other side, high levels of VEGF stimulate PDGFR which regulate cell migration and proliferation (18,24).

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Even more, MSC-secreted matrix metalloproteinases (MMPs) display crucial roles for beneficial matrix remodeling and BMSC homing at the site of injury. Under hypoxic conditions, the interactions between brain microvascular and BMSCs are enhanced (18,20,25,26).

BMSCs are able to elaborate various potentially neuroprotective growth factors such as brainderived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3), glial cell linederived neurotrophic factor (GDNF), fibroblast growth factor-2, and insulin-like growth factor type 1(28,29). Neuroprotective factors then inhibit death-inducing pathways and also activate a variety of cell survival pathways. For example, BDNF is a member of the neurotrophin family of growth factors that is widely expressed in the adult and developing nervous system. BDNF activates a number of signaling pathways in neurons specially, PI3kinase/Akt pathways. The PI3kinase/Akt signaling pathway is a critical cellular survival. This pathway can be activated by a variety of trophic factors (27).

Intracellular signaling underlying processes of neuronal cell death is complex, but evidence points to a critical role for both MAPkinase and PI3kinase/Akt signaling pathways. Members of MAPkinase pathways are known to be activated by a variety of signals in neurons and specifically p38 has been implicated in neuronal cell death pathways. Several reports have shown p38 MAPkinase activation during nitric oxide mediated neuronal death vs. trophic factors inhibit the p38 activation and promote survival in this context (30).

BMSC MECHANISMS TO ATTENUATE STRESS CONDITION AND INFLAMMATORY REACTIONS BMSCs are multipotent stem cells express endothelial phenotypes and further increase capillary density and decrease the disrupt size (31) , but also by their ability to supply large amounts of angiogenic, anti-apoptotic and mitogenic factors (32).

The trophic mediators secreted by BMSCs improve organ function by a combination of multiple mechanisms such as attenuating tissue injury, inhibiting fibrotic remodeling, promoting angiogenesis, mobilizing host tissue stem cells, and reducing inflammation (20,25,33).

For example, EAE are associated with elevated levels of nitric oxide (NO) within the central nervous system, combined with reductions in trophic support from surrounding glia (15). BMSCs protect neurons from NO-mediated damage or trophic deprivation, a process which depend on survival signaling and PI3kinase/Akt pathway. Immune activators cause NO exposure which inhibit PI3kinase/Akt signaling and activate p38 MAPkinase pathway in neurons. Whereas, BMSCs exposure significantly activate the PI3kinase/Akt pathway, while reduce p38 signaling in NO-exposed neurons (27).

Intravenous injection of autologenic or allogeneic BMSCs strongly suppresses T-lymphocyte proliferation. More recently, the immunosuppressive effects of BMSCs have been found to target T cell proliferation but not its effector function. BMSCs show immunoregulatory function at different phases of T cell responses, the ability to inhibit mitogen-activated CD4+ and CD8+ subpopulations of T cells. For

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Mesenchymal Stem Cells and Molecular Mechanisms, Feasible Candidates for Neuroprotection and Regeneration

this reason, they have the potential to be exploited in the control of unwanted immune responses, in particular graft versus host disease (GVHD) and autoimmunity(39-41).

Clinical trials of BMSCs have demonstrated no significant adverse side effects even with multiple cell administrations (36). In non-ischemic inflammatory and edema disease, BMSC transplantation attenuates the infiltration of CD68-positive inflammatory cells and MCP-1 expression in milieu, and improves organ function. Hence, MCP-1 is a chemoattractant protein and results in monocyte/macrophage infiltration into the injured tissue and provokes inflammation. In disrupted organ and in response to MCP-1, BMSCs act in a paracrine manner to secret large amounts of angiogenic and anti-apoptotic signals such as VEGF, HGF, insulin-like growth factor-1 and adrenomedullin (32, 37-39).

Beside anti-inflammatory response, the cells express CCR2 the receptor for MCP-1 which promotes the migration of BMSCs (32,37,39).

Further, capillary density is increased by BMSC transplantation associated with improved organ function and decreased lesion size. 24 h after transplantation, at least 3% of the intravenously administered MSCs will be homed at the damaged site (32).

MOLECULAR REPROGRAMMING OF THE INJURED TISSUE, NOVEL CELLULAR MECHANISMS Understanding of how MSCs evoke neuron improvement is ever more controversial. While preventing progressive inflammatory and apoptosis reaction, BMSCs also elicit reparative effects through genetic reprogramming of the microenvironment manifest by alterations in the pattern of cytokine release and attenuation of the activation of proinflammatory transcription factor, NF-κB (42,43). Proinflammatory cytokines that are downstream products of the receptor-stimulated NF-κB signaling cascade, such as TNFα, can be the ultimate harmful agents (43). Reportedly, the well characterized LPS-evoked release of TNF-α is completely blunted in co-cultures while the spontaneous release of a beneficial cytokine, IL-10 is unaltered. In consistency, MSCs block the activation of NF-κB signaling cascade in cardiac myocytes (42). BMSC-derived factors act as powerful benefactors for restoring and maintain Ca2+ signaling to the damaged site (44).

Evidence has emerged a long list of potential soluble factors underlying beneficial action of BMSCs, including stromal derived factor-1α, secreted frizzled-related protein 2 (sfrp-2), IL-10, TNFαinduced protein 6 (TSG-6), and VEGF (45). Different protocols have revealed that BMSC conditioned medium could repair stress induced abnormal Ca2+ signaling both in long term (24 h) and acute (3 h) incubations (46).

MSCs have direct action on cardiac myocyte responses to stress at the gene transcriptional level that are consistent with their beneficial effects on the organ function. BMSCs exploit the stressors; endotoxin LPS and a downstream signaling proinflammatory cytokine IL-1β implicated in damages associated with sepsis and ischemia/reperfusion (46). LPS and pro-inflammatory cytokine IL-1β has proposed to act directly through their cognate receptors TLR4 and ILR, respectively. It has been shown

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that BMSCs, microglial and myocardial cells also express TLRs. Toll-like receptors (TLRs) are a group of transmembrane proteins which play critical roles in immune responses. The ligand-mediated stimulation of TLRs family can induce apoptosis of microglial, myocardial and endothelial cells (47).

Alternatively, other studies have observed that LPS, an agonist of TLR4, protects myocytes and human dendritic cells from apoptosis through a NF-kB-dependent PI3K/Akt pathway (48). Reports reveal that LPS protect MSCs from oxidative stress-induced apoptosis and enhance proliferation of MSCs via TLR4 and PI3K/Akt pathway (48).

Conclusively, stress-stimulated secretion of a variety of growth factors and cytokines by BMSCs, markedly change the pattern of microinviroment cytokine release, all of which accompanied by a genetic reprogramming and molecular switching.

BMSCS BEHAVIOR FOR RECONSTITUTION OF HYPOXIC MICRO-VASCULAR In ischemic cerebrovascular disease, the injury of brain microvascular endothelial cells (BMEC) induces the opening of the brain barrier border (BBB), which leads to a brain edema and nerve damage.

Then, the recovery and neogenesis of ischemic penumbral microvasculature is a key point in the retrieval of injured cells (49). BMSCs have become the recent focus of intense research in the treatment of ischemic disease due to their ability to repair and rebuilt of injured microvasculature. Data indicate that under hypoxia BMECs induce BMSCs to differentiate into endothelial cells, whereas BMSCs enhance proliferation and migration of BMECs, and simultaneously increasing the permeability of the BMEC monolayer, presumably through paracrine function (50).

Following an injury, there are significantly increased numbers of BMSCs in peripheral blood and at the disrupted site of the injured subjects (51), this trend correlates with significantly increased concentrations of the cytokines VEGF and G-CSF, suggestive of a molecular mechanism for BMSCs mobilization, recruitment and homing (20). BMSC homing to the site of injury involves the arrest within the vasculature and transendothelial migration followed by chemotaxis at the injured tissue. While arresting in the proper vascular position, BMSCs secrete proteases such as MMPs, and be capable of breaking down the endothelial basement membrane and journeying presumably toward chemotactic agents (20, 26).

In terms of the second, however, the collagen matrix is essential for the efficient homing. The putative proteases released into collagen I or collagen IV matrices produce proteolytic fragments for attracting more BMSCs toward the site of injury where the medium is a result of stress (25, 26, 52).

MMPs are a family of zinc-dependent proteases and classically described in the context of extracellular matrix remodeling under physiological and pathophysiological conditions. They are very important for BMSCs recruitment, migration and differentiation. High-levels of MMPs can enhance angiogenesis in hypoxic conditions. MMP-9 known as Gelatinase B, for example plays an important role in the migration of BMSCs and capable of degrading type IV collagen (a key component of basement membranes) and gelatin. Both, VEGF and MMP-9 can increase vascular permeability (20).

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Mesenchymal Stem Cells and Molecular Mechanisms, Feasible Candidates for Neuroprotection and Regeneration

Under hypoxic condition, VEGF and MMPs have been secreted by both BMECs and BMSCs. Hypoxia induces hypoxia-inducible factor-1 (HIF-1), to autocrine more VEGF, the over activity of HIF-1 and stimulate MMP expression in BMSCs (53, 54).

BMECs are able to induce Flk-1 expression in BMSCs (20). The Flk-1 is one of the earliest markers for endothelial cells, followed in sequence by Tie-2, VE-cadherin, Tie-1, CD34, and vWF expression (55).

Hypoxia results in HIF-1 stabilization, nuclear translocation and transcription of genes containing hypoxia response elements (HRE). HIF-1 interacts with the co-activator protein p300 that results in transactivating of hypoxia responsive genes, which exemplifies the promoter of VEGF gene (56). HIF-1 taking a positive role in vascularization, is also considered important in the process of wound repair (53, 54).

Interestingly, NF-κB inflammatory mediators and HIF-1 act at the same target genes, identifying inflammatory signals antagonize the HIF-1. For example, NF-κB interferes with VEGF expression, while causes the iNOS gene activation, in the presence of LPS and TNF-α. Since both transcription factors are essential for wound healing thereby, depending on condition, inflammation and hypoxia behave in synergism or counterpart to influence out come (57, 58) . The high HIF-1 activity causes to survive hypoxic/ ischemic stress more successfully by mediating cellular adaptive response to hypoxia. Elevated HIF-1 level also up-regulates MMP1 and MMP3 expression in BMSCs (41). So, it is reasonable to speculate that BMSCs with high HIF-1 level will be less affected by low oxygen level associated with aging. Since the vascular function and blood flow are reduced with age, which lowers oxygen level in tissues and represents a risk factor of age-related diseases(59). In addition, high HIF-1 levels promote the migratory activity in the collagen-rich microenvironments, during the recruitment of BMSCs (60). These findings suggest that in spite of being a cell survival signal, higher level of HIF-1 may participate in remodeling and reconstitution for healthy tissue.

CONCLUSIONS Stem cell-based therapies to repair and replace lost neural cells are a highly promising treatment for CNS degenerative diseases. BMSCs are great promise as therapeutic agents against neurological maladies. They have the ability to differentiate into neural phenotypes and can be readily isolated and auto-transplanted with no risk of immun reaction/rejection. Although multi-lineage differentiation, BMSCs therapeutic benefits have now become attributed to secretion of multiple growth factors and cytokines (trophic action). The cells have direct action on microenvironment cell responses and the pattern of cytokine release. BMCSs induce genetic reprogramming of milieu gene expression manifest by alterations in the pattern of cytokines and attenuation of pro-inflammatory transcription factors. The change of cytokine pattern is fallowed by prevention of stress-induced apoptosis, appropriate differentiation and immunomodulatory regulation, an important protective outcome.

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