Mesenchymal stem cells (MSC) are ideal materials for stem cell-based therapy. As MSCs reside in hypoxic microenvironments (low oxygen tension of 1% to 7%), several studies have focused on the beneficial effects of hypoxic preconditioning on MSC survival; however, the mechanisms underlying such effects remain unclear. This study aimed to uncover the potential mechanism involving 78-kDa glucose-regulated protein (GRP78) to explain the enhanced MSC bioactivity and survival in hindlimb ischemia. Under hypoxia (2% O₂), the expression of GRP78 was significantly increased via hypoxia-inducible factor (HIF)-1α. Hypoxia-induced GRP78 promoted the proliferation and migration potential of MSCs through the HIF-1α-GRP78-Akt signal axis. In a murine hind-limb ischemia model, hypoxic preconditioning enhanced the survival and proliferation of transplanted MSCs through suppression of the cell death signal pathway and augmentation of angiogenic cytokine secretion. These effects were regulated by GRP78. Our findings indicate that hypoxic preconditioning promotes survival, proliferation, and angiogenic cytokine secretion of MSCs via the HIF-1α-GRP78-Akt signal pathway, suggesting that hypoxia-preconditioned MSCs might provide a therapeutic strategy for MSC-based therapies and that GRP78 represents a potential target for the development of functional MSCs.

Introduction: Endothelial progenitor cells (EPCs) play a critical role in restoration of ischemic diseases. However, the actual status of EPC development and the mechanisms of EPC dysfunctions in patients with various ischemic diseases remain unknown. Methods: To investigate the detailed function of EPCs in experimental murine models, we have established an EPC colony forming assay (EPC-CFA) in murine EPCs. The abilities of murine EPCs in differentiation, adhesive capacity, proliferative potency, and transplantation in vitro and in vivo were then examined. Results: Peripheral blood mononuclear cells (PB-MNCs), bone marrow mononuclear cells (BM-MNCs) or bone marrow c-Kit⁺/Sca-1⁺ lineage negative (BM-KSL) cells differentiated into two types of EPC colony forming units (EPC-CFUs), large sized EPC (large-EPC)-CFUs and small sized EPC (small-EPC)-CFUs. Gene expression analysis demonstrated that both EPC-CFU-derived cells expressed eNOS, Flk-1 and VE-cadherin, markers of endothelial cells (ECs), although the small-EPCs derived from small-EPC-CFU were higher in number and showed more immature features (higher population of KSL cells). Functionally, the large-EPCs derived from large-EPC-CFU had higher adhesive capacity but lower proliferative potency than small-EPCs, showing improved tubular forming capacity and incorporation potency into primary EC-derived tube formation. Importantly, hindlimb ischemia increased the frequencies of large-EPC-CFUs differentiated from PB-MNCs and bone marrow. Actually, transplantation of large-EPCs into ischemic hindlimb enhanced neovascularization in hindlimb ischemia model, although small-EPCs or murine ECs did not, suggesting that large-EPC-CFUs might play an important role in restoration of ischemic diseases. Conclusions: We demonstrated, using a murine ischemia model, that the EPC-CFA could be a useful way to investigate the differentiation levels of murine EPCs, further providing a crucial clue that large-EPC-CFU status may be more functional or effective EPCs to promote neovascularization.

Mesenchymal stem cells (MSCs) are a source for cell-based therapy. Although MSCs have the potential for tissue regeneration, their therapeutic efficacy is restricted by the uremic toxin, p-cresol, in chronic kidney disease (CKD). To address this issue, we investigated the effect of fucoidan, a marine sulfated polysaccharide, on cellular senescence in MSCs. After p-cresol exposure, MSC senescence was induced, as indicated by an increase in cell size and a decrease in proliferation capacity. Treatment of senescent MSCs with fucoidan significantly reversed this cellular senescence via regulation of SMP30 and p21, and increased proliferation through the regulation of cell cycle-associated proteins (CDK2, CDK4, cyclin D1, and cyclin E). These effects were dependent on FAK-Akt-TWIST signal transduction. In particular, fucoidan promoted the expression of cellular prion protein (PrP^C), which resulted in the maintenance of cell expansion capacity in p-cresol-induced senescent MSCs. This protective effect of fucoidan on senescence-mediated inhibition of proliferation was dependent on the TWIST-PrP^C axis. In summary, this study shows that fucoidan protects against p-cresol-induced cellular senescence in MSCs through activation of the FAK-Akt-TWIST pathway and suggests that fucoidan could be used in conjunction with functional MSC-based therapies in the treatment of CKD.

Introduction: Endothelial colony-forming cells (ECFCs) significantly improve tissue repair by providing regeneration potential within injured cardiovascular tissue. However, ECFC transplantation into ischemic tissue exhibits limited therapeutic efficacy due to poor engraftment in vivo. We established an adequate ex vivo expansion protocol and identified novel modulators that enhance functional bioactivities of ECFCs. Methods: To augment the regenerative potential of ECFCs, functional bioactivities of hypoxia-preconditioned ECFCs (hypo-ECFCs) were examined. Results: Phosphorylations of the JAK2/STAT3 pathway and clonogenic proliferation were enhanced by short-term ECFC culturing under hypoxia, whereas siRNA-targeting of STAT3 significantly reduced these activities. Expression of BCL3, a target molecule of STAT3, was increased in hypo-ECFCs. Moreover, siRNA inhibition of BCL3 markedly reduced survival of ECFCs during hypoxic stress in vitro and ischemic stress in vivo. In a hindlimb ischemia model of ischemia, hypo-ECFC transplantation enhanced blood flow ratio, capillary density, transplanted cell proliferation and survival, and angiogenic cytokine secretion at ischemic sites. Conclusions: Hypoxia preconditioning facilitates functional bioactivities of ECFCs by mediating regulation of the STAT3-BCL3 axis. Thus, a hypoxic preconditioned ex vivo expansion protocol triggers expansion and functional bioactivities of ECFCs via modulation of the hypoxia-induced STAT3-BCL3 axis, suggesting that hypo-ECFCs offer a therapeutic strategy for accelerated neovasculogenesis in ischemic diseases.

Mitochondria are considered to be the powerhouses of cells. They are the most commonly damaged organelles within dopaminergic neurons in patients with Parkinson’s disease (PD). Despite the importance of protecting neuronal mitochondria in PD patients, the detailed mechanisms underlying mitochondrial dysfunction during pathogenesis and pathophysiological progression of PD have not yet been elucidated. We investigated the protective action of fucoidan against the detrimental action of 1-methyl-4-phenyl-pyridinium (MPP⁺), a neurotoxin used to model PD, in the mitochondria of SH-SY5Y neural cells. Fucoidan increased the expression of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) and protected the cells from MPP⁺-induced apoptosis by upregulating the 5′ adenosine monophosphate-activated protein kinase (AMPK)-PGC-1α axis. These effects were blocked by the silencing of the PGC-1α axis. These results indicated that fucoidan protects SH-SY5Y cells from mitochondrial dysfunction and cell death caused by MPP⁺ treatment, via the AMPK-PGC-1α axis. These findings also suggest that fucoidan could potentially be used as a therapeutic agent for PD.

Dysregulation in mitophagy, in addition to contributing to imbalance in the mitochondrial dynamic, has been implicated in the development of renal fibrosis and progression of chronic kidney disease (CKD). However, the current understanding of the precise mechanisms behind the pathogenic loss of mitophagy remains unclear for developing cures for CKD. We found that miR-4516 is downregulated and its target SIAH3, an E3 ubiquitin protein ligase that reduces PINK1 accumulation to damaged mitochondria, is upregulated in the renal cortex of CKD mice. Here, we demonstrated that melatonin injection induces miR-4516 expression and suppresses SIAH3, and promotes PINK1/Parkin-mediated mitophagy. Furthermore, we demonstrated that melatonin injection attenuates the pathological features of CKD by improving mitochondrial homeostasis. Our data supports that mitochondrial autophagy regulation by activating miR-4516/SIAH3/PINK1 mitophagy signaling axis can be a viable new strategy for treating CKD.