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.

Anticancer drugs, such as fluorouracil (5-FU), oxaliplatin, and doxorubicin (Dox) are commonly used to treat colorectal cancer (CRC); however, owing to their low response rate and adverse effects, the development of efficient drug delivery systems (DDSs) is required. The cellular prion protein PrP^C, which is a cell surface glycoprotein, has been demonstrated to be overexpressed in CRC, however, there has been no research on the development of PrP^C-targeting DDSs for targeted drug delivery to CRC. In this study, PrP^C aptamer (Apt)-conjugated gold nanoparticles (AuNPs) were synthesized for targeted delivery of Dox to CRC. Thiol-terminated PrP^C-Apt was conjugated to AuNPs, followed by hybridization of its complementary DNA for drug loading. Finally, Dox was loaded onto the AuNPs to synthesize PrP^C-Apt-functionalized doxorubicin-oligomer-AuNPs (PrP^C-Apt DOA). The PrP^C-Apt DOA were spherical nanoparticles with an average diameter of 20 nm. Treatment of CRC cells with PrP^C-Apt DOA induced reactive oxygen species generation by decreasing catalase and superoxide dismutase activities. In addition, treatment with PrP^C-Apt DOA inhibited mitochondrial functions by decreasing the expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha, complex 4 activity, and oxygen consumption rates. Compared to free Dox, PrP^C-Apt DOA decreased proliferation and increased apoptosis of CRC cells to a greater degree. In this study, we demonstrated that PrP^C-Apt DOA targeting could effectively deliver Dox to CRC cells. PrP^C-Apt DOA can be used as a treatment for CRC, and have the potential to replace existing anticancer drugs, such as 5-FU, oxaliplatin, and Dox.

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.

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.

Human mesenchymal stem cells (hMSCs) are a potent source of cell-based regenerative therapeutics used to treat patients with ischemic disease. However, disease-induced oxidative stress disrupts mitochondrial homeostasis in transplanted hMSCs, resulting in hMSC apoptosis and reducing their efficacy post-transplantation. To address this issue, we evaluated the effects of melatonin on cellular defense mechanisms and mitophagy in hMSCs subjected to oxidative stress. H₂O₂-induced oxidative stress increases the levels of reactive oxygen species and reduces membrane potential in hMSCs, leading to mitochondrial dysfunction and cell death. Oxidative stress also decreases the expression of 70-kDa heat shock protein 1L (HSPA1L), a molecular chaperone that assists in the recruitment of parkin to the autophagosomal mitochondrial membrane. Decreased expression of HSPA1L destabilizes parkin, thereby impairing mitophagy. Our results indicate that treating hMSCs with melatonin significantly inhibited mitochondrial dysfunction induced by oxidative stress, which decreased hMSCs apoptosis. In damaged hMSCs, treatment with melatonin increased the levels of HSPA1L, which bound to parkin. The interaction between HSPA1L and parkin increased membrane potential and levels of oxidative phosphorylation, resulting in enhanced mitophagy. Our results indicate that melatonin increased the expression of HSPA1L, thereby upregulating mitophagy and prolonging cell survival under conditions of oxidative stress. In this study, we have shown that melatonin, a readily available compound, can be used to improve hMSC-based therapies for patients with pathologic conditions involving oxidative stress.

Renal fibrosis, a major risk factor for kidney failure, can lead to chronic kidney disease (CKD) and is caused by cytoskeleton reorganization and mitochondrial dysfunction. In this study, we investigated the potential of melatonin treatment to reduce renal fibrosis by recovering the cytoskeleton reorganization and mitochondrial dysfunction. We found that miR-4516 expression was downregulated in the renal cortex of CKD mice and P-cresol-treated TH1 cells. Decreased miR-4516 expression stimulated cytoskeleton reorganization and mitochondrial dysfunction, and induced renal fibrosis. Melatonin treatment suppressed fibrosis by inhibiting cytoskeleton reorganization and restoring mitochondrial function via increased miR-4516 expression. More specifically, melatonin treatment increased miR-4516 expression while decreasing ITGA9 expression, thereby inhibiting cytoskeleton reorganization. In addition, increased expression of miR-4516 by melatonin treatment reduced ROS formation and restored mitochondrial function. These findings suggest that melatonin may be a promising treatment for patients with CKD having renal fibrosis. Moreover, regulation of miR-4516 expression may be a novel strategy for the treatment of renal fibrosis.