Secretory products of DPSC mitigate inflammatory effects in microglial cells by targeting MAPK pathway.

Activated microglial cells in the central nervous system (CNS) are the main contributors to neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease. Inhibiting their activation will help in reducing inflammation and oxidative stress during pathogenesis, potentially limiting the progression of the diseases. The immunomodulation properties of dental pulp-derived stem cells (DPSC) make it a promising therapy for neurodegenerative disorders. This study aims to determine whether secretory factors of DPSC (DPSC℗) inhibit inflammation and proliferation of microglial cells and define the molecular mechanisms. Our quantitative RT-PCR analysis showed that the DPSC℗ reduced the markers of the inflammation and induced anti-inflammatory molecules in microglial cells. DPSC ℗ reduced the intracellular and mitochondrial reactive oxygen species (ROS) production and mitochondrial membrane potential in microglial cells. In addition, DPSC ℗ decreased the cellular bioenergetics parameters related to oxygen consumption rate (OCAR) and extracellular acidification rate (ECAR). We found that DPSC℗ inhibited microglial cell proliferation by activating a checkpoint molecule, Chk1 leading an arrest at the G1 phase of the cell cycle. To define the mechanism, we performed the western blot analysis and observed that the MAPK P38 pathway was inhibited by DPSC℗. Furthermore, a System biology analysis revealed that the BDNF and GDNF, secretory factors of DPSC, blocked at the phosphorylation site (Tyr 182) of the P38 molecule resulting in the inhibition of downstream signaling of inflammation. These data suggest that the DPSC℗ may be a potential therapeutic agent for neurodegenerative diseases.

KLF2 Regulates Neural Differentiation of Dental Pulp-derived Stem Cells by Modulating Autophagy and Mitophagy.

BACKGROUND: Transplantation of stem cells for treating neurodegenerative disorders is a promising future therapeutic approach. However, the molecular mechanism underlying the neuronal differentiation of dental pulp-derived stem cells (DPSC) remains inadequately explored. The current study aims to define the regulatory role of KLF2 (Kruppel-like factor 2) during the neural differentiation (ND) of DPSC. METHODS: We first investigated the transcriptional and translational expression of KLF2, autophagy, and mitophagy-associated markers during the ND of DPSC by using quantitative RT-PCR and western blot methods. After that, we applied the chemical-mediated loss- and gain-of-function approaches using KLF2 inhibitor, GGPP (geranylgeranyl pyrophosphate), and KLF2 activator, GGTI-298 (geranylgeranyl transferase inhibitor-298) to delineate the role of KLF2 during ND of DPSC. The western blot, qRT-PCR, and immunocytochemistry were performed to determine the molecular changes during ND after KLF2 deficiency and KLF2 sufficiency. We also analyzed the oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) using the Seahorse XFe24 analyzer. RESULTS: Our study demonstrated that the expression level of KLF2, autophagy, and mitophagy-associated markers were significantly elevated during the ND of DPSC. Next, we found that the KLF2 inhibitor, GGPP significantly reduced the ND of DPSC. Inversely, KLF2 overexpression accelerated the molecular phenomenon of DPSC's commitment towards ND, indicating the crucial role of KLF2 in neurogenesis. Moreover, we found that the KLF2 positively regulated autophagy, mitophagy, and the Wnt5a signaling pathway during neurogenesis. Seahorse XFe24 analysis revealed that the ECAR and OCR parameters were significantly increased during ND, and inhibition of KLF2 marginally reversed them towards DPSC's cellular bioenergetics. However, KLF2 overexpression shifted the cellular energy metabolism toward the quiescent stage. CONCLUSION: Collectively, our findings provide the first evidence that the KLF2 critically regulates the neurogenesis of DPSC by inducing autophagy and mitophagy.

Dental Pulp-Derived Stem Cells Preserve Astrocyte Health During Induced Gliosis by Modulating Mitochondrial Activity and Functions.

Astrocytes have been implicated in the onset and complication of various central nervous system (CNS) injuries and disorders. Uncontrolled astrogliosis (gliosis), while a necessary process for recovery after CNS trauma, also causes impairments in CNS performance and functions. The ability to preserve astrocyte health and better regulate the gliosis process could play a major role in controlling damage in the aftermath of acute insults and during chronic dysfunction. Here in, we demonstrate the ability of dental pulp-derived stem cells (DPSCs) in protecting the health of astrocytes during induced gliosis. First of all, we have characterized the expression of genes in primary astrocytes that are relevant to the pathological conditions of CNS by inducing gliosis. Subsequently, we found that astrocytes co-cultured with DPSCs reduced ROS production, NRF2 and GCLM expressions, mitochondrial membrane potential, and mitochondrial functions compared to the astrocytes that were not co-cultured with DPSCs in gliosis condition. In addition, hyperactive autophagy was also decreased in astrocytes that were co-cultured with DPSCs compared to the astrocytes that were not co-cultured with DPSCs during gliosis. This reversal and mitigation of gliosis in astrocytes were partly due to induction of neurogenesis in DPSCs through enhanced expressions of the neuronal genes like GFAP, NeuN, and Synapsin in DPSCs and by secretion of higher amounts of neurotropic factors, such as BDNF, GDNF, and TIMP-2. Protein-Protein docking analysis suggested that BDNF and GDNF can bind with CSPG4 and block the downstream signaling. Together these findings demonstrate novel functions of DPSCs to preserve astrocyte health during gliosis.

Epigallocatechin-3-gallate inhibits osteoclastic differentiation by modulating mitophagy and mitochondrial functions.

A natural plant product, epigallocatechin-3-gallate (EGCG), was evaluated for its effectiveness in the regulation of osteoclastogenesis. We found that EGCG inhibited the osteoclast (OC) differentiation in vitro, and in primary bone marrow cells in a dose-dependent manner. Quantitative RT-PCR studies showed that the EGCG reduced the expression of OC differentiation markers. DCFDA, MitoSOX, and JC-1 staining revealed that the EGCG attenuated the reactive oxygen species (ROS), and mitochondrial membrane potential; and flux analysis corroborated the effect of EGCG. We further found that the EGCG inhibited mRNA and protein expressions of mitophagy-related molecules. We confirmed that the OC differentiation was inhibited by EGCG by modulating mitophagy through AKT and p38MAPK pathways. Furthermore, in silico analysis revealed that the binding of RANK and RANKL was blocked by EGCG. Overall, we defined the mechanisms of osteoclastogenesis during arthritis for developing a new therapy using a natural compound besides the existing therapeutics.

Polyphenolic Compounds Inhibit Osteoclast Differentiation While Reducing Autophagy through Limiting ROS and the Mitochondrial Membrane Potential.

Polyphenolic compounds are a diverse group of natural compounds that interact with various cellular proteins responsible for cell survival, differentiation, and apoptosis. However, it is yet to be established how these compounds interact in myeloid cells during their differentiation and the molecular and intracellular mechanisms involved. Osteoclasts are multinucleated cells that originate from myeloid cells. They resorb cartilage and bone, maintain bone homeostasis, and can cause pathogenesis. Autophagy is a cellular mechanism that is responsible for the degradation of damaged proteins and organelles within cells and helps maintain intracellular homeostasis. Imbalances in autophagy cause various pathological disorders. The current study investigated the role of several polyphenolic compounds, including tannic acid (TA), gallic acid (GA), and ellagic acid (EA) in the regulation of osteoclast differentiation of myeloid cells. We demonstrated that polyphenolic compounds inhibit osteoclast differentiation in a dose-dependent manner. Quantitative real-time PCR, immunocytochemistry, and western blotting revealed that osteoclast markers, such as NFATc1, Cathepsin K, and TRAP were inhibited after the addition of polyphenolic compounds during osteoclast differentiation. In our investigation into the molecular mechanisms, we found that the addition of polyphenolic compounds reduced the number of autophagic vesicles and the levels of LC3B, BECN1, ATG5, and ATG7 molecules through the inactivation of Akt, thus inhibiting the autophagy process. In addition, we found that by decreasing intracellular calcium and decreasing ROS levels, along with decreasing mitochondrial membrane potential, polyphenolic compounds inhibit osteoclast differentiation. Together, this study provides evidence that polyphenolic compounds inhibit osteoclast differentiation by reducing ROS production, autophagy, intracellular Ca2+ level, and mitochondrial membrane potentials.

DPSC Products Accelerate Wound Healing in Diabetic Mice through Induction of SMAD Molecules.

Despite advances in diabetic wound care, many amputations are still needed each year due to their diabetic wounds, so a more effective therapy is warranted. Herein, we show that the dental pulp-derived stem cell (DPSC) products are effective in wound healing in diabetic NOD/SCID mice. Our results showed that the topical application of DPSC secretory products accelerated wound closure by inducing faster re-epithelialization, angiogenesis, and recellularization. In addition, the number of neutrophils producing myeloperoxidase, which mediates persisting inflammation, was also reduced. NFκB and its downstream effector molecules like IL-6 cause sustained pro-inflammatory activity and were reduced after the application of DPSC products in the experimental wounds. Moreover, the DPSC products also inhibited the activation of NFκB, and its translocation to the nucleus, by which it initiates the inflammation. Furthermore, the levels of TGF-ß, and IL-10, potent anti-inflammatory molecules, were also increased after the addition of DPSC products. Mechanistically, we showed that this wound-healing process was mediated by the upregulation and activation of Smad 1 and 2 molecules. In sum, we have defined the cellular and molecular mechanisms by which DPSC products accelerated diabetic wound closure, which can be used to treat diabetic wounds in the near future.

Ferutinin induces osteoblast differentiation of DPSCs via induction of KLF2 and autophagy/mitophagy.

Osteoblast differentiation is critically reduced in various bone-related pathogenesis, including arthritis and osteoporosis. For future development of effective regenerative therapeutics, herein, we reveal the involved molecular mechanisms of a phytoestrogen, ferutinin-induced initiation of osteoblast differentiation from dental pulp-derived stem cell (DPSC). We demonstrate the significantly increased expression level of a transcription factor, Kruppel-like factor 2 (KLF2) along with autophagy-related molecules in DPSCs after induction with ferutinin. The loss-of-function and the gain-of-function approaches of KLF2 confirmed that the ferutinin-induced KLF2 modulated autophagic and OB differentiation-related molecules. Further, knockdown of the autophagic molecule (ATG7 or BECN1) from DPSC resulted not only in a decreased level of KLF2 but also in the reduced levels of OB differentiation-related molecules. Moreover, mitochondrial membrane potential-related molecules were increased and induction of mitophagy was observed in DPSCs after the addition of ferutinin. The reduction of mitochondrial as well as total ROS generations; and induction of intracellular Ca2+ production were also observed in ferutinin-treated DPSCs. To test the mitochondrial respiration in DPSCs, we found that the cells treated with ferutinin showed a reduced extracellular acidification rate (ECAR) than that of their vehicle-treated counterparts. Furthermore, mechanistically, chromatin immunoprecipitation (ChIP) analysis revealed that the addition of ferutinin in DPSCs not only induced the level of KLF2, but also induced the transcriptionally active epigenetic marks (H3K27Ac and H3K4me3) on the promoter region of the autophagic molecule ATG7. These results provide strong evidence that ferutinin stimulates OB differentiation via induction of KLF2-mediated autophagy/mitophagy.

Evaluation of Urea-Based Inhibitors of the Dopamine Transporter Using the Experimental Autoimmune Encephalomyelitis Model of Multiple Sclerosis.

The dopaminergic system is involved in the regulation of immune responses in various homeostatic and disease conditions. For conditions such as Parkinson's disease and multiple sclerosis (MS), pharmacological modulation of dopamine (DA) system activity is thought to have therapeutic relevance, providing the basis for using dopaminergic agents as a treatment of relevant states. In particular, it was proposed that restoration of DA levels may inhibit neuroinflammation. We have recently reported a new class of dopamine transporter (DAT) inhibitors with high selectivity to the DAT over other G-protein coupled receptors tested. Here, we continue their evaluation as monoamine transporter inhibitors. Furthermore, we show that the urea-like DAT inhibitor (compound 5) has statistically significant anti-inflammatory effects and attenuates motor deficits and pain behaviors in the experimental autoimmune encephalomyelitis model mimicking clinical signs of MS. To the best of our knowledge, this is the first study reporting the beneficial effects of DAT inhibitor-based treatment in animals with induced autoimmune encephalomyelitis, and the observed results provide additional support to the model of DA-related neuroinflammation.

Generation of Acute Hind Limb Ischemia in NOD/SCID Mice and Treatment with Nanofiber-Expanded CD34+ Hematopoietic Stem Cells.

Critical limb ischemia (CLI) is primarily associated with a high risk of major amputation, cardiovascular events, and death. The current therapy involves direct endovascular intervention and is associated with long-term recurrence. However, patients with significant comorbidities are not eligible for this therapy. Hind limb ischemia model via femoral artery excision has commonly been used to determine therapeutic potential and for investigating cellular and molecular mechanisms. This protocol describes the ischemic model development in NOD/SCID mice and the use of human umbilical cord blood-derived and nanofiber scaffold-expanded CD34+ stem cells to investigate the efficacy of regenerative therapy.

Current advances in ischemic stroke research and therapies.

With more than 795,000 cases occurring every year, stroke has become a major problem in the United States across all demographics. Stroke is the leading cause of long-term disability and is the fifth leading cause of death in the US. Ischemic stroke represents 87% of total strokes in the US, and is currently the main focus of stroke research. This literature review examines the risk factors associated with ischemic stroke, changes in cell morphology and signaling in the brain after stroke, and the advantages and disadvantages of in vivo and in vitro ischemic stroke models. Classification systems for stroke etiology are also discussed briefly, as well as current ischemic stroke therapies and new therapeutic strategies that focus on the potential of stem cells to promote stroke recovery.