Voltage-gated sodium channel scn8a is required for innervation and regeneration of amputated adult zebrafish fins.

Teleost fishes and urodele amphibians can regenerate amputated appendages, whereas this ability is restricted to digit tips in adult mammals. One key component of appendage regeneration is reinnervation of the wound area. However, how innervation is regulated in injured appendages of adult vertebrates has seen limited research attention. From a forward genetics screen for temperature-sensitive defects in zebrafish fin regeneration, we identified a mutation that disrupted regeneration while also inducing paralysis at the restrictive temperature. Genetic mapping and complementation tests identify a mutation in the major neuronal voltage-gated sodium channel (VGSC) gene scn8ab. Conditional disruption of scn8ab impairs early regenerative events, including blastema formation, but does not affect morphogenesis of established regenerates. Whereas scn8ab mutations reduced neural activity as expected, they also disrupted axon regrowth and patterning in fin regenerates, resulting in hypoinnervation. Our findings indicate that the activity of VGSCs plays a proregenerative role by promoting innervation of appendage stumps.

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.

Generation of Myocardial Ischemic Wounds and Healing with Stem Cells.

Myocardial ischemia is a common manifestation of cardiovascular diseases (CVD) that affects the health and lives of millions of people worldwide. While numerous treatment options exist that address cardiac damage after ischemic injury, none of these can repair damaged cardiac tissue. Stem cell-mediated therapy is an emerging approach for cardiac tissue regeneration that has shown promise in preclinical models and in clinical studies. However, much more research in this field must be carried out to bring effective stem cell therapies to clinical settings. This protocol discusses the methods for generation of an animal model of myocardial ischemia in a preclinical setting, expansion of viable hematopoietic stem cells on a nanofiber scaffold, and administration of cells into the ischemic animal to verify therapeutic efficacy.

Transcriptional Regulation of Osteoclastogenesis: The Emerging Role of KLF2.

Dysregulation of osteoclastic differentiation and its activity is a hallmark of various musculoskeletal disease states. In this review, the complex molecular factors underlying osteoclastic differentiation and function are evaluated. The emerging role of KLF2 in regulation of osteoclastic differentiation is examined, specifically in the context of rheumatoid arthritis in which it has been most extensively studied among the musculoskeletal diseases. The therapies that exist to manage diseases associated with osteoclastogenesis are numerous and diverse. They are varied in their mechanisms of action and in the outcomes they produce. For this review, therapies targeting osteoclasts will be emphasized, though it should be noted that many therapies exist which bolster the action of osteoblasts. A new targeted molecular approach is under investigation for the future potential therapeutic development of rheumatoid arthritis.

Ferutinin directs dental pulp-derived stem cells towards the osteogenic lineage by epigenetically regulating canonical Wnt signaling.

Osteoporosis is a silent systemic disease that causes bone deterioration, and affects over 10 million people in the US alone. This study was undertaken to develop a potential stem cell therapy for osteoporosis. We have isolated and expanded human dental pulp-derived stem cells (DPSCs), characterized them, and confirmed their multipotential differentiation abilities. Stem cells often remain quiescent and require activation to differentiate and function. Herein, we show that ferutinin activates DPSCs by modulating the Wnt/ß-catenin signaling pathway and key osteoblast-secreted proteins osteocalcin and collagen 1A1 both mRNA and protein levels. To confirm that ferutinin modulates the Wnt pathway, we inhibited glycogen synthase kinase 3 (GSK3) and found that protein expression patterns were similar to those found in ferutinin-treated DPSCs. To evaluate the role of ferutinin in epigenetic regulation of canonical Wnt signaling, the pathway molecules Wnt3a and Dvl3 were analyzed using chromatin immunoprecipitation (ChIP)-quantitative PCR approaches. We confirmed that active marks of both H3K9 acetylation and H3K4 trimethylation were significantly enhanced in the promoter sites of the WNT3A and DVL3 genes in DPSCs after addition of ferutinin. These data provide evidence that ferutinin activates and promotes osteogenic differentiation of DPSCs, and could be used as an inducer as a potentially effective stem cell therapy for osteoporosis.

Inhibiting p21-Activated Kinase Induces Cell Death in Vestibular Schwannoma and Meningioma via Mitotic Catastrophe.

HYPOTHESIS: p21-activated kinase (PAK) regulates signaling pathways that promote cell survival and proliferation; therefore, pharmacological inhibition of PAK will induce cell death in vestibular schwannomas (VS) and meningiomas. BACKGROUND: All VS and many meningiomas result from loss of the neurofibromatosis type 2 (NF2) gene product merlin, with ensuing PAK hyperactivation and increased cell proliferation/survival. METHODS: The novel small molecule PAK inhibitors PI-8 and PI-15-tested in schwannoma and meningioma cells-perturb molecular signaling and induce cell death. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, flow cytometry, and terminal deoxynucleotidyl transferase dUTP nick end labeling assay analyzed PAK inhibitors' effect on cell viability, cell cycle, and cell death, respectively. Western blots evaluated activation and expression of cell proliferation, apoptotic, and mitotic catastrophe markers. Light microscopy evaluated cell morphology, and immunocytochemistry analyzed cellular localization of phospho-Merlin and autophagy-related protein. RESULTS: Treatment with PI-8 and PI-15 decreased cell viability at 0.65 to 3.7 µM 50% inhibitory concentration (IC50) in schwannoma and meningioma cells. Terminal deoxynucleotidyl transferase dUTP nick end labeling and immunocytochemistry studies show that PI-8 and PI-15 induce mitotic catastrophe but not apoptosis in HEI193 cells while in BenMen1 cells, PI-8 induces autophagy and mitotic catastrophe. PI-15 induces apoptosis in BenMen1 cells. PAK inhibitor treated cells show phospho-Merlin localized to over-duplicated centrosomes of dividing cells, multiple enlarged nuclei, and misaligned/missegregated chromosomes-markers for mitotic catastrophe. Increased autophagy-related protein levels in the nucleus confirmed this cell death type. PI-8 and PI-15 inhibits PAK in both cell lines. However, only PI-15 inhibits v-akt murine thymoma viral oncogene homolog in BenMen1 cells. CONCLUSION: PAK inhibitors induce cell death in schwannoma and meningioma cells, at least in part, by mitotic catastrophe.