Inhibitory effect of melatonin on Mst1 ameliorates myocarditis through attenuating ER stress and mitochondrial dysfunction Abstract Viral myocarditis has been found to be one of the leading causes of sudden death in young adults. However, no effective drugs have been developed to intervene the progression of myocarditis. Accordingly, the present study is carried out to explore the protective role played by melatonin in the setting of viral myocarditis with a focus on Mst1-Hippo pathway, mitochondrial dysfunction and ER stress. Cardiac function was determined via echocardiographic examination. Mitochondrial function and ER stress were detected via ELISA, western blots, and immunofluorescence. Our data demonstrated that virus injection induced cardiac dysfunction as evidenced by reduced contractile function in myocardium. Besides, LDH release assay and western blotting analysis demonstrated that cardiomyocyte death was activated by virus injection. Interestingly, melatonin treatment improved cardiac function and repressed virus-mediated cardiomyocyte apoptosis. At the molecular levels, mitochondrial dysfunction was induced by virus infection, as indicated by mitochondrial membrane potential reduction, mPTP opening rate elevation and caspase-9-related apoptosis activation. Besides, ER stress parameters were also elevated in virus-treated cardiomyocytes. Interestingly, melatonin treatment maintained mitochondrial dysfunction and repressed ER stress. To the end, we found that Mst1 was upregulated by virus infection; this effect was attenuated through supplementation with melatonin. However, Mst1 overexpression reduced the beneficial impact exerted by melatonin on cardiomyocyte viability, mitochondrial function and ER homeostasis. Our study illustrated that melatonin treatment attenuated viral myocarditis via sustaining cardiomyocyte viability, repressing mitochondrial dysfunction and inhibiting ER stress in a manner dependent on Mst1 inhibition.

Cytoarchitecture of the dorsal claustrum of the cat: a quantitative Golgi study Abstract The claustrum is a subcortical nucleus, found in the telencephalon of all placental mammals. Earlier Golgi studies have mostly focused on a qualitative description of the types of neurons. The aim of the present study was to describe the types of neurons found in the dorsal claustrum of the cat using the Golgi impregnation method and to perform a quantitative analysis of the following morphometric parameters: number of terminals (ends), total dendritic length, dendritic complexity, spine density (in spiny projection neurons), varicosity density (in aspiny interneurons). We used specimens from 5 healthy male cats stained according to the Golgi-Cox method. The dendritic trees of the studied neurons were then reconstructed through the Neurolucida software. Values of the studied quantitative parameters were obtained automatically and tested for statistically significant differences. Five types of spiny neurons were observed—large, medium-sized and small multipolar, bipolar and pyramidal-like. In addition, we described three types of aspiny neurons. The quantitative values and the statistical analysis were presented with tables and diagrams. In conclusion, we have presented a detailed analysis of the cytoarchitecture of the DC of the cat and have reported the first quantitative data on a number of morphometric parameters.

NFIC promotes the vitality and osteogenic differentiation of rat dental follicle cells Abstract Nuclear factor I-C (NFIC) plays critical roles in the regulation of tooth development by influencing the biological behaviors of stem cells in the dental germ. This study aimed to investigate the effect of NFIC on the vitality and osteogenic/cementogenic differentiation of rat dental follicle cells (DFCs). DFCs were isolated from dental follicles in the first molars of neonatal rats. DFCs expressed mesenchymal stromal cell markers CD29, CD44 and CD90 and had capabilities for self-renewal and multipotent differentiation. Overexpression of NFIC promoted the proliferation of DFCs without markedly influencing the apoptosis of DFCs. Moreover, NFIC increased alkaline phosphatase (ALP) activity in DFCs and upregulated the mRNA levels of osteogenic-related markers, namely, collagen type I (Col I), Runt-related transcription factor 2 (Runx2) and ALP, as well as β-catenin. In contrast, silencing NFIC by siRNA increased the apoptosis of DFCs and downregulated the expression of osteogenic-related markers. In conclusion, these results suggested that upregulation of NFIC may promote the proliferation and osteogenic/cementogenic differentiation of DFCs.

Low temperature culture enhances ameloblastic differentiation of human keratinocyte stem cells Abstract Previous studies have demonstrated that several types of human stem cells of non-dental origin can be induced to differentiate into enamel-secreting ameloblasts after recombined with mouse embryonic dental mesenchyme. However, the successful rate of ameloblastic differentiation is about rather low, which presents a major obstacle for future stem cell-based whole tooth bioengineering. Previous studies have shown that cultures at reduced temperature could improve the differentiation capability of stem cells in tissue engineering. In this study, we systematically investigated the effects of low temperature on the viability, proliferation and stemness of human keratinocytes stem cells (hKSCs) in cell culture and further examined ameloblastic differentiation of the hKSCs in human–mouse recombinant chimeric tooth germs. Our results demonstrated that low temperature indeed reduces growth rate and maintains healthy undifferentiated morphology of hKSCs without any effects on cell viability. Moreover, examination of stemness makers revealed improved stemness of hKSCs cultured at low temperature with increased expression of stemness markers K15, CD29 and p63 and decreased expression differentiation marker K10, as compared to those cultured at 37 °C. These low temperature treated hKSCs, when recombined with mouse embryonic dental mesenchyme, exhibited significantly increased rate (40%) of ameloblastic differentiation, as compared to that (17%) in tissue recombinants with those hKSCs treated at standard temperature. Our studies demonstrate that low temperature cell culture improves the stemness and plasticity of hKSCs, which in turn enhances ameloblastic differentiation capability of the stem cells in bioengineered teeth.

Tunneling nanotubes mediate intercellular communication between endothelial progenitor cells and osteoclast precursors Abstract Tunneling nanotube (TNT)-mediated cell communication play pivotal roles in a series of physiological and pathological processes in multicellular organism. This study was designed to investigate the existence of TNTs between EPCs and osteoclast precursors and evaluate their effects on the differentiation of osteoclast precursors. For these purposes, EPCs and osteoclast precursors (RAW264.7 cells) were stained with different fluorescent dyes before direct co-culture; then, the co-cultured cells were sorted by fluorescence activated cell sorter (FACS), and the differentiation of co-cultured RAW264.7 cells was evaluated. The results showed that the differentiation potential of RAW264.7 cells was significantly inhibited after their co-culture with EPCs. Additionally, the expression of macrophage migration inhibitory factor (MIF) was up-regulated in RAW264.7 cells after co-culture. Moreover, the MIF inhibitor ISO-1 could rescue the formation of TRAP-positive multinuclear osteoclasts and the expression of osteoclastogenesis-associated genes in the co-cultured RAW264.7 cells. The present study demonstrates that EPCs can affect the differentiation of osteoclast precursors through the TNT-like structures formed across these two types of cells and might inform new therapeutic strategies for osteolytic diseases.

Expression of Ihh signaling pathway in condylar cartilage after bite-raising in adult rats Abstract Temporomandibular joint osteoarthritis (TMJOA) is a complex inflammatory condition with multiple factors and degenerative processes co-occurring. However, its pathogenesis remains uncertain. The purpose of the study was to observe the expression of Indian hedgehog (Ihh) signal related molecules in TMJOA induced by bite-raising and to study the effect and mechanism of Ihh signaling. Our research indicated that Ihh signaling pathway can be activated in condylar cartilage induced by bite-raising. The histological analysis showed TMJOA-like structural changes of condylar cartilage in experiment groups. Ihh, Smoothened (Smo), and Gli zinc finger transcription factors-1 (Gli-1) were activated in the experimental groups, and the expression levels increased significantly over time, whereas the sham control groups showed no fluctuation. Additionally, the expression levels of matrix metalloproteinase-13 (MMP-13) and cysteinyl aspartate specific proteinase-3 (Caspase-3) in the experiment groups increased in a time-dependent manner compared with the matched sham control groups. In conclusion, our results indicated that the Ihh signaling pathway may activate the occurrence of TMJOA by mediating the hypertrophy of chondrocytes, which may be an important regulatory mechanism and potential therapeutic target in the repair of condylar cartilage.

Semaphorin 3A gets involved in the establishment of mouse tooth eruptive pathway Abstract The accurately establishment of the eruptive pathway is of vital importance. The mechanisms governing tooth eruption pathway remain little known. This study is to elucidate the roles of Semaphorin 3A (Sema 3A) in mouse tooth eruptive pathway. C57BL/6 mice (11–13 and 15–17 days after birth) were chosen to observe eruptive pathway of mouse lower first molar. Expressions of Sema 3A and its receptor neuropilin 1 and plexin A1 were detected. Osteoclasts were identified by TRAP staining. Co-localization of Sema 3A and osteoclast maker CD68 was detected by double immunofluorescence staining. Picrosirius red staining was applied to observe collagen fibers during mucosal penetration phase. In vitro, Bone marrow-derived macrophages (BMMs) were prepared from 4 week C57BL/6 mice to observe the effect of Sema 3A on the differentiation of BMMs into osteoclasts by TRAP staining. Expressions of Sema 3A was observed by immunofluorescence and western blotting. At osseous eruption phase, many TRAP-positive multi-nucleated cells were distributed around occlusal alveolar bone. The positive expressions of Sema 3A were observed in the multi-nucleated cells. Fluorescence double staining showed that Sema 3A and CD68 were co-expressed in osteoclasts. Its receptor neuropilin 1 and plexin A1 were also found in osteoclasts. In vitro, Sema3A negatively regulated osteoclast differentiation. At mucosal penetration, occlusal alveolar bone had been completely resorbed and collagen fires were gradually degraded for eruptive pathway. Similar positive expressions of Sema 3A and its receptor neuropilin 1 and plexin A1 were also found in the mucosal penetration pathway. Sema 3A gets involved in the establishment of mouse tooth eruptive pathway by modulating osteoclast activity. Sema3A should be considered as a novel nervous agent or a potential biomarker for mouse tooth eruptive pathway.

Tamarixetin protects against cardiac hypertrophy via inhibiting NFAT and AKT pathway Abstract Cardiac hypertrophy is a compensatory response in reaction to mechanical load that reduces wall stress by increasing wall thickness. Chronic hypertrophic remodeling involves cardiac dysfunction that will lead to heart failure and ultimately death. Studies have been carried out on cardiac hypertrophy for years, whereas the mechanisms have not been well defined. Tamarixetin (TAM), a natural flavonoid derivative of quercetin, have been demonstrated possessing anti-oxidative and anti-inflammatory effects on multiple diseases. However, little is known about the function of TAM on the development of cardiac hypertrophy. Here, we found TAM could alleviate pressure-overload-induced cardiac hypertrophy in transverse aortic constriction (TAC) mouse model, assessed by ventricular weight/body weight, lung weight/body weight, echocardiographic parameters, as well as myocyte cross-sectional area and the expression of ANP, BNP and Myh7. In vitro, TAM showed a dose dependent inhibitory effect on phenylephrine-induced hypertrophy in H9c2 cardiomyocytes. Furthermore, TAM reversed cardiac remodeling of stress overloaded heart by suppressing apoptosis and the expression of fibrotic-related genes, reduced oxidative stress and ROS production both in vivo and in vitro. In addition, TAM could negatively modulate TAC-induced nuclear translocation of NFAT and the activation of PI3K/AKT signaling pathways. Therefore, these data indicate for the first time that TAM has a protective effect on experimental cardiac hypertrophy and might be a novel candidate for the treatment of cardiac hypertrophy in clinic.

In vitro assessment of PD-L1+ microvesicles in the cyst fluid of non-syndromic odontogenic keratocysts Abstract Odontogenic keratocysts (OKCs) are jaw cystic lesions which are characterized by local invasion and high recurrence rate. The majority of OKCs are exposed to microorganisms and occur along with focal inflammatory infiltrates. Cyst fluids are biological fluids that contain a large content of cytokines and immune globulins. Inhibitory receptor such as programmed death receptor 1 (PD-1) and its ligand programmed death-ligand 1 (PD-L1), which can induce a coinhibitory signal in activated T cells, plays a vital role in the differentiation, exhaustion and apoptosis of T cells. Cell derived microvesicles, carrying a cargo of functional proteins, nucleic acids and lipids, are important communication tools in the development of diseases. However, the expression of PD-L1 in OKCs tissues and whether PD-L1 could be carried by microvesicles are unexplored. Presently, we have isolated cyst fluid microvesicles and identified cell derived PD-L1+ cyst fluid microvesicles. PD-L1 was located in the membrane of the cyst fluid microvesicles. The main cellular origins of PD-L1+ cyst fluid microvesicles were dendritic cells followed by lymphocytes. Elevated PD-L1+ cyst fluid microvesicles were detected in the OKCs compared with dentigerous cysts. Isolated cyst fluid microvesicles could bind to the membrane of activated CD8 T cells and inhibit proliferation of stimulated peripheral blood CD8 T cells. In conclusion, the present study suggests that elevated PD-L1+ cyst fluid microvesicles might be related with the cyst development of OKCs.

Impaired autophagy mediates hyperhomocysteinemia-induced HA-VSMC phenotypic switching Abstract Hyperhomocysteinemia (HHcy) is a highly-related risk factor in vascular smooth muscle cell (VSMC) phenotypic modulation and atherosclerosis. Growing evidence indicated that autophagy is involved in pathological arterial changes. However, the risk mechanisms by which homocysteine and VSMC autophagy interact with cardiovascular disease are poorly understood. This study verified the homocysteine-responsive endoplasmic reticulum protein promotion of VSMC phenotypic switching, and the formation of atherosclerotic plaque in vitro. We found that impaired autophagy, as evidenced by decreased levels of MAP1LC3B II/MAP1LC3B I, has a vital role in HHcy-induced human aortic (HA)-VSMC phenotypic switching, with a decrease in contractile proteins (SM α-actin and calponin) and an increase in osteopontin. Knockdown of the essential autophagy gene Atg7 by small interfering RNA promoted HA-VSMC phenotypic switching, indicating that impaired autophagy induces phenotypic switching in these cells. HHcy co-treatment with rapamycin triggered autophagy, which alleviated HA-VSMC phenotypic switching. Finally, we found that Krüppel-like factor 4 (KLF4), a zinc-finger transcription factor for maintaining genomic stability by resisting oxidative stress and restoring autophagy, is closely involved in this process. HHcy clearly decreased KLF4 expression. KLF4-specific siRNA aggravated defective autophagy and phenotypic switching. Mechanistically, KLF4 regulated the HHcy-induced decrease in HA-VSMC autophagy via the m-TOR signaling pathway. In conclusion, these results demonstrated that the KLF4-dependent rapamycin signaling pathway is a novel mechanism underlying HA-VSMC phenotypic switching and is crucial for HHcy-induced HA-VSMCs with defective autophagy to accelerate early atherosclerosis.