Δευτέρα, 13 Μαΐου 2019

Molecular Histology

Distribution of sperm antigen 6 (SPAG6) and 16 (SPAG16) in mouse ciliated and non-ciliated tissues

Abstract

The cilia and flagella of eukaryotic cells serve many functions, exhibiting remarkable conservation of both structure and molecular composition in widely divergent eukaryotic organisms. SPAG6 and SPAG16 are the homologous in the mice to Chlamydomonas reinhardtii PF16 and PF20. Both proteins are associated with the axonemal central apparatus and are essential for ciliary and flagellar motility in mammals. Recent data derived from high-throughput studies revealed expression of these genes in tissues that do not contain motile cilia. However, the distribution of SPAG6 and SPAG16 in ciliated and non-ciliated tissues is not completely understood. In this work, we performed a quantitative analysis of the expression of Spag6 and Spag16 genes in parallel with the immune-localization of the proteins in several tissues of adult mice. Expression of mRNA was higher in the testis and tissues bearing motile cilia than in the other analyzed tissues. Both proteins were present in ciliated and non-ciliated tissues. In the testis, SPAG6 was detected in spermatogonia, spermatocytes, and in the sperm flagella whereas SPAG16 was found in spermatocytes and in the sperm flagella. In addition, both proteins were detected in the cytoplasm of cells from the brain, spinal cord, and ovary. A small isoform of SPAG16 was localized in the nucleus of germ cells and some neurons. In a parallel set of experiments, we overexpressed EGFP-SPAG6 in cultured cells and observed that the protein co-localized with a subset of acetylated cytoplasmic microtubules. A role of these proteins stabilizing the cytoplasmic microtubules of eukaryotic cells is discussed.



Effects of Nel-like molecule-1 and bone morphogenetic protein 2 combination on rat pulp repair

Abstract

Nel-like molecule-1 (NELL-1) is a novel highly specific growth factor that can induce osteoblast differentiation and bone formation as well as odontoblast differentiation. Recent studies have suggested that NELL-1 can synergistically increase bone formation and regeneration with bone morphogenetic protein 2 (BMP2) and inhibit adverse effects induced by BMP2. This study aimed to evaluate the combined effects of NELL-1 and BMP2 on rat pulp repair. The experiment used healthy non-carious maxillary first molars from 60 Wistar rats. Exposed pulps were capped with NELL-1 plus BMP2, NELL-1 alone, and BMP2 alone, and each was absorbed onto a sterile collagen sponge. In the control samples, the collagen sponge alone and Dycal were used as capping agents. After l, 2 and 4 weeks, the rats were sacrificed. The formation of reparative dentin, as well the situation of pulp repair, was detected by hematoxylin-eosin (HE) staining; moreover, the expression of dentin specific protein-dentin sialophosphoprotein (DSPP) and the pro-inflammatory cytokines interleukin-6 (IL6) and interleukin-8 (IL8) was detected by immunohistochemical staining. Quantitative real-time PCR experiment was used to investigate the mRNA levels of IL6 and IL8. The results showed that pulp capping with NELL-1 plus BMP2 in rats had superior ability in inducing reparative dentin formation with dentin tubules and in reducing the inflammatory cell response compared with the other groups. These findings suggested that combined use of NELL-1 and BMP2 could positively regulate pulp repair.



Quantitative changes in perineuronal nets in development and posttraumatic condition

Abstract

Perineuronal net (PNN) is a highly structured portion of the CNS extracellular matrix (ECM) regulating synaptic plasticity and a range of pathologic conditions including posttraumatic regeneration and epilepsy. Here we studied Wisteria floribunda agglutinin-stained histological sections to quantify the PNN size and enrichment of chondroitin sulfates in mouse brain and spinal cord. Somatosensory cortex sections were examined during the period of PNN establishment at postnatal days 14, 21 and 28. The single cell PNN size and the chondroitin sulfate intensity were quantified for all cortex layers and specifically for the cortical layer IV which has the highest density of PNN-positive neurons. We demonstrate that the chondroitin sulfate proteoglycan staining intensity is increased between P14 and P28 while the PNN size remains unchanged. We then addressed posttraumatic changes of the PNN expression in laminae 6 and 7 of cervical spinal cord following hemisection injury. We demonstrate increase of the chondroitin sulfate content at 1.6–1.8 mm rostrally from the injury site and increase of the density of PNN-bearing cells at 0.4–1.2 mm caudally from the injury site. We further demonstrate decrease of the single cell PNN area at 0.2 mm caudally from the injury site suggesting that the PNN ECM takes part in the posttraumatic tissue rearrangement in the spinal cord. Our results demonstrate new insights on the PNN structure dynamics in the developing and posttraumatic CNS.



Changes in mechanoreceptors in rabbits' anterior cruciate ligaments with age

Abstract

At present, a few studies have been done on the changes in the distribution, morphology and quantity of mechanoreceptors in anterior cruciate ligament (ACL) with age. In this study, we observed the changes in mechanoreceptors of healthy rabbits' ACL with age. We found that rabbits' ACLs contained 5 kinds of mechanoreceptors including Ruffini corpuscles, Pacinian corpuscles, Golgitendon bodies, free nerve endings and atypical mechanoreceptors. In each ACL, free nerve endings were the most followed by Ruffini corpuscles, Pacinian corpuscles, Golgitendon bodies and atypical mechanoreceptors in the younger than one-old rabbits. Most of the mechanoreceptors were distributed in the synovium near the attachment points of ACL with the femur and tibia. The total quantity of mechanoreceptors were the most in the 3- and 6-month groups, but did not show a significant difference between the two group (P > 0.05). However, there were significant differences in the total quantity of mechanoreceptors between other groups (all P < 0.05). RT-PCR indicated that NEFM and S100B levels increased with age, and reached a peak in the 1-year group with significant differences as compared to other groups. NEFM and S100B levels were the second in 6-month and 2-year groups and the lowest in the 1-week group. We can conclude that in rabbits' ACLs, free nerve endings are the most common, followed by Ruffini corpuscles, Pacinian corpuscles and Golgitendon bodies. The total quantity of mechanoreceptors reaches a peak in 3 months, while NEFM and S100B reach a peak in 1 year.



Role of mTOR complex in IGF-1 induced neural differentiation of DPSCs

Abstract

Recent studies have demonstrated that IGF-1 modulates the pluripotent differentiation of dental pulp stem cells (DPSCs). Although mTOR pathway activation has been showed as responsible for IGF-1 induced pluripotent differentiation, the mechanism that the IGF-1–mTOR pathway induces the neural differentiation of DPSCs is still unclear. In our research, we have demonstrated that 0–10 ng/mL IGF-1 had no obvious effect on the proliferation of DPSCs, but IGF-1 nonetheless enhances the neural differentiation of DPSCs in a dose-dependent manner. Simultaneously, we found that phosphorylated mTOR was up-regulated, which indicated the involvement of mTOR in the process. Rapamycin, an inhibitor of mTOR activity, can reverse the effect of DPSCs stimulated by IGF-1. Next, we studied the role of mTORC1 and mTORC2, two known mTOR complexes, in the neural differentiation of DPSCs. We found that inhibition of mTORC1 can severely restricts the neural differentiation of DPSCs. However, inhibition of mTORC2 has the opposite effect. This latter effect disappears when both rictor and mTOR are inhibited, showing that the mTORC2 effect is mTORC1 dependent. This study has expanded the role of mTOR in DPSCs neural differentiation regulated by IGF-1.



Expression of CPNE7 during mouse dentinogenesis

Abstract

Interactions between the ectodermal and mesenchymal tissues are the basis of the central mechanism regulating tooth development. Based on this epithelial-mesenchymal interaction (EMI), we demonstrated that copine-7 (CPNE7) is secreted by preameloblasts and regulates the differentiation of mesenchymal cells of dental or non-dental origin into odontoblasts. However, the precise expression patterns of CPNE7 in the stages of tooth development have not yet been elucidated. The aim of the present study was to establish the spatiotemporal expression pattern of CPNE7 during mouse tooth development. To examine the spatiotemporal expression patterns of CPNE7 during mouse tooth development, we investigate the distribution of CPNE7 in the embryonic and postnatal developing mouse tooth. Immunohistochemistry, in situ hybridization, real-time PCR, and western blot analysis are performed to investigate the CPNE7 expression pattern during tooth development of the mandibular mouse first molar. During the initiation stage (bud stage), CPNE7 protein expression is observed in the dental epithelium but not yet in the dental mesenchyme. At E18 (bell stage), expression of CPNE7 protein and mRNA is primarily observed in ectomesenchymal cells of dental papilla. At P7 (crown formation stage), CPNE7 is localized in differentiating odontoblasts but weak expression is detected in mature ameloblasts. These findings suggest that CPNE7 secreted by dental epithelium induces the differentiation of ectomesenchymal cells into preodontoblast in concert with EMI. CPNE7 is clearly expressed in differentiating odontoblasts and the odontoblast process during dentinogenesis, but is no longer expressed in fully differentiated odontoblasts. Furthermore, CPNE7 is expressed in the Hertwig's epithelial root sheath (HERS) and in the facing preodontoblasts during root dentin formation. Taken together, these results illustrate the dynamic expression of CPNE7 during tooth development and suggest its important function in entire stages of tooth development.



Type XI collagen–perlecan–HS interactions stabilise the pericellular matrix of annulus fibrosus cells and chondrocytes providing matrix stabilisation and homeostasis

Abstract

The aim of this study was to ascertain whether, like many cell types in cartilaginous tissues if type XI collagen was a pericellular component of annulus fibrosus (AF) cells and chondrocytes. Fine fibrillar networks were visualised which were perlecan, HS (MAb 10E4) and type XI collagen positive. Heparitinase-III pre-digestion abolished the type XI collagen and 10E4 localisation in these fibrillar assemblies demonstrating a putative HS mediated interaction which localised the type XI collagen. Type XI collagen was confirmed to be present in the Heparitinase III treated AF monolayer media samples by immunoblotting. Heparitinase-III generated ΔHS stub epitopes throughout these fibrillar networks strongly visualised by MAb 3-G-10. Monolayers of murine hip articular chondrocytes from C57BL/6 and Hspg2 exon 3 null mice also displayed pericellular perlecan localisations, however type XI collagen was only evident in the Wild type mice. Perlecan was also immunolocalised in control and murine knee articular cartilage from the two mouse genotypes subjected to a medial meniscal destabilisation procedure which induces OA. This resulted in a severe depletion of perlecan levels particularly in the perlecan exon 3 null mice and was consistent with OA representing a disease of the pericellular matrix. A model was prepared to explain these observations between the NPP type XI collagen domain and HS chains of perlecan domain-I in the pericellular matrix of AF cells which likely contributed to cellular communication, tissue stabilization and the regulation of extracellular matrix homeostasis.



Expression patterns of genes critical for SHH, BMP, and FGF pathways during the lumen formation of human salivary glands

Abstract

Sjögren's syndrome or radiotherapy for head and neck cancer leads to the irreversible hypofunction of salivary gland (SG). The stem/progenitor cell-based regenerative strategy has been proven to be the most promising approach to repair the function of SG. The molecular mechanisms that regulate SG morphogenesis, especially during lumen formation, provide valuable hints for establishment of such regenerative strategies. It has been demonstrated that numerous growth factors particularly belonging to SHH, BMP, and FGF signaling pathway are involved in the regulation of lumen formation and have shown protective effects on the SG from irradiation in mouse models. However, it remains elusive whether the expression pattern and function of these signaling molecules are conserved in humans. In this study, we examined the expression patterns of the molecules critical for SHH, BMP, and FGF signaling cascades from the canalicular stage to the terminal bud stage, the key stages for lumen formation, in human SG and compared them with the expression data observed in mice. Our results manifested that genes involved in SHH signaling pathway showed identical expression patterns, while genes involved in BMP as well as FGF pathway exhibited similar but distinct expression patterns in humans to those in the mouse. We concluded that the expression patterns of genes involved in SHH, BMP, and FGF pathways in the development of human SG exhibit high similarity to that in the development of mouse SG during lumen formation, suggesting that the molecular mechanism regulating the morphogenesis of SG during lumen formation may be conserved in mice and humans. Our results will have an implication in the future establishment of stem-cell based approaches for the repair of SG function.



Hyperinsulinemia-induced KLF5 mediates endothelial angiogenic dysfunction in diabetic endothelial cells

Abstract

Reduced expression of endothelial nitric oxide synthase (eNOS) is a hallmark of endothelial dysfunction in diabetes, which predisposes diabetic patients to numerous cardiovascular complications including blunted angiogenesis. The Krüppel-like factor (KLF) five has been implicated as a central regulator of cardiovascular remodeling, but its role in endothelial cells (ECs) remains poorly understood. We show here that expression of endothelial KLF5 was significantly increased in the ECs from mouse diabetes mellitus type 2 (T2DM) model, when compared to non-diabetic or T1DM mouse. KLF5 up-regulation by insulin was dependent on activation of multiple pathways, including mammalian target of rapamycin, oxidative stress and Protein kinase C pathways. Hyperinsulinemia-induced KLF5 inhibited endothelial function and migration, and thereby compromised in vitro and in vivo angiogenesis. Mechanistically, KLF5 acted in concert with the MTA1 coregulator to negatively regulate NOS3 transcription, thereby leading to the diminished eNOS levels in ECs. Conversely, potentiation of cGMP content (the essential downstream effector of eNOS signaling) by pharmacological approaches successfully rescued the endothelial proliferation and in vitro tube formation, in the HUVECs overexpressing the exogenous KLF5. Collectively, the available data suggest that the augmentation of endothelial KLF5 expression by hyperinsulinemia may represent a novel mechanism for negatively regulating eNOS expression, and may thus help to explain for the T2DM-related endothelial dysfunction at the transcriptional level.



Oligoprotective effect of metformin through the AMPK-dependent on restoration of mitochondrial hemostasis in the cuprizone-induced multiple sclerosis model

Abstract

Oxidative stress with mitochondrial defects has a central role in the development and deterioration of Multiple sclerosis (MS). According to new findings of the effects of metformin on mitochondrial function, has attracted a lot of attention. Furthermore, it is suggested that metformin exerts its beneficial influence through AMP-activated protein kinase (AMPK) pathway. In the current study, we investigated the possible protective effects of metformin on oxidative stress and mitochondrial function by activating the AMPK pathway in the cuprizone-induced demyelination. Mice were fed with cuprizone for 6 weeks. Animals simultaneously received metformin. After sacrificing animals, myelinations, and gliosis, changes in transcription factor and biochemical analysis were assessed. Transmission electron microscopy and luxol fast blue staining revealed that the myelinated axons within corpus callosum of cuprizone-induced demyelination animals increased after administration of metformin. Metformin also upregulated the expression of mitochondrial biogenesis genes. Furthermore, the biochemical analysis demonstrated that metformin ameliorated the oxidative stress induced by cuprizone. Immunohistochemistry analysis showed that astrogliosis and microgliosis were decreased after metformin administration while it enhanced the number of oligodendrocytes. Our data implicated that metformin exerts its therapeutic effects on MS by AMPK signaling improved mitochondrial homeostasis and protected oligodendrocytes.



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