Osteopontin mRNA Expression Research Articles

Echinochrome A inhibits HMGB1-induced vascular smooth muscle cell migration by suppressing osteopontin expression.

Echinochrome A (Ech A) isolated from marine organisms is a therapeutic effector for various cardiovascular diseases, but its precise mechanisms are unclear. This study identified the role and mechanisms mediating the effects of Ech A on the migration of vascular smooth muscle cells (VSMCs) induced by high-mobility group box 1 (HMGB1). Compared to the control cells, the migration of VSMCs stimulated with HMGB1 (100 ng/ml) was markedly increased, which was significantly attenuated in cells pretreated with MPIIIB10 (100 ng/ml), a neutralizing monoclonal antibody for osteopontin (OPN). In VSMCs stimulated with HMGB1, the increased expression of OPN mRNA and protein was accompanied by an increased OPN promoter activity. In reporter gene assays using OPN promoter-luciferase constructs, the promoter region 538-234 bp of the transcription start site containing the binding sites for activator protein 1 (AP-1) was shown to be responsible for the increased transcriptional activity by HMGB1. In addition, the binding activity of AP-1 was increased in HMGB1-stimulated cells, highlighting the pivotal role of AP-1 on OPN expression in HMGB1-stimulated VSMCs. An examination of the vascular effects of Ech A showed that the increased AP-1 binding/promoter activities and OPN expression induced by HMGB1 were attenuated in cells pretreated with Ech A (3 or 10 μM). Similarly, Ech A inhibited HMGB1-induced VSMC migration in a concentration-dependent manner. These findings suggest that Ech A inhibits VSMC migration by suppressing OPN expression. Hence, Ech A is suggested as a potential therapeutic strategy for vascular remodeling in the injured vasculatures.

Sex Differences in Expression of Pro-Inflammatory Markers and miRNAs in a Mouse Model of CVB3 Myocarditis.

Myocarditis is an inflammatory disease that may lead to dilated cardiomyopathy. Viral infection of the myocardium triggers immune responses, which involve, among others, macrophage infiltration, oxidative stress, expression of pro-inflammatory cytokines, and microRNAs (miRNAs). The cardioprotective role of estrogen in myocarditis is well documented; however, sex differences in the miRNA expression in chronic myocarditis are still poorly understood, and studying them further was the aim of the present study. Male and female ABY/SnJ mice were infected with CVB3. Twenty-eight days later, cardiac tissue from both infected and control mice was used for real-time PCR and Western blot analysis. NFκB, IL-6, iNOS, TNF-α, IL-1β, MCP-1, c-fos, and osteopontin (OPN) were used to examine the inflammatory state in the heart. Furthermore, the expression of several inflammation- and remodeling-related miRNAs was analyzed. NFκB, IL-6, TNF-α, IL-1β, iNOS, and MCP-1 were significantly upregulated in male mice with CVB3-induced chronic myocarditis, whereas OPN mRNA expression was increased only in females. Further analysis revealed downregulation of some anti-inflammatory miRNA in male hearts (let7a), with upregulation in female hearts (let7b). In addition, dysregulation of remodeling-related miRNAs (miR27b and mir199a) in a sex-dependent manner was observed. Taken together, the results of the present study suggest a sex-specific expression of pro-inflammatory markers as well as inflammation- and remodeling-related miRNAs, with a higher pro-inflammatory response in male CVB3 myocarditis mice.

Osteopontin expression in dromedary camel's conceptuses during the peri-implantation period.

The reproductive efficiency of dromedary camels is hindered by challenges such as early embryonic mortality, which may be attributed to a lack of synchronization between conceptus signalling and uterine receptivity. Understanding the intricate biological processes involved in feto-maternal interactions during implantation is crucial to address these limitations. Osteopontin (OPN) is a protein involved in cell signalling and adhesion, playing a crucial role in embryonic implantation. Previous studies have shown the presence of OPN in the uterine endometrium of various mammalian species including dromedary camels. However, the expression pattern of OPN in dromedary conceptuses remains unexplored. Thus, the current study aimed, for the first time, to investigate the temporospatial expression of OPN in dromedary conceptuses during the peri-implantation period at Days 8, 10, and 12 of pregnancy. Twelve conceptuses were recovered non-surgically from pregnant females on Days 8, 10, 12 of pregnancy. Quantitative real-time PCR (qrt-PCR), immunohistochemistry (IHC), and immunofluorescence (IF) were employed for analysis of the expression of OPN mRNA and protein. The results revealed significant increases in both OPN mRNA and protein expression started on Day 10 and peaked at Day 12 of pregnancy. Immuno-localization confirmed the presence of OPN protein in the trophectoderm and endoderm of dromedary conceptuses. In conclusion, the expression and localization of OPN during the peri-implantation period in dromedary conceptuses imply its involvement as a crucial reproductive factor and its upregulation during this period, with a pronounced increase close to attachment time (Day 12 of pregnancy) further supports its role in embryo adhesion, implantation, and placentation.

P-401 Aging affects endometrial receptivity factors. Can DHEA modulate this age-related effect?

Abstract Study question Identification of age-related gene expression differences of the human endometrium during its receptive period – Dehydroepiandrosterone (DHEA) effects. Summary answer Decrease in expression of OPN and HOXA10, receptivity markers, between increasing female age groups in primary human endometrial cells was presented. DHEA altered gene expression. What is known already The increased infertility noted in the world population, has been related to increasing maternal age. Our previous work points out the role of women’s age in endometrial cell function. Endometrial proliferation and differentiation are two important steps that lead to increased cell receptivity during the window of implantation. Optimal receptivity is achieved also through changes inexpression of several factors throughout the endometrial cycle. It is well known that androgen levels, in the female reproductive system, decrease with increasing age.Therefore, DHEA an androgen precursor, found to be reduced with age is thought to be a candidate for infertility treatment. Study design, size, duration Maternal age has not been previously directly correlated with endometrial receptivity factors. We studied 39 endometrial samples divided in three different women’s age groups (25-35 years, 36-40 years and 41-46years) and changes in the mRNA and protein expression of osteopontin (OPN), CD44 and HOXA10 in human endometrium stromal cells were analyzed, in vitro. DHEA was added on stromal cells (decidualized or not) in order to observe changes in mRNA expression. Participants/materials, setting, methods Pipelle endometrial biopsies (secretory phase) were collected from ovulating, fertile, Caucasian women volunteers aged 25–46 years. Stromal cells were isolated. These samples were divided into three age groups: 25-35 years, 36-40 years and 41-46 years. Protein and mRNA expression of receptivity modulators OPN, CD44 and HOXA10 were analyzed using Real-time PCR and Western blot analysis, respectively. Stromal cells were decidualized in vitrousing bromo-8-cAMP in order to observe DHEA effects. Main results and the role of chance Data presented showed a statistically significant decrease (p ≤ 0.05) in OPN expression levels with increasing age groups. Women included in age group of 41-45y presented a significant downregulation of OPN expression compared to younger age group 25-35y (p ≤ 0.01).CD44 expressiondid not differ with age. Furthermore, a statistically significant decrease (p ≤ 0.05) was evident in HOXA10 expression levels with increasing age. Women belonging to the age group 41-45y expression presented with significant HOXA10 downregulation compared to younger age group 25-35y (p ≤ 0.05). Analysis of OPN protein levels showed that cell-extracted OPN decreased from theage of 33y, while the secreted protein gradually decreased from the age of 38y. CD44 protein expression, similarly to the mRNA expression did not show differences with age. HOXA10 protein expression was found to be decreased mainly after the age of41y. DHEA changed, with statistical significance, the expression of decidualization marker IGFBP and OPN in decidualized stromal cells. Overall, our results support our hypothesis as expected, but we must also consider specific cases where older women have the same results as younger ones and the opposite, so factors that are specific for each individual must be further investigated. Limitations, reasons for caution It must be pointed out that there is a plethora of characterized molecules like cytokines and immune cells that also participate in the optimal receptivity status of the endometrium – leading to a successful implantation procedure – that future studies need to examine, as well, in relation to women’s age. Wider implications of the findings Human endometrial aging can affect embryo receptivity markers and therefore, receptivity. Further investigation of receptivity factors that change with the age could be a useful pool in identifying targets for diagnosis and therapy of human implantation failure. Also, DHEA must be further investigated as a tool for infertility therapy. Trial registration number not applicable

Impact of hyperoxia on the phenotype of pulmonary artery smooth muscle cells

Objective: To investigate the influence of varied oxygen (O2) concentration environments on the phenotypic transformation of pulmonary artery smooth muscle cells (PASMC) and the mechanism of pulmonary hypertension. Methods: Primary rat PASMC were isolated and cultured through the process of enzymatic digestion. Following identification, the stable passaged PASMC were subjected to a 6-hour incubation in sealed containers with normal O2 content (group C) and relative O2 content comprising 55% (group H55), 75% (group H75), and 95% (group H95). mRNA and protein expression of α-Actin (α-SMA), smooth muscle 22α (SM22α), osteopontin (OPN), and matrix metalloproteinase-2 (MMP-2) were measured using real-time quantitative PCR and western blot analysis. Results: The H55 group displayed no significant difference from the C group in terms of mRNA and relative protein expression levels for α-SMA, SM22α, OPN, and MMP-2 (all P>0.05). On the other hand, groups H75 and H95 exhibited a reduction in mRNA and relative protein expression of α-SMA and SM22α, along with an increase in mRNA and relative protein expression of OPN and MMP-2 when compared with both the C and H55 groups (all P<0.05). The H95 group showed a higher relative mRNA expression of MMP-2 as compared to the H75 group (P<0.05). Conclusions: Oxygen concentration environments of 75% or higher can serve as the foundation for the pathogenesis of pulmonary hypertension, essentially by inducing a phenotypic transformation in PASMC towards adopting a robust secretory function. This induction is contingent upon the concentration of oxygen present.

Renogrit attenuates Vancomycin-induced nephrotoxicity in human renal spheroids and in Sprague-Dawley rats by regulating kidney injury biomarkers and creatinine/urea clearance

Vancomycin, is widely used against methicillin-resistant bacterial infections. However, Vancomycin accumulation causes nephrotoxicity which leads to an impairment in the filtration mechanisms of kidney. Traditional herbal medicines hold potential for treatment of drug-induced nephrotoxicity. Herein, we investigated protective properties of plant-based medicine Renogrit against Vancomycin-induced kidney injury. Phytometabolite analysis of Renogrit was performed by UHPLC. Spheroids formed from human proximal tubular cell (HK-2) were used for in vitro evaluation of Vancomycin-induced alterations in cell viability, P-gp functionality, NAG, KIM-1 levels, and mRNA expression of NGAL and MMP-7. The in vivo efficacy of Renogrit against Vancomycin-induced nephrotoxicity was further evaluated in Sprague-Dawley (SD) rats by measurement of BUN, serum creatinine, and their respective clearances. Moreover, eGFR, kidney-to-body weight ratio, GSH/GSSG ratio, KIM-1, NAG levels and mRNA expression of KIM-1 and osteopontin were also analyzed. Changes in histopathology of kidney and hematological parameters were also observed. Renogrit treatment led to an increase in cell viability, normalization of P-gp functionality, decrease in levels of NAG, KIM-1, and reduction in mRNA expression of NGAL and MMP-7. In Vancomycin-challenged SD rats, Renogrit treatment normalized altered kidney functions, histological, and hematological parameters. Our findings revealed that Renogrit holds a clinico-therapeutic potential for alleviating Vancomycin-associated nephrotoxicity.

Lactoferrin-Anchored Tannylated Mesoporous Silica Nanomaterials-Induced Bone Fusion in a Rat Model of Lumbar Spinal Fusion.

Lactoferrin (LF) is a potent antiviral, anti-inflammatory, and antibacterial agent found in cow and human colostrum which acts as an osteogenic growth factor. This study aimed to investigate whether LF-anchored tannylated mesoporous silica nanomaterials (TA-MSN-LF) function as a bone fusion material in a rat model. In this study, we created TA-MSN-LF and measured the effects of low (1 μg) and high (100 μg) TA-MSN-LF concentrations in a spinal fusion animal model. Rats were assigned to four groups in this study: defect, MSN, TA-MSN-LF-low (1 μg/mL), and TA-MSN-LF-high (100 μg/mL). Eight weeks after surgery, a greater amount of radiological fusion was identified in the TA-MSN-LF groups than in the other groups. Hematoxylin and eosin staining showed that new bone fusion was induced in the TA-MSN-LF groups. Additionally, osteocalcin, a marker of bone formation, was detected by immunohistochemistry, and its intensity was induced in the TA-MSN-LF groups. The formation of new vessels was induced in the TA-MSN-LF-high group. We also confirmed an increase in the serum osteocalcin level and the mRNA expression of osteocalcin and osteopontin in the TA-MSN-LF groups. TA-MSN-LF showed effective bone fusion and angiogenesis in rats. We suggest that TA-MSN-LF is a potent material for spinal bone fusion.

Effect of a 630 nm light on vasculogenic mimicry in A549 lung adenocarcinoma cells in vitro

ObjectiveThe objective of this study was to investigate the effect of photodynamic therapy (PDT) on the formation of vasculogenic mimicry (VM) in the human lung adenocarcinoma A549 cell line in vitro. MethodsThe participants were divided into a blank control group, a photosensitizer group, a light group, and a PDT group. Cells from each group were cultured in three dimensions using Matrigel, and vasculogenic mimicry generation was observed microscopically. Periodic Acid-Schiff (PAS) staining was used to verify the vasculogenic mimicry structure. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was used to detect the expression levels of cellular osteopontin (OPN) and vascular endothelial growth factor (VEGF) mRNA. Western blotting was used to detect the expression levels of cellular OPN and VEGF protein. ResultsA549 cells cultured on Matrigel for about six hours revealed VM on PAS staining, and the number of formations was significantly reduced in the PDT group compared with other groups (P < 0.05). The RT-PCR results showed that the PDT group downregulated OPN and VEGF mRNA expression compared with each control group (P < 0.05). Western blot results showed that OPN and VEGF protein expression was downregulated in the PDT group compared with each control group (P < 0.05). The results of RT-PCR showed that the expression of OPN and VEGF mRNA was downregulated in the PDT group compared with each control group (P < 0.05). The results of Western blotting showed that the expression of OPN and VEGF was downregulated in the protein PDT group compared with each control group (P < 0.001). ConclusionPhotodynamic therapy significantly inhibited the formation of vasculogenic mimicry in human lung adenocarcinoma A549 cells in vitro and downregulated the expression of OPN, VEGF mRNA, and protein levels.

Osteopontin promotes gastric cancer progression via phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway.

Gastric cancer (GC) is one of the most common malignant tumors. Osteopontin (OPN) is thought to be closely related to the occurrence, metastasis and prognosis of many types of tumors. To investigate the effects of OPN on the proliferation, invasion and migration of GC cells and its possible mechanism. The mRNA and protein expression of OPN in the GC cells were analyzed by real-time quantitative-reverse transcription polymerase chain reaction and western blotting, and observe the effect of varying degree expression OPN on the proliferation and other behaviors of GC. Next, the effects of OPN knockdown on GC cells migration and invasion were examined. The short hairpin RNA (shRNA) and negative control shRNA targeting OPN-shRNA were transfected into the cells according to the manufacturer's instructions. Non transfected cells were classified as control in the identical transfecting process. 24 h after RNA transfection cell proliferation activity was detected by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide assay, and cell invasiveness and migration were detected by Trans well assay. Meanwhile, the expression of protein kinase B (AKT), matrix metalloproteinase 2 (MMP-2) and vascular endothelial growth factor (VEGF) in the human GC cell lines was detected by reverse transcription polymerase chain reaction and western blotting. The results of this study revealed that OPN mRNA and protein expression levels were highly expressed in SGC-7901 cells. OPN knockdown by specific shRNA noticeably reduced the capabilities of proliferation, invasion and migration of SGC-7901 cells. Moreover, in the experiments of investigating the underlying mechanism, results showed that OPN knockdown could down-regulated the expression of MMP-2 and VEGF, it also decreased the phosphorylation of AKT. Meanwhile, the protein expression levels of MMP-2, VEGF and phosphorylated AKT was noticeable lower than that in control group in the GC cells after they were added to phosphatidylinositol-3-kinase (PI3K) inhibitor (LY294002). These results suggested that OPN though PI3K/AKT/mammalian target of rapamycin signal pathway to up-regulate MMP-2 and VEGF expression, which contribute SGC-7901 cells to proliferation, invasion and migration. Thus, our results demonstrate that OPN may serve as a novel prognostic biomarkers as well as a potential therapeutic targets for GC.

Resveratrol Promotes the Osteogenic Differentiation of Human Amniotic Epithelial Cells via Sirt-1/PPAR-α Pathway

IntroductionResveratrol has been indicated to promote the osteogenic differentiation of several types of mesenchymal stem cells (MSCs), such as gingival MSCs and bone marrow MSCs. Human amniotic epithelial cells (hAECs) possess the characteristics of stem cells and are showing great potential in regenerative medicine. However, whether resveratrol can be beneficial to promote the osteogenesis of hAECs has not been investigated. The current study aims to reveal the role of resveratrol in the osteogenic differentiation of hAECs and the underlying mechanism. MethodsAlkaline phosphatase (ALP) staining, alizarin red staining, the quantitative analysis of ALP activity and mineralisation, quantitative real-time PCR, western blotting, and small-interfering RNA technique were employed in this study. ResultsResults showed that resveratrol increased ALP activity and mineralisation during osteogenic differentiation of hAECs in a dose-dependent manner. Also, resveratrol significantly promoted the mRNA expression of osteogenesis-associated genes, including Runx2, ALP, Collagen-I, BMP-2, osterix, and osteopontin dose-dependently. Resveratrol also induced the protein levels of Sirt-1 and PPAR-α and activated Sirt-1 signalling. Furthermore, knockdown of Sirt-1 by RNAi significantly suppressed Sirt-1/PPAR-α signalling and inhibited resveratrol-induced mRNA elevation of osteogenesis-associated genes. ConclusionsCollectively, this study for the first time confirmed the promotive role of resveratrol during osteogenic differentiation of hAECs via Sirt-1/PPAR-α.

Sperm osteopontin mRNA expression levels and its correlation on semen quality and fertility in Madura bulls

Abstract. Rosyada ZNA, Yoelinda VT, Kaiin EM, Gunawan M, Ulum MF, Tumbelaka LITA, Solihin DD, Agil M, Gunawan A, Purwantara B. 2023. Sperm osteopontin mRNA expression levels and its correlation on semen quality and fertility in Madura bulls. Biodiversitas 24: 563-570. Osteopontin (OPN) gene transcripts influence spermatogenesis and germ cell development. Therefore, transcriptome analysis is needed to identify the fertility factor OPN in Madura bull sperm. Madura cattle are crosses originating from Bos indicus (zebu) and Bos javanicus (banteng). This study aims to examine sperm osteopontin (OPN) mRNA expression levels and its correlation with semen quality and fertility in Madura bulls. Frozen semen samples from Madura bulls were categorized as high-fertile (n: 4, average field conception rate: 78.28±3.25%) or sub-fertile (n: 4, average field conception rate: 66.73±5.01%). In post-thaw semen samples, sperm motility, viability, membrane, acrosome, and DNA fragmentation index were evaluated. OPN expression in sperm total RNA was analyzed using Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR). The data analysis between the two fertility groups was assessed with Student's t-test and Pearson Square. Receiver-operating characteristic analysis and diagnostic sensitivity and specificity calculations were performed. Sperm motility, acrosome intact, and DFI differ (p&lt;0.05) between groups, whereas viability and membrane plasma intact have no significance (p&gt;0.05). In high-fertile Madura bulls, OPN mRNA was upregulated (p&lt;0.05). The OPN mRNA expression had a strong correlation with the field conception rate (R: 0.807, P&lt;0.05), sperm motility, and intact acrosome (R: 0.899, p&lt;0.01; R: 0.804, p&lt;0.05) respectively. In contrast, OPN negatively correlated with sperm DFI (R: -0.764, p&lt;0.05). The OPN predicted bull fertility with 81.3% accuracy, 75% sensitivity, and 75% specificity. Thus, OPN may serve as a potential biomarker of Madura bull fertility.

Anti-Osteoporosis Effects of the Fruit of Sea Buckthorn (Hippophae rhamnoides) through Promotion of Osteogenic Differentiation in Ovariectomized Mice.

The fruit of Hippophae rhamnoides has been widely used for medicinal purposes because of its anti-inflammatory, antioxidant, antiplatelet, and antimicrobial effects. Since there are no clear reports on the therapeutic efficacy of H. rhamnoides in osteoporosis, this study aimed to confirm the potential use of H. rhamnoides for the treatment of osteoporosis through its osteogenic differentiation-promoting effect in ovariectomized mice. Through an in vitro study, we compared the effects of the EtOH extract of H. rhamnoides fruits (EHRF) on the differentiation of C3H10T1/2, a mouse mesenchymal stem cell line, into osteoblasts based on alkaline phosphatase (ALP) staining and the relative expression of osteogenesis-related mRNAs. The EHRF significantly stimulated the differentiation of mesenchymal stem cells into osteoblasts and showed 7.5 times (* p < 0.05) higher osteogenesis than in the untreated control. A solvent fractionation process of EHRF showed that the hexane-soluble fraction (HRH) showed 10.4 times (** p < 0.01) higher osteogenesis than in the untreated control. Among the subfractions derived from the active HRH by preparative HPLC fractionation, HRHF4 showed 7.5 times (* p < 0.05) higher osteogenesis than in the untreated naïve cells, and HRH and HRHF4 fractions showed 22.6 times (*** p < 0.001) stronger osteogenesis activity than in the negative control. Osteoporosis was induced by excision of both ovaries in 9-week-old female ICR mice for in vivo analysis, and two active fractions, HRH and HRHF4, were administered orally for three months. During the oral administration period, body weight was measured weekly, and bone mineral density (BMD) and body fat density were measured simultaneously using a DEXA machine once a month. In particular, during the in vivo study, the average BMD of the ovariectomized group decreased by 0.0009 g/cm2, whereas the average BMD of the HRH intake group increased by 0.0033 g/cm2 (* p < 0.05) and that of the HRHF4 intake group increased by 0.0059 g/cm2 (** p < 0.01). The HRH and HRHF4 intake groups significantly recovered the mRNA and protein expression of osteogenic genes, including ALP, Osteopontin, Runx2, and Osterix, in the osteoporosis mouse tibia. These findings suggest that the active fractions of H. rhamnoides fruit significantly promoted osteoblast differentiation in mesenchymal stem cells and increased osteogenic gene expression, resulting in an improvement in bone mineral density in the osteoporosis mouse model. Taken together, H. rhamnoides fruits are promising candidates for the prevention and treatment of osteoporosis.

Cilostazol Attenuates AngII-Induced Cardiac Fibrosis in apoE Deficient Mice.

Cardiac fibrosis is characterized by the net accumulation of extracellular matrix in the myocardium and is an integral component of most pathological cardiac conditions. Cilostazol, a selective inhibitor of phosphodiesterase type III with anti-platelet, anti-mitogenic, and vasodilating properties, is widely used to treat the ischemic symptoms of peripheral vascular disease. Here, we investigated whether cilostazol has a protective effect against Angiotensin II (AngII)-induced cardiac fibrosis. Male apolipoprotein E-deficient mice were fed either a normal diet or a diet containing cilostazol (0.1% wt/wt). After 1 week of diet consumption, the mice were infused with saline or AngII (1000 ng kg−1 min−1) for 28 days. AngII infusion increased heart/body weight ratio (p < 0.05), perivascular fibrosis (p < 0.05), and interstitial cardiac fibrosis (p < 0.0001), but were significantly attenuated by cilostazol treatment (p < 0.05, respectively). Cilostazol also reduced AngII-induced increases in fibrotic and inflammatory gene expression (p < 0.05, respectively). Furthermore, cilostazol attenuated both protein and mRNA abundance of osteopontin induced by AngII in vivo. In cultured human cardiac myocytes, cilostazol reduced mRNA expression of AngII-induced osteopontin in dose-dependent manner. This reduction was mimicked by forskolin treatment but was cancelled by co-treatment of H-89. Cilostazol attenuates AngII-induced cardiac fibrosis in mice through activation of the cAMP–PKA pathway.

Abstract P3137: Drp1-dependent Mitochondrial Fission Induces Vascular Smooth Muscle Cell Dysfunction Through Kruppel-like Factor 4 Upregulation

Introduction: Vascular smooth muscle cell (VSMC) phenotypic switching and subsequent VSMC proliferation and migration are major events that are closely associated with progression of cardiovascular diseases. Mitochondria play key roles in regulation of cell function. Recent findings have revealed that mitochondrial fission promotes proliferation and migration of VSMCs. Hypothesis: This study aimed to explore the role of a new drug candidate, ginsenoside Rh1, in Angiotensin II (Ang II)-induced VSMC migration and proliferation and its underlying mechanisms. Methods: Sulforhodamine B and wound-healing assay were used to evaluate effects on cell proliferation and migration. Molecular mechanisms were investigated using western blotting, qRT-PCR analysis, immunofluorescence staining, and luciferase assay. Reactive oxygen species (ROS) production was measured by dihydroethidium and MitoSOX staining. Results: We found that pretreatment with 50 μM Rh1 significantly suppressed Ang II-induced cell proliferation (% of decrease vs. Ang II group: 17.41 ± 2.38%, p &lt; 0.01, n = 5) and migration (% of decrease vs. Ang II group: 198.8 ± 2.67%, p &lt; 0.001, n = 5). Concomitantly, Ang II-increased both protein and mRNA levels of MMP2, MMP9, PCNA, and cyclin D1 were reduced by Rh1 pretreatment. Rh1 incubation also reversed the changes in both protein and mRNA expressions of α-SMA, vimentin, and osteopontin induced by Ang II. Besides, Ang II enhanced protein expression and promoter activity of KLF4, a key regulator of phenotypic switching, whereas pretreatment with Rh1 reversed these effects. Mechanistically, inhibitory effects of Rh1 were found to be associated with inhibition of ERK1/2 signaling pathway and ROS generation. In addition, deactivation of ERK1/2 suppressed phosphorylation of DRP1 at Ser 616. Notably, inhibition of DRP1-dependent mitochondrial fission by Mdivi-1 inhibited KLF4 expression and transcriptional activity induced by Ang II. Conclusions: In conclusion, mitochondrial fission involves Ang II-induced VSMC proliferation and migration through KLF4 upregulation, and that Rh1 inhibits VSMC dysfunction by suppressing ROS-mediated ERK1/2/DRP1/KLF4 signaling pathway.

Osteoblasts in a Perfusion Flow Bioreactor-Tissue Engineered Constructs of TiO2 Scaffolds and Cells for Improved Clinical Performance.

Combining biomaterial scaffolds with cells serves as a promising strategy for engineering critical size defects; however, homogenous cellular growth within large scaffolds is challenging. Mechanical stimuli can enhance bone regeneration by modulating cellular growth and differentiation. Here, we compare dynamic seeding in a perfusion flow bioreactor with static seeding for a synthetic bone scaffold for up to 21 days using the cell line MC3T3-E1 and primary human osteoblast, confocal laser scanning microscopy, and real-time reverse transcriptase-polymerase chain reaction. The secretion of bone-related proteins was quantified using multiplex immunoassays. Dynamic culture improved cellular distribution through the TiO2 scaffold and induced a five-fold increase in cell number after 21 days. The relative mRNA expression of osteopontin of MC3T3-E1 was 40-fold enhanced after 7 and 21 days at a flow rate of 0.08 mL/min, and that of collagen type I alpha I expression was 18-fold after 21 days. A flow rate of 0.16 mL/min was 10-fold less effective. Dynamic culture increased the levels of dickkopf-related protein 1 (60-fold), osteoprotegrin (29-fold), interleukin-6 (23-fold), interleukin-8 (36-fold), monocyte chemoattractant protein 1 (28-fold) and vascular endothelial growth factor (6-fold) in the medium of primary human osteoblasts after 21 days compared to static seeding. The proposed method may have clinical potential for bone tissue engineering.

Effect of compression on mandibular fracture haematoma-derived cells

Mechanical stress induces a variety of biochemical and morphological reactions in bone cell biology. This study aimed to investigate appropriate pressures of osteogenesis on the biological responses of 3-dimensional cultured human mandibular fracture haematoma-derived cells by compressive loading. Six patients with mandibular fractures who underwent open reduction and internal fixation were included in the study. During the operation, fracture haematomas that formed fibrin clots were manually removed before irrigation. First, pressures were applied to human mandibular fracture haematoma-derived cell-seeded collagen sponges. The sponges were subjected to mechanical compression using loading equipment applied at no compression, 0.5, or 1 mm. Compressive loading was applied to the samples prior to compression for 0, 6, 12, or 24 hours. Collagen sponge samples were collected for quantification of mRNA using several parameters including alkaline phosphatase (ALP), osteopontin (OPN), osterix (OSX), runt-related gene 2 (RUNX2), protein level, and immunocytochemistry (anti-sclerostin). Among these the 0.5 mm compression group compared with the control and 1.0 mm compression groups upregulated mRNA expression of OPN and OSX after 24 hours. Additionally, compared with the control group, a significantly higher OSX gene expression was observed in both the 0.5 mm and 1.0 mm groups after 6, 12, and 24 hours of compression (p < 0.05). However, no significant differences were observed regarding ALP and RUNX2 expression. These results indicated increased stimulation of osteogenesis of the mandibular fracture-line gap in the 0.5 mm compression group compared with the control and 1.0 mm compression groups.

Genetics of osteopontin in patients with chronic kidney disease: The German Chronic Kidney Disease study.

Osteopontin (OPN), encoded by SPP1, is a phosphorylated glycoprotein predominantly synthesized in kidney tissue. Increased OPN mRNA and protein expression correlates with proteinuria, reduced creatinine clearance, and kidney fibrosis in animal models of kidney disease. But its genetic underpinnings are incompletely understood. We therefore conducted a genome-wide association study (GWAS) of OPN in a European chronic kidney disease (CKD) population. Using data from participants of the German Chronic Kidney Disease (GCKD) study (N = 4,897), a GWAS (minor allele frequency [MAF]≥1%) and aggregated variant testing (AVT, MAF<1%) of ELISA-quantified serum OPN, adjusted for age, sex, estimated glomerular filtration rate (eGFR), and urinary albumin-to-creatinine ratio (UACR) was conducted. In the project, GCKD participants had a mean age of 60 years (SD 12), median eGFR of 46 mL/min/1.73m2 (p25: 37, p75: 57) and median UACR of 50 mg/g (p25: 9, p75: 383). GWAS revealed 3 loci (p<5.0E-08), two of which replicated in the population-based Young Finns Study (YFS) cohort (p<1.67E-03): rs10011284, upstream of SPP1 encoding the OPN protein and related to OPN production, and rs4253311, mapping into KLKB1 encoding prekallikrein (PK), which is processed to kallikrein (KAL) implicated through the kinin-kallikrein system (KKS) in blood pressure control, inflammation, blood coagulation, cancer, and cardiovascular disease. The SPP1 gene was also identified by AVT (p = 2.5E-8), comprising 7 splice-site and missense variants. Among others, downstream analyses revealed colocalization of the OPN association signal at SPP1 with expression in pancreas tissue, and at KLKB1 with various plasma proteins in trans, and with phenotypes (bone disorder, deep venous thrombosis) in human tissue. In summary, this GWAS of OPN levels revealed two replicated associations. The KLKB1 locus connects the function of OPN with PK, suggestive of possible further post-translation processing of OPN. Further studies are needed to elucidate the complex role of OPN within human (patho)physiology.

Mechanism of ring finger protein 11 regulating Akt signaling pathway to promote osteogenic differentiation of bone marrow mesenchymal stem cells

To investigate the role and regulatory mechanism of ring finger protein 11 (RNF11) on Akt signaling pathway in the process of osteogenesis of bone marrow mesenchymal stem cells (BMSCs) to provide ideas for further clarifying its osteogenesis mechanism and its use in clinical treatment in the future. BMSCs were isolated and cultured from fresh bone marrow of healthy donors and subcultured. The 4th generation cells were used in experiments after identification by flow cytometry, and osteogenic, chondrogenic, and adipogenic induction. BMSCs were cultured in osteogenic differentiation medium for 0-14 days. The degree of osteogenic differentiation was detected by Alizarin red staining and alkaline phosphatase (ALP) staining, and the protein expression of RNF11 was detected by Western blot. The 4th generation BMSCs were divided into blank control group (group A), empty lentivirus (Lv-NC) group (group B), and knockdown RNF11 (Lv-ShRNF11) group (group C). Osteogenesis was induced and cultured for 0-14 days. The expression of RNF11 protein was detected by Western blot, the degree of osteogenic differentiation was detected by Alizarin red staining and ALP staining, and the relative mRNA expressions of Runx2, osteocalcin (OCN), and osteopontin (OPN) were detected by real-time fluorescence quantitative PCR (qRT-PCR). The protein relative expressions of Akt, Smad1/5/8, and β-catenin signaling pathway were detected by Western blot, expressed as the ratio before and after phosphorylation. In order to study the effect mechanism of RNF11 on Akt signaling pathway, the 4th generation BMSCs were divided into Lv-NC transfection group (group A1), Lv-ShRNF11 transfection group (group B1), and Lv-ShRNF11 transfection supplemented with Akt signaling pathway activator SC79 group (group C1). The protein relative expressions of RNF11 and Akt signaling pathway were detected by Western blot, the related osteogenesis indexes were detected by Alizarin red staining, ALP staining, and qRT-PCR. The flow cytometry, and osteogenic, chondrogenic, adipogenic induction culture identification showed that the isolated and cultured cells were BMSCs. The protein relative expression of RNF11 increased gradually with the extension of osteogenic differentiation time ( P<0.05); after knockdown RNF11, Alizarin red and ALP stainings showed that the degree of osteogenic differentiation of BMSCs in group C were significantly lower than those in groups A and B, and qRT-PCR detection showed that the relative expression of Runx2, OCN, and OPN mRNA significantly decreased ( P<0.05). The protein relative expressions of RNF11 and Akt signaling pathway significantly increased with the extensions of osteogenic differentiation time ( P<0.05). After knockdown RNF11, the protein relative expression of Akt signaling pathway in group C was significantly lower than that in groups A and B ( P<0.05), while Smad1/5/8 and β-catenin signaling pathway had no significant effect ( P>0.05). Compared with group A1, the protein relative expression of RNF11 in groups B1 and C1 significantly decreased ( P<0.05). Compared with groups A1 and C1, the protein relative expression of Akt signaling pathway in group B1 was significantly lower ( P<0.05); Alizarin red and ALP stainings showed that the degree of osteogenic differentiation of BMSCs in group C1 were slightly lower than that of group A1 ( P>0.05), but significantly higher than that of group B1 ( P<0.05); qRT-PCR detection showed that the relative expressions of Runx2, OCN, and OPN mRNA in group C1 were slightly lower than those of group A1 ( P>0.05), but were significantly higher than those of group B1 ( P<0.05). RNF11 promotes the differentiation of BMSCs into osteoblasts by positively regulating the activation level of Akt signaling pathway. RNF11 can be used as a potential target to improve the bone repair efficacy of BMSCs and treat bone metabolic diseases.

Biocompatibility and pro-mineralization effect of tristrontium aluminate cement for endodontic use

Background/purposeTristrontium aluminate (S3A) is a hydraulic cement with setting behavior similar to that of mineral trioxide aggregate (MTA). This study examined the biological effects of S3A on mouse dental papilla cells (MDPs) in vitro and on rat exposed pulps in vivo. Materials and methodsExtracts of S3A and MTA were prepared by immersing each cement in ultrapure water. MDPs were cultured with S3A or MTA extracts, and cell proliferation was evaluated with a tetrazolium-salt assay. Attachment of MDPs on the set cements was examined with scanning electron microscopy (SEM). mRNA expression of bone morphogenic protein (Bmp2), osteocalcin (Oc) and osteopontin (Opn) in MDPs exposed to S3A or MTA extracts was determined with reverse transcription-quantitative polymerase chain reaction. Mineralized nodule formation was evaluated with Alizarin Red S staining. Simulated body fluid (SBF)-dipped S3A was examined with SEM and energy dispersive X-ray analysis (EDX). Exposed molar pulps of male Wistar rats capped with S3A or MTA were histologically examined. ResultsS3A extract did not inhibit proliferation of MDPs. Set S3A and MTA exhibited attachment of MDPs on their surface. S3A extract showed significantly higher mineralized nodule formation and mRNA expression of Bmp2, Oc, and Opn than did MTA extract. SBF-dipped S3A exhibited formation of surface precipitates, which were composed of Ca, P, Sr, and Al. Direct pulp capping with S3A and with MTA induced mineralized tissue repair of the exposed pulp. ConclusionS3A possesses biocompatibility and pro-mineralization effects comparable to those of MTA.

Spatial Expression of Osteopontin in Testis, Epididymis and Spermatozoa in Dromedary Camel

The aim of this study is to clarify the function of osteopontin (OPN) through its expression and localisation in male testis, epididymis and sperm cell of dromedary camel during rutting season. Testis and epididymis parts (caput, corpus and cauda) specimens were obtained from 8 mature male camels and semen was collected from 4 another fertile camels for sperms. OPN mRNA expression and its location was analysed using quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC) techniques, respectively. OPN mRNA expression was significantly (P < 0.05) higher in the cauda of epididymis than other epididymal parts and testis. Immunolocalisation revealed high immunoreaction intensity in the elongated spermatids in seminiferous tubules and Leydig cells in interstitial tissue. However, a faint immunostaining was seen in spermatogonia and Sertoli cells. Along the epididymis tract, intense immunoreactivity of OPN was localised in the cytoplasm of the epithelial cells with high concentration in the apical region nearby lumen and the acrosomal part of the cauda epididymal sperms. Immunofluorescence and immunochromogenic staining of OPN was detected on the acrosomal cap and neck of epididymal sperms. These findings suggested that OPN might play an important role in the sperm protection, migration and fertilisation in dromedary camels.