Assessment of the (Pro)renin Receptor Protein Expression in Organs.

The (pro)renin receptor ((P)RR) is an essential component of the renin-angiotensin system (RAS) as a specific single-pass transmembrane receptor for prorenin and renin and has now emerged as a multifunctional protein implicated in a wide variety of developmental and physio-pathological processes and pathways. The (P)RR may be of pathological significance in metabolic syndrome. The (P)RR has received much consideration; substantial efforts have been made to understand the localization, regulation, and function of the (P)RR at both a molecular and system level. (P)RR regulation of cell function depends on whether it is intact or cleaved into its constituent forms. Therefore, the present chapter describes immunohistochemical approaches to examine the expression of (P)RR in various organs. It was shown that different molecular forms of (P)RR could be present in different tissue compartments in almost all organs. Among them, the liver has high PRR activity. Our findings could elucidate more detailed distribution of different (P)RR molecular forms in different organs, which could provide useful information to further investigate the pathophysiological mechanisms of the development of various diseases in the future.

6-shogaol is a potential treatment for Head and Neck Squamous Cell Carcinoma.

Objective: Natural products in diet have shown a potential role in the prevention and treatment of cancer. Ginger (Zingiber officinale Roscoe) is a great candidate because of its properties of anti-inflammatory, antioxidant, and anti-cancer, but little is known about its effect on head and neck cancer. 6-Shogaol is an active compound derived from Ginger. Thus, this study aimed to investigate the possible anticancer effects of 6-shogaol, a major ginger derivate, on head and neck squamous cell carcinomas (HNSCCs) and the underlying mechanisms. Material and Methods: Two HNSCC cell lines, SCC4 and SCC25, were used in this study. Both SCC4 and SCC25 cells were kept as control or treated with 6-shogaol for 8 and 24 hours and then the cell apoptosis and cell cycle progression of treated cells were examined by PI and Annexin V-FITC double stain and flow cytometry analysis. The Cleaved caspase 3, phosphorylations of ERK1/2 and p38 kinases were examined by Western blot analysis. Results: The results showed that 6-shogaol significantly initiated the G2/M phase arrest of the cell cycle and apoptosis to inhibit the survival of both cell lines. Moreover, these responses could be regulated by ERK1/2 and p38 signaling. And, finally, we also demonstrated that 6-shogaol could enhance the cytotoxicity of cisplatin in HNSCC cells. Conclusion: Our data provided new insights to understand the potential pharmaceutical efficacy of a ginger derivate, 6-shogaol, in antagonizing HNSCC survival. The present study suggests that 6-shogaol is a potential novel candidate for anti-HNSCCs therapy.

Therapeutic Potential of Regorafenib in Cisplatin-Resistant Bladder Cancer with High Epithelial-Mesenchymal Transition and Stemness Properties.

Bladder cancer is becoming one of the most common malignancies across the world. Although treatment strategy has been continuously improved, which has led to cisplatin-based chemotherapy becoming the standard medication, cancer recurrence and metastasis still occur in a high proportion of patients because of drug resistance. The high efficacy of regorafenib, a broad-spectrum kinase inhibitor, has been evidenced in treating a variety of advanced cancers. Hence, this study investigated whether regorafenib could also effectively antagonize the survival of cisplatin-resistant bladder cancer and elucidate the underlying mechanism. Two types of cisplatin-resistant bladder cancer cells, T24R1 and T24R2, were isolated from T24 cisplatin-sensitive bladder cancer cells. These cells were characterized, and T24R1- and T24R2-xenografted tumor mice were created to examine the therapeutic efficacy of regorafenib. T24R1 and T24R2 cells exhibited higher expression levels of epithelial-mesenchymal transition (EMT) and stemness markers compared to the T24 cells, and regorafenib could simultaneously inhibit the viability and the expression of EMT/stemness markers of both T24R1 and T24R2 cells. Moreover, regorafenib could efficiently arrest the cell cycle, promote apoptosis, and block the transmigration/migration capabilities of both types of cells. Finally, regorafenib could significantly antagonize the growth of T24R1- and T24R2-xenografted tumors in mice. These results demonstrated the therapeutic efficacy of regorafenib in cisplatin-resistant bladder cancers. This study, thus, provides more insights into the mechanism of action of regorafenib and demonstrates its great potential in the future treatment of cisplatin-resistant advanced bladder cancer patients.

Clinical and Cellular Evidence of Glycyrrhiza glabra and Platycodon grandiflorus for Vocal Fold Nodules Complementary Treatment.

Vocal fold nodules (VFNs) are the most frequent cause of hoarseness. The management comprised medical, surgical and physical therapy but the effectiveness is not always satisfactory. In this study, we try to figure out an alternative treatment from our clinical experience summary. We retrospectively reviewed VFNs patients who received traditional Chinese medicine (TCM) treatments from July 2018 to August 2020 and traced their Chinese Voice Handicap Index-10 (VHI-C10) and multidimensional voice program (MDVP) analysis results. For further evaluation, we conducted an inflammatory response of porcine vocal fold epithelial (PVFE) cells with 50 ng/mL TNF-alpha. The inflamed PVFE cells were separately cultured in the aqueous extract of Glycyrrhiza glabra (G. glabra) and Platycodon grandifloras (P. grandifloras). In these VFNs patients (n = 22), the average VHI-C10 score decreased from 17.6 to 6.6 (p < 0.001). MDVP analysis revealed improvements in jitter, shimmer, noise-harmonic ratio, and GRBAS scoring system. Of the TCM prescription patterns, G. glabra and P. grandiflorus were used most frequently. In the MTT assay of PVFE cells, no adverse effects of our extracts were observed at doses of 1-200 µg/mL. Western blot analysis revealed downregulation of p65 and mitogen activated protein kinase pathway proteins. The results from both the clinical and in vitro aspects of this study revealed that the herbs G. glabra and P. grandiflorus may offer beneficial outcomes as alternative treatments for VFNs after precise diagnosis.

Blood Reflux-Induced Epigenetic Factors HDACs and DNMTs Are Associated with the Development of Human Chronic Venous Disease.

Blood reflux and metabolic regulation play important roles in chronic venous disease (CVD) development. Histone deacetylases (HDACs) and DNA methyltransferases (DNMTs) serve as repressors that inhibit metabolic signaling, which is induced by proatherogenic flow to promote aortic endothelial cell (EC) dysfunction and atherosclerosis. The aim of this study was to elucidate the relationship between blood reflux and epigenetic factors HDACs and DNMTs in CVD. Human varicose veins with different levels of blood reflux versus normal veins with normal venous flow were examined. The results show that HDAC-1, -2, -3, -5, and -7 are overexpressed in the endothelium of varicose veins with blood reflux. Blood reflux-induced HDACs are enhanced in the varicose veins with a longer duration time of blood reflux. In contrast, these HDACs are rarely expressed in the endothelium of the normal vein with normal venous flow. Similar results are obtained for DNMT1 and DNMT3a. Our findings suggest that the epigenetic factors, HDACs and DNMTs, are induced in venous ECs in response to blood reflux but are inhibited in response to normal venous flow. Blood reflux-induced HDACs and DNMTs could inhibit metabolic regulation and promote venous EC dysfunction, which is highly correlated with CVD pathogenesis.

γ-secretase inhibitors, DAPT and RO4929097, promote the migration of Human Glioma Cells via Smad5-downregulated E-cadherin Expression.

Malignant gliomas are a type of central nervous system cancer with extremely high mortality rates in humans. γ-secretase has been becoming a potential target for cancer therapy, including glioma, because of the involvement of its enzymatic activity in regulating the proliferation and metastasis of cancer cells. In this study, we attempted to determine whether γ-secretase activity regulates E-cadherin to affect glioma cell migration. The human glioma cell lines, including LN18 and LN229, and the γ-secretase inhibitors, including N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) and RO4929097, were used in this study. It was shown that γ-secretase activity inhibition by DAPT and RO4929097 could promote LN18 and LN229 glioma cell migration via downregulating E-cadherin mRNA and protein expressions, but not via affecting E-cadherin protein processing. In addition, γ-secretase activity inhibition was regulated by bone morphogenetic proteins-independent Smad5 activation in glioma cells. Moreover, endogenous Smad1 in glioma cells was found to play an important role in regulating E-cadherin expression and subsequent cell migration but did not affect DAPT-stimulated effects. These results help further elucidate the molecular mechanisms of γ-secretase activity regulation involved in controlling glioma cell malignancy. Information about a potential role for Smad1/5 activity upregulation and subsequent E-cadherin downregulation during inhibition of γ-secretase activity in the development of gliomas is therefore relevant for future research.

Drug Resistance of CPT-11 in Human DLD-1 Colorectal Cancer Cells through MutS Homolog 2 Upregulation.

Colorectal cancers (CRCs) is the most commonly diagnosed and deadly cancer types in the world. Despite advances in chemotherapy for CRCs, drug resistance remains a major challenge to high incurable and eventually deadly rates for patients. CPT-11 is one of the current chemotherapy agents for CRC patients and the CPT-11 resistance development of CRCs is also inevitable. Recently, accumulating data has suggested that DNA repair system might be an inducer of chemotherapy resistance in cancer cells. Thus, this study was aimed to examine whether MutS homolog (MSH) 2, one member of DNA repair system, plays a role to affect the cytotoxicity of CPT-11 to CRCs. Human DLD-1 CRC cells were used in this study. It was shown that MSH2 gene and protein expression could be upregulated in DLD-1 cells under CPT-11 treatment and this upregulation subsequently attenuates the sensitivity of DLD-1 cells to CPT-11. Moreover, ERK1/2 and Akt signaling and AP-1 transcription factor have been found to modulate these effects. These results elucidate the drug resistance role of MSH2 upregulation in the CPT-11-treated DLD-1 CRC cells. Our findings may provide a useful thought for new adjuvant drug development by controlling the DNA repair system.

Dihydroisotanshinone I as a Treatment Option for Head and Neck Squamous Cell Carcinomas.

Head and neck squamous cell carcinomas (HNSCCs) are the most common cancers of the head and neck, and their prevalence is rapidly increasing. HNSCCs present a clinical challenge because of their high recurrence rate, therapeutic resistance to radiation and chemotherapy drugs, and adverse effects. Hence, traditional Chinese herbal treatment may be advantageous to therapeutic strategies for HNSCCs. Danshen (Salvia miltiorrhiza), a well-known Chinese herb, has been extensively applied in treatments for various diseases, including cancer, because of its high degree of safety and low rate of adverse effects despite its unclear mechanism. Thus, we aimed to explore the possible anticancer effects and mechanisms of dihydroisotanshinone I (DT), a compound in danshen (extract from danshen), on HNSCCs. Three HNSCCs cell lines were used for in vitro studies, and a Detroit 562 xenograft mouse model was chosen for in vivo studies. Our in vitro results showed that DT could initiate apoptosis, resulting in cell death, and the p38 signaling partially regulated DT-initiated cell apoptosis in the Detroit 562 model. In the xenograft mouse model, DT reduced tumor size with no obvious adverse effect of hepatotoxicity. The present study suggests that DT is a promising novel candidate for anti-HNSCCs therapy.

Effects of Visfatin on Intracellular Mechanics and Catabolism in Human Primary Chondrocytes through Glycogen Synthase Kinase 3ß Inactivation.

Osteoarthritis (OA) is still a recalcitrant musculoskeletal disease on account of its complex biochemistry and mechanical stimulations. Apart from stimulation by external mechanical forces, the regulation of intracellular mechanics in chondrocytes has also been linked to OA development. Recently, visfatin has received significant attention because of the clinical finding of the positive correlation between its serum/synovial level and OA progression. However, the precise mechanism involved is still unclear. This study determined the effect of visfatin on intracellular mechanics and catabolism in human primary chondrocytes isolated from patients. The intracellular stiffness of chondrocytes was analyzed by the particle-tracking microrheology method. It was shown that visfatin damages the microtubule and microfilament networks to influence intracellular mechanics to decrease the intracellular elasticity and viscosity via glycogen synthase kinase 3ß (GSK3ß) inactivation induced by p38 signaling. Further, microtubule network destruction in human primary chondrocytes is predominantly responsible for the catabolic effect of visfatin on the cyclooxygenase 2 upregulation. The present study shows a more comprehensive interpretation of OA development induced by visfatin through biochemical and biophysical perspectives. Finally, the role of GSK3ß inactivation, and subsequent regulation of intracellular mechanics, might be considered as theranostic targets for future drug development for OA.

Sarcomeres Morphology and Z-Line Arrangement Disarray Induced by Ventricular Premature Contractions through the Rac2/Cofilin Pathway.

The most common ventricular premature contractions (VPCs) originate from the right ventricular outflow tract (RVOT), but the molecular mechanisms of altered cytoskeletons of VPC-induced cardiomyopathy remain unexplored. We created a RVOT bigeminy VPC pig model (n = 6 in each group). Echocardiography was performed. The histopathological alternations in the LV myocardium were analyzed, and next generation sequencing (NGS) and functional enrichment analyses were employed to identify the differentially expressed genes (DEGs) responsible for the histopathological alternations. Finally, a cell silencing model was used to confirm the key regulatory gene and pathway. VPC pigs had increased LV diameters in the 6-month follow-up period. A histological study showed more actin cytoskeleton disorganization and actin accumulation over intercalated disc, Z-line arrangement disarray, increased ß-catenin expression, and cardiomyocyte enlargement in the LV myocardium of the VPC pigs compared to the control pigs. The NGS study showed actin cytoskeleton signaling, RhoGDI signaling, and signaling by Rho Family GTPases and ILK Signaling presented z-scores with same activation states. The expressions of Rac family small GTPase 2 (Rac2), the p-cofilin/cofilin ratio, and the F-actin/G-actin ratio were downregulated in the VPC group compared to the control group. Moreover, the intensity and number of actin filaments per cardiomyocyte were significantly decreased by Rac2 siRNA in the cell silencing model. Therefore, the Rac2/cofilin pathway was found to play a crucial role in the sarcomere morphology and Z-line arrangement disarray induced by RVOT bigeminy VPCs.

Stearoyl-CoA Desaturase-1 Attenuates the High Shear Force Damage Effect on Human MG63 Osteosarcoma Cells.

Mechanical regulation is known as an important regulator in cancer progression and malignancy. High shear force has been found to inhibit the cell cycle progression and result in cell death in various cancer cells. Stearoyl-CoA desaturase (SCD)-1, one of the important lipogenic enzymes, has recently been indicated as a potential pharmaceutical target in cancer therapy. In this study, we determined whether the cell fate control of shear force stimulation is through regulating the SCD-1 expression in cancer cells. Human MG63 osteosarcoma cells were used in this study. 2 and 20 dynes/cm2 shear forces were defined as low and high intensities, respectively. A SCD-1 upregulation in human MG63 osteosarcoma cells under 20, but not 2, dynes/cm2 shear force stimulation was shown, and this induction was regulated by Smad1/5 and peroxisome proliferator-activated receptor δ (PPARδ) signaling. Moreover, gene knockdown of PPARδ and SCD-1 in human MG63 osteosarcoma cells attenuated the differentiation inhibition and resulted in much more cell death of high shear force initiation. The present study finds a possible auto-protective role of SCD-1 upregulation in high shear force-damaged human MG63 osteosarcoma cells. However, its detailed regulation in the cancer fate decision of high shear force should be further examined.

Hyperglycemia inhibits osteoblastogenesis of rat bone marrow stromal cells via activation of the Notch2 signaling pathway.

Background: Bone fragility and related fractures are increasingly being recognized as an important diabetic complication. Mesenchymal progenitors often serve as an important source of bone formation and regeneration. In the present study, we have evaluated the effects of diabetes on osteoblastogenesis of mesenchymal progenitors. Methods: Primary bone marrow stromal cells (BMSCs) were isolated from control and streptozotocin-induced diabetic rats. These cells were evaluated for the effects of in vivo hyperglycemia on the survival and function of mesenchymal progenitors. We concomitantly investigated the effects of different concentrations of glucose, osmolality, and advanced glycation end product (AGE) on osteogenic differentiation and matrix mineralization of rat bone marrow mesenchymal stem cells (RMSC-bm). The relationship between the expression levels of Notch proteins and the corresponding ALP levels was also examined. Results: Our results revealed that in vivo hyperglycemia increased cell proliferation rate but decreased osteogenic differentiation and matrix mineralization of primary rat BMSCs. In vitro high glucose treatment, instead of high AGE treatment, induced a dose-dependent inhibition of osteoblastogenesis of RMSC-bm cells. Activation of the Notch2 signaling pathway, instead of the Notch1 or osmotic response pathways, was associated with these diabetic effects on osteoblastogenesis of mesenchymal progenitors. Conclusions: Hyperglycemia might inhibit osteoblastogenesis of mesenchymal progenitors via activation of the Notch2 signaling pathway.

Zoledronate induces cell cycle arrest and differentiation by upregulating p21 in mouse MC3T3-E1 preosteoblasts.

Background: Increasing research has recently been focused on the supplementary use of drugs such as bisphosphonates that are known to influence bone turnover to prevent and treat periprosthetic bone loss and subsequent implant loosening following total joint replacements. However, there are still concerns about the conflicting effects of bisphosphonate treatment on osteoblastic bone formation in the literature. Methods: In this study, we investigate the role of zoledronate (ZOL) in regulating cell cycle distribution and differentiation in mouse MC3T3-E1 preosteoblasts and also explore the mechanism underlying this effect of ZOL. We examined the expression levels of osteocalcin (OCN) by quantitative polymerase chain reaction (qPCR), the total amount of CDK6, p21 and p27 proteins by Western blot analysis, and the cell cycle distribution by flow cytometric analysis in mouse MC3T3-E1 preosteoblasts to evaluate the effect of ZOL. Small interfering RNAs (siRNAs) were used to assess the individual contributions of genes to specific osteoblast phenotypes. Results: In addition to increased OCN expression, we found that ZOL treatment induces the G0/G1 arrest and results in the increase of p21 and p27 expressions and decrease of CDK6 expression in mouse MC3T3-E1 preosteoblasts. Both p21 and p27 mediates ZOL-induced cell cycle exit; however, p21, but not p27, is responsible for the increase of ZOL-induced OCN expression in these cells. Conclusions: These results endorse that ZOL might have an anabolic effect on osteoblasts. The CDK inhibitor p21 plays a key role in regulating osteoblast differentiation by controlling proliferation-related events in mouse MC3T3-E1 preosteoblasts.

Regulation of calcification in human aortic smooth muscle cells infected with high-glucose-treated Porphyromonas gingivalis.

Porphyromonas (P.) gingivalis infection leading to the periodontitis has been associated with the development of systemic diseases, including cardiovascular diseases and diabetes. However, the effect of a high concentration of glucose (HG) on the invasion efficiency of P. gingivalis and the consequent modulation of pathogenesis in vascular cells, especially in the vascular smooth muscle cells (VSMCs), remains unclear. Hence, the aim of this study was to investigate whether treating P. gingivalis with HG could change its invasion capability and result in VSMC calcification and the underlying mechanism. Human aortic SMCs (HASMCs) and P. gingivalis strain CCUG25226 were used in this study. We found that HGPg infection of HASMCs could initiate the HASMC calcification by stimulating the autocrine regulation of bone morphogenetic protein (BMP) 4 in HASMCs. The upregulation of BMP4 expression in HASMCs was mediated by toll-like receptor 4 and ERK1/2-p38 signaling after P. gingivalis infection. Moreover, the autocrine action of BMP4 in HGPg infection-initiated HASMC calcification upregulated BMP4-specific downstream smad1/5/8-runx2 signaling to increase the expressions of bone-related matrix proteins, that is, osteopontin, osteocalcin, and alkaline phosphatase. This study elucidates the detailed mechanism of HGPg infection-initiated calcification of HASMCs and indicates a possible therapeutic role of BMP4 in P. gingivalis infection-associated vascular calcification.

2 dyn/cm2 shear force upregulates kruppel-like factor 4 expression in human chondrocytes to inhibit the interleukin-1ß-activated nuclear factor-κB.

The shear force effect on human chondrocytes is time and magnitude dependent. Recently, kruppel-like factor (KLF) 4 has been identified as a pleiotropic protein and its activity in cells is dependent on different stimuli and/or cell types. The role of KLF4 in chondrocytes is still unclear and there has been no report determining whether shear force regulates KLF4 levels in chondrocytes. Hence, this study was carried out to investigate the role of KLF4 in human chondrocytes under shear force stimulation and the underlying mechanism. Human primary and SW1353 chondrocytes were used in this study. The shear forces at 2, 5, or 15 dyn/cm2 intensity were applied to both types of human chondrocytes. The specific small interfering RNAs, activators, and inhibitors were used to study the detailed mechanism of shear force. The presented results showed that 2, but not 5 and 15, dyn/cm2 shear force increases KLF4 expression in human primary and SW1353 chondrocytes. Extracellular signal-regulated kinase 5 induced peroxisome proliferator-activated receptor γ transcription activity to increase KLF4 transcription. Moreover, the KLF4 induction in human chondrocytes in response to 2 dyn/cm2 shear force could attenuate interleukin (IL)-1ß-stimulated nuclear factor-κB activation. These results elucidate the role of KLF4 in antagonizing the effect of IL-1ß in human chondrocytes under 2 dyn/cm2 shear force stimulation and provide a possible mechanism to demonstrate the protection of moderate forces or exercises in cartilage.

Effect of 5-fluorouracil on excision repair cross-complementing 1 expression and consequent cytotoxicity regulation in human gastric cancer cells.

Gastric cancer is the third leading cause of cancer mortality all over the world. The combination therapy of surgery with chemotherapy, that is, 5-fluorouracil (5-FU) and platinum-containing anticancer drugs, is becoming a current clinical strategy for patients with gastric cancer because of the lower curative rate and higher cancer recurrence rate of patients treated with only surgery. However, the development of drug resistance in cancer cells is still the most challenge in clinical chemotherapy. Excision repair cross-complementing 1 (ERCC1), an essential member of nucleotide excision repair system, recently has been suggested to be a predictive biomarker of treatment evaluation and might affect the outcomes of chemotherapy. Thus, this study was aimed to investigate whether ERCC1 expression could be regulated, and its role in gastric cancer cells treated with 5-FU and the underlying mechanism. Human AGS gastric cancer cells were used in this study. It was shown that ERCC1 expression could be upregulated in AGS cells treated with 5-FU and this upregulation could subsequently attenuate the cytotoxicity of 5-FU in AGS cells. Moreover, 5-FU-upregulated ERCC1 expression was regulated by extracellular signal-regulated kinase (ERK) 1/2 and p38 signaling through activating the transcription factor c-jun/activator protein (AP)-1. These results indicated the role of ERCC1 in the development of drug resistance to 5-FU in AGS cells. The mechanism elucidation concerning the ERK1/2 and p38 kinases and transcription factor c-jun/AP-1 might contribute another idea to the development of chemotherapy strategy for the gastric cancers in the future.

Metformin increases the cytotoxicity of oxaliplatin in human DLD-1 colorectal cancer cells through down-regulating HMGB1 expression.

Colorectal cancer (CRC) is the fourth most common cause of cancer death worldwide. Chemotherapy has been the major strategy for treating patients with advanced CRC. Oxaliplatin (OXA) is used as both an adjuvant and neoadjuvant anticancer agent available to treat advanced CRC. High-mobility group box 1 protein (HMGB1) is a critical regulator of cell death and survival. HMGB1 overexpression has been shown to be resistant to cytotoxic agents. In addition, Metformin, a widely used drug for diabetes, has emerged as a potential anticancer agent. In this study, we examined whether HMGB1 plays a role in the OXA- and/or metformin-induced cytotoxic effect on CRC cells. The results showed that treatment with OXA increased HMGB1 expression in the ERK1/2- and Akt-dependent manners in DLD-1 cells. HMGB1 gene knockdown enhanced the cytotoxicity and cell growth inhibition of OXA. Moreover, OXA-increased HMGB1 expression was by inducing NF-κB-DNA-binding activity to in DLD-1 cells. Compared to a single agent, OXA combined with metformin administration resulted in cytotoxicity and cell growth inhibition synergistically, accompanied with reduced HMGB1 level. These findings may have implications for the rational design of future drug regimens incorporating OXA and metformin for the treatment of CRC.

Leptin induces MMP1/13 and ADAMTS 4 expressions through bone morphogenetic protein-2 autocrine effect in human chondrocytes.

The induction of bone morphogenetic protein (BMP)2 in injured and arthritis articular cartilage has been proposed, but the precise mechanism has not been clearly clarified. Our previous study has found that leptin could stimulate the BMP2 autocrine effect to increase the anabolic collagen II expression when it initiates the catabolic response in human chondrocytes. It has been suggested that this BMP2 autocrine effect contributes to a reparative role in leptin-stimulated human chondrocytes. In this study, we further determined whether this BMP2 autocrine effect also affect the expressions of catabolic matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motif (ADAMTS). Human primary and SW1353 chondrocytes were used in this study. It was shown that leptin could induce the expressions of MMP1, 3, and 13 and ADAMTS4 and 5 in both human primary and SW1353 chondrocytes. Leptin-increased MMP1/13 (not MMP3) and ADAMTS4 (not ADAMTS5) expressions were affected by the leptin-upregulated BMP2 and its specific downstream Smad1/5 signaling. Moreover, both HDAC3 and 4 are involved in regulating leptin-induced BMP2 upregulation and then affect MMP1 and 13 and ADAMTS4 expression. Both HDAC3 and 4 also affect leptin-increased MMP3 mRNA expression but not through BMP2 autocrine effect of leptin induction. Our results further elucidated the role of BMP2 autocrine effect in matrix-degrading enzymes expressions under leptin stimulation. The findings in this study provide new insights into the possible mechanism of BMP2 induction in leptin-stimulated chondrocytes and in leptin-induced OA development.

The Impact of Lipid Types and Liposomal Formulations on Osteoblast Adiposity and Mineralization.

Recent studies have demonstrated that fat accumulation in bone cells is detrimental to bone mass. Both adipocytes and osteoblasts are derived from common multipotent mesenchymal stem cells (MSCs) and hence the presence of fat may increase adipocyte proliferation, differentiation and fat accumulation while inhibiting osteoblast differentiation and bone formation. Lipids are common constituents in supramolecular vesicles (e.g., micelles or liposomes) that serve as drug delivery systems. Liposomal formulations such as Meriva® were proven to decrease joint pain and improve joint function in osteoarthritis (OA) patients. In this study, we evaluated how lipid types and liposomal formulations affect osteoblast behavior including cell viability, differentiation, mineralization and inflammation. Various liposomal formulations were prepared using different types of lipids, including phosphatidylcholine (PC), 1,2-dioleoyl-sn-glycero-3-phospho-ethanolamine (DOPE), cholesterol (Chol), 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol hydrochloride (DC-cholesterol HCl), and 1,2-dioleoyl-3-trimethylammonium-propane chloride salt (DOTAP) to investigate the impact on osteoblast differentiation and inflammation. The results indicated that cationic lipids, DC-cholesterol and DOTAP, presented higher dose-dependent cytotoxicity and caused high level of inflammatory responses. Due to the natural properties of lipids, all the lipids can induce lipid droplet formation in osteoblasts but the level of lipid droplet accumulation was different. In comparison with cationic lipids, neutral lipids induced less adiposity, and maintained high osteoblast mineralization. Similar to previous researches, we also confirmed an inverse relationship between lipid droplet formation and osteoblast mineralization in 7F2 mouse osteoblasts. Importantly, PC containing liposomes (PC only and PC/DOTAP) suppressed IL-1ß-induced gene expression of COX-2 and MMP-3 but not Chol/DOTAP liposomes or DC-Chol/DOPE liposomes. Taken together, we suggested that PC contained liposomes could provide the best liposomal formulation for the treatment of bone diseases.

Low Shear Stress Attenuates COX-2 Expression Induced by Resistin in Human Osteoarthritic Chondrocytes.

Low shear stress has been proposed to play a reparative role in modulating cartilage homeostasis. Recently, epidemiological studies have found a positive correlation between the resistin level in serum and synovial fluid and osteoarthritis (OA) severity in patients. However, the effect of moderate shear stress on the catabolic stimulation of resistin in OA chondrocytes remains unclear. Hence, this study was to investigate whether low shear stress could regulate resistin-induced catabolic cyclooxygenase (COX)-2 expression in human OA chondrocytes and the underlying mechanism. Human OA chondrocytes and SW1353 chondrosarcoma cells were used in this study. Two modes of low shear stress (2 dyn/cm2 ), pre-shear and post-shear, were applied to the chondrocytes. A specific activator and siRNAs were used to investigate the mechanism of low shear stress-regulated COX-2 expression of resistin induction. We found that human OA chondrocytes exposed to different modes of low shear stress elicit an opposite effect on resistin-induced COX-2 expression: pre-shear for a short duration attenuates the resistin effect by inhibiting the transcription factor nuclear factor (NF)-κB-p65 subunit and the cAMP response element binding protein; however, post-shear over a longer duration enhances the resistin effect by activating only the NF-κB-p65 subunit. Moreover, our results demonstrated that the regulation of both shear modes in resistin-stimulated COX-2 expression occurs through increasing AMP-activated protein kinase activation and then sirtuin 1 expression. This study elucidates the detailed mechanism of low shear stress regulating the resistin-induced catabolic COX-2 expression and indicates a possible reparative role of moderate shear force in resistin-stimulated OA development. J. Cell. Physiol. 232: 1448-1457, 2017. © 2016 Wiley Periodicals, Inc.