Soluble Sema4D cleaved from osteoclast precursors by TACE suppresses osteoblastogenesis.

Bone remodelling is mediated by orchestrated communication between osteoclasts and osteoblasts which, in part, is regulated by coupling and anti-coupling factors. Amongst formally known anti-coupling factors, Semaphorin 4D (Sema4D), produced by osteoclasts, plays a key role in downmodulating osteoblastogenesis. Sema4D is produced in both membrane-bound and soluble forms; however, the mechanism responsible for producing sSema4D from osteoclasts is unknown. Sema4D, TACE and MT1-MMP are all expressed on the surface of RANKL-primed osteoclast precursors. However, only Sema4D and TACE were colocalized, not Sema4D and MT1-MMP. When TACE and MT1-MMP were either chemically inhibited or suppressed by siRNA, TACE was found to be more engaged in shedding Sema4D. Anti-TACE-mAb inhibited sSema4D release from osteoclast precursors by ~90%. Supernatant collected from osteoclast precursors (OC-sup) suppressed osteoblastogenesis from MC3T3-E1 cells, as measured by alkaline phosphatase activity, but OC-sup harvested from the osteoclast precursors treated with anti-TACE-mAb restored osteoblastogenesis activity in a manner that compensates for diminished sSema4D. Finally, systemic administration of anti-TACE-mAb downregulated the generation of sSema4D in the mouse model of critical-sized bone defect, whereas local injection of recombinant sSema4D to anti-TACE-mAb-treated defect upregulated local osteoblastogenesis. Therefore, a novel pathway is proposed whereby TACE-mediated shedding of Sema4D expressed on the osteoclast precursors generates functionally active sSema4D to suppress osteoblastogenesis.

Functional properties of thermally tampered poly(ethylene oxide).

Introduction: Poly(ethylene oxide) (PEO) is the most common polymer used in commercial abuse-deterrent tablets. Due to its vulnerability to high-temperature manipulation, we investigated abuse-deterrent capability and the toxicity of this polymer upon thermal treatments at 80°C and 180°C for 1 hour. Methods: Tablets (200 mg PEO and 300 mg Avicel®) were directly compressed under 2000 lb. The thermally manipulated PEOs were evaluated for their viscosity, crushability, structural changes, and cell toxicity. Results: Our findings showed that 180°C-treated tablets underwent some degrees of oxidative degradation with profound toxicity in both mesenchymal stem cells and MG63 cells. The 180°C-treated tablets exhibited almost no resistance against crushing and were prone to abuse. While thermal processing of PEO at around its melting temperature is a common approach to enhance crush resistance of its dosage forms, thermal manipulation at close to the PEO's oxidation temperature can lead to structural changes, dramatic loss of crush and extraction resistance, and significant cell toxicity. Conclusion: Similar to the low molecular weight PEO, when thermally manipulated at its thermo-oxidative temperature, the high molecular weight PEO loses its deterrence performance and causes severe cell toxicity.

Locally Secreted Semaphorin 4D Is Engaged in Both Pathogenic Bone Resorption and Retarded Bone Regeneration in a Ligature-Induced Mouse Model of Periodontitis.

It is well known that Semaphorin 4D (Sema4D) inhibits IGF-1-mediated osteogenesis by binding with PlexinB1 expressed on osteoblasts. However, its elevated level in the gingival crevice fluid of periodontitis patients and the broader scope of its activities in the context of potential upregulation of osteoclast-mediated periodontal bone-resorption suggest the need for further investigation of this multifaceted molecule. In short, the pathophysiological role of Sema4D in periodontitis requires further study. Accordingly, attachment of the ligature to the maxillary molar of mice for 7 days induced alveolar bone-resorption accompanied by locally elevated, soluble Sema4D (sSema4D), TNF-α and RANKL. Removal of the ligature induced spontaneous bone regeneration during the following 14 days, which was significantly promoted by anti-Sema4D-mAb administration. Anti-Sema4D-mAb was also suppressed in vitro osteoclastogenesis and pit formation by RANKL-stimulated BMMCs. While anti-Sema4D-mAb downmodulated the bone-resorption induced in mouse periodontitis, it neither affected local production of TNF-α and RANKL nor systemic skeletal bone remodeling. RANKL-induced osteoclastogenesis and resorptive activity were also suppressed by blocking of CD72, but not Plexin B2, suggesting that sSema4D released by osteoclasts promotes osteoclastogenesis via ligation to CD72 receptor. Overall, our data indicated that ssSema4D released by osteoclasts may play a dual function by decreasing bone formation, while upregulating bone-resorption.

Dual-Function Semaphorin 4D Released by Platelets: Suppression of Osteoblastogenesis and Promotion of Osteoclastogenesis.

Effects of the antiosteoblastogenesis factor Semaphorin 4D (Sema4D), expressed by thrombin-activated platelets (TPs), on osteoblastogenesis, as well as osteoclastogenesis, were investigated in vitro. Intact platelets released both Sema4D and IGF-1. However, in response to stimulation with thrombin, platelets upregulated the release of Sema4D, but not IGF-1. Anti-Sema4D-neutralizing monoclonal antibody (mAb) upregulated TP-mediated osteoblastogenesis in MC3T3-E1 osteoblast precursors. MC3T3-E1 cells exposed to TPs induced phosphorylation of Akt and ERK further upregulated by the addition of anti-sema4D-mAb, suggesting the suppressive effects of TP-expressing Sema4D on osteoblastogenesis. On the other hand, TPs promoted RANKL-mediated osteoclastogenesis in the primary culture of bone-marrow-derived mononuclear cells (BMMCs). Among the known three receptors of Sema4D, including Plexin B1, Plexin B2 and CD72, little Plexin B2 was detected, and no Plexin B1 was detected, but a high level of CD72 mRNA was detected in RANKL-stimulated BMMCs by qPCR. Both anti-Sema4D-mAb and anti-CD72-mAb suppressed RANKL-induced osteoclast formation and bone resorptive activity, suggesting that Sema4D released by TPs promotes osteoclastogenesis via ligation to a CD72 receptor. This study demonstrated that Sema4D released by TPs suppresses osteogenic activity and promotes osteoclastogenesis, suggesting the novel property of platelets in bone-remodeling processes.

Abuse-deterrent properties and cytotoxicity of poly(ethylene oxide) after thermal tampering.

Poly(ethylene oxide) (PEO) is the most common deterring agent used in the abuse-deterrent formulations (ADFs). In this study, we investigated the PEO's abuse-deterrent properties and its potential cytotoxicity after being heated at high temperatures (80 °C and 180 °C). The results indicated a significant loss in both crush and extraction resistance features of the polymer, which is primarily associated with the polymer degradation at the higher temperatures. The heat-treated PEO at the high temperature was also found to lose its controlled-release feature, upon which over 80% of the drug was released after one hour in the simulated gastric fluid. The cytotoxicity of the PEO was further assessed to evaluate the safety of the polymer following the thermal treatment. Our findings revealed a substantial loss in the viability of the cells exposed to the PEO treated at higher temperatures. Taken all, heating PEO at high temperatures can lead to a significant loss in both the crush/extraction resistance characteristics and the safety of the polymer. These findings reemphasize the fact that more appropriate and stricter test and regulations will be needed to assure that the abuse deterrent formulations are safe and effective under severe conditions of abuse.

Predifferentiated Gingival Stem Cell-Induced Bone Regeneration in Rat Alveolar Bone Defect Model.

Cleft alveolus, a common birth defect of the maxillary bone, affects one in 700 live births every year. This defect is traditionally restored by autogenous bone grafts or allografts, which may possibly cause complications. Cell-based therapies using the mesenchymal stem cells (MSCs) derived from human gingiva (gingiva-derived mesenchymal stem cells [GMSCs]) is attracting the research interest due to their highly proliferative and multilineage differentiation capacity. Undifferentiated GMSCs expressed high level of MSC-distinctive surface antigens, including CD73, CD105, CD90, and CD166. Importantly, GMSCs induced with osteogenic medium for a week increased the surface markers of osteogenic phenotypes, such as CD10, CD92, and CD140b, indicating their osteogenic potential. The objective of this study was to assess the bone regenerative efficacy of predifferentiated GMSCs (dGMSCs) toward an osteogenic lineage in combination with a self-assembling hydrogel scaffold PuraMatrix™ (PM) and/or bone morphogenetic protein 2 (BMP2), on a rodent model of maxillary alveolar bone defect. A critical size maxillary alveolar defect of 7 mm × 1 mm × 1 mm was surgically created in athymic nude rats. The defect was filled with either PM/BMP2 or PM/dGMSCs or the combination of three (PM/dGMSCs/BMP2) and the bone regeneration was evaluated at 4 and 8 weeks postsurgery. New bone formation was evaluated by microcomputed tomography and histology using Hematoxylin and Eosin staining. The results demonstrated the absence of spontaneous bone healing, either at 4 or 8 weeks postsurgery in the defect group. However, the PM/dGMSCs/BMP2 group showed significant enhancement in bone regeneration at 4 and 8 weeks postsurgery, compared with the transplantation of individual material/cells alone. Apart from developing the smallest critical size defect, results showed that PM/dGMSCs/BMP2 could serve as a promising option for the regeneration of bone in the cranio/maxillofacial region in humans.

Human gingiva-derived mesenchymal stem cells are therapeutic in lupus nephritis through targeting of CD39-CD73 signaling pathway.

Cell specific and cytokine targeted therapeutics have underperformed in systemic lupus erythematosus (SLE). Mesenchymal stem cells (MSCs) have emerged as a novel therapy to address the dysregulation in autoimmune diseases but also have limitations. Human gingiva derived MSCs (GMSCs) are superior in regulating immune responses. Here, we demonstrate that the adoptive transfer of GMSCs homes to and maintains in the kidney and has a robust therapeutic effect in a spontaneous lupus nephritis model. Specifically, GMSCs limits the development of autoantibodies as well as proteinuria, decreases the frequency of plasma cells and lupus nephritis histopathological scores by directly suppressing B cells activation, proliferation and differentiation. The blockage of CD39-CD73 pathway dramatically abrogates the suppressive capacities of GMSCs in vitro and in vivo and highlights the significance of this signaling pathway in SLE. Collectively, manipulation of GMSCs provides a promising strategy for the treatment of patients with SLE and other autoimmune diseases.

Human gingival tissue-derived MSC suppress osteoclastogenesis and bone erosion via CD39-adenosine signal pathway in autoimmune arthritis.

BACKGROUND: Bone destruction is one of many severe complications that occurs in patients with rheumatoid arthritis (RA) and current therapies are unable to cure this manifestation. This study here aims to determine whether GMSC can directly inhibit osteoclast formation and eventually attenuate osteoclastogenesis and bone erosion in an inflammatory milieu. METHOD: GMSC were co-cultured with osteoclast precursors with or without CD39 inhibitor, CD73 inhibitor or adenosine receptors inhibitors pretreatment and osteoclast formation were evaluated in vitro. 2×10^6 GMSC per mouse were transferred to CIA mice and pathology scores, the frequency of osteoclasts, bone erosion in joints were assessed in vivo. FINDING: GMSC but not control cells, markedly suppressed human or mice osteoclastogenesis in vitro. GMSC treatment also resulted in a dramatically decreased level of NF-κB p65/p50 in osteoclasts in vitro. Infusion of GMSC to CIA significantly attenuated the severity of arthritis, pathology scores, frequency of osteoclasts, particularly bone erosion, as well as a decreased expression of RANKL in synovial tissues in vivo. Blockade of CD39/CD73 or adenosine receptors has significantly abrogated the suppressive ability of GMSC in vitro and therapeutic effect of GMSC on bone erosion during CIA in vivo. INTERPRETATION: GMSC inhibit osteoclast formation in vitro and in vivo partially via CD39-CD73-adenosine signals. Manipulation of GMSC may have a therapeutic implication on rheumatoid arthritis and other bone erosion related diseases. FUND: This study was supported by grants from the National Key R&D Program of China (2017YFA0105801 to F.H); the Zhujiang Innovative and Entrepreneurial Talent Team Award of Guangdong Province (2016 ZT 06S 252 to F·H) and National Institutes of Health (R01 AR059103, R61 AR073409 and NIH Star Award to S.G.Z).

Three-dimensional macroporous materials for tissue engineering of craniofacial bone.

Repair of critical-size defects caused by trauma, removal of a tumour, or congenital abnormalities is a challenge in the craniomaxillofacial region because of the limitations associated with treatment. We have reviewed research papers and updated information relevant to the various types of macroporous scaffolds. We have included papers on several biomaterials and their use in various craniofacial defects such as mandibular, calvarial, and others, as well as the latest technological developments such as 3-dimensional printed scaffolds. We selected all papers about scaffolds, stem cells, and growth factors for review. Initial selection was by review of titles and abstracts, and the full texts of potentially suitable articles were then assessed. Methods of tissue engineering for repair of critical-size defects in the craniofacial bones seem to be viable options for surgical treatment in the future. Macroporous scaffolds with interconnected pores are of great value in regeneration of bone in the craniofacial region. In recent years, various natural or synthetic materials, or both, have been developed, on which macroporous scaffolds can be based. In this review we present a review on the various types of three-dimensional macroporous scaffolds that have been developed in recent years, and evaluate their potential for regeneration of craniofacial bone.

Injectable platelet rich fibrin (i-PRF): opportunities in regenerative dentistry?

OBJECTIVES: Platelet rich plasma (PRP) has been utilized in regenerative dentistry as a supra-physiological concentrate of autologous growth factors capable of stimulating tissue regeneration. Despite this, concerns have been expressed regarding the use of anti-coagulants, agents known to inhibit wound healing. In this study, a liquid formulation of platelet rich fibrin (PRF) termed injectable-PRF (i-PRF) without the use of anti-coagulants was investigated. MATERIALS AND METHODS: Standard PRP and i-PRF (centrifuged at 700 rpm (60G) for 3 min) were compared for growth factor release up to 10 days (8 donor samples). Furthermore, fibroblast biocompatibility at 24 h (live/dead assay); migration at 24 h; proliferation at 1, 3, and 5 days, and expression of PDGF, TGF-ß, and collagen1 at 3 and 7 days were investigated. RESULTS: Growth factor release demonstrated that in general PRP had higher early release of growth factors whereas i-PRF showed significantly higher levels of total long-term release of PDGF-AA, PDGF-AB, EGF, and IGF-1 after 10 days. PRP showed higher levels of TGF-ß1 and VEGF at 10 days. While both formulations exhibited high biocompatibility and higher fibroblast migration and proliferation when compared to control tissue-culture plastic, i-PRF induced significantly highest migration whereas PRP demonstrated significantly highest cellular proliferation. Furthermore, i-PRF showed significantly highest mRNA levels of TGF-ß at 7 days, PDGF at 3 days, and collagen1 expression at both 3 and 7 days when compared to PRP. CONCLUSIONS: i-PRF demonstrated the ability to release higher concentrations of various growth factors and induced higher fibroblast migration and expression of PDGF, TGF-ß, and collagen1. Future animal research is now necessary to further validate the use of i-PRF as a bioactive agent capable of stimulating tissue regeneration. CLINICAL RELEVANCE: The findings from the present study demonstrate that a potent formulation of liquid platelet concentrates could be obtained without use of anti-coagulants.

Osteogain® loaded onto an absorbable collagen sponge induces attachment and osteoblast differentiation of ST2 cells in vitro.

OBJECTIVES: Dimensional changes of the alveolar bone following tooth extraction are a major challenge in daily dental practice. To limit bone loss, a variety of biomaterials including bone grafts, barrier membranes, and growth factors have been utilized either alone or in combination therapies to increase the speed and quality of new bone formation. The aim of the present in vitro study was to investigate the regenerative potential of Osteogain®, a new liquid carrier system of enamel matrix derivative (EMD) in combination with an absorbable collagen sponge (ACS) specifically designed for extraction socket healing. MATERIALS AND METHODS: The potential of ACS was first investigated using ELISA to quantify total amelogenin adsorption and release from 0 to 10 days. Thereafter, the cellular effects of ST2 pre-osteoblasts were investigated for cellular attachment at 8 h and cell proliferation at 1, 3, and 5 days as well as osteoblast differentiation by real-time PCR and alizarin red staining for cells seeded on (1) tissue culture plastic, (2) ACS alone, and (3) ACS + Osteogain®. RESULTS: ACS efficiently loaded nearly 100% of the amelogenin proteins found in Osteogain® which were gradually released up to a 10-day period. Osteogain® also significantly induced a 1.5-fold increase in cell attachment and resulted in a 2-6-fold increase in mRNA levels of osteoblast differentiation markers including runt-related transcription factor 2 (Runx2), collagen1a2, alkaline phosphatase, and bone sialoprotein as well as induced alizarin red staining when combined with ACS. CONCLUSIONS: In summary, these findings suggest that Osteogain® is capable of inducing osteoblast attachment and differentiation when combined with ACS. Future animal studies and randomized human clinical trials are necessary to further support these findings. CLINICAL RELEVANCE: The use of Osteogain® in combination with ACS may provide a valuable means to limit dimensional changes following tooth extraction.

Optimized Platelet-Rich Fibrin With the Low-Speed Concept: Growth Factor Release, Biocompatibility, and Cellular Response.

BACKGROUND: Over the past decade, use of leukocyte platelet-rich fibrin (L-PRF) has gained tremendous momentum in regenerative dentistry as a low-cost fibrin matrix used for tissue regeneration. This study characterizes how centrifugation speed (G-force) along with centrifugation time influence growth factor release from fibrin clots, as well as the cellular activity of gingival fibroblasts exposed to each PRF matrix. METHODS: Standard L-PRF served as a control (2,700 revolutions per minute [rpm]-12 minutes). Two test groups using low-speed (1,300 rpm-14 minutes, termed advanced PRF [A-PRF]) and low-speed + time (1,300 rpm-8 minutes; A-PRF+) were investigated. Each PRF matrix was tested for growth factor release up to 10 days (eight donor samples) as well as biocompatibility and cellular activity. RESULTS: The low-speed concept (A-PRF, A-PRF+) demonstrated a significant increase in growth factor release of platelet-derived growth factor (PDGF), transforming growth factor (TGF)-ß1, epidermal growth factor, and insulin-like growth factor, with A-PRF+ being highest of all groups. Although all PRF formulations were extremely biocompatible due to their autogenous sources, both A-PRF and A-PRF+ demonstrated significantly higher levels of human fibroblast migration and proliferation compared with L-PRF. Furthermore, gingival fibroblasts cultured with A-PRF+ demonstrated significantly higher messenger RNA (mRNA) levels of PDGF, TGF-ß, and collagen1 at either 3 or 7 days. CONCLUSIONS: The findings from the present study demonstrate modifications to centrifugation speed and time with the low-speed concept favor an increase in growth factor release from PRF clots. This, in turn, may directly influence tissue regeneration by increasing fibroblast migration, proliferation, and collagen mRNA levels. Future animal and clinical studies are now necessary.

Recombinant human bone morphogenetic protein (rhBMP)9 induces osteoblast differentiation when combined with demineralized freeze-dried bone allografts (DFDBAs) or biphasic calcium phosphate (BCP).

OBJECTIVES: Recently, recombinant human bone morphogenetic protein 9 (rhBMP9) has been characterized as one of the most osteogenic growth factors among the 15 human BMPs. The aim of the present study was to investigate the effects of rhBMP9 in comparison to the clinically utilized rhBMP2 on in vitro cell behavior when combined with two bone graft materials including demineralized freeze-dried bone allografts (DFDBAs) and biphasic calcium phosphate (BCP). MATERIALS AND METHODS: The absorption and release kinetics of rhBMPs from DFDBA and BCP were investigated by ELISA. Moreover, murine bone stromal ST2 cell behavior was investigated on DFDBA or BCP seeded on (1) graft only, (2) rhBMP2 (10 ng/ml), (3) rhBMP2 (100 ng/ml), (4) rhBMP9 (10 ng/ml), and (5) rhBMP9 (100 ng/ml). The effects of rhBMPs on DFDBA and BCP were assessed for cell adhesion, proliferation, and osteoblast differentiation by alkaline phosphatase (ALP) activity, alizarin red staining, and real-time PCR for genes encoding Runx2, ALP, and bone sialoprotein (BSP). RESULTS: While both BMPs were gradually released from DFDBA and BCP over time, significantly higher adsorption was observed on BCP when compared to DFDBA. Cell attachment and proliferation was higher on BCP with little influence of either rhBMP2/9. Despite rhBMPs having relatively no effect on cell attachment/proliferation, a pronounced and marked effect was observed on osteoblast differentiation for both rhBMP2/9. Interestingly, it was observed that rhBMP9 induced significantly higher ALP activity, alizarin red staining, and messenger RNA (mRNA) levels of ALP and BSP when compared to rhBMP2. Our results also revealed higher differentiation for rhBMP2/9 with BCP when compared to DFDBA most likely as a result of higher growth factor adsorption. CONCLUSION: While both rhBMP2/9 combined with DFDBA or BCP induced osteoblast differentiation, rhBMP9 induced greater osteoblast differentiation when compared to rhBMP2. CLINICAL RELEVANCE: rhBMP9 may be a recombinant growth factor with higher potential to induce new bone formation when compared to rhBMP2. Further in vivo studies are necessary to characterize its regenerative potential in various animal models.

Mesenchymal stem cells and alginate microcarriers for craniofacial bone tissue engineering: A review.

Craniofacial bone is a complex structure with an intricate anatomical and physiological architecture. The defects that exist in this region therefore require a precise control of osteogenesis in their reconstruction. Unlike traditional surgical intervention, tissue engineering techniques mediate bone development with limited postoperative risk and cost. Alginate stands as the premier polymer in bone repair because of its mild ionotropic gelation and excellent biocompatibility, biodegradability, and injectability. Alginate microcarriers are candidates of choice to mediate cells and accommodate into 3-D environment. Several studies reported the use of alginate microcarriers for delivering cells, drugs, and growth factors. This review will explore the potential use of alginate microcarrier for stem cell systems and its application in craniofacial bone tissue engineering.

Angiotensin III induces signal transducer and activator of transcription 3 and interleukin-6 mRNA levels in cultured rat astrocytes.

INTRODUCTION: Recently we established that pro-inflammatory actions of angiotensin (Ang) II in astrocytes involved Janus kinase 2 (JAK2), signal transducer and activator of transcription 3 (STAT3), and interleukin-6 (IL-6). MATERIALS AND METHODS: In our current study, we determined in brainstem and cerebellum whether Ang III also activates STAT3 leading to IL-6 mRNA expression and astrocyte proliferation. RESULTS: Ang III induced STAT3 phosphorylation in a concentration- and time-dependent manner. Significant STAT3 phosphorylation was rapid and was maximal within 10 min, and with 100 nM Ang III. The Ang AT1 receptor was shown to mediate this action of Ang III. Ang III also significantly induced IL-6 mRNA expression within an hour, and maximal Ang III-mediated IL-6 mRNA expression occurred in the presence of 100 nM Ang III. Ang III-mediated IL-6 mRNA expression occurred by the interaction of the peptide with the Ang AT1 receptor and was mediated by STAT3. In addition, STAT3 was shown to mediate Ang III astrocyte proliferation. CONCLUSIONS: These findings suggest that Ang III, similar to Ang II, has pro-inflammatory effects since it induces STAT3 leading to an induction of IL-6 mRNA expression, outcomes that lend relevance to the physiological importance of central Ang III.

Bromelain-induced apoptosis in GI-101A breast cancer cells.

Bromelain is a proteolytic enzyme extracted from the stems and the immature fruits of pineapple that was found to be antitumorigenic in different in vitro models. Bromelain has been reported to promote apoptosis, particularly in breast cancer cells, with the up-regulation of c-Jun N-terminal kinase and p38 kinase. Our study was designed to determine if bromelain could induce apoptosis in GI-101A breast cancer cells. GI-101A cells were treated with increasing concentrations of bromelain for 24 hours. The effect of bromelain for inducing cell death via activation of the apoptosis mechanism in GI-101A cells was further determined by using caspase-9 and caspase-3 assays along with the M30-Apoptosense assay to measure cytokeratin 18 (CK18) levels in the cytoplasm of the cultured cancer cells. A dose-dependent increase in the activities of caspase-9 and caspase-3 coinciding with elevation of CK18 levels was found in bromelain-treated cells compared with control cells. Furthermore, the apoptosis induction by bromelain was confirmed by DNA fragmentation analysis and 4,6'-diamino-2-phenylindole dihydrochloride fluorescence staining of the nucleus. Our results indicate an increase in apoptosis-related cell death in breast cancer cells with increasing concentrations of bromelain.

Angiotensin II induces cell growth and IL-6 mRNA expression through the JAK2-STAT3 pathway in rat cerebellar astrocytes.

The pleiotrophic effects of angiotensin II (Ang II) play important roles in astrocyte growth and inflammatory responses. We investigated whether Ang II induces astrocyte growth and interleukin-6 (IL-6) mRNA expression in rat cerebellar astrocytes through Janus kinase 2-signal transduction activator of transcription (JAK2-STAT3). Ang II increased JAK2 and STAT3 phosphorylation in a time- and a dose-dependent manner. One hundred nanomolar Ang II induced maximal phosphorylation of both JAK2 and STAT3 between 15 min and 30 min. The Ang II-mediated phosphorylation of both JAK2 and STAT3 was blocked by AG490, a selective JAK2 inhibitor. Losartan, a selective AT1 receptor antagonist, inhibited Ang II-mediated JAK2 and STAT3 phosphorylation, while pretreatment with an AT2 receptor blocker, PD123319, was ineffective. Ang II increased the mRNA expression of IL-6 in a concentration-and time-dependent manner. Maximal IL-6 mRNA expression occurred with 100 nM Ang II, and the peak effect occurred in a biphasic manner at 3 h and between 12 and 24 h. Moreover, pretreatments with AG490 attenuated Ang II-induced IL-6 mRNA levels, and Ang II-induced astrocyte growth. This study has demonstrated that Ang II induced the phosphorylation of both JAK2 and STAT3 via the AT1 receptor in cerebellar astrocytes. In addition, our results suggest that JAK2 and STAT3 are upstream signals that mediate Ang II-induced IL-6 mRNA expression and astrocyte growth. These findings represent a novel non-classical mechanism of Ang II signaling in cerebellar astrocytes.

Treatment of ovarian cancer cells with nutlin-3 and resveratrol combination leads to apoptosis via caspase activation.

The current study was focused on the induction of apoptotic effects of resveratrol along with the combination treatments of nutlin-3 and transforming growth factor-ß (TGF-ß) against the human ovarian cancer cell line A2780/CP70. To determine the extent of apoptosis following the above-mentioned treatments, we assessed the execution of apoptotic events that proceed via caspase activation and cytochrome c release. We estimated the caspase-3 and -9 activities using a direct enzymatic assay that measures the cleavage of synthetic peptide substrate (N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide). Our experiments showed an increase in caspase-3 and -9 activities in the cells that were treated with the combination of resveratrol (5 µM) with nutlin-3 (5 µM) or TGF-ß (1 µg/mL). Since activation of procaspase-3 by caspase-9 requires the release of cytochrome c into the cytoplasm, we measured the levels of cytochrome c in the cytoplasm by western blot experiments. The data indicated a considerable increase in caspase-3 and cytochrome c levels when cells were treated with drugs for 24 hours. Experiments with 4,6'-diamino-2-phenylindole dihydrochloride (DAPI) staining also confirmed the induction of apoptosis in all the above-mentioned treatments done at 24 and 48 hours. These results support our hypothesis that resveratrol combination can induce programmed cell death at doses that are less than half of what is typically needed for nutlin-3 and TGF-ß to induce apoptosis.

Angiotensin II activates JAK2/STAT3 pathway and induces interleukin-6 production in cultured rat brainstem astrocytes.

We have shown that tyrosine kinases and mitogen-activated protein kinases mediate angiotensin II (Ang II) effects in cultured rat astrocytes. In this study, we investigated whether Ang II induces Janus kinase (JAK) 2, signal transducer and activators of transcription (STAT) 3 phosphorylation, and interleukin-6 (IL-6) secretion in cultured brainstem rat astrocytes. Ang II increased JAK2 phosphorylation in a time- and dose-dependent manner. Maximal phosphorylation of 1.7+/-0.4 fold above basal was observed at 15 min with 100 nM Ang II. Losartan (10 microM), an AT(1) receptor blocker, inhibited Ang II-mediated JAK2 phosphorylation, while 10 microM PD123319, an AT(2) receptor blocker, was ineffective. The JAK2 inhibitor, AG490 (50 microM), prevented Ang II JAK2 phosphorylation. Ang II also stimulated STAT3 in a concentration- and time-dependent manner. Maximal phosphorylation of 0.8+/-0.11 above basal was observed at 15 min with 100 nM Ang II. Treatment with AG490 reduced Ang II phosphorylation of STAT3 and Ang II-induced astrocyte growth suggesting that JAK2 is an upstream signal in these Ang II effects. Ang II also stimulated IL-6 secretion from brainstem astrocytes in a concentration- and time-dependent manner. Maximal IL-6 secretion of 0.7+/-0.2 above basal was observed with 100 nM Ang II after 48 h of treatment. Losartan decreased Ang II-induced IL-6 secretion while PD123319 was ineffective. Interestingly, AG490 reduced Ang II-stimulated IL-6 secretion. Our study showed for the first time that Ang II induced JAK2/STAT3 phosphorylation and IL-6 secretion through activation of the Ang II AT(1) receptor in brainstem astrocytes. In addition, Ang II stimulated IL-6 secretion and astrocyte growth through the JAK2 pathway in brainstem astrocytes. These results provide new insights into pro-inflammatory and mitogenic signaling mechanisms of Ang II in astrocytes.

Regulation of c-fos, c-jun and c-myc gene expression by angiotensin II in primary cultured rat astrocytes: role of ERK1/2 MAP kinases.

We have previously shown that angiotensin II (Ang II) stimulates astrocyte growth through activation of ERK1/2 mitogen activated protein (MAP) kinases. In the current study, we determined whether Ang II stimulates the expression of c-fos, c-jun and c-myc in brainstem astrocyte cultures. Reverse transcriptase-PCR analysis showed c-fos, c-jun, and c-myc mRNAs were induced by Ang II. The EC50 values for Ang II stimulation of c-fos, c-jun and c-myc were 1.3, 1.68 and 1.4 nM, respectively. Ang II (100 nM) induced peak stimulation for all genes by 45 min followed by a gradual decline. Inhibition of ERK1/2 by PD98059 attenuated Ang II-induced c-fos and c-myc mRNA expression (by 75% and 100%, respectively) but was ineffective in preventing Ang II induction of c-jun. These studies show for the first time in brainstem astrocytes that Ang II induces the expression of c-fos, c-myc and c-jun, and showed that ERK1/2 mediate Ang II stimulation of c-fos and c-myc. These data implicate the ERK1/2 MAP kinase pathway as a divergent point in controlling Ang II stimulation of immediate early response genes in the central nervous system.