Targeted inhibition of transforming growth factor-ß type I receptor by AZ12601011 improves paraquat poisoning-induced multiple organ fibrosis.

Paraquat (PQ) causes fatal poisoning that leads to systemic multiple organ fibrosis, and transforming growth factor (TGF)-ß1 plays a critical role in this process. In this study, we aimed to investigate the effects of AZ12601011 (a small molecular inhibitor of TGFßRI) on PQ-induced multiple organ fibrosis. We established a mouse model of PQ in vivo and used PQ-treated lung epithelial cell (A549) and renal tubular epithelial cells (TECs) in vitro. Haematoxylin-eosin and Masson staining revealed that AZ12601011 ameliorated pulmonary, hepatic, and renal fibrosis, consistent with the decrease in the levels of fibrotic indicators, alpha-smooth muscle actin (α-SMA) and collagen-1, in the lungs and kidneys of PQ-treated mice. In vitro data showed that AZ12601011 suppressed the induction of α-SMA and collagen-1 in PQ-treated A549 cells and TECs. In addition, AZ12601011 inhibited the release of inflammatory factors, interleukin (IL)-1ß, IL-6, and tumour necrosis factor-α. Mechanistically, TGF-ß and TGFßRI levels were significantly upregulated in the lungs and kidneys of PQ-treated mice. Cellular thermal shift assay and western blotting revealed that AZ12601011 directly bound with TGFßRI and blocked the activation of Smad3 downstream. In conclusion, our findings revealed that AZ12601011 attenuated PQ-induced multiple organ fibrosis by blocking the TGF-ß/Smad3 signalling pathway, suggesting its potential for PQ poisoning treatment.

14-3-3ζ: A suppressor of inflammatory arthritis.

Inflammatory arthritis (IA) is a common disease that affects millions of individuals worldwide. Proinflammatory events during IA pathogenesis are well studied; however, loss of protective immunity remains underexplored. Earlier, we reported that 14-3-3zeta (ζ) has a role in T-cell polarization and interleukin (IL)-17A signal transduction. Here, we demonstrate that 14-3-3ζ knockout (KO) rats develop early-onset severe arthritis in two independent models of IA, pristane-induced arthritis and collagen-induced arthritis. Arthritic 14-3-3ζ KO animals showed an increase in bone loss and immune cell infiltration in synovial joints. Induction of arthritis coincided with the loss of anti-14-3-3ζ antibodies; however, rescue experiments to supplement the 14-3-3ζ antibody by passive immunization did not suppress arthritis. Instead, 14-3-3ζ immunization during the presymptomatic phase resulted in significant suppression of arthritis in both wild-type and 14-3-3ζ KO animals. Mechanistically, 14-3-3ζ KO rats exhibited elevated inflammatory gene signatures at the messenger RNA and protein levels, particularly for IL-1ß. Furthermore, the immunization with recombinant 14-3-3ζ protein suppressed IL-1ß levels, significantly increased anti-14-3-3ζ antibody levels and collagen production, and preserved bone quality. The 14-3-3ζ protein increased collagen expression in primary rat mesenchymal cells. Together, our findings indicate that 14-3-3ζ causes immune suppression and extracellular remodeling, which lead to a previously unrecognized IA-suppressive function.

Human umbilical cord mesenchymal stem cell-derived small extracellular vesicles ameliorate collagen-induced arthritis via immunomodulatory T lymphocytes.

BACKGROUND: Rheumatoid arthritis (RA) is an autoimmune disease for which there are currently no effective therapies. Although mesenchymal stem cells (MSCs) can prevent arthritis through immunomodulatory mechanisms, there are several associated risks. Alternatively, MSC-derived small extracellular vesicles (sEVs) can mimic the effects of MSCs, while reducing the risk of adverse events. However, few studies have examined sEVs in the context of RA. Here, we evaluate the immunomodulatory effects of human umbilical cord MSC (hUCMSC)-derived sEVs on T lymphocytes in a collagen-induced arthritis (CIA) rat model to elucidate the possible mechanism of sEVs in RA treatment. We then compare these mechanisms to those of MSCs and methotrexate (MTX). METHODS: The arthritis index and synovial pathology were assessed. T lymphocyte proliferation and apoptosis, Th17 and Treg proportions, and interleukin (IL)-17, IL-10, and transforming growth factor (TGF)-ß expression were detected using flow cytometry. Retinoic acid receptor-related orphan receptor gamma t (RORγt) and forkhead box P3 (FOXP3), which are master transcriptional regulators of Th17 and Treg differentiation, were also assessed using immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: sEV treatment ameliorated arthritis and inhibited synovial hyperplasia in a dose-dependent manner. These effects were mediated by inhibiting T lymphocyte proliferation and promoting their apoptosis, while decreasing Th17 cell proportion and increasing that of Treg cells in the spleen, resulting in decreased serum IL-17, and enhanced IL-10 and TGF-ß expression. Transcriptionally, sEVs decreased RORγt and increased FOXP3 expression in the spleen, and decreased RORγt and FOXP3 expression in the joints. In some aspects sEVs were more effective than MSCs and MTX in treating CIA. CONCLUSIONS: hUCMSC-derived sEVs ameliorate CIA via immunomodulatory T lymphocytes, and might serve as a new therapy for RA.

Combination of LMT-28 and Metformin Improves Beneficial Anti-Inflammatory Effect in Collagen-Induced Arthritis.

OBJECTIVES: The interleukin-6 (IL-6)-mediated signaling pathway plays an essential role in the development of rheumatoid arthritis. LMT-28 suppresses the activation of the IL-6-mediated signaling by direct targeting of gp130. Although LMT-28 and metformin both possess anti-inflammatory activity, the beneficial effect of LMT-28 and metformin combination on a collagen-induced arthritis (CIA) model has not yet been investigated. This study aimed to investigate the anti-inflammatory effect and mechanism of a combination of LMT-28 and metformin in a CIA model. METHODS: In MH7A cells, cell proliferation and the IL-6-mediated signaling pathway following administration of LMT-28 and metformin combination was analyzed through MTT assay and Western blotting. The level of T helper 17 (Th17) cell differentiation from CD4+ T cells was analyzed in mouse splenocytes and human peripheral blood mononuclear cells. Arthritis score, incidence rate, inflammatory cytokine, and T-cell subsets were measured in CIA mice following administration of LMT-28 and metformin combination. RESULTS: Combination treatment with LMT-28 and metformin diminished proliferation of MH7A cells and IL-6-mediated gp130, STAT3, and ERK signaling more than in individual treatments. Furthermore, the differentiation of CD4+ T cells into Th17 cells was attenuated more by combination treatment with LMT-28 and metformin than individual treatments. The combination of LMT-28 and metformin ameliorated the arthritic score better than individual treatments. The combination significantly reduced tumor necrosis factor and IL-6 levels in the sera and had an anti-inflammatory effect on the distribution of Treg/Th17 cells in the lymph nodes. CONCLUSION: Combination treatment with LMT-28 and metformin significantly ameliorates arthritic symptoms in CIA by suppressing Th17 differentiation and IL-6 signaling.

BAD inactivation exacerbates rheumatoid arthritis pathology by promoting survival of sublining macrophages.

The resistance of synovial sublining macrophages to apoptosis has a crucial role in joint inflammation and destruction in rheumatoid arthritis (RA). However, the underlying mechanism is incompletely understood. Here we report that inactivation of the pro-apoptotic BCL-2 family protein BAD is essential for survival of synovial sublining macrophage in RA. Genetic disruption of Bad leads to more severe joint inflammation and cartilage and bone damage with reduced apoptosis of synovial sublining macrophages in collagen-induced arthritis (CIA) and TNFα transgenic (TNF-Tg) mouse models. Conversely, Bad3SA/3SA mice, in which BAD can no longer be inactivated by phosphorylation, are protected from collagen-induced arthritis. Mechanistically, phosphorylation-mediated inactivation of BAD specifically protects synovial sublining macrophages from apoptosis in highly inflammatory environment of arthritic joints in CIA and TNF-Tg mice, and in patients with RA, thereby contributing to RA pathology. Our findings put forward a model in which inactivation of BAD confers the apoptosis resistance on synovial sublining macrophages, thereby contributing to the development of arthritis, suggesting that BAD may be a potential therapeutic target for RA.

Chebulanin exerts its anti-inflammatory and anti-arthritic effects via inhibiting NF-κB and MAPK activation in collagen-induced arthritis mice.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by synovial inflammation and progressive joint destruction. Chebulanin is a natural polyphenol acid isolated from the traditional Tibetan medicine Terminalia chebula Retz that has previously been reported to possess anti-inflammatory properties. The present study aimed to investigate the anti-inflammatory and anti-arthritic effects of chebulanin and explore its underlying mechanisms in vivo and in vitro using a collagen-induced arthritis (CIA) mouse model and lipopolysaccharide (LPS) stimulated RAW264.7 cell inflammation model. Arthritis severity scores were assessed twice weekly; the levels of cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in serum were detected using enzyme-linked immunosorbent assay kits; histopathological assessment was performed using micro computed tomography and hematoxylin and eosin staining. Activation of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways were assessed using western blotting. The inhibition of translocation of cytosolic p38 and p65 into the nucleus was observed using immunofluorescence staining and western blotting in vitro. Chebulanin significantly suppressed the progression and development of RA in CIA mice by decreasing the arthritis severity scores, attenuating paw swelling and joint destruction, and reducing the levels of IL-6 and TNF-α significantly (p < 0.05). Furthermore, chebulanin reduced the levels of excised phosphorylated (p)-p38, phosphorylated-c-JUN N-terminal kinase (p-JNK), p-p65 and phosphorylated NF-κB inhibitor alpha (p-IκBα) in CIA mice, but did not affect the level of phosphorylated extracellular-signal-regulated kinase (ERK). In addition, chebulanin could inhibit the nuclear translocation of p38 and p65 in LPS-stimulated macrophages in dose-dependent manner. In conclusion, this study demonstrated that chebulanin exerts anti-inflammatory and anti-arthritic effects by inhibiting the activation of NF-κB and MAPK signaling pathways.

Effect of the Buffer on the Buildup and Stability of Tannic Acid/Collagen Multilayer Films Applied as Antibacterial Coatings.

The deposition of polyelectrolyte multilayers, obtained by the layer-by-layer (LbL) method, is a well-established technology to design biocompatible and antibacterial coatings aimed at preventing implant-associated infections. Several types of LbL films have been reported to exhibit antiadhesive and/or antibacterial (contact-killing or release-killing) properties governed not only by the incorporated compounds but also by their buildup conditions or their postbuildup treatments. Tannic acid (TA), a natural polyphenol, is known to inhibit the growth of several bacterial strains. In this work, we developed TA/collagen (TA/COL) LbL films built in acetate or citrate buffers at pH 4. Surprisingly, the used buffer impacts not only the physicochemical but also the antibacterial properties of the films. When incubated in physiological conditions, both types of TA/COL films released almost the same amount of TA depending on the last layer and showed an antibacterial effect against Staphylococcus aureus only for citrate-built films. Because of their granular topography, TA/COL citrate films exhibited an efficient release-killing effect with no cytotoxicity toward human gingival fibroblasts. Emphasis is put on a comprehensive evaluation of the physicochemical parameters driving the buildup and the antibacterial property of citrate films. Specifically, complexation strengths between TA and COL are different in the presence of the two buffers affecting the LbL deposition. This work constitutes an important step toward the use of polyphenols as an antibacterial agent when incorporated in LbL films.

ZNRF3 Regulates Collagen-Induced Arthritis Through NF-kB and Wnt Pathways.

Although the E3 ubiquitin ligase Zinc and ring finger 3 (ZNRF3) negatively regulates the Wnt signaling pathway, its function in rheumatoid arthritis (RA) is elusive. Here, the effects and the mechanism of ZNRF3 on a mouse model of collagen-induced arthritis (CIA) and human fibroblast-like synoviocytes (FLS) obtained from RA patients were determined. Our results showed that ZNRF3 was highly expressed in tissues and FLSs compared to trauma patients. Lentivirus-mediated silencing of ZNRF3 induced apoptosis decreased cell viability and significantly attenuated inflammation in RA-FLSs via tumor necrosis-α (TNF-α). Additionally, silencing of ZNRF3 reduced knee joint damage and also decreased the level of TNF-α, IL-1ß, and IL-6 in the CIA mouse model. These effects were mediated by the crosstalk between Wnt and NF-κB pathways in RA-FLS.

GRK2 Mediated Abnormal Transduction of PGE2-EP4-cAMP-CREB Signaling Induces the Imbalance of Macrophages Polarization in Collagen-Induced Arthritis Mice.

Rheumatoid arthritis (RA) is characterized by the massive infiltration of various chronic inflammatory cells in synovia. In synovial fluid of patients with RA, M1 macrophages are dominant among all subtypes of macrophages, the mechanisms of macrophages polarization imbalance in RA has not been fully illuminated. The prostaglandin E2 (PGE2) augments M2 polarization in part via the cyclic adenosine monophosphate (cAMP)-cyclic AMP responsive element binding (CREB) signaling. However, previous study found constant stimulus of PGE2 on fibroblast-like synovial cells of adjuvant arthritis rats induced the decrease of cAMP, which is primarily caused by G protein-coupled receptor kinase 2 (GRK2)-induced EP4 over- desensitization. Whether GRK2 mediated-EP4 over-desensitization reduces the level of cAMP and inhibits M2 polarization in RA is unclear. Here we observed M1 macrophages were dominant in peritoneal macrophages (PMs), bone-marrow-derived macrophages (BMMs) and synovial macrophages of collagen-induced arthritis (CIA) mice. PGE2 stimulated M2 polarization via the EP4-cAMP-CREB in normal mice, while failed to promote M2 polarization in the PMs of CIA mice. Further, we found the EP4 over-desensitization stimulated by PGE2 induced abnormal PGE2-cAMP-CREB signaling as well as the imbalance of macrophage polarization. Targeted disruption of GRK2 in Raw264.7 (RAW) through GRK2 siRNA or CRISPR/Cas9 downregulated the M1 macrophage markers, upregulated the M2 macrophage markers and the EP4 membrane localization. The reduced M1/M2 ratio and increased p-CREB expression were observed in BMMs and PMs of GRK2+/- mice. This study highlighted a novel role of GRK2 in regulating macrophages function in RA and provided new idea for precision treatment of RA.

Asperosaponin VI protects against bone destructions in collagen induced arthritis by inhibiting osteoclastogenesis.

BACKGROUND: Bone destructive diseases like rheumatoid arthritis (RA), osteoporosis and bone metastatic tumors are mainly mediated by over-activated osteoclasts. Asperosaponin VI (AVI), isolated from the rhizome of Dipsacus asper, belongs to triterpenoid saponins. It has multiple physiological activities but its effects on RA, especially on osteoclast differentiation and activation are still unclear. PURPOSE: Explore the protective role of AVI on collagen induced arthritis (CIA) in vivo and RANKL induced osteoclastogenesis in vitro. METHODS: The effects of AVI on cell viability and RANKL-induced osteoclastogenesis, actin ring formation, bone resorption activity as well as on osteoclast specific gene and protein expression were tested using bone marrow derived monocytes (BMMs). Paws from CIA mice were used for micro-CT, HE and TRAP staining, real-time PCR and western blot. Sera were used for cytokine analysis by ELISA. The signaling pathways were detected using western blot, real-time PCR and immunofluorescence assay. RESULTS: AVI significantly inhibited RANKL-induced osteoclast formation and bone resorption activity by suppressing the formation of actin ring. It also inhibited the expression of various osteoclatogenesis marker genes and signaling pathways. AVI protected arthritis in vivo by suppressing inflammation and bone loss. CONCLUSION: AVI exerts its anti-osteoclastogenic activity both in vitro and in vivo by inhibiting RANKL-induced osteoclast differentiation and function. Thus, our studies demonstrate a potential therapeutic role for AVI in preventing or inhibiting RANKL-mediated osteolytic bone diseases.

In vivo evaluation of scaffolds compatible for colonoid engraftments onto injured mouse colon epithelium.

Colon organoids (colonoids) are known to be similar to colon tissue in structure and function, which makes them useful in the treatment of intestinal de-epithelialized disease. Matrigel, which is used as a transplantation scaffold for colonoids, cannot be used in clinical applications because of its undefined composition and tumorigenicity. This study identifies clinically available scaffolds that are effective for colonoid transplantation in damaged intestinal mucosa. The colon crypt was isolated and cultured from C57BL/6-Tg[CAG enhanced green fluorescent protein (EGFP)131Osb/LeySopJ mice into EGFP + colonoids and subsequently transplanted into the EDTA colitis mouse model using gelatin, collagen, or fibrin glue scaffolds. To identify scaffolds suitable for colonoid engraftment in injured colon mucosa, the success rates of transplantation and secondary EGFP colonoid formation were measured, and the scaffolds' mediated toxicity in vitro and in vivo was observed in recipient mice. When colonoids were transplanted with gelatin, collagen, and fibrin glue into the EDTA colitis mouse model, all groups were found to be successfully engrafted. Fibrin glue, especially, showed significant increase in the engrafted area compared with Matrigel after 4 wk. The scaffolds used in the study did not induce colonic toxicity after transplantation into the recipients' colons and were thus deemed safe when locally administrated. This study suggests new methods for and provides evidence of the safety and utility of the clinical application of colonoid-based therapeutics. Furthermore, the methods introduced in this study will be helpful in developing cell treatment using the esophagus or a stomach organoid for various digestive-system diseases.-Jee, J., Jeong, S. Y., Kim, H. K., Choi, S. Y., Jeong, S., Lee, J., Ko, J. S., Kim, M. S., Kwon, M.-S., Yoo, J. In vivo evaluation of scaffolds compatible for colonoid engraftments onto injured mouse colon epithelium.

IL-35 ameliorates collagen-induced arthritis by promoting TNF-α-induced apoptosis of synovial fibroblasts and stimulating M2 macrophages polarization.

Synovitis, the chronic inflammation of the synovial membranes, is a hallmark of rheumatoid arthritis, a chronic disease with profound impact on human health. Recently, interleukin-35 (IL-35), a new member of the IL-12 family, was identified as an anti-inflammatory and immunosuppressive cytokine and was shown to ameliorate collagen-induced arthritis (CIA) in mice. However, the mechanism by which IL-35 alleviates CIA remains unknown. In this study, we investigated the effect of IL-35 on the CIA microenvironment and, specifically, the tumor necrosis factor alpha (TNF-α)-induced macrophage inflammatory response and apoptosis of fibroblast-like synoviocytes (FLSs). Firstly, using RT-PCR, western blot, and flow cytometry, we found that IL-35 suppressed TNF-α-induced inflammatory responses by down-regulating iNOS and COX-2 in peripheral blood monocyte-derived macrophages. IL-35 also activated alternative M2 macrophage polarization, as determined by evaluation of CCR7 and CD206 expression. Moreover, we showed that IL-35 enhanced TNF-α-induced FLS apoptosis. Using a panel of immunohistochemical and immunofluorescence analyses in a CIA model established in 18 DBA/1J mice, we demonstrated that IL-35 promotes synoviocyte apoptosis and alternative activation of macrophages to alleviate arthritis in vivo. Taken together, our results show that IL-35 promotes TNF-α-induced FLS apoptosis and modulates M2 macrophage polarization to ameliorate CIA inflammation both in vitro and in vivo.

Sinomenine mitigates collagen-induced arthritis mice by inhibiting angiogenesis.

OBJECTIVE: The objective of the present study is to investigate the inhibitory effects of sinomenine (SIN) on angiogenesis in a collagen-induced arthritis (CIA) mouse model. METHODS: Arthritis assessments for all mice were recorded. The histopathological assessments were performed following haematoxylin and eosin (HE) staining. Immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) analyses were used to detect the expression of hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF) and angiopoietin 1 (ANG-1) in the serum and in the membrane. Immunohistochemistry was employed to detect the synovium microvessel density (MVD). RESULTS: Compared with the CIA model group, SIN significantly ameliorated swelling and erythema extension, decreased the arthritis index, reduced inflammation, cartilage damage and bone erosion, and lessened the number of CD31 positive cells on the synovium. Moreover, the levels of HIF-1α, VEGF and ANG-1 in the synovium and in the peripheral serum were increased in the untreated CIA model group but were significantly reduced in the 30 mg/kg, 100 mg/kg and 300 mg/kg SIN treatment groups. CONCLUSION: SIN could mitigate CIA by inhibiting angiogenesis, and the mechanism may associate with the HIF-1α-VEGF-ANG-1 axis. Additionally, our study provides a referable experimental basis for the use of SIN for the treatment of rheumatoid arthritis.

Hematopoietic prostaglandin D synthase-derived prostaglandin D2 ameliorates adjuvant-induced joint inflammation in mice.

Although prostaglandins (PGs) are known to be involved in the progression of arthritis, the role of PGD2 remains unclear. In this study, we evaluated the role of PGD2 in joint inflammation using genetically modified mice. Injection of complete Freund's adjuvant (CFA) increased the production of PGD2 and induced paw swelling and cartilage erosion in wild-type (WT) mice. These phenomena were accompanied with an increase in the mRNA levels of TNF-α, IL-6, IL-1ß, and matrix-degrading metalloproteinase-9. Knockdown of hematopoietic PGD synthase (H-PGDS) abolished the PGD2 production and exacerbated all of the arthritic manifestations in the inflamed paw. Immunostaining revealed that infiltrating macrophages strongly expressed H-PGDS in the CFA-injected paw. Morphologic studies revealed vascular hyperpermeability and angiogenesis in the inflamed WT paw. H-PGDS deficiency was accelerated, whereas daily administration of a PGD2 receptor D prostanoid (DP) agonist attenuated the CFA-induced hyperpermeability and angiogenesis. We further confirmed that DP deficiency exacerbated, whereas the administration of the DP agonist improved, the CFA-induced arthritic manifestations. The findings demonstrate that H-PGDS-derived PGD2 ameliorates joint inflammation by attenuating vascular permeability and subsequent angiogenesis and indicates the therapeutic potential of a DP agonist for arthritis.-Tsubosaka, Y., Maehara, T., Imai, D., Nakamura, T., Kobayashi, K., Nagata, N., Fujii, W., Murata, T. Hematopoietic prostaglandin D synthase-derived prostaglandin D2 ameliorates adjuvant-induced joint inflammation in mice.

Modification of chitosan grafted with collagen peptide by enzyme crosslinking.

Free radicals are closely related to the occurrence and development of aging, cancer and inflammation. In this paper, the microbial transglutaminase (MTGase) was used as a catalyst to graft the collagen peptide (COP) molecules on the amino group of carboxymethyl chitosan sulfate (CMCS) to improve the antioxidant effects. FT-IR and NMR spectroscopy were used to confirm the successful grafting of COP to CMCS. Degree of substitution (DS) of CMCS-COP could be controlled by adjusting the reaction conditions. With the increase of concentration, the ability of each sample on scavenging capacity and reducibility tends to increase obviously. The results of anticoagulant experiments showed that the ability of CMCS and CMCS-COP with three different degrees of substitution on activated partial thrombin time (APTT) and prothrombin time (PT) values were all increased to compare with the control group. No relevant cytotoxicity against NIH-3T3 mouse fibroblasts was found for the copolymers. These results suggested that CMCS-COP would appear to be a promising candidate for wound dressing application.

Vasodilator-stimulated phosphoprotein (VASP) is not a major mediator of platelet aggregation, thrombogenesis, haemostasis, and antiplatelet effect of prasugrel in rats.

Vasodilator-stimulated phosphoprotein (VASP) is a member of actin regulatory proteins implicated in platelet adhesion. In addition, phosphorylation of VASP is utilised for the assessment of platelet reactivity in patients treated with P2Y12 receptor antagonists, a class of antiplatelet agents. However, the role of VASP in platelet aggregation, thrombogenesis, haemostasis, and the antiplatelet effect of P2Y12 receptor antagonists remains unclear. We investigated these effects using heterozygous and homozygous VASP knockout rats generated with a CRISPR/Cas9 system. Baseline characteristics, such as haematology and other biochemical parameters, were comparable among the genotypes. In vitro platelet aggregation stimulated by adenosine diphosphate (ADP) or collagen, P-selectin expression of rat platelets treated with ADP, and in vivo thrombocytopenia induced by collagen were also comparable among the genotypes. In addition, in vivo thrombogenesis in a ferric chloride-induced arterial thrombosis model and bleeding time were also comparable among the genotypes. Furthermore, the in vitro antiplatelet effect of prasugrel, a third-generation P2Y12 receptor antagonist, was unaffected by VASP knockout. Although phosphorylated VASP is still an important surrogate marker specific for P2Y12 antagonists, our findings demonstrate that VASP is not a major mediator of platelet aggregation, thrombogenesis, haemostasis, and the antiplatelet effect of prasugrel in rats.

Digesting a Path Forward: The Utility of Collagenase Tumor Treatment for Improved Drug Delivery.

Collagen and hyaluronan are the most abundant components of the extracellular matrix (ECM) and their overexpression in tumors is linked to increased tumor growth and metastasis. These ECM components contribute to a protective tumor microenvironment by supporting a high interstitial fluid pressure and creating a tortuous setting for the convection and diffusion of chemotherapeutic small molecules, antibodies, and nanoparticles in the tumor interstitial space. This review focuses on the research efforts to deplete extracellular collagen with collagenases to normalize the tumor microenvironment. Although collagen synthesis inhibitors are in clinical development, the use of collagenases is contentious and clinically untested in cancer patients. Pretreatment of murine tumors with collagenases increased drug uptake and diffusion 2-10-fold. This modest improvement resulted in decreased tumor growth, but the benefits of collagenase treatment are confounded by risks of toxicity from collagen breakdown in healthy tissues. In this review, we evaluate the published in vitro and in vivo benefits and limitations of collagenase treatment to improve drug delivery.

Improved collagen extraction from jellyfish (Acromitus hardenbergi) with increased physical-induced solubilization processes.

Efficiency and effectiveness of collagen extraction process contribute to huge impacts to the quality, supply and cost of the collagen produced. Jellyfish is a potential sustainable source of collagen where their applications are not limited by religious constraints and threats of transmittable diseases. The present study compared the extraction yield, physico-chemical properties and toxicology in vitro of collagens obtained by the conventional acid-assisted and pepsin-assisted extraction to an improved physical-aided extraction process. By increasing physical intervention, the production yield increased significantly compared to the conventional extraction processes (p < .05). Collagen extracted using the improved process was found to possess similar proximate and amino acids composition to those extracted using pepsin (p > .05) while retaining high molecular weight distributions and polypeptide profiles similar to those extracted using only acid. Moreover, they exhibited better appearance, instrumental colour and were found to be non-toxic in vitro and free of heavy metal contamination.

Lysyl oxidase is involved in synovial hyperplasia and angiogenesis in rats with collagen­induced arthritis.

Lysyl oxidase (LOX) serves an important role in remodeling the extracellular matrix and angiogenesis in various types of cancer; however, whether LOX is involved in the pathogenesis of rheumatoid arthritis remains unknown. In order to investigate this in the present study, ß­aminopropionitrile, an inhibitor of LOX, was injected intraperitoneally into rats with type II collagen­induced arthritis (CIA). Subsequently, synovial hyperplasia was examined by hematoxyl in and eosin staining, and the microvascular density (MVD) and expression levels of LOX, matrix metalloproteinase (MMP)­2 and MMP­9 in the synovial membrane and fluid were determined by immunohistochemistry and ELISA, respectively. The enzyme activity of LOX was evaluated by the Amplex Red Hydrogen Peroxide method. The results demonstrated an increased amount of rough synovial membranes, higher MVD in these membranes and more synovial cell layers in CIA rats compared with in the control rats. In addition, higher enzymatic activity of LOX and higher expression levels of MMP­2 and MMP­9 were revealed in CIA rats compared with in the control rats. Notably, ß­aminopropionitrile inhibited paw swelling and the decreased the arthritis index, the MVD in the synovial membranes and the expression levels of MMP­2 and MMP­9. Furthermore, the expression level of LOX in the synovial membranes was positively associated with the MVD and the expression levels of MMP­2 and MMP­9, suggesting that LOX promotes synovial hyperplasia and angiogenesis and that LOX may be a potential therapeutic target for rheumatoid arthritis.

Effects of polysaccharide fractions isolated from Caltha palustris L. on the activity of phagocytic cells & humoral immune response in mice with collagen-induced arthritis: A comparison with methotrexate.

BACKGROUND & OBJECTIVES: The extracts from Caltha palustris L. have been shown to be beneficial for treating arthritis and rheumatism. In this study, the immunomodulatory effects of polysaccharide fractions B and C of C. palustris extracts were studied, using the collagen-induced arthritis (CIA) mouse arthritis experimental model. The aim was to determine the activity of blood phagocytic cells and humoral immune response in CIA mice treated with polysaccharide fractions from C. palustris. METHODS: The effects of fractions B and C of C. palustris were explored by evaluating phagocytic activity of peripheral blood granulocytes and monocytes and humoral immune response in sheep red blood cell (SRBC)-immunized mice. The results were compared with methotrexate (MTX) treatment. Following the onset of CIA, DBA/1J mice were treated for 21 days with B or C fractions (10 mg/kg; i.p.) or MTX (every 48 h, 6.6 mg/kg; i.p.). RESULTS: The results showed that fraction B reduced the level of interleukin (IL)-1ß, boosted nitric oxide synthesis in murine peritoneal macrophages stimulated in vitro with lipopolysaccharide and enhanced the monocyte phagocytic activity. Exposure of SRBC-immunized mice to fraction B and MTX during the course of CIA resulted in decreased total anti-SRBC haemagglutinin titres. INTERPRETATION & CONCLUSIONS: Fraction B of C. palustris polysaccharides modulated macrophage function and exerted beneficial effects on the clinical course of CIA in mice. The results also suggested efficacy of fraction B was comparable to that of MTX treatment for certain parameters.