Induction of MiR133a expression by IL-19 targets LDLRAP1 and reduces oxLDL uptake in VSMC.

The transformation of vascular smooth muscle cells [VSMC] into foam cells leading to increased plaque size and decreased stability is a key, yet understudied step in atherogenesis. We reported that Interleukin-19 (IL-19), a novel, anti-inflammatory cytokine, attenuates atherosclerosis by anti-inflammatory effects on VSMC. In this work we report that IL-19 induces expression of miR133a, a muscle-specific miRNA, in VSMC. Although previously unreported, we report that miR133a can target and reduce mRNA abundance, mRNA stability, and protein expression of Low Density Lipoprotein Receptor Adaptor Protein 1, (LDLRAP1), an adaptor protein which functions to internalize the LDL receptor. Mutations in this gene lead to LDL receptor malfunction and cause the Autosomal Recessive Hypercholesterolemia (ARH) disorder in humans. Herein we show that IL-19 reduces lipid accumulation in VSMC, and LDLRAP1 expression and oxLDL uptake in a miR133a-dependent mechanism. We show that LDLRAP1 is expressed in plaque and neointimal VSMC of mouse and human injured arteries. Transfection of miR133a and LDLRAP1 siRNA into VSMC reduces their proliferation and uptake of oxLDL. miR133a is significantly increased in plasma from hyperlipidemic compared with normolipidemic patients. Expression of miR133a in IL-19 stimulated VSMC represents a previously unrecognized link between vascular lipid metabolism and inflammation, and may represent a therapeutic opportunity to combat vascular inflammatory diseases.

Osteoactivin Promotes Migration of Oral Squamous Cell Carcinomas.

Nearly 50% of patients with oral squamous cell carcinoma (OSCC) die of metastases or locoregional recurrence. Metastasis is mediated by cancer cell adhesion, migration, and invasion. Osteoactivin (OA) overexpression plays a role in metastases in several malignancies. The aims were to determine how integrin interactions modulate OA-induced OSCC cell migration; and to investigate OA effects on cell survival and proliferation. We confirmed OA mRNA and protein overexpression in OSCC cell lines. We assessed OA's interactions with integrins using adhesion inhibition assays, fluorescent immunocytochemistry and co-immunoprecipitation. We investigated OA-mediated activation of mitogen-activated protein kinases (MAPKs) and cell survival. Integrin inhibition effects on OA-mediated cell migration were determined. We assessed effects of OA knock-down on cell migration and proliferation. OA is overexpressed in OSCC cell lines, and serves as a migration-promoting adhesion molecule. OA co-localized with integrin subunits, and co-immunoprecipitated with the subunits. Integrin blocking antibodies, especially those directed against the ß1 subunit, inhibited cell adhesion (P = 0.03 for SCC15 cells). Adhesion to OA activated MAPKs in UMSCC14a cells and OA treatment promoted survival of SCC15 cells. Integrin-neutralizing antibodies enhanced cell migration with OA in the extracellular matrix. OA knock-down resulted in decreased proliferation of SCC15 and SCC25 cells, but did not inhibit cell migration. OA in the extracellular matrix promotes OSCC cell adhesion and migration, and may be a novel target in the prevention of HNSCC spread. J. Cell. Physiol. 231: 1761-1770, 2016. © 2015 Wiley Periodicals, Inc.

HIV and Bone Disease: A Perspective of the Role of microRNAs in Bone Biology upon HIV Infection.

Increased life expectancy and the need for long-term antiretroviral therapy have brought new challenges to the clinical management of HIV-infected individuals. The prevalence of osteoporosis and fractures is increased in HIV-infected patients; thus optimal strategies for risk management and treatment in this group of patients need to be defined. Prevention of bone loss is an important component of HIV care as the HIV population grows older. Understanding the mechanisms by which HIV infection affects bone biology leading to osteoporosis is crucial to delineate potential adjuvant treatments. This review focuses on HIV-induced osteoporosis within the context of microRNAs (miRNAs) by reviewing first basic concepts of bone biology as well as current knowledge of the role of miRNAs in bone development. Evidence that HIV-associated osteoporosis is in part independent of therapies employed to treat HIV (HAART) is supported by cross-sectional and longitudinal studies and is the focus of this review.

Erosive arthritis and hepatic granuloma formation induced by peptidoglycan polysaccharide in rats is aggravated by prasugrel treatment.

Administration of the thienopyridine P2Y12 receptor antagonist, clopidogrel, increased the erosive arthritis induced by peptidoglycan polysaccharide (PG-PS) in rats or by injection of the arthritogenic K/BxN serum in mice. To determine if the detrimental effects are caused exclusively by clopidogrel, we evaluated prasugrel, a third-generation thienopyridine pro-drug, that contrary to clopidogrel is mostly metabolized into its active metabolite in the intestine. Prasugrel effects were examined on the PG-PS-induced arthritis rat model. Erosive arthritis was induced in Lewis rats followed by treatment with prasugrel for 21 days. Prasugrel treated arthritic animals showed a significant increase in the inflammatory response, compared with untreated arthritic rats, in terms of augmented macroscopic joint diameter associated with significant signs of inflammation, histomorphometric measurements of the hind joints and elevated platelet number. Moreover, fibrosis at the pannus, assessed by immunofluorescence of connective tissue growth factor, was increased in arthritic rats treated with prasugrel. In addition to the arthritic manifestations, hepatomegaly, liver granulomas and giant cell formation were observed after PG-PS induction and even more after prasugrel exposure. Cytokine plasma levels of IL-1 beta, IL-6, MIP1 alpha, MCP1, IL-17 and RANTES were increased in arthritis-induced animals. IL-10 plasma levels were significantly decreased in animals treated with prasugrel. Overall, prasugrel enhances inflammation in joints and liver of this animal model. Since prasugrel metabolites inhibit neutrophil function ex-vivo and the effects of both clopidogrel and prasugrel metabolites on platelets are identical, we conclude that the thienopyridines metabolites might exert non-platelet effects on other immune cells to aggravate inflammation.

Assembly of the prothrombinase complex on the surface of human foreskin fibroblasts: Implications for connective tissue growth factor.

Activated factor X (FXa) and thrombin can up-regulate gene expression of connective tissue growth factor (CTGF/CCN2) on fibroblasts. Since tissue factor (TF) is expressed on these cells, we hypothesized that they may assemble the prothrombinase complex leading to CTGF/CCN2 upregulation. In addition, the effect of thrombospondin-1 (TSP1) on this reaction was evaluated. Human foreskin fibroblasts were incubated with purified factor VII (FVII), factor X (FX), factor V (FV), prothrombin and calcium in the presence and absence of TSP1. Generation of FXa and of thrombin were assessed using chromogenic substrates. SMAD pathway phosphorylation was detected via Western-blot analysis. Pre-incubation of fibroblasts with FVII led to its auto-activation by cell-surface expressed TF, which in turn in the presence of FX, FVa, prothrombin and calcium led to FXa (9.7±0.8nM) and thrombin (7.9±0.04 U/mL×10-3) generation. Addition of TSP1 significantly enhanced thrombin (23.3±0.7 U/mL×10-3) but not FXa (8.5±0.6nM) generation. FXa and thrombin generation leads to upregulation of CTGF/CCN2. TSP1 alone upregulated CTGF/CCN2, an effect mediated via activation of transforming growth factor beta (TGFß) as shown by phosphorylation of the SMAD pathway, an event blunted by using a TGFß receptor I inhibitor (TGFßRI). FXa- and thrombin-induced upregulation of CTGF/CCN2 was not blocked by TGFßRI. In summary, assembly of the prothrombinase complex occurs on fibroblast's surface leading to serine proteases generation, an event enhanced by TSP1 and associated with CTGF/CCN2 upregulation. These mechanisms may play an important role in human diseases associated with fibrosis.

Improving patient provider communication for Latinos at Temple University Hospital and Temple University School of Medicine.

This Report from the Field documents a series of interventions developed by Temple University Health System and School of Medicine through participation in the RWJF initiative entitled Hablamos Juntos. The report delineates outcomes to date demonstrating that these interventions have met the challenge of improving patient provider communication for Latinos.

Clopidogrel, a P2Y12 receptor antagonist, potentiates the inflammatory response in a rat model of peptidoglycan polysaccharide-induced arthritis.

The P2Y12 receptor plays a crucial role in the regulation of platelet activation by several agonists, which is irreversibly antagonized by the active metabolite of clopidogrel, a widely used anti-thrombotic drug. In this study, we investigated whether reduction of platelet reactivity leads to reduced inflammatory responses using a rat model of erosive arthritis. We evaluated the effect of clopidogrel on inflammation in Lewis rats in a peptidoglycan polysaccharide (PG-PS)-induced arthritis model with four groups of rats: 1) untreated, 2) clopidogrel-treated, 3) PG-PS-induced, and 4) PG-PS-induced and clopidogrel-treated. There were significant differences between the PG-PS+clopidogrel group when compared to the PG-PS group including: increased joint diameter and clinical manifestations of inflammation, elevated plasma levels of pro-inflammatory cytokines (IL-1 beta, interferon (IFN) gamma, and IL-6), an elevated neutrophil blood count and an increased circulating platelet count. Plasma levels of IL-10 were significantly lower in the PG-PS+clopidogrel group compared to the PG-PS group. Plasma levels of platelet factor 4 (PF4) were elevated in both the PG-PS and the PG-PS+clopidogrel groups, however PF4 levels showed no difference upon clopidogrel treatment, suggesting that the pro- inflammatory effect of clopidogrel may be due to its action on cells other than platelets. Histology indicated an increase in leukocyte infiltration at the inflammatory area of the joint, increased pannus formation, blood vessel proliferation, subsynovial fibrosis and cartilage erosion upon treatment with clopidogrel in PG-PS-induced arthritis animals. In summary, animals treated with clopidogrel showed a pro-inflammatory effect in the PG-PS-induced arthritis animal model, which might not be mediated by platelets. Elucidation of the mechanism of clopidogrel-induced cell responses is important to understand the role of the P2Y12 receptor in inflammation.

Comparison of bone morphogenetic protein-2 and osteoactivin for mesenchymal cell differentiation: effects of bolus and continuous administration.

Current osteoinductive protein therapy utilizes bolus administration of large doses of bone morphogenetic proteins (BMPs), which is costly, and may not replicate normal bone healing. The limited in vivo biologic activity of BMPs requires the investigation of growth factors that may enhance this activity. In this study, we utilized the C3H10T1/2 murine mesenchymal stem cell line to test the hypotheses that osteoactivin (OA) has comparable osteoinductive effects to bone morphogenetic protein-2 (BMP-2), and that sustained administration of either growth factor would result in increased osteoblastic differentiation as compared to bolus administration. Sustained release biodegradable hydrogels were designed, and C3H10T1/2 cells were grown on hydrogels loaded with BMP-2 or OA. Controls were grown on unloaded hydrogels, and positive controls were exposed to bolus growth factor administration. Cells were harvested at several time points to assess osteoblastic differentiation. Alkaline phosphatase (ALP) staining and activity, and gene expression of ALP and osteocalcin were assessed. Treatment with OA or BMP-2 resulted in comparable effects on osteoblastic marker expression. However, cells grown on hydrogels demonstrated osteoblastic differentiation that was not as robust as cells treated with bolus administration. This study shows that OA has comparable effects to BMP-2 on osteoblastic differentiation using both bolus administration and continuous release, and that bolus administration of OA has a more profound effect than administration using hydrogels for sustained release. This study will lead to a better understanding of appropriate delivery methods of osteogenic growth factors like OA for repair of fractures and segmental bone defects.

The axis of thrombospondin-1, transforming growth factor beta and connective tissue growth factor: an emerging therapeutic target in rheumatoid arthritis.

Biologic therapy for rheumatoid arthritis (RA) targets specific molecules that mediate and sustain the clinical manifestations of this complex illness. Compared with the general population, patients with RA die prematurely, in part due to associated cardiovascular disease. Even though the mechanisms by which premature atherosclerosis develops in RA is unknown, chronic inflammation may play a major role. This review connects current knowledge of the pathophysiology of RA with data available in the literature related to thrombospondin-1 (TSP1), transforming growth factor beta (TGFbeta and connective tissue growth factor (CTGF) and their relationship with cardiovascular disease in RA. The TSP1/TGFbeta/CTGF axis may contribute in the pro-inflammatory and pro-atherogenic state in patients affected with RA. In fact, increased TSP1 plasma levels are found in patients of RA. TGFbeta is activated by TSP1 through a non-enzymatic mechanism and is constitutively overexpressed by synovial fibroblasts from RA patients. Activation of TGFbeta pathway in synovial fibroblasts and other cells including neutrophils leads to downstream upregulation of CTGF. Overexpression of CTGF is associated with angiogenesis, fibrosis, atherosclerotic blood vessels and erosive arthritis lesions. Recent RA therapies emphasize the need for aggressive control of the activity of the disease to prevent premature atherosclerosis in RA patients. The complexity and heterogeneity of RA as judged by response to a wide spectrum of treatments mandates the elucidation of unknown pro-inflammatory pathways playing a major role in this disease. The TSP1/TGFbeta/CTFG axis represents one of these pro-inflammatory pathways that may result in the development of promising therapeutic strategies to prevent chronic inflammation and thus premature atherosclerosis in RA.

Thrombospondin-1 and transforming growth factor beta are pro-inflammatory molecules in rheumatoid arthritis.

Thrombospondin-1 (TSP1/THBS1) plays a major role in the pathophysiology of rheumatoid arthritis (RA); however, its interface with the cytokine network involved in RA has not been delineated. Correlations were performed between plasma levels of TSP1 and selected cytokines from blood samples collected from 20 patients affected by RA and 13 healthy donors (control). Plasma levels of TSP1 and tissue growth factor beta (TGFbeta) were determined by standard enzyme-linked immunosorbent assay, and cytokines were measured by protein profiling rolling-circle amplification (RCA). TSP1 circulating levels in plasma were found significantly increased in the RA patients when compared with control individuals (P = 0.039). The plasma levels of TGFbeta were also increased in the RA patients, which indicates a statistical trend. Cytokine levels of interleukin (IL)-4, IL-5, IL-12, chemokine CXC 10 (CXCL10/IP10), and chemokine CC 4 (CCL4)/MIP1beta were significantly increased in the RA patients when compared with the control group. In summary, this study demonstrates increased plasma levels of TSP1, which correlated with increased levels of proinflammatory cytokines in plasma of RA patients. More detailed research is required to explore the cytokine imprint yielded by this study and its interface with TSP1 and TGFbeta.

Amelioration of inflammation, angiogenesis and CTGF expression in an arthritis model by a TSP1-derived peptide treatment.

OBJECTIVE: To evaluate the effect of a thrombospondin 1 (TSP1)-derived peptide on inflammation and angiogenesis in an animal model of erosive arthritis and to assess the relationship between TSP1 and connective tissue growth factor (CTGF) in the pathophysiology of rheumatoid arthritis. METHODS: Erosive arthritis in Lewis rats was induced by peptidoglycan-polysaccharide (PG-PS). Animals were divided into four groups: (1) negative control and groups receiving, (2) no treatment, (3) treatment with a TSP1-derived peptide, and (4) treatment with a scrambled peptide. Samples obtained from ankle joint, spleen and liver were studied using histology, histomorphometry, immunohistochemistry and RT-PCR. RESULTS: Histological data indicated that the TSP1-derived peptide treatment decreased neovascularization, leukocyte infiltration and thickening of the synovial lining of the joint, and reduced granuloma formation in the spleen and liver when compared to control groups. Higher concentrations of CTGF and TSP1 proteins were observed in the affected areas of animals which did not receive TSP1-derived peptide treatment. Also, immunofluorescence and RT-PCR analyses showed an increase in CTGF protein expression and regulation, respectively, in the tissues of untreated animals when compared to the TSP1-derived peptide treated animals. By immunofluorescence, TSP1 expression was decreased in the TSP1-derived peptide treated animals. Moreover, macrophage/monocyte-specific staining revealed a decrease in cell infiltration in the articular tissue of the TSP1-derived peptide treated animals. CONCLUSION: Both inflammation and angiogenesis were decreased after TSP1-derived peptide treatment indicating a potential pathway by which TSP1 interaction with neutrophils induces CTGF in RA affected tissues.

A peptide from thrombospondin 1 modulates experimental erosive arthritis by regulating connective tissue growth factor.

OBJECTIVE: Rheumatoid arthritis (RA) is a chronic inflammatory disease associated with leukocyte adhesion to and extravasation through vascular endothelium into synovial tissue. Recent evidence indicates that the thrombospondin 1 gene is up-regulated in patients with RA. We have identified a region within the TSP-1 type 3 repeats that inhibits human neutrophil elastase (HNE) and binds to human neutrophils. The present study was undertaken to investigate the therapeutic benefit of this TSP-1-derived peptide sequence and its effect on connective tissue growth factor (CTGF), a protein involved in fibrotic disorders and in neovascularization, which is a hallmark of RA. METHODS: CTGF gene and protein expression, as well as protein levels of CTGF in the synovium, after treatment with the TSP-1-derived peptide were studied in the peptidoglycan-polysaccharide animal model of erosive arthritis. RESULTS: Peptide treatment prevented joint infiltration and inflammation and was associated with reduced circulating antigen levels of HNE and TSP-1. Additionally, CTGF was up-regulated in this experimental model of RA. Treatment with the TSP-1-derived peptide was associated with down-regulation of the message and protein levels of CTGF. Immunofluorescence studies showed that the mean area fraction of CTGF immunoreactivity in the peptide-treated group of animals was significantly less than that in the untreated group. CONCLUSION: These results document a role for TSP-1 in regulating CTGF gene and protein expression in synovial tissue, suggesting a link with the disease course in this model of RA. This TSP-1-derived synthetic peptide may represent an important template for drug development in RA and other inflammatory conditions associated with neutrophil activation.

Thromsbospondin-1 binds to the heavy chain of elastase activated coagulation factor V (FVaHNE) and enhances thrombin generation on the surface of a promyelocytic cell line.

INTRODUCTION: Thrombospondin 1 (TSP1) has the ability to bind to HL-60 cells and to reversibly inhibit human neutrophil elastase (HNE). Human factor V (FV) can be cleaved by HNE thereby providing FV with cofactor activity (FVa(HNE)). Experiments were performed to evaluate the ability of HNE expressed on the surface of HL-60 cells to generate FVa(HNE) to support thrombin generation, and to determine the effect of TSP1 on this reaction. RESULTS: Western blot analysis showed TSP1 forming a complex with FVa(HNE) within a region corresponding to the heavy chain of FV. Enzymatic reactions were performed to determine the role of TSP1-HNE-FVa(HNE) on the surface of HL-60 cells, namely the assembly of the prothrombinase complex. Thrombin generation was measured by the chromogenic substrate S2238. Exposure of factor V to HL-60 cells prior to the addition of prothrombin and activated factor X provided FV with cofactor activity. HL-60 cells were found capable of synthesizing factor V with cofactor activity, but HL-60 cells failed to synthesize and/or to provide factor X with enzymatic activity. The ability of HL-60 cells to synthesize FV and TSP1 was demonstrated. The addition of exogenous TSP1 enhanced both the rate and amount of thrombin generated on the HL-60 cell surface. CONCLUSION: Despite the ability of TSP1 to reversibly inhibit HNE in a purified system, TSP1 expression favors the reactions leading to thrombin generation on the HL-60 cell surface. These observations are relevant to clinical conditions where there is a prothrombotic state such as malignant tumors.

Modulation of inflammation by kininogen deficiency in a rat model of inflammatory arthritis.

OBJECTIVE: To compare inflammatory peripheral arthritis in wild-type and high molecular weight kininogen (HK)-deficient rats, both on the genetically susceptible Lewis background. METHODS: By backcrossing Brown-Norway HK-deficient rats with Lewis rats for 6 generations, 2 new strains were produced, wild-type F6 and HK-deficient F6, each with a 98.5% Lewis genome. Inflammatory arthritis was induced by intraperitoneal injection of peptidoglycan-polysaccharide (PG-PS), and the clinical, histopathologic, and biochemical responses were compared in both strains. RESULTS: Eighteen days after PG-PS injection, rats with normal concentrations of HK showed weight loss and marked increase in hind ankle diameter with severe synovial inflammation and cartilage abnormalities. In contrast, HK-deficient rats showed no weight loss (P < 0.05), no increase in hind ankle diameter (P < 0.05), and an absence of inflammatory changes (P < 0.05), as measured by the histologic and morphometric Mankin grading system for synovial and cartilage injury. CONCLUSION: Plasma HK is a key mediator of acute and chronic inflammatory arthritis in genetically susceptible Lewis rats.

A monoclonal antibody to high-molecular weight kininogen is therapeutic in a rodent model of reactive arthritis.

We reported that high-molecular weight kininogen is proangiogenic by releasing bradykinin and that a monoclonal antibody to high-molecular weight kininogen, C11C1, blocked its binding to endothelial cells. We now test if this antibody can prevent arthritis and systemic inflammation in a Lewis rat model. We studied 32 animals for 16 days. Group I (negative control) received saline intraperitoneally. Group II (disease-treated) received peptidoglycan-polysaccharide simultaneously with C11C1. Group III (disease-untreated) received peptidoglycan-polysaccharide simultaneously with isotype-matched mouse IgG. Group IV (disease-free-treated) and group V (disease-free isotype-treated) received saline and C11C1 or mouse IgG. Analysis of joint diameter changes showed a decrease in the C11C1 disease-treated group compared to the disease-untreated group. The hind paw inflammatory score showed a decrease in the intensity and extent of inflammation between the disease-untreated and the C11C1 disease-treated group. Prekallikrein, high-molecular weight kininogen, factor XI, and factor XII were decreased in the disease-untreated group compared to the C11C1 disease-treated group. T-kininogen was increased in the disease-untreated group when compared with the C11C1 disease-treated group. Disease-free groups IV and V did not show any sign of inflammation at any time. This study shows that monoclonal antibody C11C1 attenuates plasma kallikrein-kinin system activation, local and systemic inflammation, indicating therapeutic potential in reactive arthritis.