Cathepsin K Deficiency Reduces Elastase Perfusion-Induced Abdominal Aortic Aneurysms in Mice

Objective: Cathepsin K (CatK) is one of the most potent mammalian elastases. We have previously shown increased expression of CatK in human abdominal aortic aneurysm (AAA) lesions. Whether this protease participates directly in AAA formation, however, remains unknown. Methods and Results: Mouse experimental AAA was induced with aortic perfusion of a porcine pancreatic elastase. Using this experimental model, we demonstrated that absence of CatK prevented AAA formation in mice 14 days postperfusion. CatK deficiency significantly reduced lesion CD4 T-cell content, total lesion and medial cell proliferation and apoptosis, medial smooth muscle cell (SMC) loss, elastinolytic CatL and CatS expression, and elastin fragmentation, but it did not affect AAA lesion Mac-3 macrophage accumulation or CD31 microvessel numbers. In vitro studies revealed that CatK contributed importantly to CD4 T-cell proliferation, SMC apoptosis, and other cysteinyl cathepsin and matrix metalloproteinase expression and activities in SMCs and endothelial cells but played negligible roles in microvessel growth and monocyte migration. AAA lesions from CatK-deficient mice showed reduced elastinolytic cathepsin activities compared with those from wild-type control mice. Conclusion: This study demonstrates that CatK plays an essential role in AAA formation by promoting T-cell proliferation, vascular SMC apoptosis, and elastin degradation and by affecting vascular cell protease expression and activities.Other Research Uni

Mast cells modulate the pathogenesis of elastase-induced abdominal aortic aneurysms in mice

Abdominal aortic aneurysm (AAA), an inflammatory disease, involves leukocyte recruitment, immune responses, inflammatory cytokine production, vascular remodeling, neovascularization, and vascular cell apoptosis, all of which contribute to aortic dilatation. This study demonstrates that mast cells, key participants in human allergic immunity, participate in AAA pathogenesis in mice. Mast cells were found to accumulate in murine AAA lesions. Mast cell–deficient KitW-sh/KitW-sh mice failed to develop AAA elicited by elastase perfusion or periaortic chemical injury. KitW-sh/KitW-sh mice had reduced aortic expansion and internal elastic lamina degradation; decreased numbers of macrophages, CD3+ T lymphocytes, SMCs, apoptotic cells, and CD31+ microvessels; and decreased levels of aortic tissue IL-6 and IFN-γ. Activation of mast cells in WT mice via C48/80 injection resulted in enhanced AAA growth while mast cell stabilization with disodium cromoglycate diminished AAA formation. Mechanistic studies demonstrated that mast cells participated in angiogenesis, aortic SMC apoptosis, and matrix-degrading protease expression. Reconstitution of KitW-sh/KitW-sh mice with bone marrow–derived mast cells from WT or TNF-α–/– mice, but not from IL-6–/– or IFN-γ–/– mice, caused susceptibility to AAA formation to be regained. These results demonstrate that mast cells participate in AAA pathogenesis in mice by releasing proinflammatory cytokines IL-6 and IFN-γ, which may induce aortic SMC apoptosis, matrix-degrading protease expression, and vascular wall remodeling, important hallmarks of arterial aneurysms

Deficiency of Antigen Presenting Cell Invariant Chain Reduces Atherosclerosis in Mice

August 25, 2010Background: Adaptive immunity and innate immunity play important roles in atherogenesis. Invariant chain (CD74) mediates antigen-presenting cell antigen presentation and T-cell activation. This study tested the hypothesis that CD74-deficient mice have reduced numbers of active T cells and resist atherogenesis. Methods and Results: In low-density lipoprotein receptor–deficient (Ldlr[superscript −/−]) mice, CD74 deficiency (Ldlr[superscript −/−]Cd74[superscript −/−]) significantly reduced atherosclerosis and CD25+-activated T cells in the atheromata. Although Ldlr[superscript −/−]Cd74[superscript −/−] mice had decreased levels of plasma immunoglobulin (Ig) G1, IgG2b, and IgG2c against malondialdehyde-modified LDL (MDA-LDL), presumably as a result of impaired antigen-presenting cell function, Ldlr[superscript −/−]Cd74[superscript −/−] mice showed higher levels of anti–MDA-LDL IgM and IgG3. After immunization with MDA-LDL, Ldlr[superscript −/−]Cd74[superscript −/−] mice had lower levels of all anti–MDA-LDL Ig isotypes compared with Ldlr[superscript −/−] mice. As anticipated, only Ldlr[superscript −/−] splenocytes responded to in vitro stimulation with MDA-LDL, producing Th1/Th2 cytokines. Heat shock protein-65 immunization enhanced atherogenesis in Ldlr[superscript −/−] mice, but Ldlr[superscript −/−] Cd74[superscript −/−] mice remained protected. Compared with Ldlr[superscript −/−] mice, Ldlr[superscript −/−]Cd74[superscript −/−] mice had higher anti–MDA-LDL autoantibody titers, fewer lesion CD25+-activated T cells, impaired release of Th1/Th2 cytokines from antigen-presenting cells after heat shock protein-65 stimulation, and reduced levels of all plasma anti–heat shock protein-65 Ig isotypes. Cytofluorimetry of splenocytes and peritoneal cavity cells of MDA-LDL– or heat shock protein-65–immunized mice showed increased percentages of autoantibody-producing marginal zone B and B-1 cells in Ldlr[superscript −/−]Cd74[superscript −/−] mice compared with Ldlr[superscript −/−] mice. Conclusions: Invariant chain deficiency in Ldlr[superscript −/−] mice reduced atherosclerosis. This finding was associated with an impaired adaptive immune response to disease-specific antigens. Concomitantly, an unexpected increase in the number of innate-like peripheral B-1 cell populations occurred, resulting in increased IgM/IgG3 titers to the oxidation-specific epitopes

SIRT6 Promotes Osteogenic Differentiation of Adipose-Derived Mesenchymal Stem Cells Through Antagonizing DNMT1

BackgroundAdipose-derived stem cells (ADSCs) are increasingly used in regenerative medicine because of their potential to differentiate into multiple cell types, including osteogenic lineages. Sirtuin protein 6 (SIRT6) is a nicotinamide adenine dinucleotide (NAD)-dependent deacetylase that plays important roles in cell differentiation. NOTCH signaling has also been reported to involve in osteogenic differentiation. However, the function of SIRT6 in osteogenic differentiation of ADSCs and its relation to the NOTCH signaling pathways are yet to be explored.MethodsThe in vitro study with human ADSCs (hADSCs) and in vivo experiments with nude mice have been performed. Alkaline phosphatase (ALP) assays and ALP staining were used to detect osteogenic activity. Alizarin Red staining was performed to detect calcium deposition induced by osteogenic differentiation of ADSCs. Western blot, RT-qPCR, luciferase reporter assay, and co-immunoprecipitation assay were applied to explore the relationship between of SIRT6, DNA methyltransferases (DNMTs) and NOTCHs.ResultsSIRT6 promoted ALP activity, enhanced mineralization and upregulated expression of osteogenic-related genes of hADSCs in vitro and in vivo. Further mechanistic studies showed that SIRT6 deacetylated DNMT1, leading to its unstability at protein level. The decreased expression of DNMT1 prevented the abnormal DNA methylation of NOTCH1 and NOTCH2, resulting in the upregulation of their transcription. SIRT6 overexpression partially suppressed the abnormal DNA methylation of NOTCH1 and NOTCH2 by antagonizing DNMT1, leading to an increased capacity of ADSCs for their osteogenic differentiation.ConclusionThis study demonstrates that SIRT6 physical interacts with the DNMT1 protein, deacetylating and destabilizing DNMT1 protein, leading to the activation of NOTCH1 and NOTCH2, Which in turn promotes the osteogenic differentiation of ADSCs

Regulation of Endothelial Cell Adhesion Molecule Expression by Mast Cells, Macrophages, and Neutrophils

Leukocyte adhesion to the vascular endothelium and subsequent transendothelial migration play essential roles in the pathogenesis of cardiovascular diseases such as atherosclerosis. The leukocyte adhesion is mediated by localized activation of the endothelium through the action of inflammatory cytokines. The exact proinflammatory factors, however, that activate the endothelium and their cellular sources remain incompletely defined.Using bone marrow-derived mast cells from wild-type, Tnf(-/-), Ifng(-/-), Il6(-/-) mice, we demonstrated that all three of these pro-inflammatory cytokines from mast cells induced the expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), P-selectin, and E-selectin in murine heart endothelial cells (MHEC) at both mRNA and protein levels. Compared with TNF-α and IL6, IFN-γ appeared weaker in the induction of the mRNA levels, but at protein levels, both IL6 and IFN-γ were weaker inducers than TNF-α. Under physiological shear flow conditions, mast cell-derived TNF-α and IL6 were more potent than IFN-γ in activating MHEC and in promoting neutrophil adhesion. Similar observations were made when neutrophils or macrophages were used. Neutrophils and macrophages produced the same sets of pro-inflammatory cytokines as did mast cells to induce MHEC adhesion molecule expression, with the exception that macrophage-derived IFN-γ showed negligible effect in inducing VCAM-1 expression in MHEC.Mast cells, neutrophils, and macrophages release pro-inflammatory cytokines such as TNF-α, IFN-γ, and IL6 that induce expression of adhesion molecules in endothelium and recruit of leukocytes, which is essential to the pathogenesis of vascular inflammatory diseases

3B11-N, a monoclonal antibody against MERS-CoV, reduces lung pathology in rhesus monkeys following intratracheal inoculation of MERS-CoV Jordan-n3/2012

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) was identified in 2012 as the causative agent of a severe, lethal respiratory disease occurring across several countries in the Middle East. To date there have been over 1,600 laboratory confirmed cases of MERS-CoV in 26 countries with a case fatality rate of 36%. Given the endemic region, it is possible that MERS-CoV could spread during the annual Hajj pilgrimage, necessitating countermeasure development. In this report, we describe the clinical and radiographic changes of rhesus monkeys following infection with 5×106 PFU MERS-CoV Jordan-n3/2012. Two groups of NHPs were treated with either a human anti-MERS monoclonal antibody 3B11-N or E410-N, an anti-HIV antibody. MERS-CoV Jordan-n3/2012 infection resulted in quantifiable changes by computed tomography, but limited other clinical signs of disease. 3B11-N treated subjects developed significantly reduced lung pathology when compared to infected, untreated subjects, indicating that this antibody may be a suitable MERS-CoV treatment

Human anti-CAIX antibodies mediate immune cell inhibition of renal cell carcinoma in vitro and in a humanized mouse model in vivo

Background: Carbonic anhydrase (CA) IX is a surface-expressed protein that is upregulated by the hypoxia inducible factor (HIF) and represents a prototypic tumor-associated antigen that is overexpressed on renal cell carcinoma (RCC). Therapeutic approaches targeting CAIX have focused on the development of CAIX inhibitors and specific immunotherapies including monoclonal antibodies (mAbs). However, current in vivo mouse models used to characterize the anti-tumor properties of fully human anti-CAIX mAbs have significant limitations since the role of human effector cells in tumor cell killing in vivo is not directly evaluated. Methods: The role of human anti-CAIX mAbs on CAIX+ RCC tumor cell killing by immunocytes or complement was tested in vitro by antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and antibody-dependent cellular phagocytosis (ADCP) as well as on CAIX+ RCC cellular motility, wound healing, migration and proliferation. The in vivo therapeutic activity mediated by anti-CAIX mAbs was determined by using a novel orthotopic RCC xenograft humanized animal model and analyzed by histology and FACS staining. Results: Our studies demonstrate the capacity of human anti-CAIX mAbs that inhibit CA enzymatic activity to result in immune-mediated killing of RCC, including nature killer (NK) cell-mediated ADCC, CDC, and macrophage-mediated ADCP. The killing activity correlated positively with the level of CAIX expression on RCC tumor cell lines. In addition, Fc engineering of anti-CAIX mAbs was shown to enhance the ADCC activity against RCC. We also demonstrate that these anti-CAIX mAbs inhibit migration of RCC cells in vitro. Finally, through the implementation of a novel orthotopic RCC model utilizing allogeneic human peripheral blood mononuclear cells in NOD/SCID/IL2Rγ−/− mice, we show that anti-CAIX mAbs are capable of mediating human immune response in vivo including tumor infiltration of NK cells and activation of T cells, resulting in inhibition of CAIX+ tumor growth. Conclusions: Our findings demonstrate that these novel human anti-CAIX mAbs have therapeutic potential in the unmet medical need of targeted killing of HIF-driven CAIX+RCC. The orthotopic tumor xenografted humanized mouse provides an improved model to evaluate the in vivo anti-tumor capabilities of fully human mAbs for RCC therapy. Electronic supplementary material The online version of this article (doi:10.1186/s12943-015-0384-3) contains supplementary material, which is available to authorized users

Experimental Study of lncRNA RP11-815M8.1 Promoting Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells

Objective. This study is aimed at investigating the role of long noncoding RNA (lncRNA) RP11-815M8.1 in the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs). Methods. RT-PCR was used to detect the expression of lncRNA RP11-815M8.1 before and after osteogenic differentiation of hBMSCs. The lncRNA RP11-815M8.1 in hBMSCs was overexpressed or silenced via lentiviral transfection. The transfection efficiency was detected by RT-PCR, and the proliferation of hBMSCs was determined by CCK-8. After 14 days of osteogenic differentiation of transfected hBMSCs, the expression of osteogenic transcription factors (ALP, OCN, OPN, Runx2, and Osterix) was detected by alizarin red staining and RT-PCR. The mRNAs directly regulated by lncRNA RP11-815M8.1 and targeted miRNAs were analyzed according to the positional relationship between lncRNA and mRNA in the genome and miRanda software. Results. The expression of lncRNA RP11-815M8.1 enhanced with increasing osteogenic differentiation time of hBMSCs. Two days after the transfection of hBMSCs, lncRNA RP11-815M8.1 expression was significantly increased in the overexpression group and significantly decreased in the knockdown group, compared to control cells. The CCK-8 assay showed that overexpression and knockdown of lncRNA RP11-815M8.1 did not affect the proliferation of hBMSCs. After 14 days of differentiation of hBMSCs, stronger alizarin red staining was observed in the overexpression groups, and the expression of osteogenic transcription factors was increased in the overexpression group compared to the control. In the knockdown group, alizarin red staining and the expression of osteogenic transcription factors were decreased. Bioinformatics analysis showed that lncRNA RP11-815M8.1 was directly associated with one mRNA, 27 interacting miRNAs, and 20 miRNA-targeted mRNAs. Conclusion. The osteogenic differentiation of hBMSCs can be promoted by lncRNA RP11-815M8.1 in vitro