Cong-Lin Liu

Interleukin 18 function in atherosclerosis is mediated by the interleukin 18 receptor and the Na-Cl co-transporter.

Interleukin-18 (IL18) participates in atherogenesis through several putative mechanisms. Interruption of IL18 action reduces atherosclerosis in mice. Here, we show that absence of the IL18 receptor (IL18r) does not affect atherosclerosis in apolipoprotein E–deficient (Apoe−/−) mice, nor does it affect IL18 cell surface binding to or signaling in endothelial cells. As identified initially by co-immunoprecipitation with IL18, we found that IL18 interacts with the Na-Cl co-transporter (NCC; also known as SLC12A3), a 12-transmembrane-domain ion transporter protein preferentially expressed in the kidney. NCC is expressed in atherosclerotic lesions, where it colocalizes with IL18r. In Apoe−/− mice, combined deficiency of IL18r and NCC, but not single deficiency of either protein, protects mice from atherosclerosis. Peritoneal macrophages from Apoe−/− mice or from Apoe−/− mice lacking IL18r or NCC show IL18 binding and induction of cell signaling and cytokine and chemokine expression, but macrophages from Apoe−/− mice with combined deficiency of IL18r and NCC have a blunted response. An interaction between NCC and IL18r on macrophages was detected by co-immunoprecipitation. IL18 binds to the cell surface of NCC-transfected COS-7 cells, which do not express IL18r, and induces cell signaling and cytokine expression. This study identifies NCC as an IL18-binding protein that collaborates with IL18r in cell signaling, inflammatory molecule expression, and experimental atherogenesis.

Cathepsin G activity lowers plasma LDL and reduces atherosclerosis

Cathepsin G (CatG), a serine protease present in mast cells and neutrophils, can produce angiotensin-II (Ang-II) and degrade elastin. Here we demonstrate increased CatG expression in smooth muscle cells (SMCs), endothelial cells (ECs), macrophages, and T cells from human atherosclerotic lesions. In low-density lipoprotein (LDL) receptor-deficient (Ldlr(-/-)) mice, the absence of CatG reduces arterial wall elastin degradation and attenuates early atherosclerosis when mice consume a Western diet for 3months. When mice consume this diet for 6months, however, CatG deficiency exacerbates atherosclerosis in aortic arch without affecting lesion inflammatory cell content or extracellular matrix accumulation, but raises plasma total cholesterol and LDL levels without affecting high-density lipoprotein (HDL) or triglyceride levels. Patients with atherosclerosis also have significantly reduced plasma CatG levels that correlate inversely with total cholesterol (r=-0.535, P<0.0001) and LDL cholesterol (r=-0.559, P<0.0001), but not with HDL cholesterol (P=0.901) or triglycerides (P=0.186). Such inverse correlations with total cholesterol (r=-0.504, P<0.0001) and LDL cholesterol (r=-0.502, P<0.0001) remain significant after adjusting for lipid lowering treatments among this patient population. Human CatG degrades purified human LDL, but not HDL. This study suggests that CatG promotes early atherogenesis through its elastinolytic activity, but suppresses late progression of atherosclerosis by degrading LDL without affecting HDL or triglycerides.

Plasma cytokine levels and risks of abdominal aortic aneurysms: A population-based prospective cohort study

Abstract Background. Abdominal aortic aneurysm (AAA) is characterized by inflammatory cell accumulation in AAA lesions that produce inflammatory cytokines and advance its pathogenesis. Peripheral cytokines may predict the degree or risk of AAA. Methods and results. ELISA determined plasma interleukin-6 (IL6), IL10, IL17A, IFN-γ, and C-reactive protein (CRP) from 476 AAA patients and 200 controls. AAA patients had lower IL6, IFN-γ, IL10, IL17A, and higher CRP than controls. IL10 correlated positively with IFN-γ, IL17A, or IL6, but not CRP in control or AAA populations. IL10 associated negatively with systolic blood pressure, whereas CRP associated positively with diastolic blood pressure and body mass index. CRP was an independent AAA risk factor and correlated positively with aortic diameters before and after adjustments for other risk factors. IFN-γ, IL17A, and CRP correlated positively with cross-sectional AAA area after adjustment. IL10 correlated positively with AAA growth rate before and after adjustment. The risk of death doubled in AAA patients with CRP levels above the median. Conclusions. Reduced IFN-γ, IL10, and IL17A in AAA patients, positive correlations of IFN-γ and IL17A with cross-sectional AAA area, IL10 with AAA growth rate, and IL10 with IFN-γ and IL17A suggest combined Th1, Th2, and Th17 immune responses in human AAAs.

Allergic Lung Inflammation Aggravates Angiotensin II–Induced Abdominal Aortic Aneurysms in Mice

Objective— Asthma and abdominal aortic aneurysms (AAA) both involve inflammation. Patients with asthma have an increased risk of developing AAA or experiencing aortic rupture. This study tests the development of one disease on the progression of the other. Approach and Results— Ovalbumin sensitization and challenge in mice led to the development of allergic lung inflammation (ALI). Subcutaneous infusion of angiotensin II into mice produced AAA. Simultaneous production of ALI in AAA mice doubled abdominal aortic diameter and increased macrophage and mast cell content, arterial media smooth muscle cell loss, cell proliferation, and angiogenesis in AAA lesions. ALI also increased plasma IgE, reduced plasma interleukin-5, and increased bronchioalveolar total inflammatory cell and eosinophil accumulation. Intraperitoneal administration of an anti-IgE antibody suppressed AAA lesion formation and reduced lesion inflammation, plasma IgE, and bronchioalveolar inflammation. Pre-establishment of ALI also increased AAA lesion size, lesion accumulation of macrophages and mast cells, media smooth muscle cell loss, and plasma IgE, reduced plasma interleukin-5, interleukin-13, and transforming growth factor-&bgr;, and increased bronchioalveolar inflammation. Consequent production of ALI also doubled lesion size of pre-established AAA and increased lesion mast cell and T-cell accumulation, media smooth muscle cell loss, lesion cell proliferation and apoptosis, plasma IgE, and bronchioalveolar inflammation. In periaortic CaCl2 injury–induced AAA in mice, production of ALI also increased AAA formation, lesion inflammation, plasma IgE, and bronchioalveolar inflammatory cell accumulation. Conclusions— This study suggests a pathological link between airway allergic disease and AAA. Production of one disease aggravates the progression of the other.

Cathepsin S inhibition changes regulatory T-cell activity in regulating bladder cancer and immune cell proliferation and apoptosis

HighlightsCathepsin S inhibitor‐ or saline‐treated Tregs did not affect mouse bladder tumor growth.Tumor mice received inhibitor‐treated Tregs had reduced splenic and tumor Treg number and total cell proliferation.Inhibitor‐treated Tregs affected the proliferation and apoptosis of tumor cells and splenic lymphocytes differently from saline‐treated Treg.Tumor cell conditioned media affected inhibitor‐treated Treg activity in regulating lymphocyte proliferation and apoptosis. ABSTRACT Regulatory T cells (Tregs) are immune suppressive cells, but their roles in tumor growth have been elusive, depending on tumor type or site. Our prior study demonstrated a role of cathepsin S (CatS) in reducing Treg immunosuppressive activity. Therefore, CatS inhibition in Tregs may exacerbate tumor growth. Using mouse bladder carcinoma MB49 cell subcutaneous implant tumor model, we detected no difference in tumor growth, whether mice were given saline‐ or CatS inhibitor‐treated Tregs. However, mice that received inhibitor‐treated Tregs had fewer splenic and tumor Tregs, and lower levels of tumor and splenic cell proliferation than mice that received saline‐treated Tregs. In vitro, inhibitor‐treated Tregs showed lower proliferation and higher apoptosis than saline‐treated Tregs when cells were exposed to MB49. In contrast, both types of Tregs showed no difference in proliferation when they were co‐cultured with normal splenocytes. Inhibitor‐treated Tregs had less apoptosis in splenocytes, but more apoptosis in splenocytes with MB49 conditioned media than saline‐treated Tregs. In turn, we detected less proliferation and more apoptosis of MB94 cells after co‐culture with inhibitor‐treated Tregs, compared with saline‐treated Tregs. B220+ B‐cell, CD4+ T‐cell, and CD8+ T‐cell proliferation and apoptosis were also lower in splenocytes co‐cultured with inhibitor‐treated Tregs than with saline‐treated Tregs. Under the same conditions, the addition of cancer cell‐conditioned media greatly increased CD8+ T‐cell proliferation and reduced CD8+ T‐cell apoptosis. These observations suggest that CatS inhibition of Tregs may reduce overall T‐cell immunity under normal conditions, but enhance CD8+ T‐cell immunity in the presence of cancer cells.

Cysteine protease cathepsins in cardiovascular disease: from basic research to clinical trials

Cysteine protease cathepsins have traditionally been considered as lysosome-restricted proteases that mediate proteolysis of unwanted proteins. However, studies from the past decade demonstrate that these proteases are localized not only in acidic compartments (endosomes and lysosomes), where they participate in intracellular protein degradation, but also in the extracellular milieu, plasma membrane, cytosol, nucleus, and nuclear membrane, where they mediate extracellular matrix protein degradation, cell signalling, and protein processing and trafficking through the plasma and nuclear membranes and between intracellular organelles. Studies in experimental disease models and on cathepsin-selective inhibitors, as well as plasma and tissue biomarker data from animal models and humans, have verified the participation of cysteinyl cathepsins in the pathogenesis of many cardiovascular diseases, including atherosclerosis, myocardial infarction, cardiac hypertrophy, cardiomyopathy, abdominal aortic aneurysms, and hypertension. Clinical trials of cathepsin inhibitors in chronic inflammatory diseases suggest the utility of these inhibitors for the treatment of cardiovascular diseases and associated complications. Moreover, development of cell transfer technologies that enable ex vivo cell treatment with cathepsin inhibitors might limit the unwanted systemic effects of cathepsin inhibition and provide new avenues for targeting cysteinyl cathepsins. In this Review, we summarize the available evidence implicating cysteinyl cathepsins in the pathogenesis of cardiovascular diseases, discuss their potential as biomarkers of disease progression, and explore the potential of cathepsin inhibitors for the treatment of cardiovascular diseases.This Review summarizes the growing body of evidence implicating cathepsin activities in the pathogenesis of several cardiovascular diseases, outlining the potential of cathepsins as biomarkers of disease progression and discussing clinical trials of cathepsin inhibitors in other diseases that highlight opportunities for developing novel therapies targeting cathepsins in cardiovascular diseases.Key pointsCysteine protease cathepsins act beyond the lysosomes and have widespread physiological and pathological actions, although some cysteinyl cathepsins show tissue-specificity or cell-type-specificity.Cathepsin activity is generally increased in the heart and arterial wall in patients with cardiovascular diseases, and studies in mouse models have established the participation of cathepsins B, C, K, L, and S, and their endogenous inhibitor cystatin C, in various cardiovascular diseases.Cathepsin actions in cardiovascular diseases include the regulation of cell–cell interactions, intracellular signalling, protein expression, angiogenesis, cholesterol metabolism, cell migration, and apoptosis.Cathepsins contribute to cardiovascular inflammation directly and indirectly by regulating innate and adaptive immunity.Plasma cathepsins and cystatins might serve as biomarkers of cardiovascular disease in humans.The development of selective cathepsin antagonists and the results of their preliminary clinical evaluation warrant further clinical trials of cathepsin inhibitors for treatment of certain cardiovascular conditions.

Allergic lung inflammation promotes atherosclerosis in apolipoprotein E-deficient mice.

Inflammation drives asthma and atherosclerosis. Clinical studies suggest that asthmatic patients have a high risk of atherosclerosis. Yet this hypothesis remains uncertain, given that Th2 imbalance causes asthma whereas Th1 immunity promotes atherosclerosis. In this study, chronic allergic lung inflammation (ALI) was induced in mice by ovalbumin sensitization and challenge. Acute ALI was induced in mice by ovalbumin and aluminum sensitization and ovalbumin challenge. Atherosclerosis was produced in apolipoprotein E-deficient (Apoe(-/-)) mice with a Western diet. When chronic ALI and atherosclerosis were produced simultaneously, ALI increased atherosclerotic lesion size, lesion inflammatory cell content, elastin fragmentation, smooth muscle cell (SMC) loss, lesion cell proliferation, and apoptosis. Production of acute ALI before atherogenesis did not affect lesion size, but increased atherosclerotic lesion CD4(+) T cells, lesion SMC loss, angiogenesis, and apoptosis. Production of acute ALI after atherogenesis also did not change atherosclerotic lesion area, but increased lesion elastin fragmentation, cell proliferation, and apoptosis. In mice with chronic ALI and diet-induced atherosclerosis, daily inhalation of a mast cell inhibitor or corticosteroid significantly reduced atherosclerotic lesion T-cell and mast cell contents, SMC loss, angiogenesis, and cell proliferation and apoptosis, although these drugs did not affect lesion area, compared with those that received vehicle treatment. In conclusion, both chronic and acute ALI promote atherogenesis or aortic lesion pathology, regardless whether ALI occurred before, after, or at the same time as atherogenesis. Antiasthmatic medication can efficiently mitigate atherosclerotic lesion pathology.

Cathepsin K Deficiency Prevents the Aggravated Vascular Remodeling Response to Flow Cessation in ApoE -/- Mice

Cathepsin K (catK) is a potent lysosomal cysteine protease involved in extracellular matrix (ECM) degradation and inflammatory remodeling responses. Here we have investigated the contribution of catK deficiency on carotid arterial remodeling in response to flow cessation in apoE-/- and wild type (wt) background. Ligation-induced hyperplasia is considerably aggravated in apoE-/- versus wt mice. CatK protein expression was significantly increased in neointimal lesions of apoE-/- compared with wt mice, suggesting a role for catK in intimal hyperplasia under hyperlipidemic conditions. Surprisingly, CatK deficiency completely blunted the augmented hyperplastic response to flow cessation in apoE-/-, whereas vascular remodeling in wt mice was unaffected. As catK deficiency did neither alter lesion collagen content and elastic laminae fragmentation in vivo, we focused on effects of catK on (systemic) inflammatory responses. CatK deficiency significantly reduced circulating CD3 T-cell numbers, but increased the regulatory T cell subset in apoE-/- but not wt mice. Moreover, catK deficiency changed CD11b+Ly6G-Ly6C high monocyte distribution in apoE-/- but not wt mice and tended to favour macrophage M2a polarization. In conclusion, catK deficiency almost completely blunted the increased vascular remodeling response of apoE-/- mice to flow cessation, possibly by correcting hyperlipidemia-associated pro-inflammatory effects on the peripheral immune response.

MP98-14 CATHEPSIN S INHIBITION CHANGES REGULATORY T-CELL ACTIVITY IN REGULATING BLADDER CANCER AND IMMUNE CELL PROLIFERATION AND APOPTOSIS

Many of the genes affected by HOTAIR expression are in the canonical Wnt-pathway. HOTAIR is a known to facilitate EMT through the canonical Wnt-pathway in other tumors. Determining the importance of the Wnt-pathway in UBC may open up new treatment options. Here we show that HOTAIR is necessary for Wnt-responsiveness and its expression increases during Wnt-pathway activation. EMT is also regulated through intercellular communication mediated by ECVs. Given HOTAIR regulates thousands of genes, we hypothesized that ECVs from HOTAIR knockdown cells would have limited ability to facilitate EMT. In fact, HOTAIR knockdown cells produce fewer exosomes with altered protein cargo and do not facilitate migration or invasion, suggesting that targeting of HOTAIR therapeutically would affect EMT through both the Wnt-pathway and ECVs functionality. Objective: To evaluate the role of HOTAIR in WNT-mediated and EVCmediated EMT METHODS: UBCs treated with LiCl or rWNT and gene expression was analyzed by qRTPCR, western blot and immunohistochemistry. We used scratch and 3D spheroid invasion assays to measure in vitro EMT in rWNT treated or untreated UBC cells. shRNA or siRNA against HOTAIR were used and WNT target and antagonist gene expression was measured by qRT-PCR. Migration and invasion were measured using scratch wound assay and 3D spheroid assay. TCF7L2 binding sites were identified in the promoter region of HOTAIR by sequencing. siRNA against TCF7L2 or beta-catenin reduced HOTAIR expression. ECVs isolatedd by ultracentrifugation and sucrose gradient were analyzed using the Nanosight. ECVs protein analysis was performed with LC MS/MS mass spectrometry and western blot. EVC-mediated migration and invasion was evaluated by wound and 3D invasion assay. RESULTS: TCGA data reveals WNT pathway genes are affected in human UBC. LiCl or rWNT treated UBCs have increased EMT related gene expression. rWnt facilitates UBC in vitro migration and invasion in a HOTAIR-dependent fashion. Reduced HOTAIR expression correlates with decreased WNT-target and increased WNTantagonist gene expression. Importantly, HOTAIR is a target of canonical WNT signaling. Reduced HOTAIR expression affects UBC EVC number, content and in vitro migration and invasion. CONCLUSIONS: These data support a role for the canonical WNT-pathway in UBC in a manner dependent on HOTAIR expression. Therefore, therapeutic targeting of the WNT-pathway may affect UBC tumor progression through reduced HOTAIR expression. Importantly, loss of HOTAIR affects the expression of hundreds of genes that results in reduced ECVs number, content, and ability to affect in vitro migration and invasion.

Toll-like receptor 7 deficiency protects apolipoprotein E-deficient mice from diet-induced atherosclerosis

Toll-like receptor 7 (TLR7) mediates autoantigen and viral RNA-induced cytokine production. Increased TLR7 expression in human atherosclerotic lesions suggests its involvement in atherogenesis. Here we demonstrated TLR7 expression in macrophages, smooth muscle cells (SMCs), and endothelial cells from mouse atherosclerotic lesions. To test a direct participation of TLR7 in atherosclerosis, we crossbred TLR7-deficient (Tlr7−/−) mice with apolipoprotein E-deficient (Apoe−/−) mice and produced Apoe−/−Tlr7−/− and Apoe−/−Tlr7+/+ littermates, followed by feeding them an atherogenic diet to produce atherosclerosis. Compared to Apoe−/−Tlr7+/+ mice, Apoe−/−Tlr7−/− mice showed reduced aortic arch and sinus lesion areas. Reduced atherosclerosis in Apoe−/−Tlr7−/− mice did not affect lesion macrophage-positive area and CD4+ T-cell number per lesion area, but reduced lesion expression of inflammatory markers major histocompatibility complex-class II and IL6, lesion matrix-degrading proteases cathepsin S and matrix metalloproteinase-9, and systemic serum amyloid A levels. TLR7 deficiency also reduced aortic arch SMC loss and lesion intima and media cell apoptosis. However, TLR7 deficiency did not affect aortic wall elastin fragmentation and collagen contents, or plasma lipoproteins. Therefore, TLR7 contributes to atherogenesis in Apoe−/− mice by regulating lesion and systemic inflammation. A TLR7 antagonist may mitigate atherosclerosis.