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BackgroundCathepsin L and some other cathepsins have been implicated in the development of obesity in humans and mice. The functional inactivation of the proteases reduces fat accumulation during mammalian adipocyte differentiation. However, beyond degrading extracellular matrix protein fibronectin, the molecular mechanisms by which cathepsins control fat accumulation remain unclear. We now provide evidence fromCaenorhabditis elegansand mouse models to suggest a conserved regulatory circuit in which peripheral cathepsin L inhibition lowers fat accumulation through promoting central serotonin synthesis.ResultsWe established aC. elegansmodel of fat accumulation using dietary supplementation with glucose and palmitic acid. We found that nutrient supplementation elevated fat storage inC. elegans, and along with worm fat accumulation, an increase in the expression ofcpl-1was detected using real-time PCR and western blot. The functional inactivation ofcpl-1reduced fat storage inC. elegansthrough activating serotonin signaling. Further, knockdown ofcpl-1in the intestine and hypodermis promoted serotonin synthesis in worm ADF neurons and induced body fat loss inC. elegansvia central serotonin signaling. We found a similar regulatory circuit in high-fat diet-fed mice. Cathepsin L knockout promoted fat loss and central serotonin synthesis. Intraperitoneal injection of the cathepsin L inhibitor CLIK195 similarly reduced body weight gain and white adipose tissue (WAT) adipogenesis, while elevating brain serotonin level and WAT lipolysis and fatty acid β-oxidation. These effects of inhibiting cathepsin L were abolished by intracranial injection of p-chlorophenylalanine, inhibitor of a rate-limiting enzyme for serotonin synthesis.ConclusionThis study reveals a previously undescribed molecular mechanism by which peripheral CPL-1/cathepsin L inhibition induces fat loss inC. elegansand mice through promoting central serotonin signaling.

Abdominal aortic aneurysms (AAAs) elicit massive inflammatory leukocyte recruitment to the aorta. CD4

Regulatory T cells (Tregs) are a subset of CD4+T cells with their immunosuppressive activities to block abnormal or excessive immune responses to self and non-autoantigens. Tregs express the transcription factor Foxp3, maintain the immune homeostasis, and prevent the initiation of anti-tumor immune effects in various ways as their mechanisms to modulate tumor development. Recognition of different phenotypes and functions of intratumoral Tregs has offered the possibilities to develop therapeutic strategies by selectively targeting Tregs in cancers with the aim of alleviating their immunosuppressive activities from anti-tumor immune responses. Several Treg-based immunotherapeutic approaches have emerged to target cytotoxic T lymphocyte antigen-4, glucocorticoid-induced tumor necrosis factor receptor, CD25, indoleamine-2, 3-dioxygenase-1, and cytokines. These immunotherapies have yielded encouraging outcomes from preclinical studies and early-phase clinical trials. Further, dual therapy or combined therapy has been approved to be better choices than single immunotherapy, radiotherapy, or chemotherapy. In this short review article, we discuss our current understanding of the immunologic characteristics of Tregs, including Treg differentiation, development, therapeutic efficacy, and future potential of Treg-related therapies among the general cancer therapy.

Objective: To explore the associations of common mitochondrial DNA polymorphisms with chronic kidney disease (CKD). Methods: Data from 286 longevous individuals aged 95 years or older from the long...

Pressure overload and heart failure are among the leading causes of cardiovascular morbidity and mortality. Accumulating evidence suggests that inflammatory cell activation and release of inflammatory mediators are of vital importance during the pathogenesis of these cardiac diseases. Yet, the roles of innate immune cells and subsequent inflammatory events in these processes remain poorly understood. Here, we outline the possible underlying mechanisms of innate immune cell participation, including mast cells, macrophages, monocytes, neutrophils, dendritic cells, eosinophils, and natural killer T cells in these pathological processes. Although these cells accumulate in the atrium or ventricles at different time points after pressure overload, their cardioprotective or cardiodestructive activities differ from each other. Among them, mast cells, neutrophils, and dendritic cells exert detrimental function in experimental models, whereas eosinophils and natural killer T cells display cardioprotective activities. Depending on their subsets, macrophages and monocytes may exacerbate cardiodysfunction or negatively regulate cardiac hypertrophy and remodeling. Pressure overload stimulates the secretion of cytokines, chemokines, and growth factors from innate immune cells and even resident cardiomyocytes that together assist innate immune cell infiltration into injured heart. These infiltrates are involved in pro-hypertrophic events and cardiac fibroblast activation. Immune regulation of cardiac innate immune cells becomes a promising therapeutic approach in experimental cardiac disease treatment, highlighting the significance of their clinical evaluation in humans.

Summary Transforming growth factor beta (TGF-β) signaling contributes to tissue fibrosis. Here we demonstrate that TGF-β enhances CatS and CatK expression but reduces CatB and CatL expression in mouse kidney tubular epithelial cells (TECs). CatS- and CatK deficiency reduces TEC nuclear membrane importer importin-β expression, Smad-2/3 activation, and extracellular matrix (ECM) production. Yet CatB- and CatL-deficiency displays the opposite observations with reduced nuclear membrane exporter RanBP3 expression. CatS and CatK form immunocomplexes with the importin-β and RanBP3 more effectively than do CatB and CatL. On the plasma membrane, CatS and CatK preferentially form immunocomplexes with and activate TGF-β receptor-2, whereas CatB and CatL form immunocomplexes with and inactivate TGF-β receptor-1. Unilateral ureteral obstruction-induced renal injury tests differential cathepsin activities in TGF-β signaling and tissue fibrosis. CatB- or CatL-deficiency exacerbates fibrosis, whereas CatS- or CatK-deficiency protects kidneys from fibrosis. These cathepsins exert different effects in the TGF-β signaling cascade independent of their proteolytic properties., Graphical Abstract, Highlights • Cathepsins exert different activities in kidney TEC TGF-β signaling and fibrosis • Cathepsins regulate nuclear membrane transporter expression and form immunocomplexes • Cathepsins regulate plasma membrane TGF-β receptor expression and form immunocomplexes • Cathepsins play different roles in acute injury-induced kidney fibrosis, Fibrosis; Molecular Mechanism of Behavior; Functional Aspects of Cell Biology

Obstructive nephropathy is a common cause for chronic kidney disease. Surgery, which is adopted to promptly relieve the obstruction, is the most important method to save damaged kidneys. However, earlier studies have shown that renal function will continue to deteriorate until the terminal stage after the obstruction' relief. The aim of this study is to explore the renal fibrosis and investigate the effect of losartan on renal fibrosis after the obstruction' relief using an improved mouse model of relief for unilateral ureteral obstruction (RUUO). Experiments carried out using C57BL/6 mice (n = 30) were randomly divided into RUUO + Losartan group, RUUO group and sham group. Using an improved mouse RUUO model, this study revealed that the mouse kidney for 3- or 7-day unilateral ureteral obstruction undergoing the RUUO surgery was still in a state of injury and fibrosis, while losartan could effectively ameliorate renal fibrosis by upregulating the expression of CD4 + CD25 + Foxp3 + regulatory T cells (Tregs) in kidney after the surgery of RUUO.

Mast cell (MC) deficiency in KitW-sh/W-sh mice and inhibition with disodium chromoglycate (DSCG) or ketotifen reduced obesity and diabetes in mice on a high-cholesterol (1.25%) Western diet. Yet, Kit-independent MC-deficient mice and mice treated with DSCG disproved MC function in obesity and diabetes when mice are fed a high-fat diet (HFD) that contains no cholesterol. This study reproduced the obesity and diabetes inhibitory activities of DSCG and ketotifen from mice on a Western diet. Yet, such inhibitory effects were diminished in mice on the HFD. DSCG and ketotifen MC inhibitory activities were recovered from mice on the HFD supplemented with the same amount of cholesterol (1.25%) as that in the Western diet. DSCG and ketotifen effectively blunted the high-cholesterol diet-induced elevations of blood histamine and adipose tissue MC degranulation. Pearson's correlation test demonstrated significant and positive correlations between plasma histamine and total cholesterol or low-density lipoprotein-cholesterol (LDL). In cultured bone marrow-derived MCs, plasma from mice following a Western diet or a cholesterol-supplemented HFD, but not those from HFD-fed mice, induced MC degranulation and the release of β-hexosaminidase, histamine, and serotonin. IgE, LDL, very low-density lipoprotein, and high-density lipoprotein also induced MC activation, which can be inhibited by DSCG and ketotifen depending on the doses and types of MC inhibitors and cholesterol, and also the MC granule molecules of interest. DSCG or ketotifen lost their activities in inhibiting LDL-induced activation of MCs from LDL receptor-deficient mice. These results indicate that dietary cholesterol critically influences the function of mouse MCs.

Severe haemorrhagic transformation (HT), a common complication of recombinant tissue plasminogen activator (rtPA) treatment, predicts poor clinical outcomes in acute ischaemic stroke. The search for agents to mitigate this effect includes investigating biomolecules involved in neovascularization. This study examines the role of Cathepsin K (Ctsk) in rtPA-induced HT after focal cerebral ischaemia in mice. After knockout of Ctsk, the gene encoding Ctsk, the outcomes of Ctsk+/+ and Ctsk−/− mice were compared 24 h after rtPA-treated cerebral ischaemia with respect to HT severity, neurological deficits, brain oedema, infarct volume, number of apoptotic neurons and activated microglia/macrophage, blood–brain barrier integrity, vascular endothelial growth factor (VEGF) expression and Akt-mTOR pathway activation. We observed that haemoglobin levels, brain oedema and infarct volume were significantly greater and resulted in more severe neurological deficits in Ctsk−/− than in Ctsk+/+ mice. Consistent with our hypothesis, the number of NeuN-positive neurons was lower and the number of TUNEL-positive apoptotic neurons and activated microglia/macrophage was higher in Ctsk−/− than in Ctsk+/+ mice. Ctsk knockout mice exhibited more severe blood–brain barrier (BBB) disruption, with microvascular endothelial cells exhibiting greater VEGF expression and lower ratios of phospo-Akt/Akt and phospo-mTOR/mTOR than in Ctsk+/+ mice. This study is the first to provide molecular insights into Ctsk-regulated HT after cerebral ischaemia, suggesting that Ctsk deficiency may disrupt the BBB via Akt/mTOR/VEGF signalling, resulting in neurological deficits and neuron apoptosis. Ctsk administration has the potential as a novel modality for improving the safety of rtPA treatment following stroke.

Objective— Inflammation occurs during the progression of abdominal aortic aneurysm (AAA). IL (interleukin)-33 is a pleiotropic cytokine with multiple immunomodulatory effects, yet its role in AAA remains unknown. Approach and Results— Immunoblot, immunohistochemistry, and immunofluorescent staining revealed increased IL-33 expression in adventitia fibroblasts from mouse AAA lesions. Daily intraperitoneal administration of recombinant IL-33 or transgenic IL-33 expression ameliorated periaorta CaPO 4 injury- and aortic elastase exposure–induced AAA in mice, as demonstrated by blunted aortic expansion, reduced aortic wall elastica fragmentation, enhanced AAA lesion collagen deposition, attenuated T-cell and macrophage infiltration, reduced inflammatory cytokine production, skewed M2 macrophage polarization, and reduced lesion MMP (matrix metalloproteinase) expression and cell apoptosis. Flow cytometry analysis, immunostaining, and immunoblot analysis showed that exogenous IL-33 increased CD4 + Foxp3 + regulatory T cells in spleens, blood, and aortas in periaorta CaPO 4 -treated mice. Yet, ST2 deficiency muted these IL-33 activities. Regulatory T cells from IL-33–treated mice also showed significantly stronger activities in suppressing smooth muscle cell inflammatory cytokine and chemokine expression, macrophage MMP expression, and in increasing M2 macrophage polarization than those from vehicle-treated mice. In contrast, IL-33 failed to prevent AAA and lost its beneficial activities in CaPO 4 -treated mice after selective depletion of regulatory T cells. Conclusions— Together, this study established a role of IL-33 in protecting mice from AAA formation by enhancing ST2-dependent aortic and systemic regulatory T-cell expansion and their immunosuppressive activities.

Few studies have investigated the association of the neutrophil-to-lymphocyte ratio (NLR) with gait speed, but whether the NLR is predictive of slow gait speed in older adults remains unknown. The aim of this study is to examine the association of NLR levels with risk of slow gait speed development in older adults.Overall, 1753 participants (53.11% male, aged 60-92 years, with a mean age of 77.01 ± 4.27 years) from the second wave of the Rugao Longitudinal Aging Study were included. The second wave was recognized as the baseline in this study. Gait speed was measured using a 5-m walk test at baseline and at the 3.5-year follow-up. A slow gait speed was considered a walking speed less than 0.8 m/s. The NLR was calculated based on absolute blood neutrophil and lymphocyte counts. Logistic regression models were used to investigate the association between NLR levels and slow gait speed.In the cross-sectional analysis, 394 individuals were identified as having slow gait speed. We found that increased NLR levels were associated with a higher risk of slow gait speed in older adults with and without comorbidities (P-value0.05). During the 3.5-year follow-up period, 440 individuals had developed new-onset slowness. After confounding factors were controlled, increased NLR levels were significantly and independently associated with an increased risk of slow gait speed among older adults with (odds ratio [OR] 3.82, 95% confidence interval [CI] 1.87-7.89) and without (OR 3.29, 95% CI: 1.54-7.10) comorbidities.The NLR is an inexpensive and easily obtainable inflammatory biomarker that robustly and independently predicts slow gait speed risk in older adults.

Aim To explore the cross-sectional and longitudinal associations between social frailty (SF) and incident depressive symptoms in a Chinese population. Methods SF was measured with 6 questions (6 points maximum; 0-1 = non-SF, 2-3 = pre-SF, 4-6 = SF). Depressive symptoms were defined as a score of ≥6 on the Geriatric Depression Scale. Compared to baseline, participants with a ≥2-point increase in the Geriatric Depression Scale score were considered to have worsening depressive symptoms. Results At baseline, among 1764 participants, 9.9% (n = 175) had depressive symptoms, 3.6% (n = 61) were SF, and 38.2% (n = 650) were pre-SF. The percentage of depressive symptoms increased with SF status from 5.1% (non-SF) to 12.9% (pre-SF), to 41.0% (SF). In cross-sectional analysis, after adjustments for multiple covariates, depressive symptoms were significantly associated with both pre-SF (odds ratio (OR) = 2.94, 95% confidence interval (CI) 2.01-4.32) and SF (OR = 16.70, 95% CI 8.80-31.71). During the 3-year follow-up period, 10.0% (n = 117) of the participants developed depressive symptoms. In longitudinal analyses, after multiple adjustments, SF and pre-SF were associated with a 2.31-fold (95% CI 1.10-4.88) and 1.58-fold (95% CI 1.05-2.38) increased risk of incidence of depressive symptoms, respectively. Among participants without depressive symptoms at baseline, 23.2% had worsening depressive symptoms, and SF was associated with increased risk of worsening depressive symptoms (OR = 2.07, 95% CI 1.18-3.65). Conclusions Our findings suggested that SF may be a predictor of depression among Chinese community-dwelling older adults. In addition, in elders with no depressive symptoms at baseline, those with SF had greater odds of worsening depressive symptoms 3 years later.

Dysregulation of circular RNAs (circRNAs) is involved in various human diseases. Fibroblast-like synoviocytes (FLSs), which form the lining of the joint, are epigenetically imprinted with an aggressive phenotype and contribute to joint destruction in rheumatoid arthritis (RA). In the present study, we identified a novel circRNA, Circ_0088194, which was upregulated in RA fibroblast-like synoviocytes (RA-FLSs) and correlated with the disease activity score in 28 joints. Overexpression of Circ_0088194 promoted RA-FLS migration and invasion, while inhibition of Circ_0088194 had the opposite effect. Mechanistically, Circ_0088194 acted as a miR-766-3p sponge to relieve the repressive effect of miR-766-3p on its target, MMP2 (encoding matrix metalloproteinase 2), thereby promoting migration and invasion. The expression level of Circ_0088194 was inversely correlated with that of miR-766-3p in RA-FLSs. Importantly, overexpression of miR-766-3p partially blocked the migration and invasion induced by Circ_0088194 overexpression. Collectively, this study identified a novel circRNA Circ_0088194 that promotes RA-FLS invasion and migration via the miR-766-3p/MMP2 axis. Circ_0088194 might represent a novel therapeutic target to prevent and treat RA.

Introduction: The regeneration of pancreatic β cells to restore insulin production is a promising therapeutic strategy for insulin-dependent type-1 (T1D) and type-2 diabetes (T2D). Plasma IL18 levels are increased in these patients. Prior studies suggest a role for IL18 in inducing islet insulin production. IL18 administration suppressed autoimmune diabetes in non-obese diabetic mice. We recently discovered that IL18 uses two receptors IL18r and NCC (Na-Cl co-transporter) with equal binding kinetics. Hypothesis: IL18r and NCC use different mechanisms to mediate IL18 activity in islet β cell insulin production. Methods and Results: Immunoblot and immunofluorescent staining localized IL18 on islet α cells, NCC on islet β cells, and IL18r on pancreas acinar cells. Development of T1D or T2D increased the α cell IL18 expression. From high fat diet (HFD)-induced T2D, deficiency of IL18r ( Il18r -/- ) or NCC ( Ncc -/- ), or combined deficiency of both receptors ( Il18r -/- Ncc -/- ) reduced insulin secretion, exacerbated β cell failure, decreased α cell content, reduced islet size, reduced β cell proliferation, and decreased the expression of islet proliferation markers. Deficiency of IL18r in Il18r -/- and Il18r -/- Ncc -/- mice aggravated β cell content and size reductions and glucose metabolism and increased β cell apoptosis after streptozotocin (STZ)-induced T1D. Il18r -/- Ncc -/- mice also showed increased macrophage accumulation in the islets from both T1D and T2D mice. Using α cell-selective IL18-deficient Gcg CreERT2 IL18 fl/fl mice, acinar cell-selective IL18r-deficient Mist1 CreERT2 Il18r fl/fl mice, and β cell NCC-deficient Ins1 Cre Ncc fl/fl mice, we demonstrated that these mice showed impaired insulin secretion and glucose metabolism, exacerbated β cell failure, and aggravated HFD-induced T2D. Mechanistic studies suggested that IL18 uses NCC on β cells to promote insulin secretion and uses IL18r on acinar cells to assist β cell development. Conclusions: Islet α cell IL18 uses NCC on β cells to promote β cell proliferation and uses IL18r on acinar cells to produce inflammatory molecules to assist β cell generation and insulin secretion. Both the IL18r and NCC signaling pathways are essential to β cell function in diabetic patients.

Dysregulation of circular RNAs (circRNAs) is involved in various human diseases. Fibroblast-like synoviocytes (FLSs), which form the lining of the joint, are epigenetically imprinted with an aggressive phenotype and contribute to joint destruction in rheumatoid arthritis (RA). In the present study, we identified a novel circRNA, Circ_0088194, which was upregulated in RA fibroblast-like synoviocytes (RA-FLSs) and correlated with the disease activity score in 28 joints. Overexpression of Circ_0088194 promoted RA-FLS migration and invasion, while inhibition of Circ_0088194 had the opposite effect. Mechanistically, Circ_0088194 acted as a miR-766-3p sponge to relieve the repressive effect of miR-766-3p on its target, MMP2 (encoding matrix metalloproteinase 2), thereby promoting migration and invasion. The expression level of Circ_0088194 was inversely correlated with that of miR-766-3p in RA-FLSs. Importantly, overexpression of miR-766-3p partially blocked the migration and invasion induced by Circ_0088194 overexpression. Collectively, this study identified a novel circRNA Circ_0088194 that promotes RA-FLS invasion and migration via the miR-766-3p/MMP2 axis. Circ_0088194 might represent a novel therapeutic target to prevent and treat RA.

Rationale: Blood eosinophil count and ECP (eosinophil cationic protein) associate with human cardiovascular diseases. Yet, whether eosinophils play a role in cardiovascular disease remains untested. The current study detected eosinophil accumulation in human and murine abdominal aortic aneurysm (AAA) lesions, suggesting eosinophil participation in this aortic disease. Objective: To test whether and how eosinophils affect AAA growth. Methods and Results: Population-based randomized clinically controlled screening trials revealed higher blood eosinophil count in 579 male patients with AAA than in 5063 non-AAA control (0.236±0.182 versus 0.211±0.154, 10 9 /L, P P P =0.031) logistic regression analyses indicated that increased blood eosinophil count in patients with AAA served as an independent risk factor of human AAA. Immunostaining and immunoblot analyses detected eosinophil accumulation and eosinophil cationic protein expression in human and murine AAA lesions. Results showed that eosinophil deficiency exacerbated AAA growth with increased lesion inflammatory cell contents, matrix-degrading protease activity, angiogenesis, cell proliferation and apoptosis, and smooth muscle cell loss using angiotensin-II perfusion–induced AAA in Apoe −/− and eosinophil-deficient Apoe −/− ΔdblGATA mice. Eosinophil deficiency increased lesion chemokine expression, muted lesion expression of IL (interleukin) 4 and eosinophil-associated-ribonuclease-1 (mEar1 [mouse EOS-associated-ribonuclease-1], human ECP homolog), and slanted M1 macrophage polarization. In cultured macrophages and monocytes, eosinophil-derived IL4 and mEar1 polarized M2 macrophages, suppressed CD11b + Ly6C hi monocytes, and increased CD11b + Ly6C lo monocytes. mEar1 treatment or adoptive transfer of eosinophil from wild-type and Il13 −/− mice, but not eosinophil from Il4 −/− mice, blocked AAA growth in Apoe −/− ΔdblGATA mice. Immunofluorescent staining and immunoblot analyses demonstrated a role for eosinophil IL4 and mEar1 in blocking NF-κB (nuclear factor-κB) activation in macrophages, smooth muscle cells, and endothelial cells. Conclusions: Eosinophils play a protective role in AAA by releasing IL4 and cationic proteins such as mEar1 to regulate macrophage and monocyte polarization and to block NF-κB activation in aortic inflammatory and vascular cells.

Introduction: The Healthy Ageing Index (HAI) has been shown not only to have wider applicability and predictive ability but also to adequately predict mortality in Western populations. There is still a lack of studies validating the applicability of the HAI in China. Objective: To evaluate the applicability of the HAI and validate whether the HAI is suitable for monitoring ageing in the elderly population in China. Methods: Data were obtained from the Rugao Longevity and Ageing Study. The modified HAI was constructed based on systolic blood pressure, chronic pulmonary diseases, cognitive function, fasting glucose, and kidney function. It was calculated in 1719 individuals aged 70–84 years at baseline. The adverse outcomes were mortality and disability. Demographic, physiologic, and clinical data were collected. Cox proportional hazards and logistic regression models were used to analyze the relationship between the modified HAI and adverse outcomes. Results: A total of 1,719 older adults were analyzed in our study. A total of 793 (46.13%) males were recruited. The mean age was 75.69 ± 3.93 years. At the 5-year follow-up, there were 266 deaths and 275 individuals with disabilities. In the multivariable models, the modified HAI was associated with mortality (hazard ratio = 1.11, 95% confidence interval [CI]: 1.03–1.20) and disability (odds ratio = 1.11, 95% CI: 1.05–1.18). In the sensitivity analyses, similar associations remained after imputing missing data using multiple imputation and excluding participants with major cardiovascular disease at baseline. Conclusion: The modified HAI was a robust and independent predictor of adverse outcomes. It is a valid and feasible tool for monitoring ageing in older adults.

IgE-mediated activation of Na(+)-H(+) exchanger-1 (Nhe1) induces aortic cell extracellular acidification and promotes cell apoptosis. A pH-sensitive probe pHrodo identified acidic regions at positions of macrophage accumulation, IgE expression, and cell apoptosis in human and mouse abdominal aortic aneurysm (AAA) lesions. Angiotensin-II-induced AAA in Nhe1-insufficient Apoe(−/−)Nhe1(+/−) mice and Apoe(−/−)Nhe1(+/+) littermates tested Nhe1 activity in experimental AAA, because Nhe1(–/−) mice develop ataxia and epileptic-like seizures and die early. Nhe1 insufficiency reduced AAA incidence and size, lesion macrophage and T-cell accumulation, collagen deposition, elastin fragmentation, cell apoptosis, smooth muscle cell loss, and matrix metalloproteinase (MMP) activity. Nhe1 insufficiency also reduced blood pressure and the plasma apoptosis marker translationally controlled tumor protein (TCTP), but did not affect plasma IgE. While pHrodo localized the acidic regions to macrophage clusters, IgE expression, and cell apoptosis in AAA lesions from Apoe(−/−)Nhe1(+/+) mice, such acidic areas were much smaller in lesions from Apoe(−/−)Nhe1(+/–) mice. Nhe1-FcεR1 colocalization in macrophages from AAA lesions support a role of IgE-mediated Nhe1 activation. Gelatin zymography, immunoblot, and RT-PCR analyses demonstrated that Nhe1 insufficiency reduced the MMP activity, cysteinyl cathepsin expression, IgE-induced apoptosis, and NF-κB activation in macrophages and blocked IgE-induced adhesion molecule expression in endothelial cells. A near-infrared fluorescent probe (LS662) together with fluorescence reflectance imaging of intact aortas showed reduced acidity in AAA lesions from Nhe-1-insufficient mice. This study revealed extracellular acidity at regions rich in macrophages, IgE expression, and cell apoptosis in human and mouse AAA lesions and established a direct role of Nhe1 in AAA pathogenesis.

Clinical studies reveal changes in blood eosinophil counts and eosinophil cationic proteins that may serve as risk factors for human coronary heart diseases. Here we report an increase of blood or heart eosinophil counts in humans and mice after myocardial infarction (MI), mostly in the infarct region. Genetic or inducible depletion of eosinophils exacerbates cardiac dysfunction, cell death, and fibrosis post-MI, with concurrent acute increase of heart and chronic increase of splenic neutrophils and monocytes. Mechanistic studies reveal roles of eosinophil IL4 and cationic protein mEar1 in blocking H2O2- and hypoxia-induced mouse and human cardiomyocyte death, TGF-β-induced cardiac fibroblast Smad2/3 activation, and TNF-α-induced neutrophil adhesion on the heart endothelial cell monolayer. In vitro-cultured eosinophils from WT mice or recombinant mEar1 protein, but not eosinophils from IL4-deficient mice, effectively correct exacerbated cardiac dysfunctions in eosinophil-deficient ∆dblGATA mice. This study establishes a cardioprotective role of eosinophils in post-MI hearts., Blood eosinophil (EOS) counts may serve as risk factors for human coronary heart diseases. Here the authors show that increased circulating and myocardial EOS after myocardial infarction play a cardioprotective role by reducing cardiomyocyte death, cardiac fibroblast activation and fibrosis, and endothelium activation-mediated inflammatory cell accumulation.

Background Chronic psychological stress (CPS) is linked to cardiovascular disease initiation and progression. Given that cysteinyl cathepsin K (CatK) participates in vascular remodeling and atherosclerotic plaque growth in several animal models, we investigated the role of CatK in the development of experimental neointimal hyperplasia in response to chronic stress. Methods and results At first, male wild-type (CatK) mice that underwent carotid ligation injury were subjected to chronic immobilization stress. On postoperative and stressed day 14, the results demonstrated that stress accelerated injury-induced neointima hyperplasia. On day 4, stressed mice showed following: increased levels of monocyte chemoattractant protein-1, gp91phox, toll-like receptor-2 (TLR2), TLR4, and CatK mRNAs or/and proteins, oxidative stress production, aorta-derived smooth muscle cell (SMC) migration, and macrophage infiltration as well as targeted intracellular proliferating-related molecules. Stressed mice showed increased matrix metalloproteinase-2 (MMP-2) and MMP-9 mRNA expressions and activities and elastin disruption in the injured carotid arteries. Second, CatK and CatK deficiency (CatK) mice received ligation injury and stress to explore the role of CatK. The stress-induced harmful changes were prevented by CatK. Finally, CatK mice that had undergone ligation surgery were randomly assigned to one of two groups and administered vehicle or CatK inhibitor for 14 days. Pharmacological CatK intervention produced a vascular benefit. Conclusion These data indicate that CatK deletion protects against the development of experimental neointimal hyperplasia via the attenuation of inflammatory overaction, oxidative stress production, and VSMC proliferation, suggesting that CatK is a novel therapeutic target for the management of CPS-related restenosis after intravascular intervention therapies.

To explore the associations of TNF-α -308 GA (rs1800629) and TNF-β 252 AG (rs909253) with physical function and plasma B-type natriuretic peptide (BNP).Data of 1747 community-dwelling elders from the ageing arm of the Rugao Longevity and Ageing Study was used. Physical function was measured by handgrip strength, Timed Up and Go (TUG) test and 5-meter walking test (5MWT).AA genotype of the TNF-α -308 GA was associated with higher mean time of TUG test and 5MWT (multivariable adjusted β=5.75 and 5.70, respectively, p0.05), compared with GG genotype. For the TNF-β 252 AG polymorphism, GG genotype was associated with higher mean time of TUG test and 5MWT (multivariable adjusted β=1.55 and 0.83, respectively, p0.05) and lower handgrip strength (multivariable adjusted β=-0.69, p0.05), compared with AA genotype. Further, GG was associated with greater odds of low handgrip strength (OR=1.47, 95% CI=1.06-2.04), low speed of TUG test (OR=1.87, 95% CI=1.20-2.01) and elevated BNP (OR=1.30, 95% CI=1.08-1.84). GG also interacted with elevated BNP to be associated with greater odds of low handgrip strength and 5MWT.TNF-β 252 AG was associated with physical function measurements, plasma BNP level, and odds of elevated BNP in an elderly population. TNF-β 252 AG also interacted with elevated BNP to be associated with greater odds of physical function measurements.

Objective: By binding to its high-affinity receptor FcεR1, IgE activates mast cells, macrophages, and other inflammatory and vascular cells. Recent studies support an essential role of IgE in cardiometabolic diseases. Plasma IgE level is an independent predictor of human coronary heart disease. Yet, a direct role of IgE and its mechanisms in cardiometabolic diseases remain incompletely understood. Approach and Results: Using atherosclerosis prone Apoe −/− mice and IgE-deficient Ige −/− mice, we demonstrated that IgE deficiency reduced atherosclerosis lesion burden, lesion lipid deposition, smooth muscle cell and endothelial cell contents, chemokine MCP (monocyte chemoattractant protein)-1 expression and macrophage accumulation. IgE deficiency also reduced bodyweight gain and increased glucose and insulin sensitivities with significantly reduced plasma cholesterol, triglyceride, insulin, and inflammatory cytokines and chemokines, including IL (interleukin)-6, IFN (interferon)-γ, and MCP-1. From atherosclerotic lesions and peritoneal macrophages from Apoe −/− Ige −/− mice that consumed an atherogenic diet, we detected reduced expression of M1 macrophage markers (CD68, MCP-1, TNF [tumor necrosis factor]-α, IL-6, and iNOS [inducible nitric oxide synthase]) but increased expression of M2 macrophage markers (Arg [arginase]-1 and IL-10) and macrophage-sterol-responsive-network molecules (complement C3, lipoprotein lipase, LDLR [low-density lipoprotein receptor]-related protein 1, and TFR [transferrin]) that suppress macrophage foam cell formation. These IgE activities can be reproduced in bone marrow-derived macrophages from wild-type mice, but muted in cells from FcεR1-deficient mice, or blocked by anti-IgE antibody or complement C3 deficiency. Conclusions: IgE deficiency protects mice from diet-induced atherosclerosis, obesity, glucose tolerance, and insulin resistance by regulating macrophage polarization, macrophage-sterol-responsive-network gene expression, and foam cell formation.

To explore the associations of B-type natriuretic peptide (BNP) with physical frailty status as well as each domain of frailty in a general elderly population. Cross-sectional analysis of prospective cohort study. All of 31 communities in Jiang’an township. Overall 1338 participants (aged 70–89 years, mean 77.42±4.08 years) without a history of cardiovascular diseases in the third-wave of the aging arm of the Rugao Longevity and Aging Study (RuLAS). Frailty was defined as the presence of ≥3 domains among five modified Fried’s criteria (unintentional weight loss, low physical activity level, weakness (low grip strength), exhaustion, and slowness (slow gait speed)) and pre-frailty as the presence of 1–2 domains. The prevalence of frailty and pre-frailty was 10.4% and 53.3%, respectively, in this elderly population. Elevated BNP (≥100 pg/mL) was significantly associated with pre-frailty (OR: 1.61, 95% CI: 1.13-2.29) and frailty (OR: 2.63, 95% CI: 1.61-4.32) after adjustment for covariates. In addition, elevated BNP was associated with low grip strength (OR: 2.00, 95% CI: 1.41-2.82) and low gait speed (OR: 1.62, 95% CI: 1.15-2.28) after adjustment for multiple covariates. Log BNP was inversely associated with grip strength (r= -0.265, p

Background Although apoptosis and cell proliferation have been extensively investigated in atherosclerosis and restenosis postinjury, the communication between these 2 cellular events has not been evaluated. Here, we report an inextricable communicative link between apoptosis and smooth muscle cell proliferation in the promotion of vascular remodeling postinjury. Methods and Results Cathepsin K–mediated caspase‐8 maturation is a key initial step for oxidative stress–induced smooth muscle cell apoptosis. Apoptotic cells generate a potential growth‐stimulating signal to facilitate cellular mass changes in response to injury. One downstream mediator that cathepsin K regulates is PLF‐1 (proliferin‐1), which can potently stimulate growth of surviving neighboring smooth muscle cells through activation of PI3K/Akt/p38MAPK (phosphatidylinositol 3‐kinase/protein kinase B/p38 mitogen‐activated protein kinase)‐dependent and ‐independent mTOR (mammalian target of rapamycin) signaling cascades. We observed that cathepsin K deficiency substantially mitigated neointimal hyperplasia by reduction of Toll‐like receptor‐2/caspase‐8–mediated PLF ‐1 expression. Interestingly, PLF ‐1 blocking, with its neutralizing antibody, suppressed neointima formation and remodeling in response to injury in wild‐type mice. Contrarily, administration of recombinant mouse PLF ‐1 accelerated injury‐induced vascular actions. Conclusions This is the first study detailing PLF ‐1 as a communicator between apoptosis and proliferation during injury‐related vascular remodeling and neointimal hyperplasia. These data suggested that apoptosis‐driven expression of PLF ‐1 is thus a novel target for treatment of apoptosis‐based hyperproliferative disorders.

Teng Ma,1–3,* Jian Cai,4,* Yin-Sheng Zhu,5 Xue-Feng Chu,5 Yong Wang,5 Guo-Ping Shi,5 Zheng-Dong Wang,5 Shun Yao,1–3 Xiao-Feng Wang,1–3 Xiao-Yan Jiang6–8 1Unit of Epidemiology, Ministry of Education Key Laboratory of Contemporary Anthropology, Fudan University, Shanghai, People’s Republic of China; 2State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, People’s Republic of China; 3National Clinical Research Center for Aging and Medicine (Huashan), Huashan Hospital, Fudan University, Shanghai, People’s Republic of China; 4Department of Neurology, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China; 5Rugao People’s Hospital, Rugao, Jiangsu, People’s Republic of China; 6Key Laboratory of Arrhythmias of the Ministry of Education of China, Tongji University School of Medicine, Shanghai, People’s Republic of China; 7Department of Pathology and Pathophysiology, Tongji University School of Medicine, Shanghai, People’s Republic of China; 8Institute of Medical Genetics, Tongji University, Shanghai, People’s Republic of China *These authors contributed equally to this work Background: Risk factors for heart rate-corrected QT interval (QTc) proglongation should be explored to stratify high-risk individuals to aid the prevention of incident cardiovascular events and mortality. The diversity of risk factors for QTc prolongation suggests that use of the frailty index (FI), indicating general health deficits, may be an effective approach, especially in the elderly, to identify the risk of QTc prolongation. Methods: We used the data of 1,780 individuals aged 70–87years from the Rugao Longevity and Ageing Study (RuLAS), a community-based longitudinal study. The FI was constructed using 20 routine laboratory tests, plus the body mass index and measures of systolic and diastolic blood pressures (FI-Lab). Results: The mean FI-Lab value was 0.24±0.09. The mean heart rate-corrected QT interval (QTc) was 407±38ms. The prevalence of QTc prolongation was 5.2% in elderly community populations aged 70–87years. A higher FI-Lab value was associated with a higher risk for QTc prolongation. Each 10% increase in the FI-Lab value increased the odds ratio (OR) by 33% (OR: 1.33; 95% CI: 1.07–1.64). Compared with the lowest quartile, the top quartile FI-Lab score was associated with a 2.50-fold QTc prolongation risk in elderly individuals (95% CI: 1.21–5.19). Conclusion: An FI based on routine laboratory data can identify older adults at increased risk for QTc prolongation. The FI approach may therefore be useful for the risk stratification of QTc prolongation. Keywords: QTc prolongation, frailty, frailty index, Chinese elderly

Background To explore the relationship between sleep disturbances and falls in an elderly Chinese population. Methods Data from 1726 individuals aged 70–87 years from the Rugao Longevity and Ageing Study were used. The Pittsburgh Sleep Quality Index (PSQI) was used to assess sleep variables. Outcomes were falls ≥1 time per year and falls ≥2 times per year. Results A total of 22.7% of the participants experienced ≥1 fall, and 9.8% experienced ≥2 falls per year. Poor sleep quality was associated with ≥1 fall (OR 1.08, 95% CI 1.05–1.12; OR 1.27, 95% CI 1.14–1.41) and ≥2 falls (OR 1.08, 95% CI 1.03–1.14; OR 1.28, 95% CI 1.10–1.48), with an increase per PSQI score and SD PSQI score, respectively. In addition, sleep quality, sleep latency, sleep efficiency, and sleep disturbance subcomponents were associated with an increased risk of ≥1 fall with ORs of 1.44 (95% CI, 1.21–1.72), 1.23 (95%CI,1.09–1.40), 1.12 (95%CI, 1.01–1.23) and 1.70 (95% CI,1.35–2.14), respectively, and were associated with an increased risk of ≥2 falls with ORs 1.54 (95%CI, 1.22–1.96), 1.21(95%CI, 1.02–1.44), 1.17 (95% CI 1.02–1.33), and 1.78 (95%CI, 1.31–2.44), respectively. Further, participants slept ≤5 h per night had an increased risk of ≥1 fall (OR 2.34; 95%CI, 1.59–3.46) and ≥2 falls (OR 2.19; 95%CI, 1.30–3.69). Conclusions Poor sleep quality and several subcomponent sleep symptoms were consistently associated with increased risk of falls ≥1 time and ≥2 times in Chinese elderly. The identification of sleep disturbances may help identify high-risk Chinese elders who may benefit from fall prevention education.

Background Cathepsin K (CatK) is a widely expressed cysteine protease that has gained attention because of its enzymatic and non-enzymatic functions in signalling. Here, we examined whether CatK-deficiency (CatK−/−) would mitigate injury-related skeletal muscle remodelling and fibrosis in mice, with a special focus on inflammation and muscle cell apoptosis. Methods Cardiotoxin (CTX, 20 μM/200 μL) was injected into the left gastrocnemius muscle of male wild-type (CatK+/+) and CatK−/− mice, and the mice were processed for morphological and biochemical studies. Results On post-injection Day 14, CatK deletion ameliorated muscle interstitial fibrosis and remodelling and performance. At an early time point (Day 3), CatK−/− reduced the lesion macrophage and leucocyte contents and cell apoptosis, the mRNA levels of monocyte chemoattractant protein-1, toll-like receptor-2 and toll-like receptor-4, and the gelatinolytic activity related to matrix metalloproteinase-2/-9. CatK deletion also restored the protein levels of caspase-3 and cleaved caspase-8 and the ratio of the BAX to the Bcl-2. Moreover, CatK deficiency protected muscle fibre laminin and desmin disorder in response to CTX injury. These beneficial muscle effects were mimicked by CatK-specific inhibitor treatment. In vitro experiments demonstrated that pharmacological CatK inhibition reduced the apoptosis of C2C12 mouse myoblasts and the levels of BAX and caspase-3 proteins induced by CTX. Conclusions These results demonstrate that CatK plays an essential role in skeletal muscle loss and fibrosis in response to CTX injury, possibly via a reduction of inflammation and cell apoptosis, suggesting a novel therapeutic strategy for the control of skeletal muscle diseases by regulating CatK activity.

Background: Coronary heart disease (CHD) is characterized by arterial wall inflammation and matrix degradation. Matrix metalloproteinase (MMP)-22 and -29 and pro-inflammatory cytokine interleukin-18 (IL18) are present in human hearts. IL18 may regulate MMP-22 and -29 expression, which may correlate with CHD progression. Methods and results: Immunoblot analysis showed that IL18 induced MMP-22 expression in human aortic smooth muscle cells. The Mann Whitney test from a prospective study of 194 CHD patients and 68 non-CHD controls demonstrated higher plasma levels of IL18, MMP-22 and -29 in CHD patients than in the controls. A logistic regression test suggested that plasma IL18 (odds ratio (OR)=1.131, P=0.007), MMP-22 (OR=1.213, P=0.040), and MMP-29 (OR=1.198, P=0.033) were independent risk factors of CHD. Pearson’s correlation test showed that IL18 (coefficient (r)=0.214, P=0.045; r=0.246, P=0.031) and MMP-22 (r=0.273, P=0.006; r=0.286, P=0.012) were associated with the Gensini score before and after adjusting for potential confounding factors. The multivariate Pearson’s correlation test showed that plasma MMP-22 levels correlated positively with high-sensitive-C-reactive protein (hs-CRP) (r=0.167, P=0.023), and MMP-29 levels correlated negatively with triglyceride (r=−0.169, P=0.018). Spearman’s correlation test indicated that plasma IL18 levels associated positively with plasma MMP-22 (r=0.845, P

CD74 mediates MHC class-II antigenic peptide loading and presentation and plays an important role in the pathogenesis of autoimmune diseases, including systemic lupus erythematosus. C57BL/6 Faslpr mice that develop spontaneous lupus-like autoimmunity and pathology showed elevated CD74 expression in the inflammatory cell infiltrates and the adjacent tubular epithelial cells (TECs) in kidneys affected by lupus nephritis but negligible levels in kidneys from age-matched wild-type mice. The inflammatory cytokine IFN-γ or IL-6 induced CD74 expression in kidney TECs in vitro. The presence of kidney TECs from Faslpr mice, rather than from wild-type mice, produced significantly stronger histones, dsDNA, and ribonucleoprotein-Smith Ag complex–induced CD4+ T cell activation. Splenocytes from CD74-deficient FaslprCd74−/− mice had muted responses in a MLR and to the autoantigen histones. Compared with FaslprCd74+/+ mice, FaslprCd74−/− mice had reduced kidney and spleen sizes, splenic activated T cells and B cells, serum IgG and autoantibodies, urine albumin/creatinine ratio, kidney Periodic acid–Schiff score, IgG and C3 deposition, and serum IL-6 and IL-17A levels, but serum IL-2 and TGF-β levels were increased. Study of chronic graft-versus-host C57BL/6 mice that received donor splenocytes from B6.C-H2bm12/KhEg mice and those that received syngeneic donor splenocytes yielded similar observations. CD74 deficiency reduced lupus-like autoimmunity and kidney pathology in chronic graft-versus-host mice. This investigation establishes the direct participation of CD74 in autoimmunity and highlights a potential role for CD74 in kidney TECs, together with professional APCs in systemic lupus erythematosus.

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.

Cysteinyl cathepsin K (CatK) is expressed in osteoclasts to mediate bone resorption, but is also inducible under inflammatory conditions. Faslpr mice on a C57BL/6 background develop spontaneous systemic lupus erythematosus-like manifestations. Although normal mouse kidneys expressed negligible CatK, those from Faslpr mice showed elevated CatK expression in the glomeruli and tubulointerstitial space. Faslpr mice also showed elevated serum CatK levels. CatK deficiency in Faslpr mice reduced all tested kidney pathologies, including glomerulus and tubulointerstitial scores, glomerulus complement C3 and IgG deposition, chemokine expression and macrophage infiltration, and serum autoantibodies. CatK contributed to Faslpr mouse autoimmunity and pathology in part by its activity in TLR-7 proteolytic processing and consequent regulatory T (Treg) cell biology. Elevated TLR7 expression and proteolytic processing in Faslpr mouse kidneys and Tregs showed significantly reduced levels in CatK-deficient mice, leading to increased spleen and kidney Treg content. Purified CD4+CD25highFoxp3+ Tregs from CatK-deficient mice doubled their immunosuppressive activity against T effector cells, compared with those from CatK-sufficient mice. In Faslpr mice, repopulation of purified Tregs from CatK-sufficient mice reduced spleen sizes, autoantibody titers, and glomerulus C3 and IgG deposition, and increased splenic and kidney Treg contents. Tregs from CatK-deficient mice had significantly more potency than CatK-sufficient Tregs in reducing spleen sizes, serum autoantibody titers, and glomerulus C3 deposition, and in increasing splenic and kidney Treg content. This study established a possible role of CatK in TLR7 proteolytic activation, Treg immunosuppressive activity, and lupus autoimmunity and pathology.

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.

Intestinal ischemia followed by reperfusion leads to local and remote organ injury attributed to inflammatory response during the reperfusion phase. The extent to which ischemia contributes to ischemia/reperfusion injury has not been thoroughly studied. After careful evaluation of intestinal tissue following 30 min of ischemia, we noticed significant local mucosal injury in wild-type mice. This injury was drastically reduced in C3-deficient mice, suggesting C3 involvement. Depletion of circulating complement with cobra venom factor eliminated, as expected, injury recorded at the end of the reperfusion phase but failed to eliminate injury that occurred during the ischemic phase. Immunohistochemical studies showed that tissue damage during ischemia was associated with increased expression of C3/C3 fragments primarily in the intestinal epithelial cells, suggesting local involvement of complement. In vitro studies using Caco2 intestinal epithelial cells showed that in the presence of LPS or exposure to hypoxic conditions the cells produce higher C3 mRNA as well as C3a fragment. Caco2 cells were also noted to produce cathepsins B and L, and inhibition of cathepsins suppressed the release of C3a. Finally, we found that mice treated with a cathepsin inhibitor and cathepsin B–deficient mice suffer limited intestinal injury during the ischemic phase. To our knowledge, our findings demonstrate for the first time that significant intestinal injury occurs during ischemia prior to reperfusion and that this is due to activation of C3 within the intestinal epithelial cells in a cathepsin-dependent manner. Modulation of cathepsin activity may prevent injury of organs exposed to ischemia.

Background/Aims: Circular RNAs (circRNAs) compose a large class of RNAs that can be used as biomarkers in clinical blood samples. This study aimed to determine the expression of circRNAs in peripheral blood mononuclear cells (PBMCs) from rheumatoid arthritis (RA) patients to identify novel biomarkers for RA screening. Methods: We started with a microarray screening of circRNA changes in PBMCs from 5 RA patients and 5 healthy controls. We then confirmed the selected circRNA changes in PBMCs from 30 RA patients and 25 age- and sex-matched controls using the real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Spearman correlation test was performed to assess the correlation of circRNAs and clinical variables. Receiver operating characteristic (ROC) curve was calculated to evaluate the diagnostic value. Results: We identified and verified five circRNAs (092516, 003524, 103047, 104871, 101873) that were significantly elevated in PBMCs from RA patients. Among these RA patients, we detected no significant correlation between the five circRNAs and the disease severity, including disease activity score (DAS28), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and health assessment questionnaire (HAQ). Yet, ROC curve analysis suggested that circRNA_104871 has significant value of RA diagnosis (AUC=0.833, P

Regulatory T cells (Tregs) play essential roles in obesity and diabetes. Here, we report a role of Tregs in enhancing β3-adrenergic receptor agonist CL316243 (CL)-stimulated thermogenic program in subcutaneous adipose tissue (SAT), but not in visceral fat. CL treatment for 7 days increased SAT adipocyte beiging and thermogenic gene expression in male or female mice. Adoptive transfer of Tregs enhanced this CL activity. Such Treg activity lost in male epididymal white adipose tissue (eWAT) and female gonadal gWAT. Adipocyte culture yielded the same conclusion. Tregs enhanced the expression of CL-induced thermogenic genes in SAT from male and female mice. This activity of Tregs reduced or disappeared in adipocytes from eWAT or gWAT. Both CL and Tregs induced much higher UCP-1 (uncoupling protein-1) expression in SAT from females than that from males. A mechanistic study demonstrated a role of Tregs in suppressing the expression of M1 macrophage markers (Tnfa, Il6, iNos, Ip10) and promoting the expression of M2 macrophage markers (Mrc1, Arg1, Il10) in bone-marrow-derived macrophages or in SAT from male or female mice. In female mice with pre-established obesity, Treg adoptive transfer reduced the gWAT weight in 2 weeks. Together with CL treatment, Treg adoptive transfer reduced the SAT weight and further improved CL-induced glucose metabolism and insulin sensitivity in female obese mice, but did not affect CL-induced body weight loss in male or female obese mice. This study revealed a predominant role of Tregs in female mice in promoting adipocyte beiging and thermogenesis in SAT, in part by slanting M2 macrophage polarization.

Cysteinyl cathepsins are lysosomal/endosomal proteases that mediate bulk protein degradation in these intracellular acidic compartments. Yet, studies indicate that these proteases also appear in the nucleus, nuclear membrane, cytosol, plasma membrane, and extracellular space. Patients with cardiovascular diseases (CVD) show increased levels of cathepsins in the heart, aorta, and plasma. Plasma cathepsins often serve as biomarkers or risk factors of CVD. In aortic diseases, such as atherosclerosis and abdominal aneurysms, cathepsins play pathogenic roles, but many of the same cathepsins are cardioprotective in hypertensive, hypertrophic, and infarcted hearts. During the development of CVD, cathepsins are regulated by inflammatory cytokines, growth factors, hypertensive stimuli, oxidative stress, and many others. Cathepsin activities in inflammatory molecule activation, immunity, cell migration, cholesterol metabolism, neovascularization, cell death, cell signaling, and tissue fibrosis all contribute to CVD and are reviewed in this article in memory of Dr. Nobuhiko Katunuma for his contribution to the field.

The pH in atherosclerotic lesions varies between individuals. IgE activates macrophage Na+-H+ exchanger (Nhe1) and induces extracellular acidification and cell apoptosis. Here, we show that the pH-sensitive pHrodo probe localizes the acidic regions in atherosclerotic lesions to macrophages, IgE, and cell apoptosis. In Apoe–/– mice, Nhe1-deficiency or anti-IgE antibody reduces atherosclerosis and blocks lesion acidification. Reduced atherosclerosis in Apoe–/– mice receiving bone marrow from Nhe1- or IgE receptor FceR1-deficient mice, blunted foam cell formation and signaling in IgE-activated macrophages from Nhe1-deficient mice, immunocomplex formation of Nhe1 and FceR1 in IgE-activated macrophages, and Nhe1-FceR1 colocalization in atherosclerotic lesion macrophages support a role of IgE-mediated macrophage Nhe1 activation in atherosclerosis. Intravenous administration of a near-infrared fluorescent pH-sensitive probe LS662, followed by coregistered fluorescent molecular tomography-computed tomography imaging, identifies acidic regions in atherosclerotic lesions in live mice, ushering a non-invasive and radiation-free imaging approach to monitor atherosclerotic lesions in live subjects. Na+-H+ exchanger 1 (Nhe1) regulates extracellular pH by extruding protons in exchange for extracellular Na+ . Here, Liu et al. show that Nhe1 promotes the development and acidification of atherosclerotic lesions and that pH-sensitive probes can be used to monitor plaque growth and acidification.

Background Exposure to chronic psychosocial stress is a risk factor for atherosclerosis‐based cardiovascular disease. We previously demonstrated the increased expressions of cathepsin S (CatS) in atherosclerotic lesions. Whether CatS participates directly in stress‐related neointimal hyperplasia has been unknown. Methods and Results Male wild‐type and CatS‐deficient mice that underwent carotid ligation injury were subjected to chronic immobilization stress for morphological and biochemical studies at specific times. On day 14 after stress/surgery, stress enhanced the neointima formation. At the early time points, the stressed mice had increased plaque elastin disruption, cell proliferation, macrophage accumulation, mRNA and/or protein levels of vascular cell adhesion molecule‐1, angiotensin II type 1 receptor, monocyte chemoattractant protein‐1, gp91 phox , stromal cell–derived factor‐1, C‐X‐C chemokine receptor‐4, toll‐like receptor‐2, toll‐like receptor‐4, SC 35, galectin‐3, and CatS as well as targeted intracellular proliferating‐related molecules (mammalian target of rapamycin, phosphorylated protein kinase B, and p‐glycogen synthase kinase‐3α/β). Stress also increased the plaque matrix metalloproteinase‐9 and matrix metalloproteinase‐2 mRNA expressions and activities and aorta‐derived smooth muscle cell migration and proliferation. The genetic or pharmacological inhibition of CatS by its specific inhibitor (Z‐ FL ‐COCHO) ameliorated the stressed arterial targeted molecular and morphological changes and stressed aorta‐derived smooth muscle cell migration. Both the genetic and pharmacological interventions had no effect on increased blood pressure in stressed mice. Conclusions These results demonstrate an essential role of CatS in chronic stress–related neointimal hyperplasia in response to injury, possibly via the reduction of toll‐like receptor‐2/toll‐like receptor‐4–mediated inflammation, immune action, and smooth muscle cell proliferation, suggesting that CatS will be a novel therapeutic target for stress‐related atherosclerosis‐based cardiovascular disease.

Aims Obesity is a risk factor of abdominal aortic aneurysm (AAA). Inflammatory cytokine interleukin-18 (IL18) has two receptors: IL18 receptor (IL18r) and Na-Cl co-transporter (NCC). In human and mouse AAA lesions, IL18 colocalizes to its receptors at regions rich in adipocytes, suggesting a role of adipocytes in promoting IL18 actions in AAA development. Methods and results We localized both IL18r and NCC in human and mouse AAA lesions. Murine AAA development required both receptors. In mouse AAA lesions, IL18 binding to these receptors increased at regions enriched in adipocytes or adjacent to perivascular adipose tissue. 3T3-L1 adipocytes enhanced IL18 binding to macrophages, aortic smooth muscle cells (SMCs), and endothelial cells by inducing the expression of both IL18 receptors on these cells. Adipocytes also enhanced IL18r and IL18 expression from T cells and macrophages, AAA-pertinent protease expression from macrophages, and SMC apoptosis. Perivascular implantation of adipose tissue from either diet-induced obese mice or lean mice but not that from leptin-deficient ob/ob mice exacerbated AAA development in recipient mice. Further experiments established an essential role of adipocyte leptin and fatty acid-binding protein 4 (FABP4) in promoting IL18 binding to macrophages and possibly other inflammatory and vascular cells by inducing their expression of IL18, IL18r, and NCC. Conclusion Interleukin-18 uses both IL18r and NCC to promote AAA formation. Lesion adipocyte and perivascular adipose tissue contribute to AAA pathogenesis by releasing leptin and FABP4 that induce IL18, IL18r, and NCC expression and promote IL18 actions.

To explore whether frailty, defined by frailty index (FI), is associated with the risk of elevated B-type natriuretic peptide (BNP), a surrogate endpoint of cardiovascular events. Data of 1382 community-dwelling elders who had no documented cardiovascular diseases aged 70–84 years from the ageing arm of the Rugao Longevity and Ageing Study was used. Traditional risk factor index (TI) was constructed using eight established cardiovascular-related risk factors. FI was constructed using 36 health deficits. Elevated BNP was defined as BNP ≥ 100pg/mL. Cardiovascular events include incident major cardiovascular events and cardiovascular death. During a 3-year follow-up period, 97 participants had cardiovascular events. TI was not associated with the risk of elevated BNP, but was associated with cardiovascular events (HR = 1.16, 95% CI 1.01–1.34). Frailty index was not only associated with cardiovascular events (HR = 1.32, 95% CI 1.06–1.64), but also associated with elevated BNP with an OR of 1.22 (95% CI 1.02–1.47) for each 0.1 increment. Further, both frailty (OR = 1.93, 95% CI 1.67–3.17) and pre-frailty (OR = 1.54, 95% CI 1.06–2.25) were associated with increased risk of elevated BNP. FI is associated with increased risks of both cardiovascular events and surrogated endpoint of cardiovascular disease—elevated BNP. Frailty may be a non-traditional risk factor of cardiovascular diseases and frailty index may be a measurement for early identifying high risk elderly individuals of cardiovascular abnormities.

TGF-β signaling, Smad activation, and extracellular matrix (ECM) production contribute to tissue fibrosis. Here we demonstrate that TGF-β enhances the expression of cysteinyl cathepsins CatS and CatK, but reduces CatB and CatL expression in mouse kidney tubular epithelial cells (TECs). TECs from Ctss-/- and Ctsk-/- mice show reduced expression of nuclear membrane importer protein importin-β, Smad-2/3 activation, and ECM production, yet those from Ctsb-/- and Ctsl-/- mice display increased importin-I² expression, Smad-2/3 activation, and ECM production, but reduced nuclear membrane exporter RanBP3 expression. CatS and CatK form immunocomplexes with the nuclear membrane importin-β and RanBP3 more effectively than do CatB and CatL. On the plasma membrane, CatS and CatK preferentially form immunocomplexes with and activate TGF-β receptor-2, while in contrast CatB and CatL form immunocomplexes with and inactivate TGF-β receptor-1. Unilateral ureteral obstruction-induced renal injury tests differential cathepsin activities in TGF-β signaling and tissue fibrosis. TECs in fibrotic tissues exhibit decreased CatB and CatL, but increased CatS and CatK activities. CatB- or CatL-deficiency exacerbates fibrosis, while CatS- or CatK-deficiency protects kidneys from fibrosis. These results shed novel mechanistic insight into how these closely related cysteinyl cathepsins can exert opposing effects in the TGF-β signaling cascade independent of their proteolytic properties. Funding: This work is supported by grants from the National Institute of Health [HL080472 to PL, HL123568 and HL60942 to GPS], and the RRM Charitable Fund to PL. Declaration of Interest: The authors have declared no conflicts of interest. Ethical Approval: All animal procedures conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health and were approved by the Harvard Medical School Standing Committee on Animals (protocol #03759).

Objective/background: The development of an abdominal aortic aneurysm (AAA) involves extensive extracellular matrix remodelling, leading to aortic wall weakening. This process is mediated by proteases, including cysteinyl cathepsins. Cystatins are their endogenous inhibitors. This study tested whether plasma cystatin B levels in patients with AAA differed from those of healthy controls. Methods: Plasma samples from patients with AAA and age matched controls were selected from the Viborg Vascular (VIVA) screening trial for AAA. Enzyme linked immunosorbent assay determined plasma cystatin B. T-test, logistic regression, Pearson's correlation and Cox regression tested whether plasma cystatin B correlates with AAA size and growth rate, or serves as a marker for AAA. Results: Plasma cystatin B levels were significantly higher in patients with AAA than in controls (p < 0.001). Logistic regression analysis showed that cystatin B tertile at baseline was associated with the presence of AAA before (odds ratio [OR] 1.656; p < 0.001) and after adjustment for peripheral arterial disease (PAD), chronic obstructive pulmonary disease (COPD), and previous ischaemic events (OR 1.526; p < 0.001). A t-test showed a significant association between cystatin B and PAD at screening, hospital diagnosis of COPD, previous atherosclerotic events, and use of low dose aspirin. Pearson's correlation test showed positive and significant associations between cystatin B and AAA size (r = 0.15; p < 0.001). Cox regression test showed that plasma cystatin B tertile at baseline was associated with later AAA surgical repair before (hazard ratio [HR] 1.387; p < 0.001) and after adjustment for PAD, COPD, previous ischaemic event, and maximum infrarenal aortic diameter (HR 1.523; p < 0.001). Conclusion: In contrast to prior studies that showed that cystatin C is negatively associated with AAA development, this study demonstrated a positive association between cystatin B and AAA size and associations between cystatin B tertile at baseline and AAA presence and need for later surgical repair. It is possible that these two cystatins inhibit cathepsin activity and participate in AAA with different mechanisms.

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-β, 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 CaCl 2 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.

SummaryMast cells (MCs) contribute to the pathogenesis of obesity and diabetes. This study demonstrates that leptin deficiency slants MCs toward anti-inflammatory functions. MCs in the white adipose tissue (WAT) of lean humans and mice express negligible leptin. Adoptive transfer of leptin-deficient MCs expanded ex vivo mitigates diet-induced and pre-established obesity and diabetes in mice. Mechanistic studies show that leptin-deficient MCs polarize macrophages from M1 to M2 functions because of impaired cell signaling and an altered balance between pro- and anti-inflammatory cytokines, but do not affect T cell differentiation. Rampant body weight gain in ob/ob mice, a strain that lacks leptin, associates with reduced MC content in WAT. In ob/ob mice, genetic depletion of MCs exacerbates obesity and diabetes, and repopulation of ex vivo expanded ob/ob MCs ameliorates these diseases.

The widespread coronavirus SARS-CoV-2 has already infected over 4 million people worldwide, with a death toll over 280,000. Current treatment of COVID-19 patients relies mainly on antiviral drugs lopinavir/ritonavir, arbidol, and remdesivir, the anti-malarial drugs hydroxychloroquine and chloroquine, and traditional Chinese medicine. There are over 2,118 on-going clinical trials underway, but to date none of these drugs have consistently proven effective. Cathepsin L (CatL) is an endosomal cysteine protease. It mediates the cleavage of the S1 subunit of the coronavirus surface spike glycoprotein. This cleavage is necessary for coronavirus entry into human host cells, virus and host cell endosome membrane fusion, and viral RNA release for next round of replication. Here we summarize data regarding seven CatL-selective inhibitors that block coronavirus entry into cultured host cells and provide a mechanism to block SARS-CoV-2 infection in humans. Given the rapid growth of the SARS-CoV-2-positive population worldwide, ready-to-use CatL inhibitors should be explored as a treatment option. We identify ten US FDA-approved drugs that have CatL inhibitory activity. We provide evidence that supports the combined use of serine protease and CatL inhibitors as a possibly safer and more effective therapy than other available therapeutics to block coronavirus host cell entry and intracellular replication, without compromising the immune system., Key messages • There are over 2118 on-going clinical trials for COVID-19 patients, but current results are limited. • Host cell membrane CatL and TMPRSS2 promote coronavirus cell entry by removing the virus surface spike protein S1 subunit. • In host cell endosomes, CatL removes the S1 subunit and promotes viral membrane fusion, viral RNA release, and replication. • At least 7 CatL-selective inhibitors effectively block coronavirus infection of human cells. • At least 10 US FDA-approved drugs exert CatL inhibitory activity. • Combined use of TMPRSS2 inhibitor camostat and CatL inhibitors or FDA-approved CatL inhibitory drugs merits consideration for urgent testing in COVID-19 patients. • Chloroquine-induced endosomal pH increase and protease inactivation inhibit coronavirus infection, but impair human adaptive immunity. • The protease inhibitor cocktail therapy proposed here could block coronavirus infection while sparing human adaptive immunity.

Background Extracellular matrix metabolism and cardiac cell death participate centrally in myocardial infarction (MI). This study tested the roles of collagenolytic cathepsin K (CatK) in post-MI left ventricular remodeling. Methods and results Patients with acute MI had higher plasma CatK levels (20.49 ± 7.07 pmol/L, n = 26) than those in subjects with stable angina pectoris (8.34 ± 1.66 pmol/L, n = 28, P = .01) or those without coronary heart disease (6.63 ± 0.84 pmol/L, n = 93, P = .01). CatK protein expression increases in mouse hearts at 7 and 28 days post-MI. Immunofluorescent staining localized CatK expression in cardiomyocytes, endothelial cells, fibroblasts, macrophages, and CD4+ T cells in infarcted mouse hearts at 7 days post-MI. To probe the direct participation of CatK in MI, we produced experimental MI in CatK-deficient mice (Ctsk−/−) and their wild-type (Ctsk+/+) littermates. CatK-deficiency yielded worsened cardiac function at 7 and 28 days post-MI, compared to Ctsk+/+ littermates (fractional shortening percentage: 5.01 ± 0.68 vs. 8.62 ± 1.04, P Conclusion Plasma CatK levels are increased in MI patients. Heart CatK expression is also elevated post-MI, but CatK-deficiency impairs post-MI cardiac function in mice by increasing myocardial fibrosis and cardiomyocyte death.

CLCA1 is a member of the CLCA (calcium-activated chloride channel regulator) family and plays an essential role in goblet cell mucus production from the respiratory tract epithelium. CLCA1 also regulates Ca2+-dependent Cl− transport that involves the channel protein transmembrane protein 16A (TMEM16A) and its accessary molecules. CLCA1 modulates epithelial cell chloride current and participates in the pathogenesis of mucus hypersecretory-associated respiratory and gastrointestinal diseases, including asthma, chronic obstructive pulmonary disease, cystic fibrosis, pneumonia, colon colitis, cystic fibrosis intestinal mucous disease, ulcerative colitis, and gastrointestinal parasitic infection. Most studies have been focused on the expression regulation of CLCA1 in human specimens. Limited studies used the CLCA1-deficient mice and CLCA1 blocking agents and yielded inconsistent conclusions regarding its role in these diseases. CLCA1 not only regulates mucin expression, but also participates in innate immune responses by binding to yet unidentified molecules on inflammatory cells for cytokine and chemokine production. CLCA1 also targets lymphatic endothelial cells and cancer cells by regulating lymphatic cell proliferation and lymphatic sinus growth in the lymphatic organs and controlling cancer cell differentiation, proliferation, and apoptosis, all which depend on the location of the lymphatic vessels, the type of cancers, the presence of Th2 cytokines, and possibly the availability and type of CLCA1-binding proteins. Here we summarize available studies related to these different activities of CLCA1 to assist our understanding of how this secreted modifier of calcium-activated chloride channels (CaCCs) affects mucus production and innate immunity during the pathogenesis of respiratory, gastrointestinal, and malignant diseases. Keywords: CLCA1, Mucin, Innate immunity, Respiratory diseases, Gastrointestinal disease, Cancer

To explore the associations of frailty phenotype and frailty index (FI) defined frailty and pre-frailty with mortality in a Chinese elderly population.Data of 1788 community-dwelling elders aged 70-84 years from the ageing arm of Rugao Longevity and Ageing Study, a prospective cohort study, were used. Frailty phenotype was defined using modified Fried's phenotype (FP) criteria and FI was constructed using 45 health deficits. Mortality was ascertained using the Death Registry of Rugao's Civil Affairs Bureau.During 3-year follow-up, 149 (8.3%) of the 1788 elderly subjects died. For frailty phenotype, about 9.5% of the elderly were frail and 43% were pre-frail. For FI, frail (FI 0.21) was approximately 27.5%, and pre-frail (FI: 0.1-0.21) was approximately 51.3%. Highest mortality was observed among frail participants defined by both FP and FI criteria (all Log Rank P 0.05). Frailty defined by the frailty index was associated with a 2.31 fold (95% CI 1.16-4.6) risk of all-cause death compared with robust elderly. Compared with the robust elderly, not only frailty (HR 2.24, 95% CI 1.31-3.83) defined by frailty phenotype but also pre-frailty (HR 1.51, 95% CI 1.03-2.21) was associated with risk of all-cause mortality.Frailty, defined by either phenotype or index, is associated with increased risks of mortality in elderly Chinese community population.