The Role of Bone Marrow Cells in the Phenotypic Changes Associated with Diabetic Nephropathy

The aim of our study was to investigate the role of bone marrow cells in the phenotypic changes that occur in diabetic nephropathy. Bone marrow cells were obtained from either streptozotocin-induced diabetic or untreated control C3H/He mice and transplanted into control C3H/He mice. Eight weeks after bone marrow cell transplantation, renal morphologic changes and clinical parameters of diabetic nephropathy, including the urine albumin/creatinine ratio and glucose tolerance, were measured in vivo. Expression levels of the genes encoding α1 type IV collagen and transforming growth factor-β1 in the kidney were assayed. Our results demonstrated that glucose tolerance was normal in the recipients of bone marrow transplants from both diabetic and control donors. However, compared with recipients of the control bone marrow transplant, the urinary albumin/creatinine ratio, glomerular size, and the mesangial/glomerular area ratio increased 3.3-fold (p < 0.01), 1.23-fold (p < 0.01), and 2.13-fold (p < 0.001), respectively, in the recipients of the diabetic bone marrow transplant. Expression levels of the genes encoding glomerular α1 type IV collagen and transforming growth factor-β1 were also significantly increased (p < 0.01) in the recipients of the diabetic bone marrow transplant. Our data suggest that bone marrow cells from the STZ-induced diabetic mice can confer a diabetic phenotype to recipient control mice without the presence of hyperglycemia.     

Sex Bias in Experimental Immune-Mediated,Drug-Induced Liver Injury in BALB/c Mice: Suggested Roles for Tregs,Estrogen, and IL-6

Background and Aims

Immune-mediated, drug-induced liver injury (DILI) triggered by drug haptens is more prevalent in women than in men. However, mechanisms responsible for this sex bias are not clear. Immune regulation by CD4+CD25+FoxP3+ regulatory T-cells (Tregs) and 17β-estradiol is crucial in the pathogenesis of sex bias in cancer and autoimmunity. Therefore, we investigated their role in a mouse model of immune-mediated DILI.

Methods

To model DILI, we immunized BALB/c, BALB/cBy, IL-6–deficient, and castrated BALB/c mice with trifluoroacetyl chloride-haptenated liver proteins. We then measured degree of hepatitis, cytokines, antibodies, and Treg and splenocyte function.

Results

BALB/c females developed more severe hepatitis (p<0.01) and produced more pro-inflammatory hepatic cytokines and antibodies (p<0.05) than did males. Castrated males developed more severe hepatitis than did intact males (p<0.001) and females (p<0.05). Splenocytes cultured from female mice exhibited fewer Tregs (p<0.01) and higher IL-1β (p<0.01) and IL-6 (p<0.05) than did those from males. However, Treg function did not differ by sex, as evidenced by absence of sex bias in programmed death receptor-1 and responses to IL-6, anti-IL-10, anti-CD3, and anti-CD28. Diminished hepatitis in IL-6-deficient, anti-IL-6 receptor α-treated, ovariectomized, or male mice; undetectable IL-6 levels in splenocyte supernatants from ovariectomized and male mice; elevated splenic IL-6 and serum estrogen levels in castrated male mice, and IL-6 induction by 17β-estradiol in splenocytes from naïve female mice (p<0.05) suggested that 17β-estradiol may enhance sex bias through IL-6 induction, which subsequently discourages Treg survival. Treg transfer from naïve female mice to those with DILI reduced hepatitis severity and hepatic IL-6.

Conclusions

17β-estradiol and IL-6 may act synergistically to promote sex bias in experimental DILI by reducing Tregs. Modulating Treg numbers may provide a therapeutic approach to DILI.     

Sputum IgE and Cytokines in Asthma: Relationship with Sputum Cellular Profile

Background

Local IgE production may play a role in asthma pathogenesis. The aim of the study was to assess sputum total IgE and cytokines in asthmatics according to sputum cellular phenotype.

Methods

We studied 122 subjects including 22 non atopic healthy subjects, 41 eosinophilic (sputum eosinophils ≥3%), 16 neutrophilic (sputum neutrophils >76%) and 43 pauci-granulocytic asthmatics (sputum eosinophils <3% and sputum neutrophils ≤76%) recruited from the asthma clinic at CHU Liege.Sputum supernatant total IgE (tIgE) was measured by ImmunoCAP and sputum supernatant cytokines (IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, IFN-γ and TNF-α) were measured with the Luminex xMAP Technology by using commercially available Fluorokine MAP kits.

Results

After concentrating sputum samples, total IgE was detectable in the majority of subjects. Sputum IgE was raised in asthmatics when compared to healthy subjects. Overall, asthmatics did not significantly differ from healthy subjects with respect to cytokine levels. The eosinophilic asthma phenotype, however, was characterised by raised sputum tIgE, IL-5 and IL-13 compared to healthy subjects (p<0.001, p<0.001 and p<0.05 respectively) and pauci-granulocytic asthma (p<0.01, p<0.001 and p<0.05 respectively) and raised IL-5 compared to neutrophilic asthma (p<0.01). When patients were classified according to sputum IgE levels, it appeared that IL-5, IL-6, IL-17 and TNF-α sputum supernatant levels were raised in the “IgE high” asthmatics (IgE ≥0.1 kU/l) when compared to “IgE low” asthmatics (IgE<0.1 kU/l).

Conclusion

The eosinophilic asthma phenotype was associated with raised sputum IgE and a Th2 cytokine profile. Raised sputum IgE was associated with a heterogeneous cytokine overproduction.     

Voluntary Exercise Decreases Atherosclerosis in Nephrectomised ApoE Knockout Mice

Cardiovascular disease is the main cause of morbidity and mortality in patients with kidney disease. The effectiveness of exercise for cardiovascular disease that is accelerated by the presence of chronic kidney disease remains unknown. The present study utilized apolipoprotein E knockout mice with 5/6 nephrectomy as a model of combined kidney disease and cardiovascular disease to investigate the effect of exercise on aortic plaque formation, vascular function and systemic inflammation. Animals were randomly assigned to nephrectomy or control and then to either voluntary wheel running exercise or sedentary. Following 12-weeks, aortic plaque area was significantly (p<0.05, d=1.2) lower in exercising nephrectomised mice compared to sedentary nephrectomised mice. There was a strong, negative correlation between average distance run each week and plaque area in nephrectomised and control mice (r=–0.76, p=0.048 and r=–0.73, p=0.062; respectively). In vitro aortic contraction and endothelial-independent and endothelial-dependent relaxation were not influenced by exercise (p>0.05). Nephrectomy increased IL-6 and TNF-α concentrations compared with control mice (p<0.001 and p<0.05, respectively), while levels of IL-10, MCP-1 and MIP-1α were not significantly influenced by nephrectomy or voluntary exercise (p>0.05). Exercise was an effective non-pharmacologic approach to slow cardiovascular disease in the presence of kidney disease in the apolipoprotein E knockout mouse.     

The Dual Effect of Cannabinoid Receptor-1 Deficiency on the Murine Postoperative Ileus

Introduction

Intestinal inflammatory responses play a critical role in the pathogenesis of postoperative ileus (POI). As cannabinoid receptor-1 (CB1) is involved in inhibiting gastrointestinal (GI) motility and anti-inflammation, we aimed to explore its contribution to POI.

Methods

Experimental POI was induced in adult female CB1-deficient (CB1–/–) mice and wild-type littermates (C57BL/6N) by standardized small bowel manipulation. Twenty-four hours after surgery, GI transit was assessed by charcoal transport. FITC avidin, F4/80, and myeloperoxidase immunohistochemistry techniques were used to evaluate the inflammatory response in the muscularis of ileum and colon. Expressions of p38MAPK and its phosphorylated form (pp38) in the intestine were determined. Plasma levels of proinflammatory cytokines and chemokines were measured by ELISA as well.

Results

POI was characterized by decreased GI transit (p<0.01) and accompanied by a marked intestinal and systematic inflammatory response in wild-type and CB1–/– mice. Increased numbers of inflammatory cells, including macrophages, neutrophils, and mast cells were observed in the muscularis of ileum and colon (p<0.01, or p<0.05). Plasma levels of interleukin-6 (IL-6), cytokine-induced neutrophil chemoattractant-1 (CINC-1/KC), and monocyte chemoattractant protein-1 (MCP-1) were elevated (p<0.01, or p<0.05). Expression of p38 and pp38 increased in the intestine (p<0.01, or p<0.05). CB1–/– mice showed an increased inflammatory response during POI, especially the systemic inflammatory markers, such as IL-6, KC, CINC1, and pp38 expression were increased as compared to those in WT mice (p<0.05).

Conclusions

Intestinal motility was inhibited during POI. In this condition, inhibition of motility did not seem to be altered by the absence of CB1 receptors, however, an increased inflammatory response was observed in CB1–/– mice. Hence, CB1 receptor activation rather than inhibition may reduce the inflammatory response in POI, which has a remote potential to relate into reduced inhibition of intestinal motility during POI.     

Decay-Accelerating Factor 1 Deficiency Exacerbates Leptospiral-Induced Murine Chronic Nephritis and Renal Fibrosis

Leptospirosis is a global zoonosis caused by pathogenic Leptospira, which can colonize the proximal renal tubules and persist for long periods in the kidneys of infected hosts. Here, we characterized the infection of C57BL/6J wild-type and Daf1^−/− mice, which have an enhanced host response, with a virulent Leptospira interrogans strain at 14 days post-infection, its persistence in the kidney, and its link to kidney fibrosis at 90 days post-infection. We found that Leptospira interrogans can induce acute moderate nephritis in wild-type mice and is able to persist in some animals, inducing fibrosis in the absence of mortality. In contrast, Daf1^−/− mice showed acute mortality, with a higher bacterial burden. At the chronic stage, Daf1^−/− mice showed greater inflammation and fibrosis than at 14 days post-infection and higher levels at all times than the wild-type counterpart. Compared with uninfected mice, infected wild-type mice showed higher levels of IL-4, IL-10 and IL-13, with similar levels of α-smooth muscle actin, galectin-3, TGF-β1, IL-17, IFN-γ, and lower IL-12 levels at 90 days post-infection. In contrast, fibrosis in Daf1^−/− mice was accompanied by high expression of α-smooth muscle actin, galectin-3, IL-10, IL-13, and IFN-γ, similar levels of TGF-β1, IL-12, and IL-17 and lower IL-4 levels. This study demonstrates the link between Leptospira-induced murine chronic nephritis with renal fibrosis and shows a protective role of Daf1.     

RanBP9 Overexpression Accelerates Loss of Pre and Postsynaptic Proteins in the APΔE9 Transgenic Mouse Brain

There is now compelling evidence that the neurodegenerative process in Alzheimer’s disease (AD) begins in synapses. Loss of synaptic proteins and functional synapses in the amyloid precursor protein (APP) transgenic mouse models of AD is well established. However, what is the earliest age at which such loss of synapses occurs, and whether known markers of AD progression accelerate functional deficits is completely unknown. We previously showed that RanBP9 overexpression leads to robustly increased amyloid β peptide (Aβ) generation leading to enhanced amyloid plaque burden in a mouse model of AD. In this study we compared synaptic protein levels among four genotypes of mice, i.e., RanBP9 single transgenic (Ran), APΔE9 double transgenic (Dbl), APΔE9/RanBP9 triple transgenic (Tpl) and wild-type (WT) controls. We found significant reductions in the levels of synaptic proteins in both cortex and hippocampus of 5- and 6-months-old but not 3- or 4-months-old mice. Specifically, at 5-months of age, rab3A was reduced in the triple transgenic mice only in the cortex by 25% (p<0.05) and gap43 levels were reduced only in the hippocampus by 44% (p<0.01) compared to wild-type (WT) controls. Interestingly, RanBP9 overexpression in the Tpl mice reduced gap43 levels by a further 31% (p<0.05) compared to APΔE9 mice. RanBP9 also further decreased the levels of drebrin in the hippocampus by 32% (p<0.01) and chromogranin in the cortex by 24% (p<0.05) compared to APΔE9 mice. At 6-months of age, RanBP9 expression in the cortex led to further reduction of rab3A by 30% (p<0.05) and drebrin by 38% (p<0.01) compared to APΔE9 mice. RanBP9 also increased Aβ oligomers in the cortex at 6 months. Similarly, in the hippocampus, RanBP9 expression further reduced rab3A levels by 36% (p<0.01) and drebrin levels by 33% (p<0.01). Taken together these data suggest that RanBP9 overexpression accelerates loss of synaptic proteins in the mouse brain.     

Notch-1 Mediated Cardiac Protection following Embryonic and Induced Pluripotent Stem Cell Transplantation in Doxorubicin-Induced Heart Failure

Doxorubicin (DOX), an effective chemotherapeutic drug used in the treatment of various cancers, is limited in its clinical applications due to cardiotoxicity. Recent studies suggest that transplanted adult stem cells inhibit DOX-induced cardiotoxicity. However, the effects of transplanted embryonic stem (ES) and induced pluripotent stem (iPS) cells are completely unknown in DOX-induced left ventricular dysfunction following myocardial infarction (MI). In brief, C57BL/6 mice were divided into five groups: Sham, DOX-MI, DOX-MI+cell culture (CC) media, DOX-MI+ES cells, and DOX-MI+iPS cells. Mice were injected with cumulative dose of 12 mg/kg of DOX and 2 weeks later, MI was induced by coronary artery ligation. Following ligation, 5×10⁴ ES or iPS cells were delivered into the peri-infarct region. At day 14 post-MI, echocardiography was performed, mice were sacrificed, and hearts were harvested for further analyses. Our data reveal apoptosis was significantly inhibited in ES and iPS cell transplanted hearts compared with respective controls (DOX-MI+ES: 0.48±0.06% and DOX-MI+iPS: 0.33±0.05% vs. DOX-MI: 1.04±0.07% and DOX-MI+CC: 0.96±0.21%; p<0.05). Furthermore, a significant increase in levels of Notch-1 (p<0.05), Hes1 (p<0.05), and pAkt (p<0.05) were observed whereas a decrease in the levels of PTEN (p<0.05), a negative regulator of Akt, was evident following stem cell transplantation. Moreover, hearts transplanted with stem cells demonstrated decreased vascular and interstitial fibrosis (p<0.05) as well as MMP-9 expression (p<0.01) compared with controls. Additionally, heart function was significantly improved (p<0.05) in both cell-transplanted groups. In conclusion, our data show that transplantation of ES and iPS cells blunt DOX-induced adverse cardiac remodeling, which is associated with improved cardiac function, and these effects are mediated by the Notch pathway.     

Disruption of Smad7 Promotes ANG II-Mediated Renal Inflammation and Fibrosis via Sp1-TGF-β/Smad3-NF.κB-Dependent Mechanisms in Mice

Smad7 is an inhibitory Smad and plays a protective role in obstructive and diabetic kidney disease. However, the role and mechanisms of Smad7 in hypertensive nephropathy remains unexplored. Thus, the aim of this study was to investigate the role and regulatory mechanisms of Smad7 in ANG II-induced hypertensive nephropathy. Smad7 gene knockout (KO) and wild-type (WT) mice received a subcutaneous infusion of ANG II or control saline for 4 weeks via osmotic mini-pumps. ANG II infusion produced equivalent hypertension in Smad7 KO and WT mice; however, Smad7 KO mice exhibited more severe renal functional injury as shown by increased proteinuria and reduced renal function (both p<0.05) when compared with Smad7 WT mice. Enhanced renal injury in Smad7 KO mice was associated with more progressive renal fibrosis with elevated TGF-β/Smad3 signalling. Smad7 KO mice also showed more profound renal inflammation including increased macrophage infiltration, enhanced IL-1β and TNF-α expression, and a marked activation of NF-κB signaling (all p<0.01). Further studies revealed that enhanced ANG II-mediated renal inflammation and fibrosis in Smad7 KO mice were also associated with up-regulation of Sp1 but downregulation of miR-29b expression. Taken together, the present study revealed that enhanced Sp1-TGF-β1/Smad3-NF-κB signaling and loss of miR-29 may be mechanisms by which deletion of Smad7 promotes ANG II-mediated renal fibrosis and inflammation. Thus, Smad7 may play a protective role in ANG II-induced hypertensive kidney disease.     

Low Frequency Magnetic Fields Enhance Antitumor Immune Response against Mouse H22 Hepatocellular Carcinoma

Objective

Many studies have shown that magnetic fields (MF) inhibit tumor growth and influence the function of immune system. However, the effect of MF on mechanism of immunological function in tumor-bearing mice is still unclear.

Methods

In this study, tumor-bearing mice were prepared by subcutaneously inoculating Balb/c mice with hepatocarcinoma cell line H22. The mice were then exposed to a low frequency MF (0.4 T, 7.5 Hz) for 30 days. Survival rate, tumor growth and the innate and adaptive immune parameters were measured.

Results

MF treatment could prolong survival time (n = 28, p<0.05) and inhibit tumor growth (n = 9, p<0.01) in tumor-bearing mice. Moreover, this MF suppressed tumor-induced production of cytokines including interleukin-6 (IL-6), granulocyte colony- stimulating factor (G-CSF) and keratinocyte-derived chemokine (KC) (n = 9–10, p<0.05 or 0.01). Furthermore, MF exposure was associated with activation of macrophages and dendritic cells, enhanced profiles of CD4⁺ T and CD8⁺ T lymphocytes, the balance of Th17/Treg and reduced inhibitory function of Treg cells (n = 9–10, p<0.05 or 0.01) in the mice model.

Conclusion

The inhibitory effect of MF on tumor growth was related to the improvement of immune function in the tumor-bearing mice.     

IL-12p40 Deficiency Leads to Uncontrolled Trypanosoma cruzi Dissemination in the Spinal Cord Resulting in Neuronal Death and Motor Dysfunction

Chagas’ disease is a protozoosis caused by Trypanosoma cruzi that frequently shows severe chronic clinical complications of the heart or digestive system. Neurological disorders due to T. cruzi infection are also described in children and immunosuppressed hosts. We have previously reported that IL-12p40 knockout (KO) mice infected with the T. cruzi strain Sylvio X10/4 develop spinal cord neurodegenerative disease. Here, we further characterized neuropathology, parasite burden and inflammatory component associated to the fatal neurological disorder occurring in this mouse model. Forelimb paralysis in infected IL-12p40KO mice was associated with 60% (p<0.05) decrease in spinal cord neuronal density, glutamate accumulation (153%, p<0.05) and strong demyelization in lesion areas, mostly in those showing heavy protein nitrosylation, all denoting a neurotoxic degenerative profile. Quantification of T. cruzi 18S rRNA showed that parasite burden was controlled in the spinal cord of WT mice, decreasing from the fifth week after infection, but progressive parasite dissemination was observed in IL-12p40KO cords concurrent with significant accumulation of the astrocytic marker GFAP (317.0%, p<0.01) and 8-fold increase in macrophages/microglia (p<0.01), 36.3% (p<0.01) of which were infected. Similarly, mRNA levels for CD3, TNF-α, IFN-γ, iNOS, IL-10 and arginase I declined in WT spinal cords about the fourth or fifth week after infection, but kept increasing in IL-12p40KO mice. Interestingly, compared to WT tissue, lower mRNA levels for IFN-γ were observed in the IL-12p40KO spinal cords up to the fourth week of infection. Together the data suggest that impairments of parasite clearance mechanisms in IL-12p40KO mice elicit prolonged spinal cord inflammation that in turn leads to irreversible neurodegenerative lesions.     

The Uremic Toxin Adsorbent AST-120 Abrogates Cardiorenal Injury Following Myocardial Infarction

An accelerated progressive decline in renal function is a frequent accompaniment of myocardial infarction (MI). Indoxyl sulfate (IS), a uremic toxin that accumulates from the early stages of chronic kidney disease (CKD), is contributory to both renal and cardiac fibrosis. IS levels can be reduced by administration of the oral adsorbent AST-120, which has been shown to ameliorate pathological renal and cardiac fibrosis in moderate to severe CKD. However, the cardiorenal effect of AST-120 on less severe renal dysfunction in the post-MI setting has not previously been well studied. MI-induced Sprague-Dawley rats were randomized to receive either AST-120 (MI+AST-120) or were untreated (MI+Vehicle) for 16 weeks. Serum IS levels were measured at baseline, 8 and 16 weeks. Echocardiography and glomerular filtration rate (GFR) were assessed prior to sacrifice. Renal and cardiac tissues were assessed for pathological changes using histological and immunohistochemical methods, Western blot analysis and real-time PCR. Compared with sham, MI+Vehicle animals had a significant reduction in left ventricular ejection fraction (by 42%, p<0.001) and fractional shortening (by 52%, p<0.001) as well as lower GFR (p<0.05) and increased serum IS levels (p<0.05). A significant increase in interstitial fibrosis in the renal cortex was demonstrated in MI+Vehicle animals (p<0.001). Compared with MI+Vehicle, MI+AST-120 animals had increased GFR (by 13.35%, p<0.05) and reduced serum IS (p<0.001), renal interstitial fibrosis (p<0.05), and renal KIM-1, collagen-IV and TIMP-1 expression (p<0.05). Cardiac function did not change with AST-120 treatment, however gene expression of TGF-β1 and TNF-α as well as collagen-I and TIMP-1 protein expression was decreased in the non-infarcted myocardium (p<0.05). In conclusion, reduction of IS attenuates cardio-renal fibrotic processes in the post-MI kidney. KIM-1 appears to be a sensitive renal injury biomarker in this setting and is correlated with serum IS levels.     

Aberrant Activation of the WNT/β-Catenin Signaling Pathway in Lupus Nephritis

Objective

The canonical WNT pathway has been implicated as playing important roles in the pathogenesis of a variety of kidney diseases. Recently, WNT pathway activity was reported to be elevated in the renal tissue of a lupus mouse model. This study aimed to evaluate the potential role of the WNT pathway in the pathogenesis of human lupus nephritis.

Methods

The expression of β-catenin was evaluated in renal biopsy specimens from lupus nephritis patients and control kidney tissues by immunohistochemistry and western blotting. Real-time polymerase chain reaction (RT-PCR) was used to detect RNA expression of β-catenin, Dkk-1 and Axin2. Plasma concentrations of Dkk-1 were measured by ELISA.

Results

Immunohistochemistry and western blotting revealed increased expression of β-catenin in the kidneys of patients with lupus nephritis compared with control kidney tissues (p<0.05), accompanied by an increase in mRNA expression of β-catenin (p<0.01) and axin2 (p<0.05).β-catenin was significantly greater in LN patients without renal interstitial fibrosis compared with those with renal interstitial fibrosis (p<0.01) at the mRNA expression level; the increase in β-catenin mRNA positively correlated with the creatinine clearance rate (Ccr) and negatively correlated with chronicity indices of renal tissue injury. Greater plasma Dkk-1 concentrations were found in LN patients compared with controls (p<0.05). Plasma Dkk-1 concentrations also correlated negatively with anti-dsDNA antibody levels and positively with serum C3 levels.

Conclusions

The canonical WNT/β-catenin signaling pathway was activated in lupus nephritis patients, accompanied by an increase in plasma levels of Dkk-1. Altered WNT/β-catenin signaling was related to the pathogenesis of lupus nephritis and might play a role in renal fibrosis.     

Modulation of Human Valve Interstitial Cell Phenotype and Function Using a Fibroblast Growth Factor 2 Formulation

Valve interstitial cells (VICs) are fibroblastic in nature however in culture it is widely accepted that they differentiate into a myofibroblastic phenotype. This study assessed a fibroblast culture media formulation for its ability to maintain the phenotype and function of VICs as in the intact healthy valve. Normal human VICs were cultured separately in standard DMEM and in fibroblast media consisting of FGF2 (10ng/ml), insulin (50ng/ml) and 2% FCS for at least a week. Cell morphology, aspect ratio, size, levels and distribution of protein expression, proliferation, cell cycle, contraction and migration were assessed. Some VICs and some valve endothelial cells expressed FGF2 in valve tissue and this expression was increased in calcified valves. VICs in DMEM exhibited large, spread cells whereas VICs in fibroblast media were smaller, elongated and spindly. Aspect ratio and size were both significantly higher in DMEM (p<0.01). The level of expression of α-SMA was significantly reduced in fibroblast media at day 2 after isolation (p<0.01) and the expression of α-SMA, SM22 and EDA-fibronectin was significantly reduced in fibroblast media at days 7 and 12 post-isolation (p<0.01). Expression of cytoskeletal proteins, bone marker proteins and extracellular matrix proteins was reduced in fibroblast media. Proliferation of VICs in fibroblast media was significantly reduced at weeks 1 (p<0.05) and 2 (p<0.01). Collagen gel contraction was significantly reduced in fibroblast media (p<0.05). VICs were found to have significantly fewer and smaller focal adhesions in fibroblast media (p<0.01) with significantly fewer supermature focal adhesions in fibroblast media (p<0.001). Ultrastructurally, VICs in fibroblast media resembled native VICs from intact valves. VICs in fibroblast media demonstrated a slower migratory ability after wounding at 72 hours (p<0.01). Treatment of human VICs with this fibroblast media formulation has the ability to maintain and to dedifferentiate the VICs back to a fibroblastic phenotype with phenotypic and functional characteristics ascribed to cells in the intact valve. This methodology is fundamental in the study of normal valve biology, pathology and in the field of tissue engineering.     

Attenuated atherosclerosis upon IL-17R signaling disruption in LDLr deficient mice

Atherosclerosis is an inflammatory disease characterized by the influx of macrophages and T cells and IL-17 may connect innate and adaptive immune responses involved in atherogenesis. We investigated the role of IL-17 receptor signaling in atherosclerosis and transplanted LDLr deficient recipient mice with IL-17R deficient bone marrow. Induction of atherosclerosis by Western-type diet induced a 46% reduction in lesion size in the aortic root and the plaque composition revealed no significant changes in collagen content and neutrophil counts, but a reduction in mast cell number and an increase in macrophage number. In addition, we observed a decrease in anti-oxLDL antibodies of the IgG class upon IL-17R BMT, while introduction of IL-17R deficient bone marrow resulted in a reduced IL-6 production and an increased IL-10 production.In conclusion, signaling via the IL-17 receptor in bone marrow derived cells enhances the process of atherosclerosis.     

Sorafenib Ameliorates Renal Fibrosis through Inhibition of TGF-β-Induced Epithelial-Mesenchymal Transition

ObjectiveThis study was to investigate whether sorafenib can inhibit the progression of renal fibrosis and to study the possible mechanisms of this effect.MethodsEight-week-old rats were subjected to unilateral ureteral obstruction (UUO) and were intragastrically administered sorafenib, while control and sham groups were administered vehicle for 14 or 21 days. NRK-52E cells were treated with TGF-β1 and sorafenib for 24 or 48 hours. HE and Masson staining were used to visualize fibrosis of the renal tissue in each group. The expression of α-SMA and E-cadherin in kidney tissue and NRK-52E cells were performed using immunohistochemistry and immunofluorescence. The apoptosis rate of NRK-52E cells was determined by flow cytometry analysis. The protein levels of Smad3 and p-Smad3 in kidney tissue and NRK-52E cells were detected by western blot analysis.ResultsHE staining demonstrated that kidney interstitial fibrosis, tubular atrophy, and inflammatory cell infiltration in the sorafenib-treated-UUO groups were significantly decreased compared with the vehicle-treated-UUO group (p<0.05). Masson staining showed that the area of fibrosis was significantly decreased in the sorafenib-treated-UUO groups compared with vehicle-treated-UUO group (p<0.01). The size of the kidney did not significantly increase; the cortex of the kidney was thicker and had a richer blood supply in the middle-dose sorafenib group compared with the vehicle-treated-UUO group (p<0.05). Compared with the vehicle-treated-UUO and TGF-β-stimulated NRK-52E groups, the expression of a-SMA and E-cadherin decreased and increased, respectively, in the UUO kidneys and NRK-52E cells of the sorafenib-treated groups (p<0.05). The apoptotic rate of NRK-52E cells treated with sorafenib decreased for 24 hours in a dose-dependent manner (p<0.05). Compared with the vehicle-treated UUO and TGF-β-stimulated NRK-52E groups, the ratio of p-Smad3 to Smad3 decreased in the sorafenib-treated groups (p<0.05).ConclusionOur results suggest that sorafenib may useful for the treatment of renal fibrosis through the suppression of TGF-β/Smad3-induced EMT signaling.     

Effects of a High-Fat Diet on Spontaneous Metastasis of Lewis Lung Carcinoma in Plasminogen Activator Inhibitor-1 Deficient and Wild-Type Mice

This study investigated the effects of a high-fat diet on spontaneous metastasis of Lewis lung carcinoma (LLC) in plasminogen activator inhibitor-1 deficient (PAI-1^−/−) and wild-type mice. The high-fat diet increased the number of pulmonary metastases by 60% (p<0.01), tumor cross-sectional area by 82% (p<0.05) and tumor volume by 130% (p<0.05) compared to the AIN93G diet. Deficiency in PAI-1 reduced the number of metastases by 35% (p<0.01) compared to wild-type mice. In mice fed the high-fat diet, PAI-1 deficiency reduced tumor cross-sectional area by 52% (p<0.05) and tumor volume by 61% (p<0.05) compared to their wild-type counterparts; however, PAI-1 deficiency affected neither area nor volume in mice fed the AIN93G diet. Adipose and plasma concentrations of PAI-1 were significantly higher in high-fat fed wild-type mice than in their AIN93G-fed counterparts. Adipose and plasma PAI-1 were not detectable in PAI-1^−/− mice regardless of the diet. Mice deficient in PAI-1 showed significantly greater plasma concentrations of monocyte chemotactic protein-1, tumor necrosis factor-α, leptin, vascular endothelial growth factor, tissue inhibitor of metalloproteinase-1 and insulin compared to wild-type mice, indicating a compensatory overproduction of inflammatory cytokines, angiogenic factors and insulin in the absence of PAI-1. We conclude that PAI-1 produced by the host, including that by adipose tissue, promotes high-fat enhanced metastasis of LLC.     

Augmented atherogenesis in LDL receptor deficient mice lacking both macrophage ABCA1 and ApoE

Aim

ABCA1 protects against atherosclerosis by facilitating cholesterol efflux from macrophage foam cells in the arterial wall to extracellular apolipoprotein (apo) A-I. In contrast to apoA-I, apoE is secreted by macrophages and can, like apoA-I, induce ABCA1-mediated cholesterol efflux. Yet, the combined effect of macrophage ABCA1 and apoE on lesion development is unexplored.

Methods and Results

LDL receptor knockout (KO) mice were transplanted with bone marrow from ABCA1/apoE double KO (dKO) mice, their respective single KO''s, and wild-type (WT) controls and were challenged with a high-fat/high-cholesterol diet for 9 weeks. In vitro cholesterol efflux experiments showed no differences between ABCA1 KO and dKO macrophages. The serum non-HDL/HDL ratio in dKO transplanted mice was 1.7-fold and 2.4-fold (p<0.01) increased compared to WT and ABCA1 KO transplanted mice, respectively. The atherosclerotic lesion area in dKO transplanted animals (650±94×10³ µm²), however, was 1.9-fold (p<0.01) and 1.6-fold (p<0.01) increased compared to single knockouts (ABCA1 KO: 341±20×10³ µm²; apoE KO: 402±78×10³ µm², respectively) and 3.1-fold increased (p<0.001) compared to WT (211±20×10³ µm²). When normalized for serum cholesterol exposure, macrophage ABCA1 and apoE independently protected against atherosclerotic lesion development (p<0.001). Moreover, hepatic expression levels of TNFα and IL-6 were highly induced in dKO transplanted animals (3.0-fold; p<0.05, and 4.3-fold; p<0.001, respectively). In agreement, serum IL-6 levels were also enhanced in ABCA1 KO transplanted mice (p<0.05) and even further enhanced in dKO transplanted animals (3.1-fold as compared to ABCA1 KO transplanted animals; p<0.05).

Conclusions

Combined deletion of macrophage ABCA1 and apoE results in a defect in cholesterol efflux and, compared to ABCA1 KO transplanted mice, elevated serum total cholesterol levels. Importantly, these mice also suffer from enhanced systemic and hepatic inflammation, together resulting in the observed augmented atherosclerotic lesion development.     

Interleukin-19 Impairment in Active Crohn’s Disease Patients

The exact function of interleukin-19 (IL-19) on immune response is poorly understood. In mice, IL-19 up-regulates TNFα and IL-6 expression and its deficiency increases susceptibility to DSS-induced colitis. In humans, IL-19 favors a Th2 response and is elevated in several diseases. We here investigate the expression and effects of IL-19 on cells from active Crohn’s disease (CD) patient. Twenty-three active CD patients and 20 healthy controls (HC) were included. mRNA and protein IL-19 levels were analyzed in monocytes. IL-19 effects were determined in vitro on the T cell phenotype and in the production of cytokines by immune cells. We observed that unstimulated and TLR-activated monocytes expressed significantly lower IL-19 mRNA in active CD patients than in HC (logFC = −1.97 unstimulated; −1.88 with Pam3CSK4; and −1.91 with FSL-1; p<0.001). These results were confirmed at protein level. Exogenous IL-19 had an anti-inflammatory effect on HC but not on CD patients. IL-19 decreased TNFα production in PBMC (850.7±75.29 pg/ml vs 2626.0±350 pg/ml; p<0.01) and increased CTLA4 expression (22.04±1.55% vs 13.98±2.05%; p<0.05) and IL-4 production (32.5±8.9 pg/ml vs 13.5±2.9 pg/ml; p<0.05) in T cells from HC. IL-10 regulated IL-19 production in both active CD patients and HC. We observed that three of the miRNAs that can modulate IL-19 mRNA expression, were up-regulated in monocytes from active CD patients. These results suggested that IL-19 had an anti-inflammatory role in this study. Defects in IL-19 expression and the lack of response to this cytokine could contribute to inflammatory mechanisms in active CD patients.     

Accelerated nephrotoxic nephritis is exacerbated in C1q-deficient mice

C1q deficiency strongly predisposes to the development of systemic lupus erythematosus in humans and mice. We used the model of accelerated nephrotoxic nephritis in C1q-deficient mice to explore the mechanisms behind these associations. C1q-deficient mice developed severe glomerular thrombosis within 4 days of induction of disease, whereas wild-type mice developed mild injury. These findings suggest that C1q protects from immune-mediated glomerular injury. This exacerbated thrombosis was also seen in mice triply deficient in C1q, factor B, and C2, excluding a major pathogenic role for the alternative pathway of complement in this phenomenon. However, these mice did not develop elevated creatinine levels. No exacerbation of accelerated nephrotoxic nephritis was observed in mice doubly deficient in factor B and C2, suggesting a protective role for C1q against renal inflammation that is proximal to C2 activation. There were increased murine IgG deposits, neutrophil numbers, and apoptotic cells in the glomeruli of C1q-deficient mice compared with wild-type mice. Renal expression of genes encoding procoagulant proteins was also enhanced in C1q-deficient mice. The increased IgG deposits and apoptotic cells in the glomeruli of C1q-deficient mice suggest that the exacerbation of disease may be due to a defect in the clearance of immune complexes and/or apoptotic cells from their kidneys.