ONX 0914 Lacks Selectivity for the Cardiac Immunoproteasome in CoxsackievirusB3 Myocarditis of NMRI Mice and Promotes Virus-Mediated Tissue Damage

Inhibition of proteasome function by small molecules is highly efficacious in cancer treatment. Other than non-selective proteasome inhibitors, immunoproteasome-specific inhibitors allow for specific targeting of the proteasome in immune cells and the profound anti-inflammatory potential of such compounds revealed implications for inflammatory scenarios. For pathogen-triggered inflammation, however, the efficacy of immunoproteasome inhibitors is controversial. In this study, we investigated how ONX 0914, an immunoproteasome-selective inhibitor, influences CoxsackievirusB3 infection in NMRI mice, resulting in the development of acute and chronic myocarditis, which is accompanied by formation of the immunoproteasome in heart tissue. In groups in which ONX 0914 treatment was initiated once viral cytotoxicity had emerged in the heart, ONX 0914 had no anti-inflammatory effect in the acute or chronic stages. ONX 0914 treatment initiated prior to infection, however, increased viral cytotoxicity in cardiomyocytes, promoting infiltration of myeloid immune cells into the heart. At this stage, ONX 0914 completely inhibited the β5 subunit of the standard cardiac proteasome and less efficiently blocked its immunoproteasome counterpart LMP7. In conclusion, ONX 0914 unselectively perturbs cardiac proteasome function in viral myocarditis of NMRI mice, reduces the capacity of the host to control the viral burden and promotes cardiac inflammation

α1A-Adrenergic Receptor-Directed Autoimmunity Induces Left Ventricular Damage and Diastolic Dysfunction in Rats

BACKGROUND: Agonistic autoantibodies to the alpha(1)-adrenergic receptor occur in nearly half of patients with refractory hypertension; however, their relevance is uncertain. METHODS/PRINCIPAL FINDINGS: We immunized Lewis rats with the second extracellular-loop peptides of the human alpha(1A)-adrenergic receptor and maintained them for one year. Alpha(1A)-adrenergic antibodies (alpha(1A)-AR-AB) were monitored with a neonatal cardiomyocyte contraction assay by ELISA, and by ERK1/2 phosphorylation in human alpha(1A)-adrenergic receptor transfected Chinese hamster ovary cells. The rats were followed with radiotelemetric blood pressure measurements and echocardiography. At 12 months, the left ventricles of immunized rats had greater wall thickness than control rats. The fractional shortening and dp/dt(max) demonstrated preserved systolic function. A decreased E/A ratio in immunized rats indicated a diastolic dysfunction. Invasive hemodynamics revealed increased left ventricular end-diastolic pressures and decreased dp/dt(min). Mean diameter of cardiomyocytes showed hypertrophy in immunized rats. Long-term blood pressure values and heart rates were not different. Genes encoding sarcomeric proteins, collagens, extracellular matrix proteins, calcium regulating proteins, and proteins of energy metabolism in immunized rat hearts were upregulated, compared to controls. Furthermore, fibrosis was present in immunized hearts, but not in control hearts. A subset of immunized and control rats was infused with angiotensin (Ang) II. The stressor raised blood pressure to a greater degree and led to more cardiac fibrosis in immunized, than in control rats. CONCLUSIONS/SIGNIFICANCE: We show that alpha(1A)-AR-AB cause diastolic dysfunction independent of hypertension, and can increase the sensitivity to Ang II. We suggest that alpha(1A)-AR-AB could contribute to cardiovascular endorgan damage

Effects of empagliflozin and target-organ damage in a novel rodent model of heart failure induced by combined hypertension and diabetes

Type 2 diabetes mellitus and hypertension are two major risk factors leading to heart failure and cardiovascular damage. Lowering blood sugar by the sodium-glucose co-transporter 2 inhibitor empagliflozin provides cardiac protection. We established a new rat model that develops both inducible diabetes and genetic hypertension and investigated the effect of empagliflozin treatment to test the hypothesis if empagliflozin will be protective in a heart failure model which is not based on a primary vascular event. The transgenic Tet29 rat model for inducible diabetes was crossed with the mRen27 hypertensive rat to create a novel model for heart failure with two stressors. The diabetic, hypertensive heart failure rat (mRen27/tetO-shIR) were treated with empagliflozin (10 mg/kg/d) or vehicle for 4 weeks. Cardiovascular alterations were monitored by advanced speckle tracking echocardiography, gene expression analysis and immunohistological staining. The novel model with increased blood pressure und higher blood sugar levels had a reduced survival compared to controls. The rats develop heart failure with reduced ejection fraction. Empagliflozin lowered blood sugar levels compared to vehicle treated animals (182.3 ± 10.4 mg/dl vs. 359.4 ± 35.8 mg/dl) but not blood pressure (135.7 ± 10.3 mmHg vs. 128.2 ± 3.8 mmHg). The cardiac function was improved in all three global strains (global longitudinal strain − 8.5 ± 0.5% vs. − 5.5 ± 0.6%, global radial strain 20.4 ± 2.7% vs. 8.8 ± 1.1%, global circumferential strain − 11.0 ± 0.7% vs. − 7.6 ± 0.8%) and by increased ejection fraction (42.8 ± 4.0% vs. 28.2 ± 3.0%). In addition, infiltration of macrophages was decreased by treatment (22.4 ± 1.7 vs. 32.3 ± 2.3 per field of view), despite mortality was not improved. Empagliflozin showed beneficial effects on cardiovascular dysfunction. In this novel rat model of combined hypertension and diabetes, the improvement in systolic and diastolic function was not secondary to a reduction in left ventricular mass or through modulation of the afterload, since blood pressure was not changed. The mRen27/tetO-shIR strain should provide utility in separating blood sugar from blood pressure-related treatment effects

A Helminth-Derived Chitinase Structurally Similar to Mammalian Chitinase Displays Immunomodulatory Properties in Inflammatory Lung Disease

From Hindawi via Jisc Publications RouterHistory: publication-year 2021, received 2021-09-02, accepted 2021-10-25, pub-print 2021-11-25, archival-date 2021-11-25Publication status: PublishedFunder: Coronado BiosciencesFunder: FAZIT Stiftung; doi: http://dx.doi.org/10.13039/501100003099Funder: Deutsche Forschungsgemeinschaft; doi: http://dx.doi.org/10.13039/501100001659; Grant(s): GRK 1673Immunomodulation of airway hyperreactivity by excretory-secretory (ES) products of the first larval stage (L1) of the gastrointestinal nematode Trichuris suis is reported by us and others. Here, we aimed to identify the proteins accounting for the modulatory effects of the T. suis L1 ES proteins and studied six selected T. suis L1 proteins for their immunomodulatory efficacy in a murine OVA-induced allergic airway disease model. In particular, an enzymatically active T. suis chitinase mediated amelioration of clinical signs of airway hyperreactivity, primarily associated with suppression of eosinophil recruitment into the lung, the associated chemokines, and increased numbers of RELMα+ interstitial lung macrophages. While there is no indication of T. suis chitinase directly interfering with dendritic cell activation or antigen presentation to CD4 T cells, treatment of allergic mice with the worm chitinase influenced the hosts’ own chitinase activity in the inflamed lung. The three-dimensional structure of the T. suis chitinase as determined by high-resolution X-ray crystallography revealed high similarities to mouse acidic mammalian chitinase (AMCase) but a unique ability of T. suis chitinase to form dimers. Our data indicate that the structural similarities between the parasite and host chitinase contribute to the disease-ameliorating effect of the helminth-derived chitinase on allergic lung inflammation

Statins Reverse Postpartum Cardiovascular Dysfunction in a Rat Model of Preeclampsia.

Preeclampsia is associated with increased cardiovascular long-term risk; however, the underlying functional and structural mechanisms are unknown. We investigated maternal cardiac alterations after preeclampsia. Female rats harboring the human angiotensinogen gene [TGR(hAogen)L1623] develop a preeclamptic phenotype with hypertension and albuminuria during pregnancy when mated with male rats bearing the human renin gene [TGR(hRen)L10J] but behave physiologically normal before and after pregnancy. Furthermore, rats were treated with pravastatin. We tested the hypothesis that statins are a potential therapeutic intervention to reduce cardiovascular alterations due to simulated preeclamptic pregnancy. Although hypertension persists for only 8 days in pregnancy, former preeclampsia rats exhibit significant cardiac hypertrophy 28 days after pregnancy observed in both speckle tracking echocardiography and histological staining. In addition, fibrosis and capillary rarefaction was evident. Pravastatin treatment ameliorated the remodeling and improved cardiac output postpartum. Preeclamptic pregnancy induces irreversible structural changes of cardiac hypertrophy and fibrosis, which can be moderated by pravastatin treatment. This pathological cardiac remodeling might be involved in increased cardiovascular risk in later life

Regulation of body weight and energy homeostasis by neuronal cell adhesion molecule 1

Susceptibility to obesity is linked to genes regulating neurotransmission, pancreatic beta-cell function and energy homeostasis. Genome-wide association studies have identified associations between body mass index and two loci near cell adhesion molecule 1 (CADM1) and cell adhesion molecule 2 (CADM2), which encode membrane proteins that mediate synaptic assembly. We found that these respective risk variants associate with increased CADM1 and CADM2 expression in the hypothalamus of human subjects. Expression of both genes was elevated in obese mice, and induction of Cadm1 in excitatory neurons facilitated weight gain while exacerbating energy expenditure. Loss of Cadm1 protected mice from obesity, and tract-tracing analysis revealed Cadm1-positive innervation of POMC neurons via afferent projections originating from beyond the arcuate nucleus. Reducing Cadm1 expression in the hypothalamus and hippocampus promoted a negative energy balance and weight loss. These data identify essential roles for Cadm1-mediated neuronal input in weight regulation and provide insight into the central pathways contributing to human obesity.</p

Chronic Overexpression of Bradykinin in Kidney Causes Polyuria and Cardiac Hypertrophy

Acute intra-renal infusion of bradykinin increases diuresis and natriuresis via inhibition of vasopressin activity. However, the consequences of chronically increased bradykinin in the kidneys have not yet been studied. A new transgenic animal model producing an excess of bradykinin by proximal tubular cells (KapBK rats) was generated and submitted to different salt containing diets to analyze changes in blood pressure and other cardiovascular parameters, urine excretion, and composition, as well as levels and expression of renin-angiotensin system components. Despite that KapBK rats excrete more urine and sodium, they have similar blood pressure as controls with the exception of a small increase in systolic blood pressure (SBP). However, they present decreased renal artery blood flow, increased intrarenal expression of angiotensinogen, and decreased mRNA expression of vasopressin V1A receptor (AVPR1A), suggesting a mechanism for the previously described reduction of renal vasopressin sensitivity by bradykinin. Additionally, reduced heart rate variability (HRV), increased cardiac output and frequency, and the development of cardiac hypertrophy are the main chronic effects observed in the cardiovascular system. In conclusion: (1) the transgenic KapBK rat is a useful model for studying chronic effects of bradykinin in kidney; (2) increased renal bradykinin causes changes in renin angiotensin system regulation; (3) decreased renal vasopressin sensitivity in KapBK rats is related to decreased V1A receptor expression; (4) although increased renal levels of bradykinin causes no changes in mean arterial pressure (MAP), it causes reduction in HRV, augmentation in cardiac frequency and output and consequently cardiac hypertrophy in rats after 6 months of age

Silencing the CSF-1 Axis Using Nanoparticle Encapsulated siRNA Mitigates Viral and Autoimmune Myocarditis

Myocarditis is an inflammatory disease of the heart muscle most commonly caused by viral infection and often maintained by autoimmunity. Virus-induced tissue damage triggers chemokine production and, subsequently, immune cell infiltration with pro-inflammatory and pro-fibrotic cytokine production follows. In patients, the overall inflammatory burden determines the disease outcome. Following the aim to define specific molecules that drive both immunopathology and/or autoimmunity in inflammatory heart disease, here we report on increased expression of colony stimulating factor 1 (CSF-1) in patients with myocarditis. CSF-1 controls monocytes originating from hematopoietic stem cells and subsequent progenitor stages. Both, monocytes and macrophages are centrally involved in mediating tissue damage and fibrotic scarring in the heart. CSF-1 influences monocytes via engagement of CSF-1 receptor, and it is also produced by cells of the mononuclear phagocyte system themselves. Based on this, we sought to modulate the virus-triggered inflammatory response in an experimental model of Coxsackievirus B3-induced myocarditis by silencing the CSF-1 axis in myeloid cells using nanoparticle-encapsulated siRNA. siCSF-1 inverted virus-mediated immunopathology as reflected by lower troponin T levels, a reduction of accumulating myeloid cells in heart tissue and improved cardiac function. Importantly, pathogen control was maintained and the virus was efficiently cleared from heart tissue. Since viral heart disease triggers heart-directed autoimmunity, in a second approach we investigated the influence of CSF-1 upon manifestation of heart tissue inflammation during experimental autoimmune myocarditis (EAM). EAM was induced in Balb/c mice by immunization with a myocarditogenic myosin-heavy chain-derived peptide dissolved in complete Freund's adjuvant. siCSF-1 treatment initiated upon established disease inhibited monocyte infiltration into heart tissue and this suppressed cardiac injury as reflected by diminished cardiac fibrosis and improved cardiac function at later states. Mechanistically, we found that suppression of CSF-1 production arrested both differentiation and maturation of monocytes and their precursors in the bone marrow. In conclusion, during viral and autoimmune myocarditis silencing of the myeloid CSF-1 axis by nanoparticle-encapsulated siRNA is beneficial for preventing inflammatory tissue damage in the heart and preserving cardiac function without compromising innate immunity's critical defense mechanisms

Identification and characterization of new disease Genes for cardiomyopathies

GesamtdissertationDie arrhythmogene rechtsventrikuläre Kardiomyopathie (ARVC) ist gekennzeichnet durch einen progressiven Ersatz der rechtsventrikulären Kardiomyozyten durch Fett- und Bindegewebe. Diese pathologischen Veränderungen können zu ventrikulären Tachyarrhythmien und zum plötzlichen Herztod führen. Wir konnten im humanen Plakophilin-2 Gen (PKP2) bei 120 nicht verwandten ARVC Patienten 32 heterozygote Mutationen identifizierten. Plakophilin-2 ist ein essenzieller Bestandteil des Desmosoms. Die kardialen Desmosomen gewährleisten die mechanische Stabilität des Herzmuskels und übertragen die Kraft des kontraktilen Apparates. Zusätzlich können sie an der Signaltransduktion der Kardiomyozyten teilnehmen. Die Mehrzahl der identifizierten Mutationen führen zur Insertion von verfrühten Stopp-Kodons. Kardiale Expressionsanalysen von Herzbiopsien zeigen sowohl das Fehlen eines trunkierten Proteins als auch eine deutliche Reduktion des PKP2-Wildtypproteins. Weiterhin konnten wir eine Mutation in Intron 5 (c.631-2AG) im Gen des desmosomalen Cadherin Desmocollin 2 (DSC2) identifizieren, welche zu einer kryptischen Spleiß-Akzeptorstelle und zu einem verfrühten Stopp-Kodon führt. Quantitative Analysen ergaben, dass die kardiale DSC2-Expression deutlich verringert ist, bei gleichzeitigem Fehlen des mutierten Transkriptes. Der Morpholino-Knockdown des DSC2-Orthologs im Zebrafisch führt zur Reduzierung der desmosomalen Plaque-Fläche, zum Verlust der extrazellulär gelegenen elektronendichten desmosomalen Mittellinie und ist mit kardialen Kontraktilitätsstörungen assoziiert. Die Knockdown-Experimente belegen die Notwendigkeit von DSC2 für die normale Herzentwicklung und Funktion. Des Weiteren gelang es uns, einen neuen Lokus für die dilatative Kardiomyopathie (DCM) mit diffuser myokardialer Fibrose und plötzlichem Herztod auf Chromosom 10q25-q26 nachzuweisen. Die Identifizierung des verantwortlichen Krankheitsgens in diesem Intervall könnte wichtige Hinweise geben auf die zu Grunde liegenden Pathomechanismen von DCM und plötzlichem Herztod.Arrhythmogenic right ventricular cardiomyopathy (ARVC) is characterized by the replacement of right ventricular myocytes with fatty or fibrofatty tissue. These pathologic changes lead to a clinical disorder with ventricular tachyarrhythmias and sudden death. We have identified 32 heterozygous mutations in the plakophilin-2 gene (PKP2) from 120 unrelated ARVC patients. Plakophilin-2 is an essential arm repeat protein of the desmosome. Cardiac desmosomes provide mechanical stability, transmit contractile force between and participate in signal transduction in myocytes. The majority of PKP2 mutations introduce premature stop codons. Cardiac expression analysis revealed absence of the truncated, and reduction of the normal plakophilin-2 protein in a patient with a PKP2 deletion mutation. We additionally identified a heterozygous splice acceptor site mutation in intron 5 (c.631-2Aï ®G) of the human desmocollin 2 (DSC2) gene, which led to use of a cryptic splice acceptor site and the creation of a downstream premature termination codon. Quantitative analysis of cardiac DSC2 expression in patient specimens revealed a marked reduction in the abundance of the mutant transcript. Morpholino knockdown in zebrafish embryos revealed the requirement for dsc2 in the establishment of the normal myocardial structure and function with reduced desmosomal plaque area, loss of the desmosome extracellular electron-dense midlines and associated myocardial contractility defects. These data identify DSC2 mutations as a cause for ARVC in humans, and demonstrate that physiologic levels of DSC2 are crucial for normal cardiac desmosome formation, early cardiac morphogenesis and cardiac function. In another study we were able to map a novel locus for cardiomyopathy, diffuse myocardial fibrosis, and sudden death to chromosome 10q25-q26. The identification of the causative gene in this interval will be an important step in understanding the fundamental mechanisms of heart failure and sudden death

Antibody binding and functional properties of whey protein hydrolysates obtained under high pressure

7 pages, 4 figures.-- Available online May 18, 2008.This paper examines the potential of high hydrostatic pressure to produce whey protein hydrolysates that combine low immunoglobulin (Ig)G- and IgE-binding with acceptable functional properties, with the aim to produce milk-based ingredients with reduced potential allergenicity that could be used in hypoallergenic foods. Treatment with pepsin and chymotrypsin under high pressure produced, in minutes, hydrolysates in which α-lactalbumin and β-lactoglobulin were totally proteolysed, giving rise to large and hydrophobic peptides. Such hydrolysates presented reduced antigenicity and human IgE-binding properties. The hydrolysates obtained with pepsin at 400 MPa showed improved heat stability, particularly at a pH, close to the isoelectric point of the whey proteins, and their emulsion activity indexes at pH 7.0 were superior to those of the untreated whey proteins. These results suggest that the peptides present retained low antigenicity together with sufficient capacity to form emulsions.This work has been supported by the projects AGL-2004-03322, CONSOLIDER-INGENIO CSD-2007-00063 (Ministerio de Educación y Ciencia, Spain) and S-0505/AGR/0153 (Comunidad Autónoma de Madrid, Spain).Peer reviewe