IL-4-STAT6 axis amplifies histamine-induced vascular endothelial dysfunction and hypovolemic shock.

BACKGROUND: Mast cell-derived mediators induce vasodilatation and fluid extravasation, leading to cardiovascular failure in severe anaphylaxis. We previously revealed a synergistic interaction between the cytokine IL-4 and the mast cell-derived mediator histamine in modulating vascular endothelial (VE) dysfunction and severe anaphylaxis. The mechanism by which IL-4 exacerbates histamine-induced VE dysfunction and severe anaphylaxis is unknown. OBJECTIVE: We sought to identify the IL-4-induced molecular processes regulating the amplification of histamine-induced VE barrier dysfunction and the severity of IgE-mediated anaphylactic reactions. METHODS: RNA sequencing, Western blot, Ca2+ imaging, and barrier functional analyses were performed on the VE cell line (EA.hy926). Pharmacologic degraders (selective proteolysis-targeting chimera) and genetic (lentiviral short hairpin RNA) inhibitors were used to determine the roles of signal transducer and activator of transcription 3 (STAT3) and STAT6 in conjunction with in vivo model systems of histamine-induced hypovolemic shock. RESULTS: IL-4 enhancement of histamine-induced VE barrier dysfunction was associated with increased VE-cadherin degradation, intracellular calcium flux, and phosphorylated Src levels and required transcription and de novo protein synthesis. RNA sequencing analyses of IL-4-stimulated VE cells identified dysregulation of genes involved in cell proliferation, cell development, and cell growth, and transcription factor motif analyses revealed a significant enrichment of differential expressed genes with putative STAT3 and STAT6 motif. IL-4 stimulation in EA.hy926 cells induced both serine residue 727 and tyrosine residue 705 phosphorylation of STAT3. Genetic and pharmacologic ablation of VE STAT3 activity revealed a role for STAT3 in basal VE barrier function; however, IL-4 enhancement and histamine-induced VE barrier dysfunction was predominantly STAT3 independent. In contrast, IL-4 enhancement and histamine-induced VE barrier dysfunction was STAT6 dependent. Consistent with this finding, pharmacologic knockdown of STAT6 abrogated IL-4-mediated amplification of histamine-induced hypovolemia. CONCLUSIONS: These studies unveil a novel role of the IL-4/STAT6 signaling axis in the priming of VE cells predisposing to exacerbation of histamine-induced anaphylaxis.

Expression analysis of pro-apoptotic BAX and anti-apoptotic BCL-2 genes in relation to lactation performance in Deoni and Holstein Friesian crossbred cows.

The study was designed with the objective of expression analysis of pro-apoptotic BAX and anti-apoptotic BCL-2 genes on lactation performance in Bos indicus and HF crossbred cows during early lactation. BAX/BCL-2 mRNA expression ratio in HF crossbreds showed a steady increase from 30th day to 90th day, but in Deoni cows the ratio exhibited a different pattern, which increased from day 30 to day 60, decreased on day 75, and then increased on day 90. BAX/BCL-2 expression ratio in Deoni and HF crossbreds were lowest on day 30 and highest on day 90. On contrary, the milk yield was highest on day 30 and lowest on day 90 suggesting BCL-2 gene favors milk production and BAX gene oppose milk production. In comparison to HF crossbreds, Deoni cows exhibited highest BAX/BCL-2 ratio at the end of early lactation, indicating Bos indicus cows were more sensitive to apoptosis than HF crossbreds. Comparison of daily milk yield with BAX/BCL-2 mRNA expression ratio revealed significant negative correlation with a correlation coefficient of -0.98 (P < 0.01) and -0.95 (P < 0.05) in Deoni and HF crossbred cows, respectively. Our study provides new insights into understanding the genetic control of mammary apoptosis between Bos indicus and HF crossbreds.

The role of the glypican and syndecan families of heparan sulfate proteoglycans in cardiovascular function and disease.

Heparan sulfate proteoglycans (HSPGs) are proteoglycans formed by a core protein to which one or multiple heparan sulfate chains are covalently bound. They are ubiquitously expressed in cellular surfaces and can be found in the extracellular matrix and secretory vesicles. The cellular effects of HSPGs comprehend multiple functionalities that include 1) the interaction with other membrane surface proteins to act as a substrate for cellular migration, 2) acting as a binding site for circulating molecules, 3) to have a receptor role for proteases, 4) to act as a coreceptor that can provide finetuning of growth factor receptor activity threshold, and 5) to activate intracellular signaling pathways (Sarrazin S, Lamanna WC, Esko JD. Cold Spring Harb Perspect Biol 3: a004952, 2011). Among the different families of HSPGs, the syndecan and glypican families of HSPGs have gained increased attention in relation to their effects on cardiovascular cells and potential role in disease progression. In this review, we will summarize the effects of syndecan and glypican homologs on the different cardiovascular cell types and discuss their contribution to common processes found in cardiovascular diseases (inflammation, hypertrophy, and vascular remodeling) as well as their potential role in the development and progression of specific diseases including hypertension, heart failure, and atherosclerosis.

Deep Proteome Profiling of Semen of Indian Indigenous Malnad Gidda (Bos indicus) Cattle.

Malnad Gidda is a dwarf indigenous cattle breed of India, which is known for its uniqueness of calving every year under a low input grazing system of rearing. Bulls of Malnad Gidda are known to be highly fertile even in stress conditions. However, the proteomic profiling of semen of this breed has not been investigated so far, which might provide a platform for a better understanding of its semen quality and male fertility. Therefore, we made an effort to characterize and quantify the proteome of seminal plasma and spermatozoa components of Malnad Gidda semen using a high-resolution mass spectrometry platform. We identified 2814 proteins from spermatozoa and 1974 proteins from the seminal plasma of this breed. Furthermore, >90% of proteins from each fraction were quantified using the intensity-based absolute quantification. We observed signal peptides in 33% of seminal plasma proteins, indicating their secretory nature. Gene Ontology analysis revealed their involvement in cytoskeletal assembly associated with sperm head, sperm motility, acrosome reaction, seminal plasma binding, and spermatogenesis-associated protein. An in-depth proteome profiling of semen of a unique indigenous cattle breed of India was carried out. Our findings could provide a reference for further studies on sperm functions, semen quality, and reproductive health of Bos indicus cattle. Mass spectrometry data generated in this study is deposited and publicly made available through ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD014172.

Long-Term Efficacy and Safety of Immunomodulatory Therapy for Atherosclerosis.

BACKGROUND AND AIMS: The long-term effect of immune tolerance has not been explored so far in atherosclerosis. In the present study, we assessed the effect of mucosal tolerance to a multi antigenic construct expressing three peptides from ApoB, HSP60, and outer membrane protein from Chlamydia pneumonia (AHC) for 30 weeks at every 6-week interval to understand the kinetics of immune modulation in disease progression. The safety profile of the molecule was also evaluated in mice. METHODS: Apobtm2SgyLdlrtm1Her/J mice (5-6 weeks) were orally dosed with multi antigenic construct (AHC) molecule on alternate days, followed by high-fat diet feeding to initiate atherosclerosis. RESULTS: Treated animals showed an efficient reduction in plaque growth and lipid accumulation at 6 weeks (49%, p < 0.01) and 12 weeks (42.3%, p < 0.01) which decreased to 29% (p = 0.0001) at 18 weeks and at later time points. Macrophage accumulation was significantly lower at all time points (53% at 12 weeks to 27% at 30 weeks). Regulatory T cells increased in the spleen following treatment until 12 weeks (week 0 (2.57 ± 0.18 vs. 6.36 ± 0.03, p = 0.02), week 6 (4.52 ± 0.2 vs. 8.87 ± 0.32, p = 0.02), and week 12 (8.74 ± 0.37 vs. 15.4 ± 0.27, p = 0.02)) but showed a decline later. A similar trend was observed with tolerogenic dendritic cells. We observed an increase in antibody levels to low-density lipoprotein and oxidized LDL at later stages. AHC molecule was found to be safe in acute and repeated dose toxicity studies. CONCLUSIONS: Our results suggest that immune tolerance to AHC protein by oral administration is able to provide efficient atheroprotection up to 18 weeks and moderately at later stages. Apart from immune regulatory cells, protective antibodies may also have a role in controlling atherosclerosis.

Author Correction: Immune regulation by oral tolerance induces alternate activation of macrophages and reduces markers of plaque destabilization in Apobtm2Sgy/Ldlrtm1Her/J mice.

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Immune regulation by oral tolerance induces alternate activation of macrophages and reduces markers of plaque destabilization in Apobtm2Sgy/Ldlrtm1Her/J mice.

Atherosclerosis is the leading cause for cardiovascular mortality. We determined the effect of multi-antigenic construct expressing three peptides AHC (ApoB100, HSP60 and outer membrane protein of chlamydia pneumonia) in stabilizing advanced atherosclerosis in Apobtm2Sgy/Ldlrtm1Her/J mice. Atherosclerosis was induced by feeding high fat diet (HFD) to mice for 10 weeks, followed by five oral dosing with purified AHC or ovalbumin on alternate days and continued on HFD for another 10 weeks. Tolerance was associated with significantly higher numbers of regulatory T cells both in aortic sinus and spleen with higher mRNA expression of CTLA4 (3 fold), Foxp3 (1.4 folds) and TGF-ß (1.62) in aorta. Tregs cells were found to induce alternate activation of macrophages to M2 phenotype, with a reduction in plaque inflammation. AHC treatment showed evidence of plaque stabilization as observed by reduction in plaque necrosis in aortic sinus (35.8%) and in brachiocephalic artery (26%), with reduced expression of Tissue factor and MMP9. Macrophage apoptosis was reduced and collagen content was enhanced by treatment. Our results suggest that tolerance to atherogenic peptides increases regulatory T cells which activate M2 macrophages, prevent T cell proliferation and reduce plaque destabilization and inflammatory markers thus providing evidences for plaque stabilization in mice with advanced atherosclerosis.

Oral administration of recombinant Mycobacterium smegmatis expressing a tripeptide construct derived from endogenous and microbial antigens prevents atherosclerosis in ApoE(-/-) mice.

INTRODUCTION: Immunotherapy by inducing oral tolerance to atherogenic self-antigens is gaining importance as an alternative treatment modality for atherosclerosis. The use of live bacterial vectors to express the recombinant antigen in vivo will obviate the need for large-scale purification of recombinant protein and may also augment the efficacy of oral tolerance induction. AIM: The objective of the study was to explore the use of recombinant Mycobacterium smegmatis as a live vector for oral delivery of antigens to induce immune tolerance. METHOD AND RESULTS: We developed a M. smegmatis vector to secrete a recombinant tripeptide construct (AHC; peptides from Apolipoprotein B, Heat-shock protein 60 and Chlamydia pneumoniae outer membrane protein) expressed in a dendroaspin protein scaffold in pJH154 background. Immune response and oral tolerance to the cloned peptides were studied in C57/BL6 mice. The efficacy of this live vaccine to control atherosclerosis was studied in ApoE(-/-) knockout mice in C57/BL6 background. Oral administration of M. smegmatis secreting the cloned AHC antigen was found to induce tolerance to cloned protein and reduce the development of atherosclerosis by 24.0% compared to control. Protection against atherosclerosis was associated with increase in expression of regulatory T cell-associated markers including CTLA4 (1.8-fold), Foxp3 (2.6-fold), TGF-ß (2.8-fold), IL10 (2.9-fold), and reduction in lipids, macrophage infiltration, and expression of inflammatory mediators in aorta. CONCLUSIONS: Our results suggest that M. smegmatis can be developed as an oral carrier of recombinant proteins to treat inflammatory autoimmune diseases.

Understanding the progression of atherosclerosis through gene profiling and co-expression network analysis in Apob(tm2Sgy)Ldlr(tm1Her) double knockout mice.

The objective of the study was to gain molecular insights into the progression of atherosclerosis in Apob(tm2Sgy)Ldlr(tm1Her) mice, using transcriptome profiles. Weighted gene co network analysis (WGCNA) and time course analysis using limma were used to study disease progression from 0 to 20weeks. Five co-expression modules were identified by WGCNA using the expression values of 2153 genes. Genes associated with autophagy, endoplasmic reticulum stress, inflammation and lipid metabolism were differentially expressed at early stages of atherosclerosis. Time course analysis highlighted activation of inflammatory gene signaling at 4weeks, cell proliferation and calcification at 8weeks, amyloid like structures and oxidative stress at 14weeks and enhanced production of inflammatory cytokines at 20weeks. Our results suggest that maximum gene perturbations occur during early atherosclerosis which could be the danger signals associated with subclinical disease. Understanding these genes and associated pathways can help in improvement of diagnostic and therapeutic targets for atherosclerosis.