| BackgroundHeart failure is the leading cause of death in developing and developed countries. It is generally preceded by remodeling processes to adapt to alterations in wall tension in the myocardium or stress by external (hormonal) stimuli. In this regard, cardiac remodeling describes the heart's fascinating capability to respond and adapt to various stimuli such as neurohumoral factors, pressure or volume overload, biomechanical stress, myocarditis, or inherited mutations. Initial adaption to these alterations are ultimate goals of cardiac remodeling and facilitating preservation or even augmentation of cardiac pump function. While continuous cardiac remodeling will ultimately lead to heart failure. Cardiac hypertrophy is the pathological manifestations of cardiac remodeling induced by pressure overload which is characterized by multiple facets of ultimately maladaptive mechanisms such as increase in cell size, increase protein synthesis, altered calcium handling, changes in metabolic patterns and gene expression reprogram, affection of individual cell survival as well as modifications of the extracellular environment by fibrosis. Various factors influence cardiac hypertrophy through their actions on membrane receptors, which translate these stimuli into respective stress response pathways such as mitogen-activated protein kinases (MPAKs), PI3K-AKT, NFAT. Recent studies have proved that immune cells (Macrophages, T lymphocytes) coordinate cardiomyocyte and noncardiomyocyte responses during cardiac hypertrophy through multiple cytokines. Thus, the regulation of immune cell phenotypes represents an important pharma-cological approach.OX40 (CD 134) belongs to the tumour necrosis factor (TNF) receptor family, also named TNF-receptor super family-4 (TNFrsf-4). OX40 was initially identified on in vitro activated CD4+ T cells, but subsequent studies revealed that OX40 is also expressed on activated CD8+ T cells and neutrophils. OX40 expression peaks 48-72 hours after TCR stimulation and is down-regulated by approximately 120 hours after the initial TCR stimulation. Interaction between OX40 and its ligand, OX40L, dramatically increases the proliferation, cytokine production, survival and differentiation of CD4+ and CD8+ T cells. OX40 is also constitutively expressed on the majority of FoxP3+CD25+CD4+ regulatory T cells (Treg). Recent work suggests that OX40-specific signaling can abrogate the suppressive activity of Treg. T cells are recruited to the heart during cardiac hypertrophy. T cells have the ability to regulate inflmmation through interactions with various immune cells and can activate, modulate, or suppress other leukocyte inflmmatory responses.Therefore, we hypothesized that whether by changing the ratio of anti-inflammatory factors and proinflammatory cytokine secreted by CD4+ T lymphocytes, cardiac remodeling induced by pressure overload can be ameliorated. Whether OX40 is involved in the regulation of cardiac remodeling induced by pressure overload by modulation CD4+ T cell activation, proliferation and differentiation? In this study, we investigated one KO mouse line lacking OX40 to explore the role of OX40 in pressure overload induced cardiac remodeling.MethodsPart one:Using male C57BL/6 mice (8 to 10 weeks,23.5?27.5g), we established pressure overload-induced cardiac hypertrophy in mice. The mice were randomly divided into 4 groups, respectively, including the Sham operation group (Sham), AB-1 week,2 weeks,4 weeks groups (n=10 in each group). For the in vitro studies, primary cultures of rat ventricular cardiomyocytes were prepared. After starvation with serum-free DMEM/F12 for 12 h, the cultured cardiomyocytes were stimulated with 1 ?M Ang ? for 48h. The expression levels of OX40 was detected by Western blotting and PCR. Part two:For the in vivo studies, the male OX40 global knockout (KO) mice (Sham, n=15; AB, n=30) and their respective wild-type littermates (Sham, n=15; AB, n=30), aged 8 to 10 weeks and body weight ranged from 23.5-27.5g were included. The mice were randomly divided into AB group and Sham group.8 weeks after AB, cardiac function of mice was examined by echocardiography and hemodynamics, and the HW/BW, LW/BW, and HW/TL, LW/TL were compared in mice of each group. HE, PSR staining, and immunofluorescence were used for morphology analysis. Molecular markers of cardiac hypertrophy and fibrosis were determined by real-time quantitative RT-PCR. The inflammation response was also evaluated after 2 weeks of AB including the release of inflammatory cytokines determined by real-time quantitative RT-PCR, the infiltration of inflammatory cells determined by immunofluorescence staining.Part three:CD4+ T lymphocytes from WT mice and OX40 knockout mice spleen were separated by using immunomagnetic beads and cells were cultured in RIMP-1640 medium. After activated by anti-CD3? (5?g/mL) and anti-CD28 (1?g/mL) antibody for 48h, CD4+ T lymphocyte proliferation was detected by cell counting Kit 8. The supernatant was collected after activation of CD4+ T lymphocytes, and the released cytokines were detected by ELISA. After starved for 12 hours, neonatal rat cardiomyocytes were co-cultured with the activated supernatant of CD4+ T lymphocyte from OX40 KO mice spleen or WT mice spleen and stimulated with 1 ?M Ang ? for 48 hours. The extend of cardiomyocyte hypertrophy was determined by immunofluorescence staining for cardiac a-actinin and the expression levels of hypertrophic markers by Western Blotting.ResultsPart one:Western blotting and PCR revealed that the OX40 expression level was increased in mouse hearts at week 1 after AB, peaked at week 2 after AB, and decreased to the baseline at week 4 after AB, compared with sham-operated hearts (P<0.05 vs. Sham). The expression of OX40 in Ang ?-treated cardiomyocytes after 48h was no significant differences with PBS group (P> 0.05 vs. PBS). The immunohistochemistry results showed that OX40 was mainly expressed on cardiac interstitial cells and that its expression was upregulated in hypertrophic hearts.Part two:OX40-KO mice were found to have decreased ratios of heart weight to body weight or tibia length, and ratios of lung weight to body weight or tibia length. In addition, as shown by decreased left ventricular end-diastolic diameter (LVEDd), left ventricular end-systolic diameter (LVESd), and decreased hematoxylin and eosin-stained heart sections, the ameliorated effect of OX40 deficiency on AB induced hypertrophic response were confirmed. And,8 weeks afer AB, OX40-KO mice showed improved cardiac contractility as indicated by the increased fractional shorterning (FS), ejection fraction (EF), maximal rate of pressure development (dP/dTmax), cardiac output (CO), and improved cardiac diastolic function as indicated by the increased minimum rate of pressure development (dP/dT min) and decreased end-diastolic pressure (EDP). These changes were accompanied by an reduced induction of interstitial fibrosis and perivascular fibrosis. Consistently, the expression of cardiac fetal genes (ANP, BNP,P-MHC) and fibrotic markers (CTGF, Col??, Col?? and Fibronectin) were significantly decreased in OX40-KO mice after AB, while ?-MHC and SERCA2a augmented significantly in OX40-KO mice after AB.The mRNA expressions of TNF-?, IL-1?, and IL-6 were significantly decreased in OX40 KO mice heart compared with WT mice hearts. Immunohistochemistry staining of CD68, CD45, CD4, CD8, the molecular marker of macrophages, leukocytes, CD4+ and CD8+ T lymphocytes respectively, revealed that infiltration of inflammatory cells was suppressed in OX40 KO mice heart compared with WT mice hearts.Part three:CD4+ T lymphocytes were isolated from OX40 KO mice and WT mice spleen. Our results showed that OX40 deficiency decreased CD4+ T lymphocytes proliferation as well as the production of pro-inflammatory cytokines TNF-?, IFN-?, IL-2, while increased the release of anti-inflammatiory cytokine IL-10 after activated with anti-CD3? (5?g/mL) and anti-CD28 (1?g/mL) antibody for 48h. NRCMs were co-cultured with activated CD4+ T lymphocytes supernatant from either OX40 KO or WT mice spleen and stimulated with Ang ? for 48h. These experiments clearly showed that cell surface area and the hypertrophic markers (ANP, P-MHC) were significantly increased by the CD4+ T cells supernatant from WT mice spleen but were limited after co-cultured with the CD4+ T cells supernatant from OX40 KO mice spleen.Conclusions1. The expression of OX40 initially increased then decreased and peaked at week 2 after AB in mice heart tissue, which located in the mesenchyme. In in vitro Ang ?-induced cardiomyocyte hypertrophy model, OX40 expression had no obvious change.2. OX40 deficiency ameliorates pressure overload-induced cardiac hypertrophy, fibrosis, dysfunction, and inflammation.3. OX40 deficiency retards the proliferation of CD4+ T cells and the release of pro-inflammatory cytokines but enhances the release of the anti-inflammatory cytokine, which affects Ang ? induced cardiomyocyte hypertrophy. OX40 may regulate pressure overload induced cardiac remodeling by modulation the function of CD4+ T lymphocytes. |
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