Epigenetic regulation of histone H3K27 methylation has recently emerged as a key step during alternative immunoregulatory M2-like macrophage polarization; known to impact cardiac repair after ...Myocardial Infarction (MI). We hypothesized that EZH2, responsible for H3K27 methylation, could act as an epigenetic checkpoint regulator during this process. We demonstrate for the first time an ectopic EZH2, and putative, cytoplasmic inactive localization of the epigenetic enzyme, during monocyte differentiation into M2 macrophages in vitro as well as in immunomodulatory cardiac macrophages in vivo in the post-MI acute inflammatory phase. Moreover, we show that pharmacological EZH2 inhibition, with GSK-343, resolves H3K27 methylation of bivalent gene promoters, thus enhancing their expression to promote human monocyte repair functions. In line with this protective effect, GSK-343 treatment accelerated cardiac inflammatory resolution preventing infarct expansion and subsequent cardiac dysfunction in female mice post-MI in vivo. In conclusion, our study reveals that pharmacological epigenetic modulation of cardiac-infiltrating immune cells may hold promise to limit adverse cardiac remodeling after MI.
Abstract
Background and Aims
Calcific aortic valve disease (CAVD) is highly prevalent in chronic kidney disease (CKD) patients and is associated with a poor prognosis. To date, there is no ...pharmacological treatment to slow down this process. Our group recently reported that indoxyl-sulfate (IS) -induced IL-6 secretion in human interstitial valvular cells (hVICs) promotes their osteogenic transition and calcification in an autocrine manner. Preliminary data suggest that IS also influences macrophage secretion of IL-6. Whether this phenomenon affects the hVICs mineralization remains to be elucidated. In this context, the epigenetic enzyme Enhancer of Zeste Homolog 2 (EZH2), a key modulator of macrophages’ inflammation, may represent an interesting therapeutic target.
Thus, this work aimed to verify whether IS-induced macrophages’ secretion of IL-6 modulates hVICs mineralization, and to determine whether EZH2 is involved in this inflammatory process.
Method
THP1-derived macrophages were exposed to increasing concentrations of IS (IS normal Isn: 0.5 μg/mL, IS uremic Isu: 37 μg/mL, IS intermediate int: 100 μg/mL or IS maximum Ismax: 233 μg/mL) in the presence or absence of an EZH2 inhibitor called GSK-343 (5µM). EZH2 expression and activity (evidenced by the trimethylation of the lysine 27 of the histones H3 (H3K27me3)) were assessed by western blot. Macrophages’ inflammatory potential was checked by RT-qPCR, western blot and Elisa. The impact of macrophages exposed or not to IS and/or GSK-343 on hVICs osteogenic transition was evaluated using conditioned media. HVICs osteogenic transition was assessed following runx2 expression by qRT-PCR and western blot. Mineralization was quantified by the o-cresolphthalein method.
Results
IS induced macrophages secretion of IL-1β, IL-6, CCL2 and TNF-α. Conditioned media from IS-polarized macrophages promoted a 6-fold increase in hVICs calcification and favored their osteogenic transition as evidenced by increased runx2 expression. IL-6 was the only cytokine able to promote runx2 expression in hVICs, confirming the importance of this specific cytokine in the process. Exposure to IS upregulated macrophages’ expression EZH2, an effect blocked by GSK-343. If GSK-343 did not affect IS-induced macrophages’ secretion of IL-1β, CCL2 and TNF-α, it efficiently blocked the secretion of IL-6 and subsequent induction of runx2 in hVICs. In this model, neither IS, nor GSK-343 modulated macrophages’ H3K27me3, suggesting that EZH2 may act as a transcriptional factor for IL-6 through its non-canonical pathway. In this latter, EZH2 is thought to promote inflammation by binding to p65, rather than through its trimethylation activity. In line with this hypothesis, exposure to IS promoted EZH2 and p65 nuclear translocation in the meantime as well as their co-immunoprecipitation, two phenomena blocked by GSK343.
Conclusion
This work demonstrates that IS-induced macrophages’ secretion of IL-6 promotes hVICs osteogenic transition and mineralization. Our data suggest that IL-6 secretion in response to IS depends on the EZH2-p65 pathway. The fact that GSK-343 can block this mechanism sheds light on EZH2 as a new therapeutic target to prevent CAVD in CKD patients.
Aortic Stenosis (AS) is highly prevalent in chronic kidney disease (CKD) patients and is associated with a poor prognosis. We recently reported that indoxyl-sulfate (IS), a toxin that accumulates in ...the serum as kidney function declines, promotes macrophage's inflammation and subsequent mineralization of human valvular interstitial cells (hVICs). In this context, the epigenetic enzyme Enhancer of Zeste Homolog 2 (EZH2), a key modulator of macrophage inflammation, may represent an interesting therapeutic target.
This work aimed to determine whether EZH2 is involved in IS-induced macrophage's polarization and subsequent hVICs mineralization.
THP1-derived macrophages were exposed to increasing concentrations of IS in the presence or absence of an EZH2 inhibitor called GSK-343 (5μM). EZH2 expression and activity (evidenced by the trimethylation of the lysine 27 of the histones H3 (H3K27me3)) were assessed by western blot. Macrophages’ inflammatory potential was checked by RT-qPCR and Elisa. The impact of macrophages exposed or not to IS and/or GSK-343 on hVICs osteogenic transition was evaluated using conditioned media. HVICs osteogenic transition was assessed following runx2 expression by qRT-PCR and western blot. Mineralization was quantified by the o-cresolphthalein method.
Exposure to IS upregulated macrophage's expression EZH2, an effect blocked by GSK-343. The treatment with GSK-343 decreased macrophage's secretion of IL-6 (but not that of TNF-α and IL1-β), thereby preventing the induction of runx2 and subsequent calcification of hVICs. Neither IS, nor GSK-343 modulated macrophages’ H3K27me3, suggesting that EZH2 may act as a transcriptional factor for IL-6 through its non-canonical pathway. In this latter, EZH2 is thought to promote inflammation by binding to p65, rather than through its trimethylation activity. In line with this hypothesis, exposure to IS promoted EZH2 and p65 nuclear translocation as well as their coimmunoprecipitation, two phenomena blocked by GSK343.
This work demonstrates that IS induces macrophage's secretion of IL-6 through the EZH2-p65 pathway, thereby promoting hVICs osteogenic transition and mineralization. The fact that GSK-343 can block this mechanism sheds light on EZH2 as a new therapeutic target to prevent AS in CKD patients.
Chagas disease (CD) is still a neglected disease. Infected individuals are diagnosed late, being treated in worse clinical conditions. Thus, this study aimed to analyze the prevalence and the factors ...associated with new confirmed cases of CD identified by serological screening in an endemic region of Minas Gerais State, Brazil. This is an analytical cross- sectional study with data from a project of the Research Center in Tropical Medicine of Sao Paulo- Minas Gerais (SaMi-Trop) conducted in two municipalities. Data collection included a questionnaire with closed questions, a venous blood collection and an ELISA serological test for CD. A total of 2,038 individuals with no previous diagnosis of CD participated in the study. The result of the serological test for CD was adopted as the dependent variable. The independent variables addressed personal issues, health conditions and lifetime housing. A descriptive analysis of individual variables was performed. Subsequently, a bivariate analysis was performed using the Pearson’s chi-square test. Households sheltering individuals positive for CD were georeferenced, and the analysis of spatial distribution was performed using the quartic function to estimate the density of the nucleus. Among the participants, 188 (9.2 %) were positive for CD. The profile of participants with CD was associated with place of residence, age, relative/family member with CD and living conditions. It is noteworthy that there are still patients with CD who are unaware of their diagnosis in both, rural and urban areas.
This study aimed to assess the prevalence of non-use of health services in the last year by people with Chagas disease (CD) in an endemic area in Brazil and the contextual and individual factors ...associated with this non-use. This is a multilevel study that considered contextual and individual data. Contextual data were collected from official publicly accessible databases of the Brazilian government, at the municipal level. The individual data came from the first follow-up of a Brazilian cohort that assessed patients with CD in 21 municipalities in endemic area for the disease. The sample consisted of 1,160 individuals with CD. The dependent variable "use of health services in the last year" was categorized as yes vs. no. The analysis was performed using Poisson regression with robust variance. The prevalence of non-use of health services in the last year was 23.5% (IC95%: 21.1-25.9). The contextual factor "larger population" (PR: 1.6; 95% CI = 1.2-2.0) and individual factors related to the lower severity of the disease as a functional class without limitations (PR: 1.6; 95% CI = 1.2-2.1) and unaltered N-terminal pro b-type natriuretic peptide levels (PR: 2.2; 95% CI = 1.3-3.6) increased the prevalence of non-use of the health service in the last year by people with CD. The results of this study showed that individual determinants are not isolated protagonists of the non-use of health services in the last year by people with CD, which reinforces the need for public policies that consider the contextual determinants of the use of health services by populations affected by the disease.
The mammalian brain stores and distinguishes among episodic memories, i.e. memories formed during the personal experience, through a mechanism of pattern separation computed in the hippocampal ...dentate gyrus. Decision-making for food-related behaviors, such as the choice and intake of food, might be affected in obese subjects by alterations in the retrieval of episodic memories. Adult neurogenesis in the dentate gyrus regulates the pattern separation. Several molecular factors affect adult neurogenesis and exert a critical role in the development and plasticity of newborn neurons. Orexin-A/hypocretin-1 and downstream endocannabinoid 2-arachidonoylglycerol signaling are altered in obese mice. Here, we show that excessive orexin-A/2-arachidonoylglycerol/cannabinoid receptor type-1 signaling leads to the dysfunction of adult hippocampal neurogenesis and the subsequent inhibition of plasticity and impairment of pattern separation. By inhibiting orexin-A action at orexin-1 receptors we rescued both plasticity and pattern separation impairment in obese mice, thus providing a molecular and functional mechanism to explain alterations in episodic memory in obesity.
Although vaccines are greatly mitigating the worldwide pandemic diffusion of SARS-Cov-2, therapeutics should provide many distinct advantages as complementary approach to control the viral spreading. ...Here, we report the development of new tripeptide derivatives of AT1001 against SARS-CoV-2 Mpro. By molecular modeling, a small compound library was rationally designed and filtered for enzymatic inhibition through FRET assay, leading to the identification of compound 4. X-ray crystallography studies provide insights into its binding mode and confirm the formation of a covalent bond with Mpro C145. In vitro antiviral tests indicate the improvement of biological activity of 4 respect to AT1001. In silico and X-ray crystallography analysis led to 58, showing a promising activity against three SARS-CoV-2 variants and a valuable safety in Vero cells and human embryonic lung fibroblasts. The drug tolerance was also confirmed by in vivo studies, along with pharmacokinetics evaluation. In summary, 58 could pave the way to develop a clinical candidate for intranasal administration.
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•X-ray crystallography studies provide new structural elements for drug design.•Improved enzymatic inhibition from partial activity to very low micromolar range.•58 has a promising activity against three SARS-CoV-2 variants and a valuable safety.•58 could pave the way to develop a clinical candidate for intranasal administration.
There have been efforts around the globe to track individuals of many marine species and assess their movements and distribution, with the putative goal of supporting their conservation and ...management. Determining whether, and how, tracking data have been successfully applied to address real-world conservation issues is, however, difficult. Here, we compile a broad range of case studies from diverse marine taxa to show how tracking data have helped inform conservation policy and management, including reductions in fisheries bycatch and vessel strikes, and the design and administration of marine protected areas and important habitats. Using these examples, we highlight pathways through which the past and future investment in collecting animal tracking data might be better used to achieve tangible conservation benefits.
The value of animal tracking data to inform policy is illustrated by case studies from around the world and with a broad range of taxa.
Application of tracking data to policy and management can take various pathways, and engagement with stakeholders might often not be made by the original data collectors.
The impact of tracking data on policy and management can be improved if data collection and analyses target specific needs for management outcomes.
Early engagement among the data collectors and the stakeholders involved in policy development and implementation is important to help translate tracking data into conservation outcomes.
Chagas disease (CD) is recognized by the World Health Organization as one of the thirteen most neglected tropical diseases in the world. Self-perceived health is considered a better predictor of ...mortality than objective measures of health status, and the context in which one lives influences this predictor. This study aimed to evaluate the prevalence and individual and contextual factors associated with poor self-rated health among CD patients from an endemic region in Brazil. It is a multilevel cross-sectional study. The individual data come from a cross-section of a cohort study named SaMi-Trop. Contextual data was collected from publicly accessible institutional information systems and platforms. The dependent variable was self-perceived health. The analysis was performed using multilevel binary logistic regression. The study included 1,513 patients with CD, where 335 (22.1%) had Poor self-rated health. This study revealed the influence of the organization/offer of the Brazilian public health service and of individual characteristics on the self-perceived health of patients with CD.
Abstract Introduction Alzheimer's disease (AD) progression follows a specific spreading pattern, emphasizing the need to characterize those brain areas that degenerate first. The brainstem's locus ...coeruleus (LC) is the first area to develop neurofibrillary changes (neurofibrillary tangles NFTs). Methods The methods include unbiased stereological analyses in human brainstems to estimate LC volume and neuronal population in controls and individuals across all AD stages. Results As the Braak stage increases by 1 unit, the LC volume decreases by 8.4%. Neuronal loss started only midway through AD progression. Age-related changes spare the LC. Discussion The long gap between NFT accumulation and neuronal loss suggests that a second trigger may be necessary to induce neuronal death in AD. Imaging studies should determine whether LC volumetry can replicate the stage-wise atrophy observed here and how these changes are specific to AD. LC volumetry may develop into a screening biomarker for selecting high-yield candidates to undergo expensive and less accessible positron emission tomography scans and to monitor AD progression from presymptomatic stages.
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