Comprometimento da homeostase telomérica e indução do fenótipo senescente no contexto da obesidade

O envelhecimento é um processo biológico caracterizado pela deterioração progressiva das funções fisiológicas do organismo. A perda de função e o acúmulo de dano tecidual estão relacionados a uma série de marcadores celulares, dentre eles, o encurtamento acelerado de telômeros, acúmulo de células senescentes e o comprometimento da função mitocondrial. O estabelecimento do fenótipo senescente pode ser caracterizado pelo aumento na transcrição do gene CDKN1A (P21) e pela atividade da enzima β-galactosidase (β-gal), além de um conjunto de citocinas que constituem um secretoma capaz de contribuir para um estado de inflamação crônico, observado em doenças relacionadas à aceleração do envelhecimento. A obesidade é uma condição fisiológica caracterizada pela alteração na composição e a estrutura do tecido adiposo e uma desregulação metabólica. É uma doença associada a um estado de inflamação crônica, compartilhando aspectos relacionados ao envelhecimento e ao aparecimento de características de senescência de forma antecipada. Dessa maneira, o objetivo principal desta tese foi avaliar parâmetros relacionados à homeostasia do complexo telomérico e o impacto do ambiente oxidativo, característico da obesidade, na indução do fenótipo senescente. Para isso, relacionamos a homeostase do complexo telomérico de células mononucleares de sangue periférico (PBMC) com a indução do fenótipo senescente relacionado à disfunção mitocondrial em células tronco adipo-derivadas (ADSC) expostas ao plasma de indivíduos portadores de obesidade após 10 dias de tratamento. Nossos resultados demonstraram que indivíduos portadores de obesidade possuem telômeros mais curtos e uma desregulação na expressão dos componentes do complexo shelterin em PBMC, independente da ocorrência de comorbidades associadas à doença. Além disso, observamos um aumento do dano oxidativo relacionado ao aumento da lipoperoxidação e o conteúdo de proteínas carboniladas no plasma de indivíduos portadores de obesidade. A Análise de Componentes Principais (PCA) ainda sugere que o comprometimento da homeostase telomérica pode ser parcialmente explicado pela desregulação de TRF1, o qual não pôde ser revertido pelo aumento na resposta antioxidante não enzimática, decorrente de uma possível resposta celular adaptativa. Os efeitos crônicos do plasma de indivíduos portadores de obesidade sugeriram o gatilho da indução do fenótipo senescente em ADSC pelo aumento da atividade da enzima β-gal e um aumento na expressão de CDKN1A. Observamos um acúmulo de mitocôndrias danificadas e indícios de inibição da autofagia celular, características que podem contribuir na indução do fenótipo senescente no contexto da obesidade. Complementando os achados obtidos no sistema periférico (PBMC), nossos resultados ainda demonstram um aumento na expressão de TRF1 nas ADSC no início do fenótipo senescente, sugerindo novamente que TRF1 tem um papel importante na progressão do envelhecimento, tanto a nível de tecido adiposo quando a nível sistêmico. Os dados obtidos nessa tese sugerem que a obesidade é uma doença que está associada à aceleração do fenótipo senescente em diferentes contextos celulares, induzindo a ativação de respostas moleculares antecipadas, similares aos mecanismos observados no envelhecimento.Aging is a biological process characterized by the progressive deterioration of the physiological functions of the organism. Loss of function and accumulation of tissue damage are related to several cell hallmarks, such as accelerated telomere shortening, senescent cell accumulation, and mitochondrial dysfunction. The establishment of the senescent phenotype can be characterized by the increase in CDKN1A (P21) gene transcription and the enzyme β-galactosidase (β-gal) activity, as well as a set of cytokines that constitute a secretome that contributes to a chronic inflammation state, observed in age-related diseases. Obesity is a multifactorial condition characterized by changes in the composition and structure of adipose tissue and excessive metabolic dysregulation. It is associated with a chronic low-grade inflammatory condition that accompanies metabolic dysfunction, sharing aspects related to anticipation of aging characteristics. Thus, the aim of this thesis was to evaluate parameters related to homeostasis of the telomeric complex and the impact of the obesity-related oxidative environment on the induction of senescent phenotype. To this end, we evaluated the telomere homeostasis of the peripheral blood mononuclear cell (PBMC) and the senescence induction on adipose-derived stem cells (ADSC) by chronic exposure to an obesogenic environment. We demonstrated that obese individuals have shorter telomeres and dysregulated gene expression of shelterin components in PBMC, regardless of the occurrence of disease-associated comorbidities. In addition, we observed increased oxidative damage related to augmented lipoperoxidation and carbonyl protein content in plasma of obese individuals. Also, Principal Component Analysis (PCA) suggests that the impairment of telomeric homeostasis may be partially explained by the positive regulation of TRF1 and could not be reversed by the increase in non-enzymatic antioxidant response, observed as an adaptive cellular response. The chronic effects of obesogenic environment were observed after plasma incubation for 10 days. Our data sugested that the onset of senescent phenotype induction in ADSC was observed by increased β-gal enzyme activity and an increase in CDKN1A expression. After plasma incubation for 10 days. Furthermore, the accumulation of damaged mitochondria may be a response due to autophagy machinery impairment, so that it may contribute to the induction of senescent phenotype in the context of obesity. Our data further demonstrated an increase in TRF1 expression in ADSC at the onset of the senescent phenotype, suggesting that TRF1 plays an important role in the progression of aging, both at adipose and systemic levels. The data obtained in this thesis suggest that obesity is a disease that is associated with acceleration of the senescent phenotype in different cellular contexts, contributing to the activation of molecular responses that trigger characteristics observed in aging

Eotaxin-1/CCL11 promotes cellular senescence in human-derived fibroblasts through pro-oxidant and pro-inflammatory pathways

Introduction: Eotaxin-1/CCL11 is a pivotal chemokine crucial for eosinophil homing to the lungs of asthmatic patients. Recent studies also suggest that CCL11 is involved in the aging process, as it is upregulated in elderly, and correlated with shorter telomere length in leukocytes from asthmatic children. Despite its potential pro-aging effects, the precise contribution of CCL11 and the underlying mechanisms involved in the promotion of cellular senescence remains unclear. Therefore, the primary goal of this study was to explore the role of CCL11 on senescence development and the signaling pathways activated by this chemokine in lung fibroblasts. Methods: To investigate the targets potentially modulated by CCL11, we performed an in silico analysis using PseudoCell. We validated in vitro the activation of these targets in the human lung fibroblast cell line MRC-5 following rhCCL11 exposure. Finally, we performed differential gene expression analysis in human airway epithelial cells of asthmatic patients to assess CCL11 signaling and activation of additional senescent markers. Results: Our study revealed that eotaxin-1/CCL11 promote reactive oxygen secretion (ROS) production in lung fibroblasts, accompanied by increased activation of the DNA damage response (DDR) and p-TP53 and γH2AX. These modifications were accompanied by cellular senescence promotion and increased secretion of senescence-associated secretory phenotype inflammatory cytokines IL-6 and IL-8. Furthermore, our data show that airway epithelial lung cells from atopic asthmatic patients overexpress CCL11 along with aging markers such as CDKN2A (p16INK4a) and SERPINE1. Discussion: These findings provide new insights into the mechanisms underlying the pro-aging effects of CCL11 in the lungs of asthmatic patients. Understanding the role of CCL11 on senescence development may have important implications for the treatment of age-related lung diseases, such as asthma

Retinoic acid downregulates thiol antioxidant defences and homologous recombination while promotes A549 cells sensitization to cisplatin

Recent studies have investigated the use of retinoic acid (RA) molecule in combined chemotherapies to cancer cells as an attempt to increase treatment efficiency and circumvent cell resistance. Positive results were obtained in clinical trials from lung cancer patients treated with RA and cisplatin. Meanwhile, the signalling process that results from the interaction of both molecules remains unclear. One of the pathways that RA is able to modulate is the activity of NRF2 transcription factor, which is highly associated with tumour progression and resistance. Therefore, the aim of this work was to investigate molecular mechanism of RA and cisplatin co-treatment in A549 cells, focusing in NRF2 pathway. To this end, we investigated NRF2 and NRF2-target genes expression, cellular redox status, cisplatin-induced apoptosis, autophagy and DNA repair through homologous recombination. RA demonstrated to have an inhibitory effect over NRF2 activation, which regulates the expression of thiol antioxidants enzymes. Moreover, RA increased reactive species production associated with increased oxidation of thiol groups within the cells. The expression of proteins associated with DNA repair through homologous recombination was also suppressed by RA pre-treatment. All combined, these effects appear to create a more sensitive cellular environment to cisplatin treatment, increasing apoptosis frequency. Interestingly, autophagy was also increased by combination therapy, suggesting a resistance mechanism by A549 cells. In conclusion, these results provided new information about molecular mechanisms of RA and cisplatin treatment contributing to chemotherapy optimization

Telomere length and epigenetic age acceleration in adolescents with anxiety disorders

Evidence on the relationship between genetics and mental health are flourishing. However, few studies are evaluating early biomarkers that might link genes, environment, and psychopathology. We aimed to study telomere length (TL) and epigenetic age acceleration (AA) in a cohort of adolescents with and without anxiety disorders (N = 234). We evaluated a representative subsample of participants at baseline and after 5 years (n = 76) and categorized them according to their anxiety disorder diagnosis at both time points: (1) control group (no anxiety disorder, n = 18), (2) variable group (anxiety disorder in one evaluation, n = 38), and (3) persistent group (anxiety disorder at both time points, n = 20). We assessed relative mean TL by real-time quantitative PCR and DNA methylation by Infinium HumanMethylation450 BeadChip. We calculated AA using the Horvath age estimation algorithm and analyzed differences among groups using generalized linear mixed models. The persistent group of anxiety disorder did not change TL over time (p = 0.495). The variable group had higher baseline TL (p = 0.003) but no accelerated TL erosion in comparison to the non-anxiety control group (p = 0.053). Furthermore, there were no differences in AA among groups over time. Our findings suggest that adolescents with chronic anxiety did not change telomere length over time, which could be related to a delay in neuronal development in this period of life

Shorter telomeres in children with severe asthma, an indicative of accelerated aging

Severe therapy-resistant asthma (STRA) is closely associated with distinct clinical and inflammatory pheno-endotypes, which may contribute to the development of age-related comorbidities. Evidence has demonstrated a contribution of accelerated telomere shortening on the poor prognosis of respiratory diseases in adults. Eotaxin-1 (CCL11) is an important chemokine for eosinophilic recruitment and the progression of asthma. In the last years has also been proposed as an age-promoting factor. This study aimed to investigate the association of relative telomere length (rTL) and eotaxin-1 in asthmatic children. Children aged 8-14 years (n=267) were classified as healthy control (HC, n=126), mild asthma (MA, n=124) or severe therapy-resistant asthma (STRA, n=17). rTL was performed by qPCR from peripheral blood. Eotaxin-1 was quantified by ELISA from fresh-frozen plasma. STRA had shorter telomeres compared to HC (p=0.02) and MA (p=0.006). Eotaxin-1 levels were up-regulated in STRA [median; IQR25-75)] [(1,190 pg/mL; 108–2,510)] compared to MA [(638 pg/mL; 134–1,460)] (p=0.03) or HC [(627 pg/mL; 108–1,750)] (p<0.01). Additionally, shorter telomeres were inversely correlated with eotaxin-1 levels in STRA (r=-0.6, p=0.013). Our results suggest that short telomeres and up-regulated eotaxin-1, features of accelerated aging, could prematurely contribute to a senescent phenotype increasing the risk for early development of age-related diseases in asthma

Telomere length and epigenetic age acceleration in adolescents with anxiety disorders

Evidence on the relationship between genetics and mental health are flourishing. However, few studies are evaluating early biomarkers that might link genes, environment, and psychopathology. We aimed to study telomere length (TL) and epigenetic age acceleration (AA) in a cohort of adolescents with and without anxiety disorders (N = 234). We evaluated a representative subsample of participants at baseline and after 5 years (n = 76) and categorized them according to their anxiety disorder diagnosis at both time points: (1) control group (no anxiety disorder, n = 18), (2) variable group (anxiety disorder in one evaluation, n = 38), and (3) persistent group (anxiety disorder at both time points, n = 20). We assessed relative mean TL by real-time quantitative PCR and DNA methylation by Infinium HumanMethylation450 BeadChip. We calculated AA using the Horvath age estimation algorithm and analyzed differences among groups using generalized linear mixed models. The persistent group of anxiety disorder did not change TL over time (p = 0.495). The variable group had higher baseline TL (p = 0.003) but no accelerated TL erosion in comparison to the non-anxiety control group (p = 0.053). Furthermore, there were no differences in AA among groups over time. Our findings suggest that adolescents with chronic anxiety did not change telomere length over time, which could be related to a delay in neuronal development in this period of life

Telomere length in healthy newborns is not affected by adverse intrauterine environments

Different intrauterine exposures are associated with different metabolic profiles leading to growth and development characteristics in children and also relate to health and disease patterns in adult life. The objective of this work was to evaluate the impact of four different intrauterine environments on the telomere length of newborns. This is a longitudinal observational study using a convenience sample of 222 mothers and their term newborns (>37 weeks of gestational age) from hospitals in Porto Alegre, Rio Grande do Sul (Brazil), from September 2011 to January 2016. Sample was divided into four groups: pregnant women with Gestational Diabetes Mellitus (DM) (n=38), smoking pregnant women (TOBACCO) (n=52), mothers with small-for-gestational age (SGA) children due to idiopathic intrauterine growth restriction (n=33), and a control group (n=99). Maternal and newborn genomic DNA were obtained from epithelial mucosal cells. Telomere length was assessed by qPCR, with the calculation of the telomere and single copy gene (T/S ratio). In this sample, there was no significant difference in telomere length between groups (p>0.05). There was also no association between childbirth weight and telomere length in children (p>0.05). For term newborns different intrauterine environments seems not to influence telomere length at birth

Obesity drives adipose-derived stem cells into a senescent and dysfunctional phenotype associated with P38MAPK/NF-KB axis

Background: Adipose-derived stem cells (ADSC) are multipotent cells implicated in tissue homeostasis. Obesity represents a chronic inflammatory disease associated with metabolic dysfunction and age-related mechanisms, with progressive accumulation of senescent cells and compromised ADSC function. In this study, we aimed to explore mechanisms associated with the inflammatory environment present in obesity in modulating ADSC to a senescent phenotype. We evaluated phenotypic and functional alterations through 18 days of treatment. ADSC were cultivated with a conditioned medium supplemented with a pool of plasma from eutrophic individuals (PE, n = 15) or with obesity (PO, n = 14), and compared to the control. Results: Our results showed that PO-treated ADSC exhibited decreased proliferative capacity with G2/M cycle arrest and CDKN1A (p21WAF1/Cip1) up-regulation. We also observed increased senescence-associated β-galactosidase (SA-β-gal) activity, which was positively correlated with TRF1 protein expression. After 18 days, ADSC treated with PO showed augmented CDKN2A (p16INK4A) expression, which was accompanied by a cumulative nuclear enlargement. After 10 days, ADSC treated with PO showed an increase in NF-κB phosphorylation, while PE and PO showed an increase in p38MAPK activation. PE and PO treatment also induced an increase in senescence-associated secretory phenotype (SASP) cytokines IL-6 and IL-8. PO-treated cells exhibited decreased metabolic activity, reduced oxygen consumption related to basal respiration, increased mitochondrial depolarization and biomass, and mitochondrial network remodeling, with no superoxide overproduction. Finally, we observed an accumulation of lipid droplets in PO-treated ADSC, implying an adaptive cellular mechanism induced by the obesogenic stimuli. Conclusions: Taken together, our data suggest that the inflammatory environment observed in obesity induces a senescent phenotype associated with p38MAPK/NF-κB axis, which stimulates and amplifies the SASP and is associated with impaired mitochondrial homeostasis