Myeloid-derived suppressor cells and associated events in urethane-induced lung cancer

OBJECTIVES: Myeloid-derived suppressor cells contribute to the immunosuppressive microenvironment during tumor development and limit the efficacy of cancer immunotherapy. Identifying myeloid-derived suppressor cells and associated factors is the first step in creating strategies to reverse the suppressive effects of these cells on the immune system. METHODS: To induce lung cancer, we administered 2 doses of urethane to BALB/c mice and observed these animals for 120 days. After this period, we evaluated the percentage of myeloid-derived suppressor cells in the blood, lung and bone marrow. The expression of alpha-smooth muscle actin, transforming growth factor-β, Toll-like receptor 2, Toll-like receptor 4, and interleukin-6 was also determined in the lung tissue. RESULTS: Myeloid-derived suppressor cells were increased in all evaluated tissues after lung cancer development in association with increased Toll-like receptor 4 expression and decreased interleukin-6 expression in the lung. We observed alpha-smooth muscle actin and transforming growth factor-β expression in lung nodules. CONCLUSIONS: We believe that the early diagnosis of cancer through determining the blood levels of myeloid-derived suppressor cells followed by the depletion of these cells should be further investigated as a possible approach for cancer treatment.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESPConselho Nacional de Pesquisa (CNPq)Federal University of São Paulo Immunology DivisionFederal University of São Paulo Pathology DivisionFederal University of São Paulo Psychobiology DivisionFederal University of São Paulo Physiology DivisionUNIFESP, Immunology DivisionUNIFESP, Pathology DivisionUNIFESP, Psychobiology DivisionUNIFESP, Physiology DivisionSciEL

Deficiency of MicroRNA-181a results in transcriptome-wide cell-specific changes in the kidney and increases blood pressure

MicroRNA miR-181a is downregulated in the kidneys of hypertensive patients and hypertensive mice. In vitro, miR-181a is a posttranslational inhibitor of renin expression, but pleiotropic mechanisms by which miR-181a may influence blood pressure (BP) are unknown. Here, we determined whether deletion of miR-181a/b-1 in vivo changes BP and the molecular mechanisms involved at the single-cell level. We developed a KO (knockout) mouse model lacking miR-181a/b-1 genes using CRISPR/Cas9 technology. Radiotelemetry probes were implanted in 12-week-old C57BL/6J WT (wild type) and miR-181a/b-1 KO mice. Systolic and diastolic BP were 4- to 5-mm Hg higher in KO compared with WT mice over 24 hours (P<0.01). Compared with WT mice, renal renin was higher in the juxtaglomerular cells of KO mice. BP was similar in WT mice on a high- (3.1%) versus low- (0.3%) sodium diet (+0.4 +/- 0.8 mm Hg), but KO mice showed salt sensitivity (+3.3 +/- 0.8 mm Hg; P<0.001). Since microRNAs can target several mRNAs simultaneously, we performed single-nuclei RNA sequencing in 6699 renal cells. We identified 12 distinct types of renal cells, all of which had genes that were dysregulated. This included genes involved in renal fibrosis and inflammation such as Stat4, Col4a1, Cd81, Flt3l, Cxcl16, and Smad4. We observed upregulation of pathways related to the immune system, inflammatory response, reactive oxygen species, and nerve development, consistent with higher tyrosine hydroxylase in the kidney. In conclusion, downregulation of the miR-181a gene led to increased BP and salt sensitivity in mice. This is likely due to an increase in renin expression in juxtaglomerular cells, as well as microRNA-driven pleiotropic effects impacting renal pathways associated with hypertension

Chronic Sleep Restriction during Pregnancy - Repercussion on Cardiovascular and Renal Functioning of Male Offspring

Changes in the maternal environment can induce fetal adaptations that result in the progression of chronic diseases in the offspring. the objective of the present study was to evaluate the effects of maternal chronic sleep restriction on blood pressure, renal function and cardiac baroreflex response on male offspring at adult age. Female 3-month-old Wistar rats were divided in two experimental groups: control (C) and chronic sleep restricted (CSR). Pregnancy was confirmed by vaginal smear. Chronic sleep restricted females were subjected to sleep restriction by the multiple platform technique for 20 h daily, between the 1st and 20th day of pregnancy. After birth, the litters were reduced to 6 rats per mother, and were designated as offspring from control (OC) and offspring from chronic sleep restricted (OCSR). Indirect blood pressure (BPi tail cuff) was measured by plethysmography in male offspring at 3 months old. Following, the renal function and cardiac baroreflex response were analyzed. Values of BPi in OCSR were significantly higher compared to OC [OC: 127 +/- 2.6 (19); OCSR: 144 +/- 2.5 (17) mmHg]. the baroreflex sensitivity to the increase of blood pressure was reduced in OCSR [Slope: OC: -2.6 +/- 0.15 (9); OCRS: -1.6 +/- 0.13 (9)]. Hypothalamic activity of ACE2 was significantly reduced in OCSR compared to OC [OC: 97.4 +/- 15 (18); OSR: 60.2 +/- 3.6 (16) UAF/min/protein mg]. Renal function alteration was noticed by the increase in glomerular filtration rate (GFR) observed in OCSR [OC: 6.4 +/- 0.2 (10); OCSR: 7.4 +/- 0.3 (7)]. Chronic sleep restriction during pregnancy caused in the offspring hypertension, altered cardiac baroreflex response, reduced ACE-2 activity in the hypothalamus and renal alterations. Our data suggest that the reduction of sleeping time along the pregnancy is able to modify maternal homeostasis leading to functional alterations in offspring.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Associacao Fundo de Incentivo a Pesquisa (AFIP)Universidade Federal de São Paulo, Dept Fisiol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Psicobiol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biociencias, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Fisiol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Psicobiol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biociencias, São Paulo, BrazilFAPESP: FAPESP-10/51665-4Web of Scienc

Kallikrein-kinin system and vascular endothelial growth factor interaction in intermittent hypoxia models

Low levels of oxygen in the tissues (hypoxia) occur in a number of pathophysiologic conditions such as the growth of tumors, heart disease and obstructive sleep apnea. During hypoxia, blood vessel growth (angiogenesis) is stimulated, leading to increased tissue O2 uptake. The vascular endothelial growth factor (VEGF) and the kallikrein-kinin system are essential for this process. As in humans, in intermittent hypoxia, an experimental model of obstructive sleep apnea, increased blood pressure, decreased cardiac output, left ventricular hypertrophy and pulmonary hypertension were described. However, the evidence of cardiac angiogenesis in this model are not clearly established. In order to investigate in vivo angiogenesis, left ventricular male mice C57BI6/J and knockouts for the bradykinin B2 receptor were subjected to intermittent hypoxia during 1, 2, 3, 4 or 5 months with subsequent histological evaluation of vascularization after the implantation of a Matrigel plug. The gene and protein expressions of vascular endothelial growth factor and components of the kallikrein-kinin system were also evaluated. A decrease in cardiac angiogenesis in vivo after 2, 3 and 4 weeks of intermittent hypoxia was found. Consistent with this data, although there is an increase in the expression of hypoxia induced factor since the first week, VEGF only increased after the third week (isoform 165) and the fourth week (isoform 121) and its VEGF-R2 receptor in the fifth week of intermittent hypoxia. None of these changes was observed in its B2 receptor knockout animals. In wild type animals, the B2 receptor had an increased expression since the first week of intermittent hypoxia that remained until the fifth week. Unexpectedly the gene expression of B1 receptor in wild type was decreased in the first week, while there was no change in protein expression. The expression of intracellular proteins (Akt and c-Src) had no change in left ventricular wild animals or knockouts. These results suggest that the intermittent hypoxia, unlike that seen in other models of hypoxia, the classic interaction of VEGF with the components of the kallikreinkinin system is not enough to stimulate the formation of new vessels. Additionally, the B2 receptor appears to be essential for the observed changes to occur in the model. Our results suggest a mechanism for the cardiac effects of obstructive sleep apnea.Baixos níveis de oxigênio nos tecidos (hipóxia) ocorrem em uma série de condições fisiopatológicas como no crescimento de tumores, doenças cardíacas e na apneia obstrutiva do sono. Durante a hipóxia é estimulado o crescimento de vasos sanguíneos (angiogênese) que leva ao aumento no aporte de O2 tecidual. O fator de crescimento vascular endotelial (VEGF) e o sistema calicreína-cininas são essenciais para esse processo. Assim como em humanos, na hipóxia intermitente, modelo experimental da apneia obstrutiva do sono, foram descritos aumento da pressão arterial, diminuição do débito cardíaco, hipertrofia no ventrículo esquerdo e hipertensão pulmonar. No entanto, as evidências de angiogênese cardíaca nesse modelo não estão ainda claramente estabelecidas. A fim de investigar a angiogênese in vivo no ventrículo esquerdo, camundongos machos da linhagem C57Bl6/J e nocautes para o receptor de bradicinina B2 foram submetidos à hipóxia intermitente por 1, 2, 3, 4, ou 5 semanas, com posterior avaliação histológica de vascularização após implante de um plug de Matrigel. Também foram avaliadas as expressões gênica e proteica do fator de crescimento vascular endotelial e componentes do sistema calicreínas-cininas. Encontramos diminuição na angiogênese cardíaca in vivo em 2, 3 e 4 semanas de hipóxia intermitente. Coerente com esse dado, embora haja aumento da expressão do fator induzido por hipóxia na primeira semana, VEGF apenas aumentou após a terceira semana (isoforma 165) e a quarta semana (isoforma 121) e seu receptor VEGF-R2 na quinta semana de hipóxia intermitente. Nenhuma dessas alterações foi observada nos animais nocautes do receptor B2. Nos animais selvagens, o receptor B2 teve a expressão aumentada a partir da primeira semana de hipóxia intermitente, mantendo-se até a quinta semana. Inesperadamente a expressão gênica do receptor B1 nos animais selvagens diminuiu na primeira semana, enquanto não houve alteração na expressão proteica. A expressão das proteínas intracelulares (Akt e Src) não houve alteração no ventrículo esquerdo dos animais selvagens ou nocautes. Esses resultados sugerem que na hipóxia intermitente, ao contrário do observado em outros modelos de hipóxia, a clássica interação do VEGF com os componentes do sistema calicreínas-cininas não é suficiente para estimular a formação de novos vasos. Adicionalmente, o receptor B2 parece ser essencial para que as modificações observadas ocorram no modelo. Nossos resultados indicam um mecanismo para as consequências cardíacas da apneia obstrutiva do sono.Fundação de Amparo e Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Pesquisa e Desenvolvimento (CNPq)Associação de Fundo e Incentivo à Pesquisa (AFIP)FAPESP: 2012/02123-0Dados abertos - Sucupira - Teses e dissertações (2013 a 2016

Paradoxical Sleep Deprivation and Metallopeptidases Activities Changes in Rat Central Nervous System

Neuropeptides have a fundamental role on sleep-wake cycle control and their actions are regulated by proteolytic processing. Proteolytic enzymes are essential for the metabolism of neuropeptides, either by release from active or inactive precursor protein, or the inactivation of active neuropeptides. Using fluorescence resonance energy transfer substrates, Western blot, specific inhibitors and real time PCR, we have demonstrated changes in the expression and activity of angiotensin I-converting enzyme (ACE - EC 3.4.15.1) and thimet oligopeptidase (EP24.15 – EC 3.4.24.15) in the central nervous system of rats submitted to paradoxical sleep deprivation. Male rats were distributed in 5 groups: control, paradoxical sleep deprivation during 96h (PSD96), and sleep recovery (recovery after sleep deprivation 24, 48 and 96h after PSD96). Alterations in the activities, expression and mRNA levels of ACE and EP24.15 were found in extracts of the hypothalamus, hippocampus, brainstem, cortex and striatum. Significant changes in these parameters (activity, mRNA levels, protein expression) were observed in groups PSD96 and recovery. Modifications on ACE and EP24.15 activities can result in alterations in the metabolism of angiotensins, bradykinin, opioid peptides (dynorphins and enkephalins), substance P and GnRH and be related to some of the physiological changes (stress, memory and cognition, nociception and endocrine changes) observed during and after sleep deprivation.Os neuropeptídeos desempenham um papel fundamental no controle do ciclo vigília-sono e as suas ações podem ser reguladas pela atividade das enzimas proteolíticas. Enzimas proteolíticas são essenciais para metabolização de neuropeptídeos, seja pela liberação a partir de precursores protéicos inativos ou ativos, ou pela inativação de neuropeptídeos ativos. Utilizando substratos com supressão interna de fluorescência, Western blot, inibidores específicos e PCR em tempo real, avaliamos as alterações na expressão e atividade da enzima conversora de angiotensina-I (ECA - EC 3.4.15.1) e thimet oligopeptidase (EP24.15 - EC 3.4.24.15) no sistema nervoso central de ratos em modelos de privação de sono paradoxal. Ratos machos foram distribuídos em cinco grupos: controle, privação de sono paradoxal por 96h (PSP96) e recuperação do sono (rebote após privação 24, 48 e 96 horas após PSP96). Encontrou-se variações nas atividades, na expressão e nos níveis de RNAm da ECA e EP24.15 em extratos do hipotálamo, hipocampo, tronco encefálico, córtex e estriado. Mudanças significativas nos parâmetros avaliados (atividade, níveis de RNAm e expressão protéica) foram observadas nos grupos PSP96 e nos rebotes. As alterações de atividade da ECA e EP24.15 podem implicar em mudanças no metabolismo das angiotensinas, bradicinina, peptídeos opióides (dinorfinas e encefalinas), substância P e GnRH e relacionar-se a algumas das mudanças fisiológicas (estresse, memória e cognição, nocicepção e alterações endócrinas) observadas durante e após a privação do sono.Associação Fundo de Incentivo à Psicofarmacologia (AFIP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)TEDEBV UNIFESP: Teses e dissertaçõe

Serum Amyloid A Production Is Triggered by Sleep Deprivation in Mice and Humans: Is That the Link between Sleep Loss and Associated Comorbidities?

Serum amyloid A (SAA) was recently associated with metabolic endotoxemia, obesity and insulin resistance. Concurrently, insufficient sleep adversely affects metabolic health and is an independent predisposing factor for obesity and insulin resistance. In this study we investigated whether sleep loss modulates SAA production. The serum SAA concentration increased in C57BL/6 mice subjected to sleep restriction (SR) for 15 days or to paradoxical sleep deprivation (PSD) for 72 h. Sleep restriction also induced the upregulation of Saa1.1/Saa2.1 mRNA levels in the liver and Saa3 mRNA levels in adipose tissue. SAA levels returned to the basal range after 24 h in paradoxical sleep rebound (PSR). Metabolic endotoxemia was also a finding in SR. Increased plasma levels of SAA were also observed in healthy human volunteers subjected to two nights of total sleep deprivation (Total SD), returning to basal levels after one night of recovery. The observed increase in SAA levels may be part of the initial biochemical alterations caused by sleep deprivation, with potential to drive deleterious conditions such as metabolic endotoxemia and weight gain