Role of Membrane Cholesterol in Modulating Actin Architecture and Cellular Contractility

Atherosclerosis is a chronic inflammatory process that initiates with accumulation of apolipoprotein B containing lipoproteins (LPs) in the subendothelium (intima), especially in areas where the laminar flow is disturbed. LP retention triggers an inflammatory response leading to activation of endothelial and vascular smooth muscle cells that culminates with recruitment of leukocytes. Atherosclerosis is the leading cause of vascular disease worldwide being its major clinical manifestations ischemic heart disease, ischemic stroke, and peripheral arterial disease. Even though a lot has been done to unravel the role of turbulent flow and mechanotransduction for atherosclerosis development, little is known about the role of plasma membrane (PM) cholesterol in this process. This chapter is going to be focused on exploring what has been done so far to decipher the role of PM cholesterol in regulating actin architecture, cellular mechanical properties, and cellular contractility in muscle and nonmuscle cells

Lysosomes: How Plasma Membrane Repair Route Can Be Hijacked by Parasites?

Lysosomes are acidic organelles that are not only involved in degradation processes but also participated in other cellular functions, such as specialized secretion and plasma membrane (PM) resealing. When the PM is ruptured, Ca2+ flows from the extracellular milieu toward the cytoplasm potentially triggering cell death. In order to escape from the apoptotic route, cells developed an elegant mechanism in which lysosomes are recruited to the sites of injuries in a Ca2+-dependent fashion. Lysosomes, fuse with the PM releasing their enzymatic content. Acid sphingomyelinase (ASM), one of the secreted enzymes, cleaves sphingomyelin into ceramide, inducing compensatory endocytosis and internalization of the membrane-damaged site. Trypanosoma cruzi, the etiological agent of Chagas disease, relies heavily on lysosomes to successfully invade mammalian cells. By mechanically injuring the host PM, T. cruzi evokes lysosome exocytosis, and subsequently, compensatory endocytosis. The latter drives the parasite into the host cell, where it can replicate. This early association with lysosomes prevents T. cruzi evasion from the host cells allowing colonization of host intracellular milieu. This review chapter will summarize the main contributions in the field exploring the crosstalk between PM repair and T. cruzi invasion and how the understanding of these mechanisms evolved throughout the years

Canine distemper virus induces apoptosis in cervical tumor derived cell lines

Apoptosis can be induced or inhibited by viral proteins, it can form part of the host defense against virus infection, or it can be a mechanism for viral spread to neighboring cells. Canine distemper virus (CDV) induces apoptotic cells in lymphoid tissues and in the cerebellum of dogs naturally infected. CDV also produces a cytopathologic effect, leading to apoptosis in Vero cells in tissue culture. We tested canine distemper virus, a member of the Paramyxoviridae family, for the ability to trigger apoptosis in HeLa cells, derived from cervical cancer cells resistant to apoptosis. To study the effect of CDV infection in HeLa cells, we examined apoptotic markers 24 h post infection (pi), by flow cytometry assay for DNA fragmentation, real-time PCR assay for caspase-3 and caspase-8 mRNA expression, and by caspase-3 and -8 immunocytochemistry. Flow cytometry showed that DNA fragmentation was induced in HeLa cells infected by CDV, and immunocytochemistry revealed a significant increase in the levels of the cleaved active form of caspase-3 protein, but did not show any difference in expression of caspase-8, indicating an intrinsic apoptotic pathway. Confirming this observation, expression of caspase-3 mRNA was higher in CDV infected HeLa cells than control cells; however, there was no statistically significant change in caspase-8 mRNA expression profile. Our data suggest that canine distemper virus induced apoptosis in HeLa cells, triggering apoptosis by the intrinsic pathway, with no participation of the initiator caspase -8 from the extrinsic pathway. In conclusion, the cellular stress caused by CDV infection of HeLa cells, leading to apoptosis, can be used as a tool in future research for cervical cancer treatment and control

Canagliflozin and renal outcomes in type 2 diabetes and nephropathy

BACKGROUND Type 2 diabetes mellitus is the leading cause of kidney failure worldwide, but few effective long-term treatments are available. In cardiovascular trials of inhibitors of sodium–glucose cotransporter 2 (SGLT2), exploratory results have suggested that such drugs may improve renal outcomes in patients with type 2 diabetes. METHODS In this double-blind, randomized trial, we assigned patients with type 2 diabetes and albuminuric chronic kidney disease to receive canagliflozin, an oral SGLT2 inhibitor, at a dose of 100 mg daily or placebo. All the patients had an estimated glomerular filtration rate (GFR) of 30 to <90 ml per minute per 1.73 m2 of body-surface area and albuminuria (ratio of albumin [mg] to creatinine [g], >300 to 5000) and were treated with renin–angiotensin system blockade. The primary outcome was a composite of end-stage kidney disease (dialysis, transplantation, or a sustained estimated GFR of <15 ml per minute per 1.73 m2), a doubling of the serum creatinine level, or death from renal or cardiovascular causes. Prespecified secondary outcomes were tested hierarchically. RESULTS The trial was stopped early after a planned interim analysis on the recommendation of the data and safety monitoring committee. At that time, 4401 patients had undergone randomization, with a median follow-up of 2.62 years. The relative risk of the primary outcome was 30% lower in the canagliflozin group than in the placebo group, with event rates of 43.2 and 61.2 per 1000 patient-years, respectively (hazard ratio, 0.70; 95% confidence interval [CI], 0.59 to 0.82; P=0.00001). The relative risk of the renal-specific composite of end-stage kidney disease, a doubling of the creatinine level, or death from renal causes was lower by 34% (hazard ratio, 0.66; 95% CI, 0.53 to 0.81; P<0.001), and the relative risk of end-stage kidney disease was lower by 32% (hazard ratio, 0.68; 95% CI, 0.54 to 0.86; P=0.002). The canagliflozin group also had a lower risk of cardiovascular death, myocardial infarction, or stroke (hazard ratio, 0.80; 95% CI, 0.67 to 0.95; P=0.01) and hospitalization for heart failure (hazard ratio, 0.61; 95% CI, 0.47 to 0.80; P<0.001). There were no significant differences in rates of amputation or fracture. CONCLUSIONS In patients with type 2 diabetes and kidney disease, the risk of kidney failure and cardiovascular events was lower in the canagliflozin group than in the placebo group at a median follow-up of 2.62 years

Design and baseline characteristics of the finerenone in reducing cardiovascular mortality and morbidity in diabetic kidney disease trial

Background: Among people with diabetes, those with kidney disease have exceptionally high rates of cardiovascular (CV) morbidity and mortality and progression of their underlying kidney disease. Finerenone is a novel, nonsteroidal, selective mineralocorticoid receptor antagonist that has shown to reduce albuminuria in type 2 diabetes (T2D) patients with chronic kidney disease (CKD) while revealing only a low risk of hyperkalemia. However, the effect of finerenone on CV and renal outcomes has not yet been investigated in long-term trials. Patients and Methods: The Finerenone in Reducing CV Mortality and Morbidity in Diabetic Kidney Disease (FIGARO-DKD) trial aims to assess the efficacy and safety of finerenone compared to placebo at reducing clinically important CV and renal outcomes in T2D patients with CKD. FIGARO-DKD is a randomized, double-blind, placebo-controlled, parallel-group, event-driven trial running in 47 countries with an expected duration of approximately 6 years. FIGARO-DKD randomized 7,437 patients with an estimated glomerular filtration rate >= 25 mL/min/1.73 m(2) and albuminuria (urinary albumin-to-creatinine ratio >= 30 to <= 5,000 mg/g). The study has at least 90% power to detect a 20% reduction in the risk of the primary outcome (overall two-sided significance level alpha = 0.05), the composite of time to first occurrence of CV death, nonfatal myocardial infarction, nonfatal stroke, or hospitalization for heart failure. Conclusions: FIGARO-DKD will determine whether an optimally treated cohort of T2D patients with CKD at high risk of CV and renal events will experience cardiorenal benefits with the addition of finerenone to their treatment regimen. Trial Registration: EudraCT number: 2015-000950-39; ClinicalTrials.gov identifier: NCT02545049

Importância do colesterol para a mecânica celular e para a entrada do Trypanosoma cruzi na célula hospedeira

Exportado OPUSMade available in DSpace on 2019-08-13T00:20:40Z (GMT). No. of bitstreams: 1 tese_barbara_hissa_2013.pdf: 8047431 bytes, checksum: 65cecf67ee815487d723f58e2c80b8c9 (MD5) Previous issue date: 23No presente trabalho, investigamos a importância do colesterol na modulação das propriedades mecânicas celulares e como isto interfere na entrada do parasito flagelado, Trypanosoma cruzi, em sua célula hospedeira. Para isto, realizamos estudos de dinâmica celulares, utilizando experimentos de pinça óptica (OT) e de microscopia dedesfocalização (DM), bem como ensaios de exocitose lisossomal e invasão celular pelo T. cruzi em fibroblastos imortalizados de camundongos (WTCl3) e culturas primárias de cardiomiócitos murinos.Verificamos, através da extração de amarras celulares em células WTCL3, pela técnica de OT que tanto a rigidez de flexão quanto a tensão superficial eram maiores quanto menor a quantidade de colesterol remanescente nos fibroblastos. Já nos experimentos de DM, verificamos que o tratamento com a ciclodextrina capturadora de colesterol diminuiu a amplitude de oscilação da superfície celular, mostrando um enrijecimento celular. Já o tempo de relaxação da mesma aumentou, nos primeiros estágios do tratamento, retornando para valores semelhantes aos valores controle. Essa mudança na dinâmica de celular mediada pelo sequestro de colesterol foi gerada tanto pela reorganização do citoesqueleto cortical de actina quanto pela sua polimerização de novo, com formação de fibras de estresse viaativação de Rho. Demonstramos, em cardiomiócitos e em fibroblastos, que o tratamento com MCD, mas não com uma ciclodextrina não-depletora de colesterol, promove a exocitose de lisossomos nestas células e que este processo, em cardiomiócitos, era independente do cálcio intra e extracelular. Em células epiteliais imortalizadas, NRK, verificamos que a exocitose lisossomal induzida pela diminuição do colesterol ocorria mesmo na ausência da proteína sensora de cálcio presente nesta organela, a sinaptotagmina- VII, mostrando que este evento realmente era não dependente deste íon. A exocitose mediada pela diminuição do colesterol de membrana levou à secreção principalmente de lisossomos localizados próximos ao córtex celular, diferente do pool recrutado mediante o tratamento com drogasdespolimerizadoras do citoesqueleto, como a latrunculina-A. Estes dados sugerem a existência de dois pools distintos de lisossomos com diferentes dinâmicas de exocitose. Sabemos que o T. cruzi necessita tanto de um citoesqueleto cortical relativamente maleável quanto delisossomos localizados na região próxima ao córtex celular para que consiga infectar a sua célula hospedeira de forma irreversível. Embora haja estudos que apontem para a importância do colesterol de membrana na infectividade do T. cruzi na célula hospedeira, a qual diminui consideravelmente após tratamento com ciclodextrinas, em nenhum deles foi determinado o mecanismo pelo qual estes microdomínios de membrana contribuem para a invasão e estabelecimento da infecção pelo T. cruzi. Neste trabalho, utilizando cardiomiócitos, células importantes durante a colonização do hospedeiro por este parasito, mostramos que a depleção de colesterol diminui tanto a entrada dos tripomastigotas quanto a associação desses a lisossomos e sugerimos que essa redução da infectividade do parasito está diretamente relacionada à uma diminuição de uma população de lisossomos corticais e a um aumento da rigidez celular dos cardiomiócitos.In the present work we have investigated the importance of cholesterol in controlling mechanical properties of cells and how this modulates Trypanosoma cruzi entry in host cells. For this we performed studies of cell membrane dynamics using tether extraction with optical tweezers (OT) and defocusing microscopy (DM), as well as lysosomal exocytosis and T. cruzi invasion assays in a mouse fibroblast cell line (WTCL3) and primary cultures of neonatal murine cardiomyocytes. We observed, by OT, that both bending rigidity and surface tension increased when WTCL3 was treated with MCD, a drug that sequesters cholesterol from cell membranes. Additionally, by DM, we demonstrated that membrane-cytoskeleton relaxation time increased in the beginning of MCD treatment and decreased in the end, assuming values similar to control. With this technique we have also proved that the amplitude of oscillation diminished during cholesterol depletion, showing that these cells become stiffer. Together, these changes in membrane dynamics involved not only the actin cytoskeleton rearrangement, but also its polymerization de novo and stress fiber formation through Rho activation.Additionally, for cardiomyocytes and fibroblasts, we have proved that treatment with MCD, but not with a similar cyclodextrin with less affinity for cholesterol, triggered lysosome secretion and that this phenomenon was independent of extracellular and intracellular calcium, at least for cardiomyocytes. By using NRK cells we have also shown that lysosome exocytosis, in the absence of cholesterol, continued to happen even in the absence of synaptotagmin-VII, the calcium sensor protein present in these organelles. Exocytosis triggered by cholesterol removal led to the secretion of lysosomes located near the cell cortex, different from the pool recruited by actin depolymerizing drugs, such as latrunculin-A. These data suggest the existence of at least two different pools with different exocytosis dynamics. It is well known that T. cruzi needs both a relatively malleable actin cytoskeleton and a reservoir of cortical lysosomes in order to invade cells successfully. Although there are evidences that plasma membrane cholesterol is important for T. cruzi invasion, since it diminishes severely after cyclodextrin treatment, in none of them wasestablished a mechanism by which cholesterol interferes with T. cruzi entry. In this work we showed that for cardiomyocytes, an important cell during host infection, cholesterol depletion not only diminishes T. cruzi invasion, but also its association with lysosomal markers at the initial steps of host cell entry. The membrane dynamics change (increasing cortical energy barrier) and lysosomal exocytic events (depleting the cortical lysosome reservoir), triggered by cholesletrol sequestration, are most likely the mechanisms y which cholesterol depletion interferes withparasite host cell invasion

GAS2L1 Is a Potential Biomarker of Circulating Tumor Cells in Pancreatic Cancer

Pancreatic cancer is a malignant disease with high mortality and a dismal prognosis. Circulating tumor cell (CTC) detection and characterization have emerged as essential techniques for early detection, prognostication, and liquid biopsy in many solid malignancies. Unfortunately, due to the low EPCAM expression in pancreatic cancer CTCs, no specific marker is available to identify and isolate this rare cell population. This study analyzed single-cell RNA sequencing profiles of pancreatic CTCs from a genetically engineered mouse model (GEMM) and pancreatic cancer patients. Through dimensionality reduction analysis, murine pancreatic CTCs were grouped into three clusters with different biological functions. CLIC4 and GAS2L1 were shown to be overexpressed in pancreatic CTCs in comparison with peripheral blood mononuclear cells (PBMCs). Further analyses of PBMCs and RNA-sequencing datasets of enriched pancreatic CTCs were used to validate the overexpression of GAS2L1 in pancreatic CTCs. A combinatorial approach using both GAS2L1 and EPCAM expression leads to an increased detection rate of CTCs in PDAC in both GEMM and patient samples. GAS2L1 is thus proposed as a novel biomarker of pancreatic cancer CTCs

Differential Effects of Trp53 Alterations in Murine Colorectal Cancer

Background: Colorectal cancer (CRC) development is a multi-step process resulting in the accumulation of genetic alterations. Despite its high incidence, there are currently no mouse models that accurately recapitulate this process and mimic sporadic CRC. We aimed to develop and characterize a genetically engineered mouse model (GEMM) of Apc/Kras/Trp53 mutant CRC, the most frequent genetic subtype of CRC. Methods: Tumors were induced in mice with conditional mutations or knockouts in Apc, Kras, and Trp53 by a segmental adeno-cre viral infection, monitored via colonoscopy and characterized on multiple levels via immunohistochemistry and next-generation sequencing. Results: The model accurately recapitulates human colorectal carcinogenesis clinically, histologically and genetically. The Trp53 R172H hotspot mutation leads to significantly increased metastatic capacity. The effects of Trp53 alterations, as well as the response to treatment of this model, are similar to human CRC. Exome sequencing revealed spontaneous protein-modifying alterations in multiple CRC-related genes and oncogenic pathways, resulting in a genetic landscape resembling human CRC. Conclusions: This model realistically mimics human CRC in many aspects, allows new insights into the role of TP53 in CRC, enables highly predictive preclinical studies and demonstrates the value of GEMMs in current translational cancer research and drug development