Cross-talk between autophagy and KLF2 determines endothelial cell phenotype and microvascular function in acute liver injury.

BACKGROUND & AIMS: The transcription factor Krüppel-like factor 2 (KLF2), inducible by simvastatin, confers endothelial vasoprotection. Considering recent data suggesting activation of autophagy by statins, we aimed to: 1) characterize the relationship between autophagy and KLF2 in the endothelium, 2) assess this relationship in acute liver injury (cold ischemia/reperfusion) and 3) study the effects of modulating KLF2-autophagy in vitro and in vivo. METHODS: Autophagic flux, the vasoprotective KLF2 pathway, cell viability and microvascular function were assessed in endothelial cells and in various pre-clinical models of acute liver injury (cold storage and warm reperfusion). RESULTS: Positive feedback between autophagy and KLF2 was observed in the endothelium: KLF2 inducers, pharmacological (statins, resveratrol, GGTI-298), biomechanical (shear stress) or genetic (adenovirus containing KLF2), caused endothelial KLF2 overexpression through a Rac1-rab7-autophagy dependent mechanism, both in the specialized liver sinusoidal endothelial cells (LSEC) and in human umbilical vein endothelial cells. In turn, KLF2 induction promoted further activation of autophagy. Cold ischemia blunted autophagic flux. Upon reperfusion, LSEC stored in University of Wisconsin solution did not reactivate autophagy, which resulted in autophagosome accumulation probably due to impairment in autophagosome-lysosome fusion, ultimately leading to increased cell death and microvascular dysfunction. Simvastatin pretreatment maintained autophagy (through the upregulation of rab7), resulting in increased KLF2, improved cell viability, and ameliorated hepatic damage and microvascular function. CONCLUSIONS: We herein describe for the first time the complex autophagy-KLF2 relationship, modulating the phenotype and survival of the endothelium. These results help understanding the mechanisms of protection conferred by KLF2-inducers, such as simvastatin, in hepatic vascular disorders. LAY SUMMARY: Autophagy and the transcription factor KLF2 share a common activation pathway in the endothelium, being able to regulate each other. Statins maintain microvascular function through the inhibition of Rac1, which consequently liberates Rab7, activates autophagy and increments the expression of KLF2.

A survey on the use of mice, pigs, dogs and monkeys as animal models in biomedical research in Spain.

BACKGROUND: The use of animals in biomedical science remains controversial. An individual's level of concern is generally influenced by their culture, previous or current experience with animals, and the specific animal species in question. In this study we aimed to explore what people in Spain who had never or who no longer worked with laboratory animals thought of the use of mice, pigs, dogs and monkeys for biomedical research purposes. We also aimed to determine whether or not people currently involved in biomedical research with the aforementioned species felt their work was justified. RESULTS: The study comprised a total of 807 participants (never worked = 285, used to work = 56, currently working = 466), almost two thirds of whom were women. Our results revealed that the phylogenetic scale is an important factor in people's opinions of the use of certain species in research. The percentage of people who were against the use of dogs or monkeys was higher than that of those who were against the use of mice or pigs. The main reasons given for having stopped working with laboratory animals were change of professional career and change in research project. Participants who were currently working with animals believed that their work was justified, but said they did not talk about it with people outside their immediate social circle. CONCLUSIONS: Our findings suggest that there is a difference in moral status between monkeys and mice, as well as between companion animals (dogs) and farm animals (pigs). Our results support the idea that working with laboratory animals is a sensitive issue in Spain.

Working with laboratory rodents in Spain: a survey on welfare and wellbeing.

BACKGROUND: Replacement, reduction and refinement, the 3R principles, provide a framework to minimize the use and suffering of animals in science. In this context, we aimed to determine the actual perception that individuals working with laboratory rodents in biomedical research have on animal welfare and on their interaction with the animals, as well as how they perceive its impact on their social relations. To this end, we designed an anonymous on-line survey for people working with rodents, at three responsibility levels, in Spain. RESULTS: Of the 356 participants, 239 were women (67 %); 263 were researchers (74 %), and 93 animal facility staff (26 %), of which 55 were caretakers/technicians (15 %), and 38 welfare officer/veterinarians (11 %). Animal facility staff indicated environmental enrichment to be a universal practice. About half of the participants reported that, in their opinion, animals suffer "little to none" or "minor" stress and pain. Animal caretakers/technicians and researchers perceived higher levels of stress and pain than welfare officers/veterinarians. Participants judged decapitation the most unpleasant method to kill rodents, whereas anaesthetic overdose was the least one. A sizable proportion - 21 % of animal caretakers/technicians and 11.4 % of researchers - stated that they were never given the choice not to euthanize the rodents they work with. Overall, women reported higher interactions with animals than men. Nevertheless, we could detect a significant correlation between time spent with the animals and interaction scores. Notably, 80 % of animal facility staff and 92 % of researchers rarely talked about their work with laboratory rodents with people outside their inner social circle. CONCLUSIONS: Overall, the participants showed high awareness and sensitivity to rodent wellbeing; animal facility staff reported a similar perception on welfare questions, independently of their category, while researchers, who spent less time with the animals, showed less awareness and manifested lower human-animal interaction and less social support. Regarding the perception on social acceptance of laboratory animal work, all groups were cautious and rarely talked about their job, suggesting that it is considered a sensitive issue in Spain.

Professional Quality of Life in Research Involving Laboratory Animals.

Many workers contribute to the success of animal welfare and study outcomes in biomedical research. However, the professional quality of life (ProQoL) of those who work with laboratory animals has not been explored in Spain. To this end, we adapted the ProQoL scale to the Spanish population working with laboratory animals. Participants were contacted by email and asked to complete an anonymous on-line questionnaire. The study comprised a total of 498 participants, 12.4% welfare officers/veterinarians, 19.5% caretaker/technicians, 13.9% principal investigators, 20.7% investigators, 13.6% research technicians, and 19.9% PhD students. The adapted scale revealed very good reliability and internal validity, providing information about two different subscales, compassion satisfaction and compassion fatigue. Animal-facility personnel showed higher total ProQoL and compassion-satisfaction scores than researchers; PhD students showed the lowest scores. Thus, our results indicate that job category is a contributing factor in perceived professional quality of life. We observed that compassion satisfaction is negatively associated with the perceived animal stress/pain. Participants reporting poorer compassion satisfaction also reported lower social-support scores. Overall, our ProQoL scale is a useful tool for analyzing the professional quality of life in the Spanish population, and may help to design future interventions to improve workplace wellbeing in Spain and other Spanish-speaking populations.

Effects of aging on liver microcirculatory function and sinusoidal phenotype.

The socioeconomic and medical improvements of the last decades have led to a relevant increase in the median age of worldwide population. Although numerous studies described the impact of aging in different organs and the systemic vasculature, relatively little is known about liver function and hepatic microcirculatory status in the elderly. In this study, we aimed at characterizing the phenotype of the aged liver in a rat model of healthy aging, particularly focusing on the microcirculatory function and the molecular status of each hepatic cell type in the sinusoid. Moreover, major findings of the study were validated in young and aged human livers. Our results demonstrate that healthy aging is associated with hepatic and sinusoidal dysfunction, with elevated hepatic vascular resistance and increased portal pressure. Underlying mechanisms of such hemodynamic disturbances included typical molecular changes in the cells of the hepatic sinusoid and deterioration in hepatocyte function. In a specific manner, liver sinusoidal endothelial cells presented a dysfunctional phenotype with diminished vasodilators synthesis, hepatic macrophages exhibited a proinflammatory state, while hepatic stellate cells spontaneously displayed an activated profile. In an important way, major changes in sinusoidal markers were confirmed in livers from aged humans. In conclusion, our study demonstrates for the first time that aging is accompanied by significant liver sinusoidal deregulation suggesting enhanced sinusoidal vulnerability to chronic or acute injuries.

Study of CD27 and CCR4 Markers on Specific CD4+ T-Cells as Immune Tools for Active and Latent Tuberculosis Management.

The immunological characterization of different cell markers has opened the possibility of considering them as immune tools for tuberculosis (TB) management, as they could correlate with TB latency/disease status and outcome. CD4+ T-cells producing IFN-γ+ with a low expression of CD27 have been described as an active TB marker. In addition, there are unknown homing receptors related to TB, such as CCR4, which might be useful for understanding TB pathogenesis. The aim of our study is focused on the assessment of several T-cell subsets to understand immune-mechanisms in TB. This phenotypic immune characterization is based on the study of the specific immune responses of T-cells expressing CD27 and/or CCR4 homing markers. Subjects enrolled in the study were: (i) 22 adult patients with active TB, and (ii) 26 individuals with latent TB infection (LTBI). Blood samples were drawn from each patient. The expression of CD27 and/or CCR4 markers were analyzed within CD4+ T-cells producing: (i) IFN-γ+, (ii) TNF-α+, (iii) TNF-α+IFN-γ+, and (iv) IFN-γ+ and/or TNF-α+. The percentage of CD27- within all CD4+ T-cell populations analyzed was significantly higher on active TB compared to LTBI after PPD or ESAT-6/CFP-10 stimulation. As previously reported, a ratio based on the CD27 median fluorescence intensity (MFI) was also explored (MFI of CD27 in CD4+ T-cells over MFI of CD27 in IFN-γ+CD4+ T-cells), being significantly increased during disease (p < 0.0001 after PPD or ESAT-6/CFP-10 stimulation). This ratio was also assessed on the other CD4+ T-cells functional profiles after specific stimulation, being significantly associated with active TB. Highest diagnostic accuracies for active TB (AUC ≥ 0.91) were achieved for: (i) CD27 within IFN-γ+TNF-α+CD4+ T-cells in response to ESAT-6/CFP-10, (ii) CD27 and CCR4 markers together within IFN-γ+CD4+ T-cells in response to PPD, and (iii) CD27 MFI ratio performed on IFN-γ+TNF-α+CD4+ T-cells after ESAT-6/CFP-10 stimulation. The lowest diagnostic accuracy was observed when CCR4 marker was evaluated alone (AUC ≤ 0.77). CD27 and CCR4 expression detection could serve as a good method for immunodiagnosis. Moreover, the immunological characterization of markers/subset populations could be a promising tool for understanding the biological basis of the disease.

Liraglutide improves liver microvascular dysfunction in cirrhosis: Evidence from translational studies.

Hepatic stellate cells (HSC) play a key role in the development of chronic liver disease (CLD). Liraglutide, well-established in type 2 diabetes, showed anti-inflammatory and anti-oxidant properties. We evaluated the effects of liraglutide on HSC phenotype and hepatic microvascular function using diverse pre-clinical models of CLD. Human and rat HSC were in vitro treated with liraglutide, or vehicle, and their phenotype, viability and proliferation were evaluated. In addition, liraglutide or vehicle was administered to rats with CLD. Liver microvascular function, fibrosis, HSC phenotype and sinusoidal endothelial phenotype were determined. Additionally, the effects of liraglutide on HSC phenotype were analysed in human precision-cut liver slices. Liraglutide markedly improved HSC phenotype and diminished cell proliferation. Cirrhotic rats receiving liraglutide exhibited significantly improved liver microvascular function, as evidenced by lower portal pressure, improved intrahepatic vascular resistance, and marked ameliorations in fibrosis, HSC phenotype and endothelial function. The anti-fibrotic effects of liraglutide were confirmed in human liver tissue and, although requiring further investigation, its underlying molecular mechanisms suggested a GLP1-R-independent and NF-κB-Sox9-dependent one. This study demonstrates for the first time that liraglutide improves the liver sinusoidal milieu in pre-clinical models of cirrhosis, encouraging its clinical evaluation in the treatment of chronic liver disease.

Resistance to Taxanes in Triple-Negative Breast Cancer Associates with the Dynamics of a CD49f+ Tumor-Initiating Population.

Taxanes are a mainstay of treatment for breast cancer, but resistance often develops followed by metastatic disease and mortality. Aiming to reveal the mechanisms underlying taxane resistance, we used breast cancer patient-derived orthoxenografts (PDX). Mimicking clinical behavior, triple-negative breast tumors (TNBCs) from PDX models were more sensitive to docetaxel than luminal tumors, but they progressively acquired resistance upon continuous drug administration. Mechanistically, we found that a CD49f+ chemoresistant population with tumor-initiating ability is present in sensitive tumors and expands during the acquisition of drug resistance. In the absence of the drug, the resistant CD49f+ population shrinks and taxane sensitivity is restored. We describe a transcriptional signature of resistance, predictive of recurrent disease after chemotherapy in TNBC. Together, these findings identify a CD49f+ population enriched in tumor-initiating ability and chemoresistance properties and evidence a drug holiday effect on the acquired resistance to docetaxel in triple-negative breast cancer.

A novel modular bioreactor to in vitro study the hepatic sinusoid.

We describe a unique, versatile bioreactor consisting of two plates and a modified commercial porous membrane suitable for in vitro analysis of the liver sinusoid. The modular bioreactor allows i) excellent control of the cell seeding process; ii) cell culture under controlled shear stress stimulus, and; iii) individual analysis of each cell type upon completion of the experiment. The advantages of the bioreactor detailed here are derived from the modification of a commercial porous membrane with an elastomeric wall specifically moulded in order to define the cell culture area, to act as a gasket that will fit into the bioreactor, and to provide improved mechanical robustness. The device presented herein has been designed to simulate the in vivo organization of a liver sinusoid and tested by co-culturing endothelial cells (EC) and hepatic stellate cells (HSC). The results show both an optimal morphology of the endothelial cells as well as an improvement in the phenotype of stellate cells, most probably due to paracrine factors released from endothelial cells. This device is proposed as a versatile, easy-to-use co-culture system that can be applied to biomedical research of vascular systems, including the liver.

RANK induces epithelial-mesenchymal transition and stemness in human mammary epithelial cells and promotes tumorigenesis and metastasis.

Paracrine signaling through receptor activator of NF-κB (RANK) pathway mediates the expansion of mammary epithelia that occurs during pregnancy, and activation of RANK pathway promotes mammary tumorigenesis in mice. In this study we extend these previous data to human cells and show that the RANK pathway promotes the development of mammary stem cells and breast cancer. Overexpression of RANK (FL-RANK) in a panel of tumoral and normal human mammary cells induces the expression of breast cancer stem and basal/stem cell markers. High levels of RANK in untransformed MCF10A cells induce changes associated with both stemness and transformation, including mammary gland reconstitution, epithelial-mesenchymal transition (EMT), increased migration, and anchorage-independent growth. In addition, spheroids of RANK overexpressing MCF10A cells display disrupted acinar formation, impair growth arrest and polarization, and luminal filling. RANK overexpression in tumor cells with nonfunctional BRCA1 enhances invasiveness in acinar cultures and increases tumorigenesis and metastasis in immunodeficient mice. High levels of RANK were found in human primary breast adenocarcinomas that lack expression of the hormone receptors, estrogen and progesterone, and in tumors with high pathologic grade and proliferation index; high RANK/RANKL expression was significantly associated with metastatic tumors. Together, our findings show that RANK promotes tumor initiation, progression, and metastasis in human mammary epithelial cells by increasing the population of CD44(+)CD24(-) cells, inducing stemness and EMT. These results suggest that RANK expression in primary breast cancer associates with poor prognosis.