The gluconeogenesis enzyme PCK2 has a non-enzymatic role in proteostasis in endothelial cells.

Endothelial cells (ECs) are highly glycolytic, but whether they generate glycolytic intermediates via gluconeogenesis (GNG) in glucose-deprived conditions remains unknown. Here, we report that glucose-deprived ECs upregulate the GNG enzyme PCK2 and rely on a PCK2-dependent truncated GNG, whereby lactate and glutamine are used for the synthesis of lower glycolytic intermediates that enter the serine and glycerophospholipid biosynthesis pathways, which can play key roles in redox homeostasis and phospholipid synthesis, respectively. Unexpectedly, however, even in normal glucose conditions, and independent of its enzymatic activity, PCK2 silencing perturbs proteostasis, beyond its traditional GNG role. Indeed, PCK2-silenced ECs have an impaired unfolded protein response, leading to accumulation of misfolded proteins, which due to defective proteasomes and impaired autophagy, results in the accumulation of protein aggregates in lysosomes and EC demise. Ultimately, loss of PCK2 in ECs impaired vessel sprouting. This study identifies a role for PCK2 in proteostasis beyond GNG.

Adipose tissue as a therapeutic target for vascular damage in Alzheimer's disease.

Adipose tissue has recently been recognized as an important endocrine organ that plays a crucial role in energy metabolism and in the immune response in many metabolic tissues. With this regard, emerging evidence indicates that an important crosstalk exists between the adipose tissue and the brain. However, the contribution of adipose tissue to the development of age-related diseases, including Alzheimer's disease, remains poorly defined. New studies suggest that the adipose tissue modulates brain function through a range of endogenous biologically active factors known as adipokines, which can cross the blood-brain barrier to reach the target areas in the brain or to regulate the function of the blood-brain barrier. In this review, we discuss the effects of several adipokines on the physiology of the blood-brain barrier, their contribution to the development of Alzheimer's disease and their therapeutic potential. LINKED ARTICLES: This article is part of a themed issue From Alzheimer's Disease to Vascular Dementia: Different Roads Leading to Cognitive Decline. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.6/issuetoc.

Molecular and metabolic orchestration of the lymphatic vasculature in physiology and pathology.

Lymphangiogenesis refers to the generation of new lymphatic vessels from pre-existing ones. During development and particular adult states, lymphatic endothelial cells (LEC) undergo reprogramming of their transcriptomic and signaling networks to support the high demands imposed by cell proliferation and migration. Although there has been substantial progress in identifying growth factors and signaling pathways controlling lymphangiogenesis in the last decades, insights into the role of metabolism in lymphatic cell functions are just emerging. Despite numerous similarities between the main metabolic pathways existing in LECs, blood ECs (BEC) and other cell types, accumulating evidence has revealed that LECs acquire a unique metabolic signature during lymphangiogenesis, and their metabolic engine is intertwined with molecular regulatory networks, resulting in a tightly regulated and interconnected process. Considering the implication of lymphatic dysfunction in cancer and lymphedema, alongside other pathologies, recent findings hold promising opportunities to develop novel therapeutic approaches. In this review, we provide an overview of the status of knowledge in the molecular and metabolic network regulating the lymphatic vasculature in health and disease.

Immunomodulatory properties of the lymphatic endothelium in the tumor microenvironment.

The tumor microenvironment (TME) is an intricate complex and dynamic structure composed of various cell types, including tumor, stromal and immune cells. Within this complex network, lymphatic endothelial cells (LECs) play a crucial role in regulating immune responses and influencing tumor progression and metastatic dissemination to lymph node and distant organs. Interestingly, LECs possess unique immunomodulatory properties that can either promote or inhibit anti-tumor immune responses. In fact, tumor-associated lymphangiogenesis can facilitate tumor cell dissemination and metastasis supporting immunoevasion, but also, different molecular mechanisms involved in LEC-mediated anti-tumor immunity have been already described. In this context, the crosstalk between cancer cells, LECs and immune cells and how this communication can shape the immune landscape in the TME is gaining increased interest in recent years. In this review, we present a comprehensive and updated report about the immunomodulatory properties of the lymphatic endothelium within the TME, with special focus on primary tumors and tumor-draining lymph nodes. Furthermore, we outline emerging research investigating the potential therapeutic strategies targeting the lymphatic endothelium to enhance anti-tumor immune responses. Understanding the intricate mechanisms involved in LEC-mediated immune modulation in the TME opens up new possibilities for the development of innovative approaches to fight cancer.

Danthron, an Anthraquinone Isolated from a Marine Fungus, Is a New Inhibitor of Angiogenesis Exhibiting Interesting Antitumor and Antioxidant Properties.

The role played by a sustained angiogenesis in cancer and other diseases stimulates the interest in the search for new antiangiogenic drugs. In this manuscript, we provide evidence that 1,8- dihydroxy-9,10-anthraquinone (danthron), isolated from the fermentation broth of the marine fungus Chromolaenicola sp. (HL-114-33-R04), is a new inhibitor of angiogenesis. The results obtained with the in vivo CAM assay indicate that danthron is a potent antiangiogenic compound. In vitro studies with human umbilical endothelial cells (HUVEC) reveal that this anthraquinone inhibits certain key functions of activated endothelial cells, including proliferation, proteolytic and invasive capabilities and tube formation. In vitro studies with human breast carcinoma MDA-MB231 and fibrosarcoma HT1080 cell lines suggest a moderate antitumor and antimetastatic activity of this compound. Antioxidant properties of danthron are evidenced by the observation that it reduces the intracellular reactive oxygen species production and increases the amount of intracellular sulfhydryl groups in endothelial and tumor cells. These results support a putative role of danthron as a new antiangiogenic drug with potential application in the treatment and angioprevention of cancer and other angiogenesis-dependent diseases.

Metabolic Reprogramming in Tumor Endothelial Cells.

The dynamic crosstalk between the different components of the tumor microenvironment is critical to determine cancer progression, metastatic dissemination, tumor immunity, and therapeutic responses. Angiogenesis is critical for tumor growth, and abnormal blood vessels contribute to hypoxia and acidosis in the tumor microenvironment. In this hostile environment, cancer and stromal cells have the ability to alter their metabolism in order to support the high energetic demands and favor rapid tumor proliferation. Recent advances have shown that tumor endothelial cell metabolism is reprogrammed, and that targeting endothelial metabolic pathways impacts developmental and pathological vessel sprouting. Therefore, the use of metabolic antiangiogenic therapies to normalize the blood vasculature, in combination with immunotherapies, offers a clinical niche to treat cancer.

Characterization of the activity and the mechanism of action of a new toluquinol derivative with improved potential as an antiangiogenic drug.

The inhibition of sustained angiogenesis is an attractive approach for the treatment of cancer, blindness and other angiogenesis-dependent diseases. Encouraged by our previous finding that toluquinol, a methyl hydroquinone isolated from a marine fungus, exhibited an interesting antiangiogenic activity, we further explored structural modifications of this natural compound in order to develop improved drug candidates. Our results indicate that although the methyl group plays a relevant role in the cytotoxic activity of toluquinol, some derivatives in which this methyl was replaced by another substituent, could keep the antiangiogenic activity, whereas exhibiting a lower cytotoxicity in vitro. This is the case of (E)- 2-(3-methoxyprop-1-en-1-yl) benzene-1,4-diol, which exhibits a decreased toxicity, whereas maintaining or even improving the antiangiogenic activity of toluquinol, as demonstrated by a number of in vitro (endothelial cells proliferation, migration and tube formation) and in vivo (chick embryo chrorioallantoic membrane vascularization and murine corneal neovascularization) experimental approaches. Our results point to a mechanism of action that could be related to an induction of apoptosis, as well as to an increase in the reactive oxygen species levels, a reduction of the redox capacity and the inhibition of the VEGFR2, Akt and ERK phosphorylation in VEGF-activated endothelial cells. The biological activity of this new angiogenesis inhibitor, along with its lower undesired toxicity, suggests that it is a promising drug candidate with improved potential for the treatment of angiogenesis-related diseases.

Paragastric Autonomic Neural Blockade to Prevent Early Visceral Pain and Associated Symptoms After Laparoscopic Sleeve Gastrectomy: a Randomized Clinical Trial.

BACKGROUND: Visceral pain (VP) following laparoscopic sleeve gastrectomy remains a substantial problem. VP is associated with autonomic symptoms, especially nausea and vomiting, and is unresponsive to traditional pain management algorithms aimed at alleviating somatic (incisional) pain. The present study was performed to evaluate the safety and effectiveness of laparoscopic paragastric autonomic neural blockade (PG-ANB) in managing the symptoms associated with VP following sleeve gastrectomy. STUDY DESIGN: This prospective, double-blinded, randomized clinical trial involved patients undergoing laparoscopic sleeve gastrectomy at two high-volume institutions. The patients were randomized to laparoscopic transversus abdominis plane block with or without PG-ANB. The primary outcome was patient-reported pain scores assessed at 1, 8, and 24 h postoperatively. The secondary outcome measures were analgesic requirements, nausea, vomiting, hiccups, and hemodynamic changes immediately after PG-ANB and postoperatively. RESULTS: In total, 145 patients (block group, n = 72; control group, n = 73) were included in the study. The heart rate and mean arterial pressure significantly decreased 10 min after PG-ANB. The visual analog scale score for pain was significantly lower in the PG-ANB than in the control group at 1 h postoperatively (p < 0.001) and 8 h postoperatively (p < 0.001). Vomiting, nausea, sialorrhea, and hiccups were significantly less prevalent in the PG-ANB group. Patients in the PG-ANB group received fewer cumulative doses of analgesics at 1 h postoperatively (p = 0.003) and 8 h postoperatively (p < 0.001). No differences between the groups were detected at 24 h (p = 0.298). No complications related to PG-ANB occurred. CONCLUSION: PG-ANB safely and effectively reduces early VP, associated autonomic symptoms, and analgesic requirements after laparoscopic sleeve gastrectomy.

A course-based undergraduate research experience to illustrate the early stages of the drug discovery process.

We have implemented at the University of Málaga (Spain) a new course-based undergraduate research experience (CURE) to involve undergraduate students of Science in a real-world scientific problem. Within the topic "Let's find acetylcholinesterase inhibitors as new drug candidates for the treatment of Alzheimer's", students have been engaged into the early stages of the drug discovery process. Working in groups of 4-5 persons, they have searched information in databases, proposed solutions to the driving question and designed protocols to carry them out in vitro and in silico. Overall, the implementation of this experience has been very satisfactory in terms of academic performance and students' perception. This article reports a session from the virtual international 2021 IUBMB/ASBMB workshop, "Teaching Science on Big Data".

Lipid droplet degradation by autophagy connects mitochondria metabolism to Prox1-driven expression of lymphatic genes and lymphangiogenesis.

Autophagy has vasculoprotective roles, but whether and how it regulates lymphatic endothelial cells (LEC) homeostasis and lymphangiogenesis is unknown. Here, we show that genetic deficiency of autophagy in LEC impairs responses to VEGF-C and injury-driven corneal lymphangiogenesis. Autophagy loss in LEC compromises the expression of main effectors of LEC identity, like VEGFR3, affects mitochondrial dynamics and causes an accumulation of lipid droplets (LDs) in vitro and in vivo. When lipophagy is impaired, mitochondrial ATP production, fatty acid oxidation, acetyl-CoA/CoA ratio and expression of lymphangiogenic PROX1 target genes are dwindled. Enforcing mitochondria fusion by silencing dynamin-related-protein 1 (DRP1) in autophagy-deficient LEC fails to restore LDs turnover and lymphatic gene expression, whereas supplementing the fatty acid precursor acetate rescues VEGFR3 levels and signaling, and lymphangiogenesis in LEC-Atg5-/- mice. Our findings reveal that lipophagy in LEC by supporting FAO, preserves a mitochondrial-PROX1 gene expression circuit that safeguards LEC responsiveness to lymphangiogenic mediators and lymphangiogenesis.

Burnout syndrome and associated factors among dentists working in hospitals of the Ministry of Health in the Lima Metropolitan Area, Peru / Síndrome de burnouty factores asociados en odontólogos que trabajan en hospitales del Ministerio de Salud de Lima Metropolitana, Perú

Abstract Introduction: Worldwide, burnout syndrome is becoming increasingly frequent among dentists, so it is necessary to identify its possible causes. Objective: To describe the prevalence of burnout syndrome and the factors associated with its development among dentists working in hospitals of the Ministry of Health (MINSA) in the Lima Metropolitan Area, Peru. Materials and methods: Analytical cross-sectional study carried out between December 2019 and January 2020 in 105 dentists working in the 11 MINSA hospitals in the Lima Metropolitan Area. The Maslach Burnout Inventory instrument was used to measure burnout. Factors such as sex, age, marital status, type of shift, years of experience as a dentist, whether they performed any other dentistry-related work activity, and hours of physical activity and leisure per day were evaluated. Crude and adjusted prevalence ratios (PR) were obtained to analyze the association between the presence of the syndrome and the factors. Results: The prevalence of burnout syndrome was 28.57%. Emotional exhaustion, depersonalization and personal accomplishment levels were high in 90.47%, 98.09% and 35.23% of the participants, respectively. The presence of the syndrome was 47% lower in women (aPR=0.53, p=0.044) compared to men, and 70% lower in those who reported having worked as dentists between 11 and 20 years (aPR=0.30, p=0.017), compared to those with less than 11 years of work experience as dentists. Conclusions: Almost one third of the participants had burnout syndrome. In addition, the majority of dentists showed high levels of emotional exhaustion and depersonalization. Being a woman and having 11 to 20 years of work experience as dentists were protective factors for burnout syndrome.

Resumen Introducción. En el mundo, el síndrome de burnout es cada vez más frecuente en odontólogos, por lo que es necesario identificar sus posibles causas. Objetivo. Describir la prevalencia del síndrome de burnout y los factores asociados a su desarrollo en odontólogos que trabajan en hospitales del Ministerio de Salud (MINSA) de Lima Metropolitana, Perú. Materiales y métodos. Estudio transversal analítico realizado entre diciembre de 2019 y enero de 2020 en 105 odontólogos de los 11 hospitales del MINSA de Lima Metropolitana. Se usó el instrumento Maslach Burnout Inventory para medir el burnout. Se evaluaron factores como sexo, edad, estado civil, tipo de jornada laboral, años de ejercicio laboral, si realizaba otra actividad laboral relacionada y horas diarias de ejercicio físico y de ocio. Se obtuvieron razones de prevalencia (RP) crudas (c) y ajustadas (a) para analizar la asociación entre presencia del síndrome y los factores. Resultados. La prevalencia del síndrome de burnout fue de 28.57%. Los niveles de agotamiento emocional, despersonalización y realización personal fueron altos en 90.47%, 98.09% y 35.23% de los participantes, respectivamente. La presencia del síndrome fue 47.00% menor en las mujeres (RPa=0.53, p=0.044) en comparación con los hombres, y 70.00% menor en quienes reportaron tener entre 11 y 20 años de ejercicio laboral (RPa=0.30, p=0.017), comparado con aquellos con menos de 11 años de experiencia laboral. Conclusiones. Casi un tercio de los participantes tuvieron síndrome de burnout; además, la mayoría de los odontólogos presentó altos niveles de agotamiento emocional y despersonalización. Ser mujer y tener entre 11 y 20 años de ejercicio laboral se comportaron como factores protectores.

Angioprevention of Urologic Cancers by Plant-Derived Foods.

The number of cancer cases worldwide keeps growing unstoppably, despite the undeniable advances achieved by basic research and clinical practice. Urologic tumors, including some as prevalent as prostate, bladder or kidney tumors, are no exceptions to this rule. Moreover, the fact that many of these tumors are detected in early stages lengthens the duration of their treatment, with a significant increase in health care costs. In this scenario, prevention offers the most cost-effective long-term strategy for the global control of these diseases. Although specialized diets are not the only way to decrease the chances to develop cancer, epidemiological evidence support the role of certain plant-derived foods in the prevention of urologic cancer. In many cases, these plants are rich in antiangiogenic phytochemicals, which could be responsible for their protective or angiopreventive properties. Angiogenesis inhibition may contribute to slow down the progression of the tumor at very different stages and, for this reason, angiopreventive strategies could be implemented at different levels of chemoprevention, depending on the targeted population. In this review, epidemiological evidence supporting the role of certain plant-derived foods in urologic cancer prevention are presented, with particular emphasis on their content in bioactive phytochemicals that could be used in the angioprevention of cancer.

The Chemokine-Based Peptide, CXCL9(74-103), Inhibits Angiogenesis by Blocking Heparan Sulfate Proteoglycan-Mediated Signaling of Multiple Endothelial Growth Factors.

Growth factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and epidermal growth factor (EGF) are important angiogenesis-mediating factors. They exert their effects not only through their respective receptor tyrosine kinases (RTKs), but they also require molecular pairing with heparan sulfate proteoglycans (HSPGs). Angiogenic growth factors and their signaling pathways are commonly targeted in current anti-angiogenic cancer therapies but have unfortunately insufficient impact on patient survival. Considering their obvious role in pathological angiogenesis, HS-targeting drugs have become an appealing new strategy. Therefore, we aimed to reduce angiogenesis through interference with growth factor-HS binding and downstream signaling using a CXCL9-derived peptide with a high affinity for glycosaminoglycans (GAGs), CXCL9(74-103). We showed that CXCL9(74-103) reduced EGF-, VEGF165- and FGF-2-mediated angiogenic processes in vitro, such as endothelial cell proliferation, chemotaxis, adhesion and sprouting, without exerting cell toxicity. CXCL9(74-103) interfered with growth factor signaling in diverse ways, e.g., by diminishing VEGF165 binding to HS and by direct association with FGF-2. The dependency of CXCL9(74-103) on HS for binding to HMVECs and for exerting its anti-angiogenic activity was also demonstrated. In vivo, CXCL9(74-103) attenuated neovascularization in the Matrigel plug assay, the corneal cauterization assay and in MDA-MB-231 breast cancer xenografts. Additionally, CXCL9(74-103) reduced vascular leakage in the retina of diabetic rats. In contrast, CXCL9(86-103), a peptide with low GAG affinity, showed no overall anti-angiogenic activity. Altogether, our results indicate that CXCL9(74-103) reduces angiogenesis by interfering with multiple HS-dependent growth factor signaling pathways.

Non-targeted metabolomics characterization of Annona muricata leaf extracts with anti-angiogenic activity.

The tropical plant Annona muricata has been widely used for traditional ethnobotanic and pharmacologic applications. Extracts from different parts of this plant have been shown to have a wide range of biological activities. In the present study, we carry out a metabolomic study of both aqueous and DMSO extracts from Annona muricata leaves that has allowed us to identify 33 bioactive compounds. Furthermore, we have shown that aqueous extracts are able to inhibit endothelial cell migration and both aqueous and DMSO extracts inhibit the formation of tubule-like structures by endothelial cells cultured on Matrigel. We conclude that extracts of Annona muricata leaves have great potential as anti-angiogenic natural combinations of bioactive compounds.

Protocols for endothelial cell isolation from mouse tissues: brain, choroid, lung, and muscle.

Endothelial cells (ECs) harbor distinct phenotypical and functional characteristics depending on their tissue localization and contribute to brain, eye, lung, and muscle diseases such as dementia, macular degeneration, pulmonary hypertension, and sarcopenia. To study their function, isolation of pure ECs in high quantities is crucial. Here, we describe protocols for rapid and reproducible blood vessel EC purification established for scRNA sequencing from murine tissues using mechanical and enzymatic digestion followed by magnetic and fluorescence-activated cell sorting. For complete details on the use and execution of these protocol, please refer to Kalucka et al. (2020), Rohlenova et al. (2020), and Goveia et al. (2020).

Protocols for endothelial cell isolation from mouse tissues: kidney, spleen, and testis.

Endothelial cells (ECs) exhibit phenotypic and functional tissue specificities, critical for studies in the vascular field and beyond. Thus, tissue-specific methods for isolation of highly purified ECs are necessary. Kidney, spleen, and testis ECs are relevant players in health and diseases such as chronic kidney disease, acute kidney injury, myelofibrosis, and cancer. Here, we provide tailored protocols for rapid and reproducible EC purification established for scRNA sequencing from these adult murine tissues using the combination of magnetic- and fluorescence-activated cell sorting. For complete details on the use and execution of these protocols, please refer to Kalucka et al. (2020) and Dumas et al. (2020).

Tumor vessel co-option probed by single-cell analysis.

Tumor vessel co-option is poorly understood, yet it is a resistance mechanism against anti-angiogenic therapy (AAT). The heterogeneity of co-opted endothelial cells (ECs) and pericytes, co-opting cancer and myeloid cells in tumors growing via vessel co-option, has not been investigated at the single-cell level. Here, we use a murine AAT-resistant lung tumor model, in which VEGF-targeting induces vessel co-option for continued growth. Single-cell RNA sequencing (scRNA-seq) of 31,964 cells reveals, unexpectedly, a largely similar transcriptome of co-opted tumor ECs (TECs) and pericytes as their healthy counterparts. Notably, we identify cell types that might contribute to vessel co-option, i.e., an invasive cancer-cell subtype, possibly assisted by a matrix-remodeling macrophage population, and another M1-like macrophage subtype, possibly involved in keeping or rendering vascular cells quiescent.

Protocols for endothelial cell isolation from mouse tissues: small intestine, colon, heart, and liver.

Endothelial cells (ECs) from the small intestine, colon, liver, and heart have distinct phenotypes and functional adaptations that are dependent on their physiological environment. Gut ECs adapt to low oxygen, heart ECs to contractile forces, and liver ECs to low flow rates. Isolating high-purity ECs in sufficient quantities is crucial to study their functions. Here, we describe protocols combining magnetic and fluorescent activated cell sorting for rapid and reproducible EC purification from four adult murine tissues. For complete details on the use and execution of these protocols, please refer to Kalucka et al. (2020).