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Discrimination; Gender equity; Oncology Discriminació; Equitat de gènere; Oncologia Discriminación; Equidad de género; Oncología Background Following a European Society for Medical Oncology Women for Oncology (ESMO W4O) survey in 2016 showing severe under-representation of female oncologists in leadership roles, ESMO launched a series of initiatives to address obstacles to gender equity. A follow-up survey in October 2021 investigated progress achieved. Materials and methods The W4O questionnaire 2021 expanded on the 2016 survey, with additional questions on the impact of ethnicity, sexual orientation and religion on career development. Results were analysed according to respondent gender and age. Results The survey sample was larger than in 2016 (n = 1473 versus 482), especially among men. Significantly fewer respondents had managerial or leadership roles than in 2016 (31.8% versus 51.7%). Lack of leadership development for women and unconscious bias were considered more important in 2021 than in 2016. In 2021, more people reported harassment in the workplace than in 2016 (50.3% versus 41.0%). In 2021, ethnicity, sexual orientation and religion were considered to have little or no impact on professional career opportunities, salary setting or related potential pay gap. However, gender had a significant or major impact on career development (25.5% of respondents), especially in respondents ≤40 years of age and women. As in 2016, highest ranked initiatives to foster workplace equity were promotion of work–life balance, development and leadership training and flexible working. Significantly more 2021 respondents (mainly women) supported the need for culture and gender equity education at work than in 2016. Conclusions Gender remains a major barrier to career progression in oncology and, although some obstacles may have been reduced since 2016, we are a long way from closing the gender gap. Increased reporting of discrimination and inappropriate behaviour in the workplace is a major, priority concern. The W4O 2021 survey findings provide new evidence and highlight the areas for future ESMO interventions to support equity and diversity in oncology career development. This work was supported by the European Society for Medical Oncology (no grant number).

Subcellular membrane systems are highly enriched in dolichol, whose role in organelle homeostasis and endosomal-lysosomal pathway remains largely unclear besides being involved in protein glycosylation. DHDDS encodes for the catalytic subunit (DHDDS) of the enzyme cis-prenyltransferase (cis-PTase), involved in dolichol biosynthesis and dolichol-dependent protein glycosylation in the endoplasmic reticulum. An autosomal recessive form of retinitis pigmentosa (retinitis pigmentosa 59) has been associated with a recurrent DHDDS variant. Moreover, two recurring de novo substitutions were detected in a few cases presenting with neurodevelopmental disorder, epilepsy and movement disorder. We evaluated a large cohort of patients (n = 25) with de novo pathogenic variants in DHDDS and provided the first systematic description of the clinical features and long-term outcome of this new neurodevelopmental and neurodegenerative disorder. The functional impact of the identified variants was explored by yeast complementation system and enzymatic assay. Patients presented during infancy or childhood with a variable association of neurodevelopmental disorder, generalized epilepsy, action myoclonus/cortical tremor and ataxia. Later in the disease course, they experienced a slow neurological decline with the emergence of hyperkinetic and/or hypokinetic movement disorder, cognitive deterioration and psychiatric disturbances. Storage of lipidic material and altered lysosomes were detected in myelinated fibres and fibroblasts, suggesting a dysfunction of the lysosomal enzymatic scavenger machinery. Serum glycoprotein hypoglycosylation was not detected and, in contrast to retinitis pigmentosa and other congenital disorders of glycosylation involving dolichol metabolism, the urinary dolichol D18/D19 ratio was normal. Mapping the disease-causing variants into the protein structure revealed that most of them clustered around the active site of the DHDDS subunit. Functional studies using yeast complementation assay and in vitro activity measurements confirmed that these changes affected the catalytic activity of the cis-PTase and showed growth defect in yeast complementation system as compared with the wild-type enzyme and retinitis pigmentosa-associated protein. In conclusion, we characterized a distinctive neurodegenerative disorder due to de novo DHDDS variants, which clinically belongs to the spectrum of genetic progressive encephalopathies with myoclonus. Clinical and biochemical data from this cohort depicted a condition at the intersection of congenital disorders of glycosylation and inherited storage diseases with several features akin to of progressive myoclonus epilepsy such as neuronal ceroid lipofuscinosis and other lysosomal disorders.

Background: Cross-talk between sterol metabolism and inflammatory pathways has been demonstrated to significantly affect the development of atherosclerosis. Cholesterol biosynthetic intermediates and derivatives are increasingly recognized as key immune regulators of macrophages in response to innate immune activation and lipid overloading. 25-Hydroxycholesterol (25-HC) is produced as an oxidation product of cholesterol by the enzyme cholesterol 25-hydroxylase (CH25H) and belongs to a family of bioactive cholesterol derivatives produced by cells in response to fluctuating cholesterol levels and immune activation. Despite the major role of 25-HC as a mediator of innate and adaptive immune responses, its contribution during the progression of atherosclerosis remains unclear. Methods: The levels of 25-HC were analyzed by liquid chromatography-mass spectrometry, and the expression of CH25H in different macrophage populations of human or mouse atherosclerotic plaques, respectively. The effect of CH25H on atherosclerosis progression was analyzed by bone marrow adoptive transfer of cells from wild-type or Ch25h –/– mice to lethally irradiated Ldlr –/– mice, followed by a Western diet feeding for 12 weeks. Lipidomic, transcriptomic analysis and effects on macrophage function and signaling were analyzed in vitro from lipid-loaded macrophage isolated from Ldlr –/– or Ch25h–/–;Ldlr–/– mice . The contribution of secreted 25-HC to fibrous cap formation was analyzed using a smooth muscle cell lineage–tracing mouse model, Myh11 ERT2CRE mT/mG;Ldlr –/– , adoptively transferred with wild-type or Ch25h –/– mice bone marrow followed by 12 weeks of Western diet feeding. Results: We found that 25-HC accumulated in human coronary atherosclerotic lesions and that macrophage-derived 25-HC accelerated atherosclerosis progression, promoting plaque instability through autocrine and paracrine actions. 25-HC amplified the inflammatory response of lipid-loaded macrophages and inhibited the migration of smooth muscle cells within the plaque. 25-HC intensified inflammatory responses of lipid-laden macrophages by modifying the pool of accessible cholesterol in the plasma membrane, which altered Toll-like receptor 4 signaling, promoted nuclear factor-κB–mediated proinflammatory gene expression, and increased apoptosis susceptibility. These effects were independent of 25-HC–mediated modulation of liver X receptor or SREBP (sterol regulatory element–binding protein) transcriptional activity. Conclusions: Production of 25-HC by activated macrophages amplifies their inflammatory phenotype, thus promoting atherogenesis.

Bone morphogenetic protein-9 (BMP-9) is a circulating cytokine that is known to play an essential role in the endothelial homeostasis and the binding of BMP-9 to the receptor activin-like kinase 1 (ALK-1) promotes endothelial cell quiescence. Previously, using an unbiased screen, we identified ALK-1 as a high-capacity receptor for low-density lipoprotein (LDL) in endothelial cells that mediates its transcytosis in a nondegradative manner. Here we examine the crosstalk between BMP-9 and LDL and how it influences their interactions with ALK-1. Treatment of endothelial cells with BMP-9 triggers the extensive endocytosis of ALK-1, and it is mediated by caveolin-1 (CAV-1) and dynamin-2 (DNM2) but not clathrin heavy chain. Knockdown of CAV-1 reduces BMP-9-mediated internalization of ALK-1, BMP-9-dependent signaling and gene expression. Similarly, treatment of endothelial cells with LDL reduces BMP-9-induced SMAD1/5 phosphorylation and gene expression and silencing of CAV-1 and DNM2 diminishes LDL-mediated ALK-1 internalization. Interestingly, BMP-9-mediated ALK-1 internalization strongly re-duces LDL transcytosis to levels seen with ALK-1 deficiency. Thus, BMP-9 levels can control cell surface levels of ALK-1, via CAV-1, to regulate both BMP-9 signaling and LDL transcytosis.

Supplemental Digital Content is available in the text., Objective: Endothelial Cav-1 (caveolin-1) expression plays a relevant role during atherogenesis by controlling NO production, vascular inflammation, LDL (low-density lipoprotein) transcytosis, and extracellular matrix remodeling. Additional studies have identified cholesterol-rich membrane domains as important regulators of autophagy by recruiting ATGs (autophagy-related proteins) to the plasma membrane. Here, we investigate how the expression of Cav-1 in the aortic endothelium influences autophagy and whether enhanced autophagy contributes to the atheroprotective phenotype observed in Cav-1–deficient mice. Approach and Results: To analyze the impact of Cav-1 deficiency on regulation of autophagy in the aortic endothelium during the progression of atherosclerosis, we fed Ldlr−/− and Cav-1−/−Ldlr−/− mice a Western diet and assessed autophagy in the vasculature. We observe that the absence of Cav-1 promotes autophagy activation in athero-prone areas of the aortic endothelium by enhancing autophagic flux. Mechanistically, we found that Cav-1 interacts with the ATG5-ATG12 complex and influences the cellular localization of autophagosome components in lipid rafts, which controls the autophagosome formation and autophagic flux. Pharmacological inhibition of autophagy attenuates the atheroprotection observed in Cav-1−/− mice by increasing endothelial inflammation and macrophage recruitment, identifying a novel molecular mechanism by which Cav-1 deficiency protects against the progression of atherosclerosis. Conclusions: These results identify Cav-1 as a relevant regulator of autophagy in the aortic endothelium and demonstrate that pharmacological suppression of autophagic flux in Cav-1–deficient mice attenuates the atheroprotection observed in Cav-1−/− mice. Additionally, these findings suggest that activation of endothelial autophagy by blocking Cav-1 might provide a potential therapeutic strategy for cardiovascular diseases including atherosclerosis.

Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) is a critical mediator of vascular function. eNOS is tightly regulated at various levels, including transcription, co- and posttranslational modifications, and by various protein-protein interactions. Using stable isotope labeling with amino acids in cell culture (SILAC) and mass spectrometry (MS), we identified several eNOS interactors, including the protein plasminogen activator inhibitor-1 (PAI-1). In cultured human umbilical vein endothelial cells (HUVECs), PAI-1 and eNOS colocalize and proximity ligation assays demonstrate a protein-protein interaction between PAI-1 and eNOS. Knockdown of PAI-1 or eNOS eliminates the proximity ligation assay (PLA) signal in endothelial cells. Overexpression of eNOS and HA-tagged PAI-1 in COS7 cells confirmed the colocalization observations in HUVECs. Furthermore, the source of intracellular PAI-1 interacting with eNOS was shown to be endocytosis derived. The interaction between PAI-1 and eNOS is a direct interaction as supported in experiments with purified proteins. Moreover, PAI-1 directly inhibits eNOS activity, reducing NO synthesis, and the knockdown or antagonism of PAI-1 increases NO bioavailability. Taken together, these findings place PAI-1 as a negative regulator of eNOS and disruptions in eNOS-PAI-1 binding promote increases in NO production and enhance vasodilation in vivo.

Vascular smooth muscle cell (VSMC) phenotypic switching contributes to cardiovascular diseases. Epigenetic regulation is emerging as a key regulatory mechanism, with the methyl cytosine dioxygenase TET2 acting as a master regulator of SMC phenotype. The histone acetyltransferases (HATs) p300 and CBP are highly homologous and often considered to be interchangeable, and their roles in SMC phenotypic regulation are not known. We assessed the roles of p300 and CBP in human VSMC with knockdown, in inducible smooth muscle-specific knockout mice ( p300 iKO or CBP iKO ), and in human intimal hyperplasia. P300, CBP, and histone acetylation were differently regulated in VSMCs undergoing phenotypic switching and in injury-induced vessel remodeling. Knockdown experiments revealed opposing effects of p300 and CBP in VSMC phenotype: p300 promoted contractile protein expression and inhibited migration, but CBP inhibited contractile genes and enhanced migration. p300 iKO mice exhibited severe intimal hyperplasia following arterial injury while CBP iKO mice were entirely protected. In normal aorta, p300 iKO reduced, but CBP iKO enhanced, contractile proteins and contractility. Mechanistically, we found that these HATs oppositely regulate histone acetylation, DNA hydroxy methylation, and RNA PolII binding to promoters of differentiation-specific contractile genes. We report p300 and TET2 function together, as p300 was required for TET2-dependent hydroxy methylation of contractile promoters, and TET2 was required for p300-dependent acetylation of these loci. TET2 co-immunoprecipitated with p300 and this interaction was enhanced by rapamycin but repressed by PDGF, with p300 promoting TET2 protein stability. CBP did not associate with TET2, instead facilitated recruitment of histone deacetylases (HDAC2, HDAC5) to contractile gene promoters. Immunostaining revealed, p300 expression is repressed but CBP is induced in human intimal hyperplasia. This work reveals that p300 and CBP serve non-redundant and opposing functions in VSMC phenotypic switching and coordinately regulate chromatin modifications through distinct functional interactions with TET2 or HDACs. Targeting specific HATs may hold therapeutic promise for cardiovascular diseases.

ObjectiveWe have previously demonstrated thein vivoimportance of the Akt-eNOS substrate-kinase relationship, as defective postnatal angiogenesis characteristic of global Akt1-null mice is rescued when bred to ‘gain-of-function’ eNOS S1176D mutant mice. While multiple studies support the cardioprotective role of endothelial NO generation, the causal role of Akt1-dependent eNOS S1176 phosphorylation during atherosclerotic plaque formation is not yet clear.Approach & ResultsWe herein bred congenic ‘loss-of-function’ eNOS S1176A and ‘gain-of-function’ eNOS S1176D mutant mice to the proatherogenic Akt1−/−; ApoE−/−double knockout mice to definitively test the importance of Akt-mediated eNOS S1176 phosphorylation during atherogenesis. We find that a single amino acid substitution at the eNOS S1176 phosphorylation site yields divergent effects on atherosclerotic plaque formation, as an eNOS phospho-mimic aspartate (D) substitution at S1176 leads to decreased indices of atherosclerosis, even when on a proatherogenic Akt1 global deletion background. Conversely, mice harboring an unphosphorylatable mutation to alanine (S1176A) result in increased lipid deposition and cellular apoptosis, phenocopying the physiological consequence of eNOS deletion and/or impaired enzyme function. Furthermore, gene expression analyses of whole aortas indicate a combinatorial detriment from NO deficiency and Western Diet challenge, as ‘loss-of-function’ eNOS SA mice on a high-fat and high-cholesterol diet present a unique expression pattern indicative of augmented T-cell activity when compared to eNOS S1176D mice.ConclusionsBy using genetic epistasis approaches, we conclusively demonstrate that Akt-mediated eNOS S1176 phosphorylation and subsequent activation remains to be the most physiologically relevant method of NO production to promote cardioprotective effects.

Artworks are complex objects that merit study and preservation. Far-infrared spectroscopy in ATR mode appears to be a suitable technique for this purpose because it enables information to be obtained regarding the material’s composition in a non-destructive way. The use of Far-infrared is especially interesting because most organic compounds do not absorb in this energy range, suggesting the possibility of identifying inorganic pigments. Based on works performed by two research groups from the University of Bologna and the University of Tartu, this study attempts to obtain additional information regarding the capabilities and limitations of Far-infrared spectroscopy when it is applied to objects as complex as artworks. This article first studies the capability of the technique for identifying pigments by following the stability of the position of their absorption bands when mixed with linseed oil, the minimum amount of pigment necessary to be detected and how this amount changes when it is part of a paint layer. The consequences of the pigment: linseed oil interaction and the ageing process are also studied through changes in the linseed oil signal absorptions related to the acid carboxylic and carboxylate bands. The entire study leads to the conclusion that Far-Infrared in ATR mode is an interesting option for the selective identification of some inorganic pigments, but their potential application depends on each case considered.

Introduction/Background* In the randomized phase 3 JAVELIN Ovarian 200 trial (N=566), avelumab alone or combined with pegylated liposomal doxorubicin (PLD) did not significantly prolong progression-free survival (PFS; blinded independent central review) or overall survival (OS) vs PLD alone in patients with platinum-resistant/refractory ovarian cancer (PRROC). Here, we report exploratory biomarker analyses associated with outcomes in the avelumab alone arm. Methodology Biomarkers analyzed in tumour tissue or blood were somatic and germline mutations by whole-exome sequencing and whole-transcriptomic profiling by RNAseq. Associations were assessed using the Cox proportional hazards model. For continuous biomarkers, hazard ratios (HRs) and 95% CIs were reported using a median cutoff. Result(s)* Within the avelumab alone arm, higher expression of CD8+ T-cell signature correlated with longer PFS (HR, 0.61 [95% CI 0.43-0.87]), and higher tumour mutational burden (>0.6 mut/Mb) and presence of the high-affinity FCGR2A allele correlated with longer OS (HR [95% CI], 0.63 [0.42-0.96] and 0.53 [0.34-0.83], respectively), while presence of APOBEC and homologous recombination deficiency (HRD) mutational signatures were associated with shorter OS (HR [95% CI], 2.17 [1.22-3.89] and 1.68 [1.07-2.62], respectively). In patients with PFS of Conclusion* Although JAVELIN Ovarian 200 did not meet its primary endpoints, these analyses indicate potential subgroups of patients who could have improved outcomes with immunotherapy alone and provide insights into the biology of PRROC that may inform future trials.

Introduction/Background* In the randomized phase 3 JAVELIN Ovarian 200 trial (N=566), avelumab alone or combined with pegylated liposomal doxorubicin (PLD) did not significantly prolong progression-free survival (PFS; blinded independent central review) or overall survival (OS) vs PLD alone in patients with platinum-resistant/refractory ovarian cancer (PRROC). Here, we report exploratory biomarker analyses associated with outcomes in the avelumab alone arm. Methodology Biomarkers analyzed in tumour tissue or blood were somatic and germline mutations by whole-exome sequencing and whole-transcriptomic profiling by RNAseq. Associations were assessed using the Cox proportional hazards model. For continuous biomarkers, hazard ratios (HRs) and 95% CIs were reported using a median cutoff. Result(s)* Within the avelumab alone arm, higher expression of CD8+ T-cell signature correlated with longer PFS (HR, 0.61 [95% CI 0.43-0.87]), and higher tumour mutational burden (>0.6 mut/Mb) and presence of the high-affinity FCGR2A allele correlated with longer OS (HR [95% CI], 0.63 [0.42-0.96] and 0.53 [0.34-0.83], respectively), while presence of APOBEC and homologous recombination deficiency (HRD) mutational signatures were associated with shorter OS (HR [95% CI], 2.17 [1.22-3.89] and 1.68 [1.07-2.62], respectively). In patients with PFS of Conclusion* Although JAVELIN Ovarian 200 did not meet its primary endpoints, these analyses indicate potential subgroups of patients who could have improved outcomes with immunotherapy alone and provide insights into the biology of PRROC that may inform future trials.

Nogo-B, a member of the reticulon 4 family of proteins, is the dominant isoform expressed in endothelial cells (EC). We have shown that Nogo-B is necessary for blood flow recovery in ischemia. Mice lacking Nogo-B exhibit reduced arteriogenesis and angiogenesis that are linked to a decrease in macrophage infiltration and inflammatory gene expression in vivo. However, whether endothelial Nogo-B regulates arteriogenesis and/or angiogenesis is unknown. We generated an inducible EC-specific mouse overexpressing Nogo-B (Ng ECtg ) and investigated the arteriogenesis and angiogenesis in limb ischemia. Ischemia increased endothelial and serum Nogo-B expression. Blood flow recovery was markedly diminished after femoral artery ligation (FAL) in Ng ECtg compared to WT mice, in association with lower collateral density accessed by micro-CT arteriography. There was no reduction in capillary density or decrease in smooth muscle/pericyte and macrophage recruitment in Ng ECtg mice, suggesting EC Nogo-B overexpression regulates arteriogenesis but not angiogenesis. While Ng ECtg mice have normal number of native collateral artery, the early remodeling of the collateral artery was impaired after ischemia. Furthermore, in line with impaired remodeling, Ng ECtg mice have decreased functional (exercise) hyperemia response in the non-ischemic limb in vivo, and resistant arteries from Ng ECtg have diminished flow induced vasodilatation ex vivo compared to WT mice. Moreover, endothelial reconstitution of Nogo-B in global Nogo knockout background (NogoKO ECrc ) restored blood flow recovery after ischemia in vivo, further suggesting the EC specific function of Nogo-B in arteriogenesis. Mechanistically, we have shown that EC isolated from Ng ECtg mice have decreased nitric oxide release. Nogo-B overexpression negatively regulates eNOS phosphorylation, expression and golgi localization in EC. Future parabiosis study is warranted to elucidate the contribution of paracrine or EC intrinsic Nogo-B in modulating arteriogenesis and angiogenesis. In conclusion, our data showed for the first time that endothelial Nogo-B plays important roles in regulating collateral artery remodeling and blood flow through, at least in part, regulating NO bioavailability.

Background: Atherosclerosis is driven by synergistic interactions between pathological, biomechanical, inflammatory, and lipid metabolic factors. Our previous studies demonstrated that absence of caveolin-1 (Cav1)/caveolae in hyperlipidemic mice strongly inhibits atherosclerosis, which was attributed to activation of endothelial nitric oxide (NO) synthase (eNOS) and increased production of NO and reduced inflammation and low-density lipoprotein trafficking. However, the contribution of eNOS activation and NO production in the athero-protection of Cav1 and the exact mechanisms by which Cav1/caveolae control the pathogenesis of diet-induced atherosclerosis are still not clear. Methods: Triple-knockout mouse lacking expression of eNOS , Cav1 , and Ldlr were generated to explore the role of NO production in Cav1-dependent athero-protective function. The effects of Cav1 on lipid trafficking, extracellular matrix remodeling, and vascular inflammation were studied both in vitro and in vivo with a mouse model of diet-induced atherosclerosis. The expression of Cav1 and distribution of caveolae regulated by flow were analyzed by immunofluorescence staining and transmission electron microscopy. Results: We found that absence of Cav1 significantly suppressed atherogenesis in Ldlr −/− eNOS −/− mice, demonstrating that athero-suppression is independent of increased NO production. Instead, we find that the absence of Cav1/caveolae inhibited low-density lipoprotein transport across the endothelium and proatherogenic fibronectin deposition and disturbed flow-mediated endothelial cell inflammation. Consistent with the idea that Cav1/caveolae may play a role in early flow-dependent inflammatory priming, distinct patterns of Cav1 expression and caveolae distribution were observed in athero-prone and athero-resistant areas of the aortic arch even in wild-type mice. Conclusions: These findings support a role for Cav1/caveolae as a central regulator of atherosclerosis that links biomechanical, metabolic, and inflammatory pathways independently of endothelial eNOS activation and NO production.

© 2021 American Heart Association, Inc., Background: Activin receptor-like kinase 1 (ALK1) is an endothelial transmembrane serine threonine kinase receptor for BMP family ligands that plays a critical role in cardiovascular development and pathology. Loss-of-function mutations in the ALK1 gene cause type 2 hereditary hemorrhagic telangiectasia, a devastating disorder that leads to arteriovenous malformations. Here, we show that ALK1 controls endothelial cell polarization against the direction of blood flow and flow-induced endothelial migration from veins through capillaries into arterioles. Methods: Using Cre lines that recombine in different subsets of arterial, capillary-venous, or endothelial tip cells, we show that capillary-venous Alk1 deletion was sufficient to induce arteriovenous malformation formation in the postnatal retina. Results: ALK1 deletion impaired capillary-venous endothelial cell polarization against the direction of blood flow in vivo and in vitro. Mechanistically, ALK1-deficient cells exhibited increased integrin signaling interaction with vascular endothelial growth factor receptor 2, which enhanced downstream YAP/TAZ nuclear translocation. Pharmacologic inhibition of integrin or YAP/TAZ signaling rescued flow migration coupling and prevented vascular malformations in Alk1-deficient mice. Conclusions: Our study reveals ALK1 as an essential driver of flow-induced endothelial cell migration and identifies loss of flow-migration coupling as a driver of arteriovenous malformation formation in hereditary hemorrhagic telangiectasia disease. Integrin-YAP/TAZ signaling blockers are new potential targets to prevent vascular malformations in patients with hereditary hemorrhagic telangiectasia., This work was supported by grants from the Leducq Foundation (TNE ATTRACT to Drs Eichmann and Franco) and the US National Institutes of Health (P30 EY026878 and R01EY025979 to Dr Eichmann and R01 HL135582 to Dr Schwartz). Dr Franco was supported by European Research Council starting grant (679368) and Fundação para a Ciência e a Tecnologia funding (CEECIND/02589/2018).

Although tissue uptake of fatty acids from chylomicrons is primarily via lipoprotein lipase (LpL) hydrolysis of triglycerides (TGs), studies of patients with genetic LpL deficiency suggest additional pathways deliver dietary lipids to tissues. Despite an intact endothelial cell (EC) barrier, hyperchylomicronemic patients accumulate chylomicron-derived lipids within skin macrophages, leading to the clinical finding eruptive xanthomas. We explored whether an LpL-independent pathway exists for transfer of circulating lipids across the EC barrier. We found that LpL-deficient mice had a marked increase in aortic EC lipid droplets before and after a fat gavage. Cultured ECs internalized chylomicrons, which were hydrolyzed within lysosomes. The products of this hydrolysis fueled lipid droplet biogenesis in ECs and triggered lipid accumulation in cocultured macrophages. EC chylomicron uptake was inhibited by competition with HDL and knockdown of the scavenger receptor-BI (SR-BI). In vivo, SR-BI knockdown reduced TG accumulation in aortic ECs and skin macrophages of LpL-deficient mice. Thus, ECs internalize chylomicrons, metabolize them in lysosomes, and either store or release their lipids. This latter process may allow accumulation of TGs within skin macrophages and illustrates a pathway that might be responsible for creation of eruptive xanthomas.

BackgroundActivin receptor-like kinase 1 (ACVRL1, hereafter ALK1) is an endothelial transmembrane serine threonine kinase receptor for BMP family ligands that plays a critical role in cardiovascular development and pathology. Loss-of-function mutations in the ALK1 gene cause type 2 hereditary hemorrhagic telangiectasia (HHT), a devastating disorder that leads to arteriovenous malformations (AVMs). Here we show that ALK1 controls endothelial cell polarization against the direction of blood flow and flow-induced endothelial migration from veins through capillaries into arterioles.MethodsUsing Cre lines that recombine in different subsets of arterial, capillary-venous or endothelial tip cells, we showed that capillary-venous Alk1 deletion was sufficient to induce AVM formation in the postnatal retina.ResultsALK1 deletion impaired capillary-venous endothelial cell polarization against the direction of blood flow in vivo and in vitro. Mechanistically, ALK1 deficient cells exhibited increased integrin signaling interaction with VEGFR2, which enhanced downstream YAP/TAZ nuclear translocation. Pharmacological inhibition of integrin or YAP/TAZ signaling rescued flow migration coupling and prevented vascular malformations in Alk1 deficient mice.ConclusionsOur study reveals ALK1 as an essential driver of flow-induced endothelial cell migration and identifies loss of flow-migration coupling as a driver of AVM formation in HHT disease. Integrin-YAP/TAZ signaling blockers are new potential targets to prevent vascular malformations in HHT patients.

Background Licensed systemic treatment options for platinum-sensitive recurrent ovarian cancer are platinum-based chemotherapy and maintenance treatment with bevacizumab and poly (ADP-ribose) polymerase inhibitors. For platinum-resistant disease, several non-platinum options are available. We aimed to assess the clinical benefit of these treatments according to the European Society of Medical Oncology (ESMO)-Magnitude of Clinical Benefit Scale (MCBS). Materials and methods A PubMed search was carried out including all studies evaluating systemic treatment of recurrent epithelial ovarian cancer, from 1990 onwards. Randomised trials with an adequate comparator and design showing a statistically significant benefit of the study arm were independently scored by two blinded observers using the ESMO-MCBS. Results A total of 1127 papers were identified, out of which 61 reported results of randomised trials of sufficient quality. Nineteen trials showed statistically significant results and the studied treatments were graded according to ESMO-MCBS. Only three treatments showed substantial benefit (score of 4 on a scale of 1-5) according to the ESMO-MCBS: platinum-based chemotherapy with paclitaxel in the platinum-sensitive setting and the addition of bevacizumab to chemotherapy in the platinum-resistant setting. The WEE1 inhibitor adavosertib (not licensed) also scores a 4, based on a recent small phase II study. Assessment of quality-of-life data and toxicity using the ESMO-MCBS showed to be complex, which should be taken into account in using this score for clinical decision making. Conclusion Only a few licensed systemic therapies for recurrent ovarian cancer show substantial clinical benefit based on ESMO-MCBS scores. Trials demonstrating overall survival benefit are sparse., Highlights • Benefit of systemic treatments for recurrent ovarian cancer was scored on the ESMO-MCBS. • From 1127 identified papers, only 3 trials out of 19 that were suitable for grading showed substantial clinical benefit. • Trials demonstrating overall survival benefit are sparse.

DHX15 is a downstream substrate for Akt1, which is involved in key cellular processes affecting vascular biology. Here, we explored the vascular regulatory function of DHX15. Homozygous DHX15 gene deficiency was lethal in mouse and zebrafish embryos. DHX15 zebrafish also showed downregulation of VEGF-C and reduced formation of lymphatic structures during development. DHX15 mice depicted lower vascular density and impaired lymphatic function postnatally. RNAseq and proteome analysis of DHX15 silenced endothelial cells revealed differential expression of genes involved in the metabolism of ATP biosynthesis. The validation of these results demonstrated a lower activity of the Complex I in the mitochondrial membrane of endothelial cells, resulting in lower intracellular ATP production and lower oxygen consumption. After injection of syngeneic LLC1 tumor cells, DHX15 mice showed partially inhibited primary tumor growth and reduced lung metastasis. Our results revealed an important role of DHX15 in vascular physiology and pave a new way to explore its potential use as a therapeutical target for metastasis treatment., This study was supported by grants from MCIN/AEI/10.13039/501100011033 (SAF2016-75358-R and PDI2019-105502RB-100 to MM-R, SAF2017-85877R, PID2019-111669RB-100, PID2020-115055RB-I00). CIBERehd, CIBERonc and CIBERbbn are financed by the Instituto de Salud Carlos III. I.P. was recipient of a predoctoral fellowship supported by MCIN/AEI/10.13039/501100011033 y FSE “El FSE invierte en tu futuro” (BES-2017-080823). A.E-.C. is funded by ISCIII of the MINECO (reference PT17/0009/0019) and co-financed by FEDER. J.R-.V. was a recipient of a BIOTRACK Postdoctoral Fellowship supported by the European Community’s Seventh Framework Programme and the MINECO (Contract 229673). The CRG/UPF Proteomics Unit is part of the Spanish Infrastructure for Omics Technologies (ICTS OmicsTech) and it is a member of the ProteoRed PRB3 consortium which is supported by grant PT17/0019 of the PE I + D + i 2013-2016 from the Instituto de Salud Carlos III (ISCIII) and ERDF. The center grant P50AA011999 Southern California Research Center for ALPD and Cirrhosis funded by NIAAA/NIH. We acknowledge support from the Spanish Ministry of Science, Innovation and Universities, “Centro de Excelencia Severo Ochoa 2013-2017”, SEV-2012-0208, and “Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat de Catalunya” (2017SGR595). The authors would like to thank Drs. Simone Calzolari, Rafael Miñana and Javier Terriente from ZeClinics for their helpful collaboration with the zebrafish model generation and experimentation.

Background: Bevacizumab is approved in combination with chemotherapy for the treatment of ovarian cancer, either in first-line therapy or for patients with recurrent disease not previously treated with the same drug. We aimed to test the value of continuing bevacizumab beyond progression after first-line treatment with the same drug. Methods: In our open-label, randomised, phase 3 trial done at 82 sites in four countries, we enrolled women (aged ≥18 years) who had previously received first-line platinum-based therapy including bevacizumab, and had recurrent (≥6 months since last platinum dose), International Federation of Gynaecology and Obstetrics stage IIIB–IV ovarian cancer with an Eastern Cooperative Oncology Group performance status 0–2. Patients were randomly assigned (1:1) to receive a carboplatin-based doublet intravenously (carboplatin area under the concentration curve [AUC] 5 on day 1 plus paclitaxel 175 mg/m2 on day 1, every 21 days; carboplatin AUC 4 on day 1 plus gemcitabine 1000 mg/m2 on days 1 and 8, every 21 days; or carboplatin AUC 5 on day 1 plus pegylated liposomal doxorubicin 30 mg/m2 on day 1, every 28 days), or a carboplatin-based doublet plus bevacizumab (10 mg/kg intravenous every 14 days combined with pegylated liposomal doxorubicin–carboplatin, or 15 mg/kg every 21 days combined with gemcitabine–carboplatin or paclitaxel–carboplatin). Evaluable disease according to RECIST 1.1 guidelines was required before randomisation. Randomisation was done through the trial website with a minimisation procedure, stratified by centre, time of recurrence, performance status, and type of second-line chemotherapy. The primary endpoint was investigator-assessed progression-free survival, analysed on an intention-to-treat basis. Safety was assessed in all participants who received at least one dose. This trial is registered with ClinicalTrials.gov, NCT01802749 and EudraCT 2012-004362-17. Findings: Between Dec 6, 2013, and Nov 11, 2016, 406 patients were recruited (203 [50%] assigned to the bevacizumab group and 203 [50%] to the standard chemotherapy group). 130 patients (64%) in the bevacizumab group and 131 (65%) in the standard chemotherapy group had progressed after receiving a last dose of platinum more than 12 months before, and 146 patients (72%) in the bevacizumab group and 147 (72%) in the standard chemotherapy group had progressed after completion of first-line bevacizumab maintenance. 161 participants (79%) progressed in the standard chemotherapy group, as did 143 (70%) in the bevacizumab group. Median progression-free survival was 8·8 months (95% CI 8·4–9·3) in the standard chemotherapy group and 11·8 months (10·8–12·9) in the bevacizumab group (hazard ratio 0·51, 95% CI 0·41–0·65; log-rank p

Background: Hypoxia and consequent production of vascular endothelial growth factor A (VEGFA) promote blood vessel leakiness and edema in ocular diseases. Anti-VEGFA therapeutics may aggravate hypoxia; therefore, therapy development is needed. Methods: Oxygen-induced retinopathy was used as a model to test the role of nitric oxide (NO) in pathological neovascularization and vessel permeability. Suppression of NO formation was achieved chemically using L-NMMA, or genetically, in endothelial NO synthase serine to alanine (S1176A) mutant mice. Results: Suppression of NO formation resulted in reduced retinal neoangiogenesis. Remaining vascular tufts exhibited reduced vascular leakage through stabilized endothelial adherens junctions, manifested as reduced phosphorylation of vascular endothelial (VE)-cadherin Y685 in a c-Src-dependent manner. Treatment with a single dose of L-NMMA in established retinopathy restored the vascular barrier and prevented leakage. Conclusions: We conclude that NO destabilizes adheren junctions, resulting in vascular hyperpermeability, by converging with the VEGFA/VEGFR2/c-Src/VE-cadherin pathway. Funding: This study was supported by the Swedish Cancer foundation (19 0119 Pj ), the Swedish Research Council (2020-01349), the Knut and Alice Wallenberg foundation (KAW 2020.0057) and a Fondation Leducq Transatlantic Network of Excellence Grant in Neurovascular Disease (17 CVD 03). KAW also supported LCW with a Wallenberg Scholar grant (2015.0275). WCS was supported by Grants R35 HL139945, P01 HL1070205, AHA MERIT Award. DV was supported by grants from the Deutsche Forschungsgemeinschaft, SFB1450, B03, and CRU342, P2.

DHX15 is an ATP-dependent RNA helicase involved in pre-mRNA splicing and a downstream substrate for Akt1, which plays a significant role in vascular biology. The aim of this study was to explore the regulatory function of DHX15 over the vasculature and endothelial cell biology. Results: DHX15-/- was lethal in mouse and zebrafish embryos. DHX15-/- zebrafish also showed an undeveloped parachordal line, which leads to the formation of lymphatic structures. DHX15+/- mice triggered lower vascular network density and impaired lymphatic function postnatally. Transcriptome and proteome analysis of DHX15 silenced LEC revealed alterations in the glycolysis and gluconeogenesis pathways. The validation of these results demonstrated an uncoupling of the glycolysis with the oxidation of pyruvate in the mitochondria and a lower activity of the Complex I, resulting in lower cellular ATP production. Noteworthy, DHX15+/- mice partially inhibited primary tumor growth and reduced lung metastasis after injection of LLC1 tumor cells.

The lymphatic vasculature is involved in the pathogenesis of acute cardiac injuries, but little is known about its role in chronic cardiac dysfunction. Here, we demonstrate that angiotensin II infusion induced cardiac inflammation and fibrosis at 1 week and caused cardiac dysfunction and impaired lymphatic transport at 6 weeks in mice, while co-administration of VEGFCc156s improved these parameters. To identify novel mechanisms underlying this protection, RNA sequencing analysis in distinct cell populations revealed that VEGFCc156s specifically modulated angiotensin II-induced inflammatory responses in cardiac and peripheral lymphatic endothelial cells. Furthermore, telemetry studies showed that while angiotensin II increased blood pressure acutely in all animals, VEGFCc156s-treated animals displayed a delayed systemic reduction in blood pressure independent of alterations in angiotensin II-mediated aortic stiffness. Overall, these results demonstrate that VEGFCc156s had a multifaceted therapeutic effect to prevent angiotensin II-induced cardiac dysfunction by improving cardiac lymphatic function, alleviating fibrosis and inflammation, and ameliorating hypertension.

We established an international consortium to review and discuss relevant clinical evidence in order to develop expert consensus statements related to cancer management during the severe acute respiratory syndrome coronavirus 2-related disease (COVID-19) pandemic. The steering committee prepared 10 working packages addressing significant clinical questions from diagnosis to surgery. During a virtual consensus meeting of 62 global experts and one patient advocate, led by the European Society for Medical Oncology, statements were discussed, amended and voted upon. When consensus could not be reached, the panel revised statements until a consensus was reached. Overall, the expert panel agreed on 28 consensus statements that can be used to overcome many of the clinical and technical areas of uncertainty ranging from diagnosis to therapeutic planning and treatment during the COVID-19 pandemic.

Background & Aims Liver sinusoidal endothelial cell (LSEC) dysfunction has been reported in alcohol-related liver disease, yet it is not known whether LSECs metabolize alcohol. Thus, we investigated this, as well as the mechanisms of alcohol-induced LSEC dysfunction and a potential therapeutic approach for alcohol-induced liver injury. Methods Primary human, rat and mouse LSECs were used. Histone deacetylase 6 (HDAC6) was overexpressed specifically in liver ECs via adeno-associated virus (AAV)-mediated gene delivery to decrease heat shock protein 90 (Hsp90) acetylation in ethanol-fed mice. Results LSECs expressed CYP2E1 and alcohol dehydrogenase 1 (ADH1) and metabolized alcohol. Ethanol induced CYP2E1 in LSECs, but not ADH1. Alcohol metabolism by CYP2E1 increased Hsp90 acetylation and decreased its interaction with endothelial nitric oxide synthase (eNOS) leading to a decrease in nitric oxide (NO) production. A non-acetylation mutant of Hsp90 increased its interaction with eNOS and NO production, whereas a hyperacetylation mutant decreased NO production. These results indicate that Hsp90 acetylation is responsible for decreases in its interaction with eNOS and eNOS-derived NO production. AAV8-driven HDAC6 overexpression specifically in liver ECs deacetylated Hsp90, restored Hsp90’s interaction with eNOS and ameliorated alcohol-induced liver injury in mice. Conclusion Restoring LSEC function is important for ameliorating alcohol-induced liver injury. To this end, blocking acetylation of Hsp90 specifically in LSECs via AAV-mediated gene delivery has the potential to be a new therapeutic strategy. Lay summary Alcohol metabolism in liver sinusoidal endothelial cells (LSECs) and the mechanism of alcohol-induced LSEC dysfunction are largely unknown. Herein, we demonstrate that LSECs can metabolize alcohol. We also uncover a mechanism by which alcohol induces LSEC dysfunction and liver injury, and we identify a potential therapeutic strategy to prevent this.

BackgroundIt is unknown whether liver sinusoidal endothelial cells (LSECs) metabolize alcohol. Chronic alcohol consumption decreases endothelial nitric oxide synthase (eNOS)-derived NO production typical of LSEC dysfunction. Heat shock protein 90 (Hsp90) interacts with eNOS to increase its activity. Cytochrome P450 2E1 (CYP2E1) is a key enzyme in alcohol metabolism and facilitates protein acetylation via acetyl-CoA, but its expression in LSECs is unknown. This study investigates alcohol metabolism by LSECs, the mechanism of alcohol-induced LSEC dysfunction and a potential therapeutic approach for alcohol-induced liver injury.MethodsPrimary human, rat and mouse LSECs were used. Histone deacetylase 6 (HDAC6) was overexpressed specifically in liver ECs using an adeno-associated virus (AAV)-mediated gene delivery system to decrease Hsp90 acetylation in ethanol fed mice.ResultsLSECs expressed CYP2E1 and alcohol dehydrogenase 1 (ADH1) and metabolized alcohol. Ethanol induced CYP2E1 in LSECs, but not ADH1. Alcohol metabolism by CYP2E1 increased Hsp90 acetylation and decreased its interaction with eNOS along with a decrease in NO production. A non-acetylation mutant of Hsp90 increased its interaction with eNOS and NO production, whereas a hyper-acetylation mutant decreased NO production, compared with wildtype Hsp90. These results indicate that Hsp90 acetylation is responsible for decreases in its interaction with eNOS and eNOS-derived NO production. Adeno-associated virus 8 (AAV8)-driven HDAC6 overexpression specifically in liver ECs deacetylated Hsp90, restored Hsp90’s interaction with eNOS and ameliorated alcohol-induced liver injury in mice.ConclusionRestoring LSEC function is important for ameliorating alcohol-induced liver injury. To this end, blocking acetylation of Hsp90 specifically in LSECs via AAV-mediated gene delivery has the potential to be a new therapeutic strategy.

Cis-prenyltransferase (cis-PTase) catalyzes the rate-limiting step in the synthesis of glycosyl carrier lipids required for protein glycosylation in the lumen of endoplasmic reticulum. Here, we report the crystal structure of the human NgBR/DHDDS complex, which represents an atomic resolution structure for any heterodimeric cis-PTase. The crystal structure sheds light on how NgBR stabilizes DHDDS through dimerization, participates in the enzyme's active site through its C-terminal -RXG- motif, and how phospholipids markedly stimulate cis-PTase activity. Comparison of NgBR/DHDDS with homodimeric cis-PTase structures leads to a model where the elongating isoprene chain extends beyond the enzyme's active site tunnel, and an insert within the α3 helix helps to stabilize this energetically unfavorable state to enable long-chain synthesis to occur. These data provide unique insights into how heterodimeric cis-PTases have evolved from their ancestral, homodimeric forms to fulfill their function in long-chain polyprenol synthesis.

Most organs use fatty acids (FAs) as a key nutrient, but little is known of how blood-borne FAs traverse the endothelium to reach underlying tissues. We conducted a small-molecule screen and identified niclosamide as a suppressor of endothelial FA uptake and transport. Structure/activity relationship studies demonstrated that niclosamide acts through mitochondrial uncoupling. Inhibitors of oxidative phosphorylation and the ATP/ADP translocase also suppressed FA uptake, pointing principally to ATP production. Decreasing total cellular ATP by blocking glycolysis did not decrease uptake, indicating that specifically mitochondrial ATP is required. Endothelial FA uptake is promoted by fatty acid transport protein 4 (FATP4) via its ATP-dependent acyl-CoA synthetase activity. Confocal microscopy revealed that FATP4 resides in the endoplasmic reticulum (ER), and that endothelial ER is intimately juxtaposed with mitochondria. Together, these data indicate that mitochondrial ATP production, but not total ATP levels, drives endothelial FA uptake and transport via acyl-CoA formation in mitochondrial/ER microdomains.

Progressive myoclonus epilepsies (PMEs) comprise a group of clinically and genetically heterogeneous rare diseases. Over 70% of PME cases can now be molecularly solved. Known PME genes encode a variety of proteins, many involved in lysosomal and endosomal function. We performed whole-exome sequencing (WES) in 84 (78 unrelated) unsolved PME-affected individuals, with or without additional family members, to discover novel causes. We identified likely disease-causing variants in 24 out of 78 (31%) unrelated individuals, despite previous genetic analyses. The diagnostic yield was significantly higher for individuals studied as trios or families (14/28) versus singletons (10/50) (OR = 3.9, p value = 0.01, Fisher's exact test). The 24 likely solved cases of PME involved 18 genes. First, we found and functionally validated five heterozygous variants in NUS1 and DHDDS and a homozygous variant in ALG10, with no previous disease associations. All three genes are involved in dolichol-dependent protein glycosylation, a pathway not previously implicated in PME. Second, we independently validate SEMA6B as a dominant PME gene in two unrelated individuals. Third, in five families, we identified variants in established PME genes; three with intronic or copy-number changes (CLN6, GBA, NEU1) and two very rare causes (ASAH1, CERS1). Fourth, we found a group of genes usually associated with developmental and epileptic encephalopathies, but here, remarkably, presenting as PME, with or without prior developmental delay. Our systematic analysis of these cases suggests that the small residuum of unsolved cases will most likely be a collection of very rare, genetically heterogeneous etiologies. Refereed/Peer-reviewed

SUMMARYOne of the first events in the development of the cardiovascular system is morphogenesis of the main embryonic artery, the dorsal aorta (DA). The DA forms via a conserved genetic process mediated by the migration, specification, and organization of endothelial progenitor cells into a distinct arterial lineage and vessel type. Several angiogenic factors activate different signaling pathways to control DA formation, however the physiological relevance of distinct kinases in this complex process remains unclear. Here, we identify the role of Akt during early vascular development by generating mutant zebrafish lines that lack expression of akt isoforms. Live cell imaging coupled with single cell RNA sequencing of akt mutants reveal that Akt is required for proper development of the DA by sustaining arterial cell progenitor specification and segregation. Mechanistically, inhibition of active FOXO in akt mutants rescues impaired arterial development but not the expression of arterial markers, whereas Notch activation rescues arterial marker expression. Our work suggests that Akt activity is critical for early artery development, in part via FOXO and Notch-mediated regulation.

Acute lung injury (ALI) causes high mortality and lacks any pharmacological intervention. Here, we found that pazopanib ameliorated ALI manifestations and reduced mortality in mouse ALI models and reduced edema in human lung transplantation recipients. Pazopanib inhibits mitogen-activated protein kinase kinase kinase 2 (MAP3K2)–and MAP3K3-mediated phosphorylation of NADPH oxidase 2 subunit p47(phox) at Ser(208) to increase reactive oxygen species (ROS) formation in myeloid cells. Genetic inactivation of MAP3K2 and MAP3K3 in myeloid cells or hematopoietic mutation of p47(phox) Ser(208) to alanine attenuated ALI manifestations and abrogates anti-ALI effects of pazopanib. This myeloid MAP3K2/MAP3K3-p47(phox) pathway acted via paracrine H(2)O(2) to enhance pulmonary vasculature integrity and promote lung epithelial cell survival and proliferation, leading to increased pulmonary barrier function and resistance to ALI. Thus, pazopanib has the potential to be effective for treating ALI.