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Aims: Risk assessment is essential in the prevention of cardiovascular disease. In patients with recent acute coronary syndrome (ACS) or coronary revascularization, risk prediction tools, like the European Society of Cardiology guideline recommended SMART-REACH risk score, are increasingly used to predict the risk of recurrent cardiovascular events enabling risk-based personalized prevention. However, little is known about the association between risk stratification and the social and healthcare costs at a population level. This study evaluated the associations between baseline SMART-REACH risk scores, long-term recurrent clinical events, cumulative costs, and post-index event LDL-C goal attainment in patients with recent ACS and/or revascularization. Methods and results: This retrospective study used electronic health records and was conducted in the North Karelia region of Finland. The study cohort included all patients aged 45-85 admitted to a hospital for ACS or who underwent percutaneous coronary intervention or coronary artery bypass surgery between 1 January 2017 and 31 December 2021. Patients were divided into quintiles based on their baseline SMART-REACH risk scores to examine the associations between predicted 5-year scores and selected clinical and economic outcomes. In addition, simple age-based stratification was conducted as a sensitivity analysis. The observed 5-year cumulative incidence of recurrent events ranged from 20% in the lowest to 41% in the highest risk quintile, whereas the corresponding predicted risks ranged from 13% to 51%, and cumulative 5-year mean total costs per patient ranged from 15 827 to 46 182, respectively. Both monitoring and attainment of low LDL-C values were suboptimal. Conclusion: The use of the SMART-REACH quintiles as a population-level risk stratification tool successfully stratified patients into subgroups with different cumulative numbers of recurrent events and cumulative total costs. However, more research is needed to define clinically and economically optimal threshold values for a population-level stratification.
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Mitochondria are dynamic organelles essential for cell survival whose structural and functional integrity rely on selective and regulated transport of lipids from/to the endoplasmic reticulum (ER) and across the mitochondrial intermembrane space. As they are not connected by vesicular transport, the exchange of lipids between ER and mitochondria occurs at membrane contact sites. However, the mechanisms and proteins involved in these processes are only beginning to emerge. Here, we show that the main physiological localization of the lipid transfer proteins ORP5 and ORP8 is at mitochondria-associated ER membrane (MAM) subdomains, physically linked to the mitochondrial intermembrane space bridging (MIB)/mitochondrial contact sites and cristae junction organizing system (MICOS) complexes that bridge the two mitochondrial membranes. We also show that ORP5/ORP8 mediate non-vesicular transport of phosphatidylserine (PS) lipids from the ER to mitochondria by cooperating with the MIB/MICOS complexes. Overall our study reveals a physical and functional link between ER-mitochondria contacts involved in lipid transfer and intra-mitochondrial membrane contacts maintained by the MIB/MICOS complexes.
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Proteínas Mitocondriais , Fosfatidilserinas , Retículo Endoplasmático/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Fosfatidilserinas/metabolismoRESUMO
BACKGROUND AND AIMS: Lipoprotein (a) (Lp(a)) is a causal risk factor for cardiovascular diseases and its levels are under strict genetic control. Therefore, it is hypothesized that the concentration of Lp(a) remains stable throughout life. Finns have lower Lp(a) levels than central Europeans, but it is unknown whether there are differences within Finland, especially between the eastern and western parts of the country with known genetic duality and persistent differences in cardiovascular disease rates. We have examined the long-term stability of Lp(a) levels over 25 years in the Cardiovascular Risk in Young Finns Study (YFS), and the characteristics of individuals with different Lp(a) levels, including their geographical origin within Finland. METHODS: In YFS, the first large baseline examination was conducted in 1980 (baseline age, 3-18 years). Several follow-ups during the past 40 years have been conducted to investigate the determinants of cardiometabolic health. Lp(a) levels have been measured in study years 1986 (N = 2464, ages 9-24 years), 2001 (N = 2281, ages 24-39 years), 2007 (N = 2204, ages 35-45 years) and 2011 (N = 2044, ages 39-49 years). Tracking of Lp(a) was estimated by calculating Spearman's rank order correlations between the study years, and by cross-tabulating how many individuals diagnosed with either elevated or non-elevated Lp(a) levels in 1986, 2001 and 2007 remained in the same category in the latest follow-up in 2011. RESULTS: Spearman's correlation coefficients varied between r = 0.84-0.96. Most individuals (87-94%) who had a high Lp(a) level (>30 mg/dl) in any of the previous study years had a high level also in 2011. On average, the median Lp(a) levels were consistently â¼20% higher in the individuals originating from eastern Finland compared to those from western Finland, but there were no differences in the distribution of known genetic determinants between eastern and western Finns that would have explained the observed difference. CONCLUSIONS: These data confirm that Lp(a) levels remain very stable over the life-course. In line with the genetic duality between eastern and western parts of Finland, we observed about 20% higher Lp(a) levels in individuals originating from eastern Finland compared to those originating from western Finland.
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Doenças Cardiovasculares , Lipoproteína(a) , Adolescente , Adulto , Doenças Cardiovasculares/diagnóstico , Doenças Cardiovasculares/epidemiologia , Criança , Pré-Escolar , Finlândia/epidemiologia , Fatores de Risco de Doenças Cardíacas , Humanos , Lipoproteína(a)/genética , Pessoa de Meia-Idade , Fatores de Risco , Adulto JovemRESUMO
During angiogenesis, endothelial cells form protrusive sprouts and migrate towards the angiogenic stimulus. In this study, we investigate the role of the endoplasmic reticulum (ER)-anchored protein, Protrudin, in endothelial cell protrusion, migration and angiogenesis. Our results demonstrate that Protrudin regulates angiogenic tube formation in primary endothelial cells, Human umbilical vein endothelial cells (HUVECs). Analysis of RNA sequencing data and its experimental validation revealed cell migration as a prominent cellular function affected in HUVECs subjected to Protrudin knockdown. Further, our results demonstrate that knockdown of Protrudin inhibits focal adhesion kinase (FAK) activation in HUVECs and human aortic endothelial cells (HAECs). This is associated with a loss of polarized phospho-FAK distribution upon Protrudin knockdown as compared to Protrudin expressing HUVECs. Reduction of Protrudin also results in a perinuclear accumulation of mTOR and a decrease in VEGF-mediated S6K activation. However, further experiments suggest that the observed inhibition of angiogenesis in Protrudin knockdown cells is not affected by mTOR disturbance. Therefore, our findings suggest that defects in FAK activation and its abnormal subcellular distribution upon Protrudin knockdown are associated with a detrimental effect on endothelial cell migration and angiogenesis. Furthermore, mice with global Protrudin deletion demonstrate reduced retinal vascular progression. To conclude, our results provide evidence for a novel key role of Protrudin in endothelial cell migration and angiogenesis.
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Neovascularização Patológica , Neovascularização Fisiológica , Animais , Movimento Celular/genética , Proteína-Tirosina Quinases de Adesão Focal/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Neovascularização Patológica/genética , Neovascularização Fisiológica/genética , Proteínas de Transporte VesicularRESUMO
AIMS: Oxidized phospholipids and microRNAs (miRNAs) are increasingly recognized to play a role in endothelial dysfunction driving atherosclerosis. NRF2 transcription factor is one of the key mediators of the effects of oxidized phospholipids, but the gene regulatory mechanisms underlying the process remain obscure. Here, we investigated the genome-wide effects of oxidized phospholipids on transcriptional gene regulation in human umbilical vein endothelial cells and aortic endothelial cells with a special focus on miRNAs. METHODS AND RESULTS: We integrated data from HiC, ChIP-seq, ATAC-seq, GRO-seq, miRNA-seq, and RNA-seq to provide deeper understanding of the transcriptional mechanisms driven by NRF2 in response to oxidized phospholipids. We demonstrate that presence of NRF2 motif and its binding is more prominent in the vicinity of up-regulated transcripts and transcriptional initiation represents the most likely mechanism of action. We further identified NRF2 as a novel regulator of over 100 endothelial pri-miRNAs. Among these, we characterize two hub miRNAs miR-21-5p and miR-100-5p and demonstrate their opposing roles on mTOR, VEGFA, HIF1A, and MYC expressions. Finally, we provide evidence that the levels of miR-21-5p and miR-100-5p in exosomes are increased upon senescence and exhibit a trend to correlate with the severity of coronary artery disease. CONCLUSION: Altogether, our analysis provides an integrative view into the regulation of transcription and miRNA function that could mediate the proatherogenic effects of oxidized phospholipids in endothelial cells.
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Aterosclerose/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , MicroRNAs/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Fosfatidilcolinas/toxicidade , Transcriptoma , Aterosclerose/genética , Aterosclerose/patologia , Células Cultivadas , Senescência Celular , Bases de Dados Genéticas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , MicroRNAs/genética , Fator 2 Relacionado a NF-E2/genética , Oxirredução , Placa AteroscleróticaRESUMO
BACKGROUND: Antiangiogenic therapy, although part of standard treatment in ovarian cancer, has variable efficacy. Furthermore, little is known about the prognostic biomarkers and factors influencing angiogenesis in cancer tissue. We evaluated the expression of angiopoietin-2 and two endothelial tyrosine kinase receptors, Tie-1 and Tie-2, and assessed their value in the prediction of survival in patients with malignant epithelial ovarian cancer. We also compared the expression of these factors between primary high grade serous tumors and their distant metastasis. MATERIALS AND METHODS: We evaluated 86 women with primary epithelial ovarian cancer. Matched distal omental metastasis were investigated in 18.6% cases (N = 16). The expression levels of angiogenic factors were evaluated by immunohistochemistry in 306 specimens and by qRT-PCR in 111 samples. RESULTS: A high epithelial expression level of Tie-2 is a significant prognostic factor in primary high grade serous ovarian cancer. It predicted significantly shorter overall survival both in univariate (p<0.001) and multivariate survival analyses (p = 0.022). Low angiopoietin-2 expression levels in primary ovarian tumors were significantly associated with shorter overall survival (p = 0.015) in the univariate survival analysis. A low expression of angiopoietin-2 was also significantly related to high grade tumors, size of residual tumor after primary surgery and the recurrence of cancer (p = 0.008; p = 0.012; p = 0.018) in the whole study population. The expression of angiopoietin-2 and Tie-2 was stronger in distal omental metastasis than in primary high grade serous tumors in matched-pair analysis (p = 0.001; p = 0.002). CONCLUSIONS: The angiogenic factor, angiopoietin-2, and its receptor Tie-2 seem to be significant prognostic factors in primary epithelial ovarian cancer. Their expression levels are also increased in metastatic lesions in comparison with primary tumors.
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Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/patologia , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Receptor TIE-2/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Indutores da Angiogênese/metabolismo , Angiopoietina-2/metabolismo , Cistadenocarcinoma Seroso/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Gradação de Tumores , Metástase Neoplásica , Neoplasias Ovarianas/genética , Prognóstico , Intervalo Livre de Progressão , Receptor de TIE-1/metabolismoRESUMO
Oxysterol-binding protein-related protein 2 (ORP2), a cholesterol-PI(4,5)P2 countercurrent transporter, was recently identified as a novel regulator of plasma membrane (PM) cholesterol and PI(4,5)P2 content in HeLa cells. Here, we investigate the role of ORP2 in endothelial cell (EC) cholesterol and PI(4,5)P2 distribution, angiogenic signaling, and angiogenesis. We show that ORP2 knock-down modifies the distribution of cholesterol accessible to a D4H probe, between late endosomes and the PM. Depletion of ORP2 from ECs inhibits their angiogenic tube formation capacity, alters the gene expression of angiogenic signaling pathways such as VEGFR2, Akt, mTOR, eNOS, and Notch, and reduces EC migration, proliferation, and cell viability. We show that ORP2 regulates the integrity of VEGFR2 at the PM in a cholesterol-dependent manner, the depletion of ORP2 resulting in proteolytic cleavage by matrix metalloproteinases, and reduced activity of VEGFR2 and its downstream signaling. We demonstrate that ORP2 depletion increases the PM PI(4,5)P2 coincident with altered F-actin morphology, and reduces both VEGFR2 and cholesterol in buoyant raft membranes. Moreover, ORP2 knock-down suppresses the expression of the lipid raft-associated proteins VE-cadherin and caveolin-1. Analysis of the retinal microvasculature in ORP2 knock-out mice generated during this study demonstrates the subtle alterations of morphology characterized by reduced vessel length and increased density of tip cells and perpendicular sprouts. Gene expression changes in the retina suggest disturbance of sterol homeostasis, downregulation of VE-cadherin, and a putative disturbance of Notch signaling. Our data identifies ORP2 as a novel regulator of EC cholesterol and PI(4,5)P2 homeostasis and cholesterol-dependent angiogenic signaling.
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Células Endoteliais da Veia Umbilical Humana/metabolismo , Neovascularização Fisiológica , Receptores de Esteroides/metabolismo , Transdução de Sinais , Actinas/metabolismo , Antígenos CD/metabolismo , Caderinas/metabolismo , Caveolinas/metabolismo , Membrana Celular/metabolismo , Movimento Celular , Endossomos/metabolismo , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Metaloproteinases da Matriz/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Notch/metabolismo , Receptores de Esteroides/genética , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
BACKGROUND: In many malignancies including ovarian cancer, different angiogenic factors have been related to poor prognosis. However, data on their relations to each other or importance as a prognostic factor in ovarian cancer is missing. Therefore, we investigated the expressions of VEGF-A, VEGF-C, and VEGF-D, and the receptors VEGFR1, VEGFR2, and VEGFR3 in patients with malignant epithelial ovarian neoplasms. We further compared expression levels between primary tumors and related distant omental metastases. METHODS: This study included 86 patients with malignant ovarian epithelial tumors and 16 related distant metastases. Angiogenic factor expression was evaluated using immunohistochemistry (n = 102) and qRT-PCR (n = 29). RESULTS: Compared to primary high grade serous ovarian tumors, the related omental metastases showed higher expressions of VEGF-A (p = 0.022), VEGF-D (p = 0.010), and VEGFR1 (p = 0.046). In univariate survival analysis, low epithelial expression of VEGF-A in primary tumors was associated with poor prognosis (p = 0.024), and short progression-free survival was associated with high VEGF-C (p = 0.034) and low VEGFR3 (p = 0.002). The relative expressions of VEGF-D, VEGFR1, VEGFR2, and VEGFR3 mRNA determined by qRT-PCR analyses were significantly correlated with the immunohistochemically detected levels of these proteins in primary high grade serous ovarian cancer and metastases (p = 0.004, p = 0.009, p = 0.015, and p = 0.018, respectively). CONCLUSIONS: The expressions of VEGF receptors and their ligands significantly differed between malignant ovarian tumors and paired distant metastases. VEGF-A, VEGF-D, and VEGFR1 protein expressions seem to be higher in distant metastases than in the primary high grade serous ovarian cancer lesions.
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Carcinoma Epitelial do Ovário/metabolismo , Neoplasias Ovarianas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator D de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Carcinoma Epitelial do Ovário/patologia , Carcinoma Epitelial do Ovário/secundário , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/patologia , Cistadenocarcinoma Seroso/secundário , Feminino , Seguimentos , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Neovascularização Patológica/diagnóstico , Neoplasias Ovarianas/patologia , Prognóstico , Estudos Retrospectivos , Transdução de Sinais , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
ORP2 is a sterol-binding protein with documented functions in lipid and glucose metabolism, Akt signaling, steroidogenesis, cell adhesion, migration and proliferation. Here we investigate the interactions of ORP2 with phosphoinositides (PIPs) by surface plasmon resonance (SPR), its affinity for cholesterol with a pull-down assay, and its capacity to transfer sterol in vitro. Moreover, we determine the effects of wild-type (wt) ORP2 and a mutant with attenuated PIP binding, ORP2(mHHK), on the subcellular distribution of cholesterol, and analyze the interaction of ORP2 with the related cholesterol transporter ORP1L. ORP2 showed specific affinity for PI(4,5)P2, PI(3,4,5)P3 and PI(4)P, with suggestive Kd values in the µM range. Also binding of cholesterol by ORP2 was detectable, but a Kd could not be determined. Wt ORP2 was in HeLa cells mainly detected in the cytosol, ER, late endosomes, and occasionally on lipid droplets (LDs), while ORP2(mHHK) displayed an enhanced LD localization. Overexpression of wt ORP2 shifted the D4H cholesterol probe away from endosomes, while ORP2(mHHK) caused endosomal accumulation of the probe. Although ORP2 failed to transfer dehydroergosterol in an in vitro assay where OSBP is active, its knock-down resulted in the accumulation of cholesterol in late endocytic compartments, as detected by both D4H and filipin probes. Interestingly, ORP2 was shown to interact and partially co-localize on late endosomes with ORP1L, a cholesterol transporter/sensor at ER-late endosome junctions. Our data demonstrates that ORP2 binds several phosphoinositides, both PI(4)P and multiply phosphorylated species. ORP2 regulates the subcellular distribution of cholesterol dependent on its PIP-binding capacity. The interaction of ORP2 with ORP1L suggests a concerted action of the two ORPs.
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Colesterol/metabolismo , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Gotículas Lipídicas/metabolismo , Fosfatidilinositóis/metabolismo , Receptores de Esteroides/metabolismo , Colesterol/genética , Retículo Endoplasmático/genética , Endossomos/genética , Células HeLa , Humanos , Fosfatidilinositóis/genética , Receptores de Esteroides/genéticaRESUMO
ORP2 is a ubiquitously expressed OSBP-related protein previously implicated in endoplasmic reticulum (ER)-lipid droplet (LD) contacts, triacylglycerol (TG) metabolism, cholesterol transport, adrenocortical steroidogenesis, and actin-dependent cell dynamics. Here, we characterize the role of ORP2 in carbohydrate and lipid metabolism by employing ORP2-knockout (KO) hepatoma cells (HuH7) generated by CRISPR-Cas9 gene editing. The ORP2-KO and control HuH7 cells were subjected to RNA sequencing, analyses of Akt signaling, carbohydrate and TG metabolism, the extracellular acidification rate, and the lipidome, as well as to transmission electron microscopy. The loss of ORP2 resulted in a marked reduction of active phosphorylated Akt(Ser473) and its target Glycogen synthase kinase 3ß(Ser9), consistent with defective Akt signaling. ORP2 was found to form a physical complex with the key controllers of Akt activity, Cdc37, and Hsp90, and to co-localize with Cdc37 and active Akt(Ser473) at lamellipodial plasma membrane regions, in addition to the previously reported ER-LD localization. ORP2-KO reduced glucose uptake, glycogen synthesis, glycolysis, mRNA-encoding glycolytic enzymes, and SREBP-1 target gene expression, and led to defective TG synthesis and storage. ORP2-KO did not reduce but rather increased ER-LD contacts under basal culture conditions and interfered with their expansion upon fatty acid loading. Together with our recently published work (Kentala et al. in FASEB J 32:1281-1295, 2018), this study identifies ORP2 as a new regulatory nexus of Akt signaling, cellular energy metabolism, actin cytoskeletal function, cell migration, and proliferation.
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Transporte Biológico/genética , Metabolismo Energético/genética , Proteínas Proto-Oncogênicas c-akt/genética , Receptores de Esteroides/genética , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular , Movimento Celular/genética , Proliferação de Células/genética , Chaperoninas/genética , Técnicas de Inativação de Genes , Proteínas de Choque Térmico HSP90 , Humanos , Metabolismo dos Lipídeos/genética , Organelas/genética , Organelas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , Transdução de Sinais/genéticaRESUMO
ORP2 is implicated in cholesterol transport, triglyceride metabolism, and adrenocortical steroid hormone production. We addressed ORP2 function in hepatocytes by generating ORP2-knockout (KO) HuH7 cells by CRISPR-Cas9 gene editing, followed by analyses of transcriptome, F-actin morphology, migration, adhesion, and proliferation. RNA sequencing of ORP2-KO cells revealed >2-fold changes in 579 mRNAs. The Ingenuity Pathway Analysis (IPA) uncovered alterations in the following functional categories: cellular movement, cell-cell signaling and interaction, cellular development, cellular function and maintenance, cellular growth and proliferation, and cell morphology. Many pathways in these categories involved actin cytoskeleton, cell migration, adhesion, or proliferation. Analysis of the ORP2 interactome uncovered 109 putative new partners. Their IPA analysis revealed Ras homolog A (RhoA) signaling as the most significant pathway. Interactions of ORP2 with SEPT9, MLC12, and ARHGAP12 were validated by independent assays. ORP2-KO resulted in abnormal F-actin morphology characterized by impaired capacity to form lamellipodia, migration defect, and impaired adhesion and proliferation. Rescue of the migration phenotype and generation of typical cell surface morphology required an intact ORP2 phosphoinositide binding site, suggesting that ORP2 function involves phosphoinositide binding and transport. The results point at a novel function of ORP2 as a lipid-sensing regulator of the actin cytoskeleton, with impacts on hepatocellular migration, adhesion, and proliferation.-Kentala, H., Koponen, A., Kivelä, A. M., Andrews, R., Li, C., Zhou, Y., Olkkonen, V. M. Analysis of ORP2-knockout hepatocytes uncovers a novel function in actin cytoskeletal regulation.
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Citoesqueleto de Actina/fisiologia , Movimento Celular , Hepatócitos/fisiologia , Receptores de Esteroides/metabolismo , Sistemas CRISPR-Cas , Adesão Celular , Proliferação de Células , Técnicas de Inativação de Genes , Hepatócitos/citologia , Humanos , Receptores de Esteroides/genética , Transdução de SinaisRESUMO
OBJECTIVES: We aimed to investigate whether apparent diffusion coefficients (ADCs) measured by 3.0T diffusion-weighted magnetic resonance imaging (DWI) associate with histological aggressiveness of ovarian cancer (OC) or predict the clinical outcome. This prospective study enrolled 40 patients with primary OC, treated 2011-2014. METHODS: DWI was performed prior to surgery. Two observers used whole lesion single plane region of interest (WLsp-ROI) and five small ROIs (S-ROI) to analyze ADCs. Samples from tumours and metastases were collected during surgery. Immunohistochemistry and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used to measure the expression of vascular endothelial growth factor (VEGF) and its receptors. RESULTS: The interobserver reliability of ADC measurements was excellent for primary tumours ICC 0.912 (WLsp-ROI). Low ADCs significantly associated with poorly differentiated OC (WLsp-ROI P = 0.035). In primary tumours, lower ADCs significantly associated with high Ki-67 (P = 0.001) and low VEGF (P = 0.001) expression. In metastases, lower ADCs (WLsp-ROI) significantly correlated with low VEGF receptors mRNA levels. ADCs had predictive value; 3-year overall survival was poorer in patients with lower ADCs (WLsp-ROI P = 0.023, S-ROI P = 0.038). CONCLUSION: Reduced ADCs are associated with histological severity and worse outcome in OC. ADCs measured with WLsp-ROI may serve as a prognostic biomarker of OC. KEY POINTS: ⢠Reduced ADCs correlate with prognostic markers: poor differentiation and high Ki-67 expression ⢠ADCs also significantly correlated with VEGF protein expression in primary tumours ⢠Lower ADC values are associated with poorer survival in ovarian cancer ⢠Whole lesion single plane-ROI ADCs may be used as a prognostic biomarker in OC.
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Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/patologia , Idoso , Idoso de 80 Anos ou mais , Carcinoma Epitelial do Ovário , Imagem de Difusão por Ressonância Magnética/métodos , Intervalo Livre de Doença , Feminino , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/mortalidade , Recidiva Local de Neoplasia/patologia , Neoplasias Epiteliais e Glandulares/mortalidade , Variações Dependentes do Observador , Neoplasias Ovarianas/mortalidade , Estudos Prospectivos , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Reprodutibilidade dos Testes , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
INTRODUCTION: Despite considerable improvements in therapies, atherosclerotic cardiovascular diseases remain the leading cause of death worldwide. Therefore, in addition to current treatment options, new therapeutic approaches are still needed. AREAS COVERED: In this review, novel gene and RNA interference-based therapy approaches and promising target genes for treating atherosclerosis are addressed. In addition, relevant animal models for the demonstration of the efficacy of different gene therapy applications, and current progress toward more efficient, targeted and safer gene transfer vectors are reviewed. EXPERT OPINION: Atherosclerosis represents a complex multifactorial disease that is dependent on the interplay between lipoprotein metabolism, cellular reactions and inflammation. Recent advances and novel targets, especially in the field of RNA interference-based therapies, are very promising. However, it should be noted that the modulation of a particular gene is not as clearly associated with a complex polygenic disease as it is in the case of monogenic diseases. A deeper understanding of molecular mechanisms of atherosclerosis, further progress in vector development and the demonstration of treatment efficacy in relevant animal models will be required before gene therapy of atherosclerosis meets its clinical reality.
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Aterosclerose/genética , Aterosclerose/terapia , Terapia Genética/tendências , Animais , Aterosclerose/diagnóstico , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Humanos , Interferência de RNA/fisiologia , Resultado do TratamentoRESUMO
Lipolytic modification of LDL particles by SMase generates LDL aggregates with a strong affinity for human arterial proteoglycans and may so enhance LDL retention in the arterial wall. Here, we evaluated the effects of apoA-I mimetic peptide 4F on structural and functional properties of the SMase-modified LDL particles. LDL particles with and without 4F were incubated with SMase, after which their aggregation, structure, and proteoglycan binding were analyzed. At a molar ratio of L-4F to apoB-100 of 2.5 to 20:1, 4F dose-dependently inhibited SMase-induced LDL aggregation. At a molar ratio of 20:1, SMase-induced aggregation was fully blocked. Binding of 4F to LDL particles inhibited SMase-induced hydrolysis of LDL by 10% and prevented SMase-induced LDL aggregation. In addition, the binding of the SMase-modified LDL particles to human aortic proteoglycans was dose-dependently inhibited by pretreating LDL with 4F. The 4F stabilized apoB-100 conformation and inhibited SMase-induced conformational changes of apoB-100. Molecular dynamic simulations showed that upon binding to protein-free LDL surface, 4F locally alters membrane order and fluidity and induces structural changes to the lipid layer. Collectively, 4F stabilizes LDL particles by preventing the SMase-induced conformational changes in apoB-100 and so blocks SMase-induced LDL aggregation and the resulting increase in LDL retention.
Assuntos
Apolipoproteína A-I/farmacologia , Apolipoproteína B-100/metabolismo , Lipoproteínas LDL/metabolismo , Peptídeos/farmacologia , Esfingomielina Fosfodiesterase/metabolismo , Aorta/efeitos dos fármacos , Aorta/metabolismo , Aorta/patologia , Apolipoproteína A-I/metabolismo , Biomimética , Humanos , Lipólise/efeitos dos fármacos , Peptídeos/metabolismo , Esfingomielina Fosfodiesterase/antagonistas & inibidoresRESUMO
Vascular endothelial growth factor A (VEGF-A) is a master regulator of angiogenesis, vascular development and function. In this study we investigated the transcriptional regulation of VEGF-A-responsive genes in primary human aortic endothelial cells (HAECs) and human umbilical vein endothelial cells (HUVECs) using genome-wide global run-on sequencing (GRO-Seq). We demonstrate that half of VEGF-A-regulated gene promoters are characterized by a transcriptionally competent paused RNA polymerase II (Pol II). We show that transition into productive elongation is a major mechanism of gene activation of virtually all VEGF-regulated genes, whereas only â¼40% of the genes are induced at the level of initiation. In addition, we report a comprehensive chromatin interaction map generated in HUVECs using tethered conformation capture (TCC) and characterize chromatin interactions in relation to transcriptional activity. We demonstrate that sites of active transcription are more likely to engage in chromatin looping and cell type-specific transcriptional activity reflects the boundaries of chromatin interactions. Furthermore, we identify large chromatin compartments with a tendency to be coordinately transcribed upon VEGF-A stimulation. We provide evidence that these compartments are enriched for clusters of regulatory regions such as super-enhancers and for disease-associated single nucleotide polymorphisms (SNPs). Collectively, these findings provide new insights into mechanisms behind VEGF-A-regulated transcriptional programs in endothelial cells.
Assuntos
Ativação Transcricional , Fator A de Crescimento do Endotélio Vascular/farmacologia , Compartimento Celular , Células Cultivadas , Cromatina/metabolismo , Elementos Facilitadores Genéticos , Genoma Humano , Humanos , Fenótipo , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
AIMS: The role of vascular endothelial growth factor (VEGF-A) in atherogenesis has remained controversial. We addressed this by comparing the effects of adenoviral VEGF-A gene transfer on atherosclerosis and lipoproteins in ApoE(-/-), LDLR(-/-), LDLR(-/-)ApoE(-/-), and LDLR(-/-)ApoB(100/100) mice. METHODS AND RESULTS: After 4 weeks on western diet, systemic adenoviral gene transfer was performed with hVEGF-A or control vectors. Effects on atherosclerotic lesion area and composition, lipoprotein profiles, and plasma lipoprotein lipase (LPL) activity were examined. On day 4, VEGF-A induced alterations in lipoprotein profiles and a significant negative correlation was observed between plasma LPL activity and VEGF-A levels. One month after gene transfer, no changes in atherosclerosis were observed in LDLR(-/-) and LDLR(-/-)ApoB(100/100) models, whereas both ApoE(-/-) models displayed increased en face lesion areas in thoracic and abdominal aortas. VEGF-A also reduced LPL mRNA in heart and white adipose tissue, whereas Angptl4 was increased, potentially providing further mechanistic explanation for the findings. CONCLUSION: VEGF-A gene transfer induced pro-atherogenic changes in lipoprotein profiles in all models. As a novel finding, VEGF-A also reduced LPL activity, which might underlie the observed changes in lipid profiles. However, VEGF-A was observed to increase atherosclerosis only in the ApoE(-/-) background, clearly indicating some mouse model-specific effects.
Assuntos
Aterosclerose/etiologia , Terapia Genética , Hiperlipidemias/terapia , Lipase Lipoproteica/metabolismo , Lipoproteínas/sangue , Fator A de Crescimento do Endotélio Vascular/genética , Adenoviridae/genética , Animais , Apolipoproteínas B/deficiência , Apolipoproteínas B/fisiologia , Modelos Animais de Doenças , Feminino , Hiperlipidemias/sangue , Hiperlipidemias/patologia , Lipídeos/sangue , Lipase Lipoproteica/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores de LDL/fisiologiaRESUMO
OBJECTIVE: Endothelial lipase (EL) regulates HDL cholesterol levels and in inflammatory states, like atherosclerosis, EL expression is increased contributing to low HDL cholesterol. The regulation of EL expression is poorly understood and has mainly been attributed to inflammatory stimuli. As sterol regulatory element binding proteins (SREBPs) are regulators of genes involved in lipid metabolism, we hypothesized that EL is regulated by SREBPs and that EL expression is modified by the SREBP activator vascular endothelial growth factor A (VEGF-A). METHODS: and results: Quantitative PCR and Western blot results demonstrated that starvation increased EL expression in human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAECs). Also, 25-hydroxycholesterol (25HC), an inhibitor of SREBP activation inhibited EL expression. With siRNA-mediated inhibition of SREBPs the effect of starvation was shown to be SREBP-2 dependent. VEGF-A decreased EL expression in both endothelial cell lines used, most likely via inhibition of SREBP-2 binding determined by chromatin immunoprecipitation (ChIP). Furthermore, in atherosclerosis prone LDLR(-/-)ApoB(100/100) mice, systemic adenoviral gene transfer with human VEGF-A decreased EL mRNA in peripheral tissues and increased plasma HDL cholesterol. CONCLUSIONS: These results identify SREBPs as novel regulators of EL expression. VEGF-A as an endogenous EL inhibitor could be therapeutically relevant in atherosclerosis by increasing systemic HDL cholesterol levels.
Assuntos
HDL-Colesterol/sangue , Células Endoteliais/enzimologia , Lipase/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Adenoviridae/genética , Animais , Apolipoproteína B-100/deficiência , Apolipoproteína B-100/genética , Aterosclerose/sangue , Aterosclerose/enzimologia , Aterosclerose/genética , Sítios de Ligação , Western Blotting , Células Cultivadas , Imunoprecipitação da Cromatina , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica , Técnicas de Transferência de Genes , Vetores Genéticos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/enzimologia , Humanos , Hidroxicolesteróis/farmacologia , Lipase/genética , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Interferência de RNA , RNA Mensageiro/metabolismo , Receptores de LDL/deficiência , Receptores de LDL/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/antagonistas & inibidores , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Fatores de Tempo , Transfecção , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
OBJECTIVE: Endothelial lipase (EL) is a new member of triacylglycerol lipase family that has been shown to decrease high-density lipoprotein (HDL) cholesterol levels leading to increased risk of atherosclerosis. Its expression is increased during inflammation and by inflammatory cytokines. Sulforaphane (SFN) is a naturally occurring isothiocyanate present in cruciferous vegetables that has antioxidant and anti-inflammatory effects. Nuclear factor (NF)-κB is one of the molecular targets for SFN-mediated protective effects. Our aim was therefore to assess whether SFN could impact on EL expression via modulation of NF-κB pathway. METHODS AND RESULTS: Quantitative PCR and Western blot results demonstrated that SFN inhibited tumor necrosis factor (TNF)-α-mediated induction of EL in human umbilical vein endothelial cells (HUVEC). Lentiviral transduction of HUVEC with mutated form of IκB-α (IκBM) as well as silencing of NF-κB subunit p65 using RNA interference revealed that TNF-α-mediated induction of EL is mediated through NF-κB pathway. In addition, a total of five NF-κB binding sites were found in LIPG gene, which encodes EL. SFN inhibited binding of NF-κB to these sites analyzed by chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA). SFN also inhibited TNF-α mediated phosphorylation of IκB kinase (IKK) 1/2 and IκB-α. CONCLUSIONS: Collectively, these results indicate that SFN inhibits EL expression via inhibition of NF-κB which may have a beneficial effect on HDL cholesterol levels.
Assuntos
Células Endoteliais/citologia , Regulação Enzimológica da Expressão Gênica , Lipase/antagonistas & inibidores , NF-kappa B/metabolismo , Tiocianatos/farmacologia , Antioxidantes/química , Aterosclerose/metabolismo , Células Cultivadas , Humanos , Quinase I-kappa B/metabolismo , Isotiocianatos/química , Lipase/biossíntese , Modelos Biológicos , Ligação Proteica , Sulfóxidos , Veias Umbilicais/citologia , VerdurasRESUMO
Electrophilic fatty acid derivatives, including nitrolinoleic acid and nitro-oleic acid (OA-NO(2)), can mediate anti-inflammatory and pro-survival signaling reactions. The transcription factor Nrf2, activated by electrophilic fatty acids, suppresses redox-sensitive pro-inflammatory gene expression and protects against vascular endothelial oxidative injury. It was therefore postulated that activation of Nrf2 by OA-NO(2) accounts in part for its anti-inflammatory actions, motivating the characterization of Nrf2-dependent and -independent effects of OA-NO(2) on gene expression using genome-wide transcriptional profiling. Control and Nrf2-small interfering RNA-transfected human endothelial cells were treated with vehicle, oleic acid, or OA-NO(2), and differential gene expression profiles were determined. Although OA-NO(2) significantly induced the expression of Nrf2-dependent genes, including heme oxygenase-1 and glutamate-cysteine ligase modifier subunit, the majority of OA-NO(2)-regulated genes were regulated by Nrf2-independent pathways. Moreover, gene set enrichment analysis revealed that the heat shock response is the major pathway activated by OA-NO(2), with robust induction of a number of heat shock genes regulated by the heat shock transcription factor. Inasmuch as the heat shock response mediates anti-inflammatory and cytoprotective actions, this mechanism is proposed to contribute to the protective cell signaling functions of nitro-fatty acids and other electrophilic fatty acid derivatives.
Assuntos
Células Endoteliais/efeitos dos fármacos , Ácidos Linoleicos/farmacologia , Fator 2 Relacionado a NF-E2/genética , Nitrocompostos/farmacologia , Ácidos Oleicos/farmacologia , Western Blotting , Linhagem Celular , Células Cultivadas , Análise por Conglomerados , Relação Dose-Resposta a Droga , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Glutamato-Cisteína Ligase/genética , Glutamato-Cisteína Ligase/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Temperatura Alta , Humanos , NAD(P)H Desidrogenase (Quinona)/genética , NAD(P)H Desidrogenase (Quinona)/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacosRESUMO
The J series of cyclopentenone prostaglandins (PGs) such as 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) are electrophilic lipid signaling mediators derived from the nonenzymatic dehydration of PGD(2), a major product of the cyclooxygenase pathway. The biological actions of 15d-PGJ(2) are attributed to its ability to form covalent adducts with thiol residues within specific signaling proteins, thus triggering redox-sensitive cell signaling pathways. One of the signaling pathways potently activated by 15d-PGJ(2) is the Keap1-Nrf2-ARE system, which has a well-appreciated role in protecting cells from endogenous and exogenous stresses as well as anti-inflammatory effects. In this review, we give an overview of the mechanisms by which 15d-PGJ(2) activates the Keap1-Nrf2-ARE system, focusing particularly on the role of Keap1 in sensing electrophilic stress. In addition, the Nrf2-dependent anti-inflammatory and cytoprotective effects of 15d-PGJ(2) are discussed.