RESUMO
BACKGROUND: Extracellular vesicles (EVs) contain bioactive cargo including miRNAs and proteins that are released by cells during cell-cell communication. Endothelial cells (ECs) form the innermost lining of all blood vessels, interfacing with cells in the circulation and vascular wall. It is unknown whether ECs release EVs capable of governing recipient cells within these 2 separate compartments. Given their boundary location, we propose ECs use bidirectional release of distinct EV cargo in quiescent (healthy) and activated (atheroprone) states to communicate with cells within the circulation and blood vessel wall. METHODS: EVs were isolated from primary human aortic ECs (plate and transwell grown; ±IL [interleukin]-1ß activation), quantified, visualized, and analyzed by miRNA transcriptomics and proteomics. Apical and basolateral EC-EV release was determined by miRNA transfer, total internal reflection fluorescence and electron microscopy. Vascular reprogramming (RNA sequencing) and functional assays were performed on primary human monocytes or smooth muscle cells±EC-EVs. RESULTS: Activated ECs increased EV release, with miRNA and protein cargo related to atherosclerosis. EV-treated monocytes and smooth muscle cells revealed activated EC-EV altered pathways that were proinflammatory and atherogenic. ECs released more EVs apically, which increased with activation. Apical and basolateral EV cargo contained distinct transcriptomes and proteomes that were altered by EC activation. Notably, activated basolateral EC-EVs displayed greater changes in the EV secretome, with pathways specific to atherosclerosis. In silico analysis determined compartment-specific cargo released by the apical and basolateral surfaces of ECs can reprogram monocytes and smooth muscle cells, respectively, with functional assays and in vivo imaging supporting this concept. CONCLUSIONS: Demonstrating that ECs are capable of polarized EV cargo loading and directional EV secretion reveals a novel paradigm for endothelial communication, which may ultimately enhance the design of endothelial-based therapeutics for cardiovascular diseases such as atherosclerosis where ECs are persistently activated.
Assuntos
Aterosclerose , Vesículas Extracelulares , MicroRNAs , Humanos , Células Endoteliais/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Vesículas Extracelulares/metabolismo , Comunicação Celular , Aterosclerose/metabolismoRESUMO
BACKGROUND: Individuals with type 1 diabetes (T1D) generally have normal or even higher HDL (high-density lipoprotein)-cholesterol levels than people without diabetes yet are at increased risk for atherosclerotic cardiovascular disease (CVD). Human HDL is a complex mixture of particles that can vary in cholesterol content by >2-fold. To investigate if specific HDL subspecies contribute to the increased atherosclerosis associated with T1D, we created mouse models of T1D that exhibit human-like HDL subspecies. We also measured HDL subspecies and their association with incident CVD in a cohort of people with T1D. METHODS: We generated LDL receptor-deficient (Ldlr-/-) mouse models of T1D expressing human APOA1 (apolipoprotein A1). Ldlr-/-APOA1Tg mice exhibited the main human HDL subspecies. We also generated Ldlr-/-APOA1Tg T1D mice expressing CETP (cholesteryl ester transfer protein), which had lower concentrations of large HDL subspecies versus mice not expressing CETP. HDL particle concentrations and sizes and proteins involved in lipoprotein metabolism were measured by calibrated differential ion mobility analysis and targeted mass spectrometry in the mouse models of T1D and in a cohort of individuals with T1D. Endothelial transcytosis was analyzed by total internal reflection fluorescence microscopy. RESULTS: Diabetic Ldlr-/-APOA1Tg mice were severely hyperglycemic and hyperlipidemic and had markedly elevated plasma APOB levels versus nondiabetic littermates but were protected from the proatherogenic effects of diabetes. Diabetic Ldlr-/-APOA1Tg mice expressing CETP lost the atheroprotective effect and had increased lesion necrotic core areas and APOB accumulation, despite having lower plasma APOB levels. The detrimental effects of low concentrations of larger HDL particles in diabetic mice expressing CETP were not explained by reduced cholesterol efflux. Instead, large HDL was more effective than small HDL in preventing endothelial transcytosis of LDL mediated by scavenger receptor class B type 1. Finally, in humans with T1D, increased concentrations of larger HDL particles relative to APOB100 negatively predicted incident CVD independently of HDL-cholesterol levels. CONCLUSIONS: Our results suggest that the balance between APOB lipoproteins and the larger HDL subspecies contributes to atherosclerosis progression and incident CVD in the setting of T1D and that larger HDLs exert atheroprotective effects on endothelial cells rather than by promoting macrophage cholesterol efflux.
Assuntos
Apolipoproteína A-I , Aterosclerose , Diabetes Mellitus Tipo 1 , Receptores de LDL , Adulto , Animais , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Apolipoproteína A-I/sangue , Apolipoproteína A-I/metabolismo , Apolipoproteína B-100/metabolismo , Apolipoproteína B-100/genética , Apolipoproteína B-100/sangue , Aterosclerose/metabolismo , Aterosclerose/genética , Aterosclerose/sangue , Aterosclerose/patologia , Proteínas de Transferência de Ésteres de Colesterol/genética , Proteínas de Transferência de Ésteres de Colesterol/metabolismo , Proteínas de Transferência de Ésteres de Colesterol/sangue , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/sangue , Modelos Animais de Doenças , Lipoproteínas HDL/sangue , Lipoproteínas HDL/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptores de LDL/genética , Receptores de LDL/deficiência , Receptores de LDL/metabolismoRESUMO
BACKGROUND: In early atherosclerosis, circulating LDLs (low-density lipoproteins) traverse individual endothelial cells by an active process termed transcytosis. The CANTOS trial treated advanced atherosclerosis using a blocking antibody for IL-1ß (interleukin-1ß); this significantly reduced cardiovascular events. However, whether IL-1ß regulates early disease, particularly LDL transcytosis, remains unknown. METHODS: We used total internal reflection fluorescence microscopy to quantify transcytosis by human coronary artery endothelial cells exposed to IL-1ß. To investigate transcytosis in vivo, we injected wild-type and knockout mice with IL-1ß and LDL to visualize acute LDL deposition in the aortic arch. RESULTS: Exposure to picomolar concentrations of IL-1ß induced transcytosis of LDL but not of albumin by human coronary artery endothelial cells. Surprisingly, expression of the 2 known receptors for LDL transcytosis, ALK-1 (activin receptor-like kinase-1) and SR-BI (scavenger receptor BI), was unchanged or decreased. Instead, IL-1ß increased the expression of the LDLR (LDL receptor); this was unexpected because LDLR is not required for LDL transcytosis. Overexpression of LDLR had no effect on basal LDL transcytosis. However, knockdown of LDLR abrogated the effect of IL-1ß on transcytosis rates while the depletion of Cav-1 (caveolin-1) did not. Since LDLR was necessary but overexpression had no effect, we reasoned that another player must be involved. Using public RNAseq data to curate a list of Rab GTPases affected by IL-1ß, we identified Rab27a. Overexpression of Rab27a alone had no effect on basal transcytosis, but its knockdown prevented induction by IL-1ß. This was phenocopied by depletion of the Rab27a effector JFC1. In vivo, IL-1ß increased LDL transcytosis in the aortic arch of wild-type but not Ldlr-/- or Rab27a-deficient mice. The JFC1 inhibitor nexinhib20 also blocked IL-1ß-induced LDL accumulation in the aorta. CONCLUSIONS: IL-1ß induces LDL transcytosis by a distinct pathway requiring LDLR and Rab27a; this route differs from basal transcytosis. We speculate that induction of transcytosis by IL-1ß may contribute to the acceleration of early disease.
RESUMO
Atherosclerosis results from the deposition and oxidation of LDL and immune cell infiltration in the sub-arterial space leading to arterial occlusion. Studies have shown that transcytosis transports circulating LDL across endothelial cells lining blood vessels. LDL transcytosis is initiated by binding to either scavenger receptor B1 (SR-B1) or activin A receptor-like kinase 1 on the apical side of endothelial cells leading to its transit and release on the basolateral side. HDL is thought to partly protect individuals from atherosclerosis due to its ability to remove excess cholesterol and act as an antioxidant. Apolipoprotein A1 (APOA1), an HDL constituent, can bind to SR-B1, raising the possibility that APOA1/HDL can compete with LDL for SR-B1 binding, thereby limiting LDL deposition in the sub-arterial space. To examine this possibility, we used in vitro approaches to quantify the internalization and transcytosis of fluorescent LDL in coronary endothelial cells. Using microscale thermophoresis and affinity capture, we find that SR-B1 and APOA1 interact and that binding is enhanced when using the cardioprotective variant of APOA1 termed Milano (APOA1-Milano). In male mice, transiently increasing the levels of HDL reduced the acute deposition of fluorescently labeled LDL in the atheroprone inner curvature of the aorta. Reduced LDL deposition was also observed when increasing circulating wild-type APOA1 or the APOA1-Milano variant, with a more robust inhibition from the APOA1-Milano. The results suggest that HDL may limit SR-B1-mediated LDL transcytosis and deposition, adding to the mechanisms by which it can act as an atheroprotective particle.
Assuntos
Apolipoproteína A-I , Lipoproteínas HDL , Lipoproteínas LDL , Transcitose , Animais , Humanos , Masculino , Camundongos , Apolipoproteína A-I/metabolismo , Aterosclerose/metabolismo , Células Endoteliais/metabolismo , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/metabolismo , Ligação Proteica , Receptores Depuradores Classe B/metabolismoRESUMO
AIMS/HYPOTHESIS: A hallmark chronic complication of type 2 diabetes mellitus is vascular hyperpermeability, which encompasses dysfunction of the cerebrovascular endothelium and the subsequent development of associated cognitive impairment. The present study tested the hypothesis that during type 2 diabetes circulating small extracellular vesicles (sEVs) exhibit phenotypic changes that facilitate pathogenic disruption of the vascular barrier. METHODS: sEVs isolated from the plasma of a mouse model of type 2 diabetes and from diabetic human individuals were characterised for their ability to disrupt the endothelial cell (EC) barrier. The contents of sEVs and their effect on recipient ECs were assessed by proteomics and identified pathways were functionally interrogated with small molecule inhibitors. RESULTS: Using intravital imaging, we found that diabetic mice (Leprdb/db) displayed hyperpermeability of the cerebrovasculature. Enhanced vascular leakiness was recapitulated following i.v. injection of sEVs from diabetic mice into non-diabetic recipient mice. Characterisation of circulating sEV populations from the plasma of diabetic mice and humans demonstrated increased quantity and size of sEVs compared with those isolated from non-diabetic counterparts. Functional experiments revealed that sEVs from diabetic mice or humans induced the rapid and sustained disruption of the EC barrier through enhanced paracellular and transcellular leak but did not induce inflammation. Subsequent sEV proteome and recipient EC phospho-proteome analysis suggested that extracellular vesicles (sEVs) from diabetic mice and humans modulate the MAPK/MAPK kinase (MEK) and Rho-associated protein kinase (ROCK) pathways, cell-cell junctions and actin dynamics. This was confirmed experimentally. Treatment of sEVs with proteinase K or pre-treatment of recipient cells with MEK or ROCK inhibitors reduced the hyperpermeability-inducing effects of circulating sEVs in the diabetic state. CONCLUSIONS/INTERPRETATION: Diabetes is associated with marked increases in the concentration and size of circulating sEVs. The modulation of sEV-associated proteins under diabetic conditions can induce vascular leak through activation of the MEK/ROCK pathway. These data identify a new paradigm by which diabetes can induce hyperpermeability and dysfunction of the cerebrovasculature and may implicate sEVs in the pathogenesis of cognitive decline during type 2 diabetes.
Assuntos
Permeabilidade Capilar , Diabetes Mellitus Tipo 2 , Vesículas Extracelulares , Animais , Vesículas Extracelulares/metabolismo , Camundongos , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/complicações , Humanos , Masculino , Diabetes Mellitus Experimental/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Proteômica , Camundongos Endogâmicos C57BLRESUMO
In acute lung injury, the lung endothelial barrier is compromised. Loss of endothelial barrier integrity occurs in association with decreased levels of the tight junction protein claudin-5. Restoration of their levels by gene transfection may improve the vascular barrier, but how to limit transfection solely to regions of the lung that are injured is unknown. We hypothesized that thoracic ultrasound in combination with intravenous microbubbles (USMBs) could be used to achieve regional gene transfection in injured lung regions and improve endothelial barrier function. Since air blocks ultrasound energy, insonation of the lung is only achieved in areas of lung injury (edema and atelectasis); healthy lung is spared. Cavitation of the microbubbles achieves local tissue transfection. Here we demonstrate successful USMB-mediated gene transfection in the injured lungs of mice. After thoracic insonation, transfection was confined to the lung and only occurred in the setting of injured (but not healthy) lung. In a mouse model of acute lung injury, we observed downregulation of endogenous claudin-5 and an acute improvement in lung vascular leakage and in oxygenation after claudin-5 overexpression by transfection. The improvement occurred without any impairment of the immune response as measured by pathogen clearance, alveolar cytokines, and lung histology. In conclusion, USMB-mediated transfection targets injured lung regions and is a novel approach to the treatment of lung injury.NEW & NOTEWORTHY Acute respiratory distress syndrome is characterized by spatial heterogeneity, with severely injured lung regions adjacent to relatively normal areas. This makes targeting treatment to the injured regions difficult. Here we use thoracic ultrasound and intravenous microbubbles (USMBs) to direct gene transfection specifically to injured lung regions. Transfection of the tight junction protein claudin-5 improved oxygenation and decreased vascular leakage without impairing innate immunity. These findings suggest that USMB is a novel treatment for ARDS.
Assuntos
Lesão Pulmonar Aguda , Síndrome do Desconforto Respiratório , Animais , Camundongos , Lesão Pulmonar Aguda/patologia , Claudina-5/genética , Claudina-5/metabolismo , Imunidade Inata , Pulmão/metabolismo , Síndrome do Desconforto Respiratório/patologia , Proteínas de Junções Íntimas/metabolismo , Junções Íntimas/metabolismo , Transfecção , Ultrassonografia de IntervençãoRESUMO
PURPOSE OF REVIEW: The accumulation of LDL in the arterial intima is an initiating event in atherosclerosis. After decades of controversy, it is now clear that transcytosis of LDL across an intact endothelial monolayer contributes to its intimal deposition. We review recent observations in this field and address the question of whether LDL transcytosis can be manipulated therapeutically. RECENT FINDINGS: The development of a live-cell imaging method for studying transcytosis using total internal reflection fluorescence (TIRF) microscopy has catalyzed recent discoveries. LDL transcytosis is mediated by SR-BI and ALK1. Estrogen down-regulates SR-BI and inhibits LDL transcytosis, while the nuclear structural protein HMGB1 promotes LDL transcytosis. LDL transcytosis by ALK1 is independent of the receptor's kinase activity and is antagonized by BMP9, ALK1's canonical ligand. Inflammation stimulates LDL transcytosis. Identifying the function and mechanisms of LDL transcytosis may ultimately permit its therapeutic manipulation.
Assuntos
Aterosclerose , Lipoproteínas LDL , Humanos , Lipoproteínas LDL/metabolismo , Células Endoteliais/metabolismo , Transcitose , Aterosclerose/metabolismo , Endotélio Vascular/metabolismoRESUMO
RATIONALE: Bone marrow transplantation (BMT) is used frequently to study the role of hematopoietic cells in atherosclerosis, but aortic arch lesions are smaller in mice after BMT. OBJECTIVE: To identify the earliest stage of atherosclerosis inhibited by BMT and elucidate potential mechanisms. METHODS AND RESULTS: Ldlr-/- mice underwent total body γ-irradiation, bone marrow reconstitution, and 6-week recovery. Atherosclerosis was studied in the ascending aortic arch and compared with mice without BMT. In BMT mice, neutral lipid and myeloid cell topography were lower in lesions after feeding a cholesterol-rich diet for 3, 6, and 12 weeks. Lesion coalescence and height were suppressed dramatically in mice post-BMT, whereas lateral growth was inhibited minimally. Targeted radiation to the upper thorax alone reproduced the BMT phenotype. Classical monocyte recruitment, intimal myeloid cell proliferation, and apoptosis did not account for the post-BMT phenotype. Neutral lipid accumulation was reduced in 5-day lesions, thus we developed quantitative assays for LDL (low-density lipoprotein) accumulation and paracellular leakage using DiI-labeled human LDL and rhodamine B-labeled 70 kD dextran. LDL accumulation was dramatically higher in the intima of Ldlr-/- relative to Ldlr+/+ mice, and was inhibited by injection of HDL mimics, suggesting a regulated process. LDL, but not dextran, accumulation was lower in mice post-BMT both at baseline and in 5-day lesions. Since the transcript abundance of molecules implicated in LDL transcytosis was not significantly different in the post-BMT intima, transcriptomics from whole aortic arch intima, and at single-cell resolution, was performed to give insights into pathways modulated by BMT. CONCLUSIONS: Radiation exposure inhibits LDL entry into the aortic intima at baseline and the earliest stages of atherosclerosis. Single-cell transcriptomic analysis suggests that LDL uptake by endothelial cells is diverted to lysosomal degradation and reverse cholesterol transport pathways. This reduces intimal accumulation of lipid and impacts lesion initiation and growth.
Assuntos
Aterosclerose/metabolismo , Raios gama , Lipoproteínas LDL/metabolismo , Túnica Íntima/efeitos da radiação , Animais , Aorta/metabolismo , Aorta/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , Receptores de LDL/deficiência , Receptores de LDL/genética , Transcriptoma , Túnica Íntima/metabolismoRESUMO
Breastfeeding (BF) has been identified as a protective factor against childhood obesity. However, evidence of the association between BF duration and adiposity remains inconclusive. Few studies have been conducted among Southeast Asian infants that have measured body composition during infancy using the gold standard stable isotope method. This study aimed to evaluate the association between BF duration and body composition during infancy. Healthy full-term Thai infants aged 6-8 months (n = 60) receiving exclusive or predominant BF for at least 3 months were recruited. Skinfold thickness (SFT) was measured by well-trained investigators. Body composition was assessed by the deuterium dilution technique. Infants with longer BF duration (>6 months; mean 7.5 ± 0.5 months, n = 29) had a higher subscapular SFT z-score than those with shorter BF duration (≤6 months; mean 5.3± 0.9 months, n = 31) by 0.48 (95% confidence interval [CI]: 0.01-0.94). After adjustment for age and sex, BF duration and age at introduction of complementary feeding (CF) were positively associated with fat mass and fat mass index at 6-8 months. One month increase in BF duration and CF age was associated with a 0.37 (95% CI: 0.05, 0.69) kg/m2 and 0.76 (95% CI: 0.18, 1.34) kg/m2 increase in the fat mass index, respectively. After adjusting for infant body mass index (BMI) during the earlier infancy period, the strength of the association was attenuated. This finding may reflect reverse causality where infants with lower BMI received formula or CF earlier. A longitudinal study with follow-up into childhood is warranted to confirm the effects of BF on adiposity in infancy and childhood.
Assuntos
Adiposidade , Obesidade Infantil , Lactente , Feminino , Criança , Humanos , Aleitamento Materno , Estudos Longitudinais , Obesidade Infantil/epidemiologia , Índice de Massa Corporal , Composição CorporalRESUMO
The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other coronaviruses mediates host cell entry and is S-acylated on multiple phylogenetically conserved cysteine residues. Multiple protein acyltransferase enzymes have been reported to post-translationally modify spike proteins; however, strategies to exploit this modification are lacking. Using resin-assisted capture MS, we demonstrate that the spike protein is S-acylated in SARS-CoV-2-infected human and monkey epithelial cells. We further show that increased abundance of the acyltransferase ZDHHC5 associates with increased S-acylation of the spike protein, whereas ZDHHC5 knockout cells had a 40% reduction in the incorporation of an alkynyl-palmitate using click chemistry detection. We also found that the S-acylation of the spike protein is not limited to palmitate, as clickable versions of myristate and stearate were also labelled the protein. Yet, we observed that ZDHHC5 was only modified when incubated with alkyne-palmitate, suggesting it has specificity for this acyl-CoA, and that other ZDHHC enzymes may use additional fatty acids to modify the spike protein. Since multiple ZDHHC isoforms may modify the spike protein, we also examined the ability of the FASN inhibitor TVB-3166 to prevent S-acylation of the spike proteins of SARS-CoV-2 and human CoV-229E. We show that treating cells with TVB-3166 inhibited S-acylation of expressed spike proteins and attenuated the ability of SARS-CoV-2 and human CoV-229E to spread in vitro. Our findings further substantiate the necessity of CoV spike protein S-acylation and demonstrate that de novo fatty acid synthesis is critical for the proper S-acylation of the spike protein.
Assuntos
COVID-19 , Glicoproteína da Espícula de Coronavírus , Acilação , Aciltransferases/metabolismo , Alcinos , Azetidinas , Coenzima A/metabolismo , Cisteína , Ácido Graxo Sintase Tipo I/metabolismo , Humanos , Miristatos , Nitrilas , Palmitatos , Pirazóis , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/metabolismo , EstearatosRESUMO
BACKGROUND: Cigarette smokers are at increased risk of acquiring influenza, developing severe disease and requiring hospitalisation/intensive care unit admission following infection. However, immune mechanisms underlying this predisposition are incompletely understood, and therapeutic strategies for influenza are limited. METHODS: We used a mouse model of concurrent cigarette smoke exposure and H1N1 influenza infection, colony-stimulating factor (CSF)3 supplementation/receptor (CSF3R) blockade and single-cell RNA sequencing (scRNAseq) to investigate this relationship. RESULTS: Cigarette smoke exposure exacerbated features of viral pneumonia such as oedema, hypoxaemia and pulmonary neutrophilia. Smoke-exposed infected mice demonstrated an increase in viral (v)RNA, but not replication-competent viral particles, relative to infection-only controls. Interstitial rather than airspace neutrophilia positively predicted morbidity in smoke-exposed infected mice. Screening of pulmonary cytokines using a novel dysregulation score identified an exacerbated expression of CSF3 and interleukin-6 in the context of smoke exposure and influenza. Recombinant (r)CSF3 supplementation during influenza aggravated morbidity, hypothermia and oedema, while anti-CSF3R treatment of smoke-exposed infected mice improved alveolar-capillary barrier function. scRNAseq delineated a shift in the distribution of Csf3 + cells towards neutrophils in the context of cigarette smoke and influenza. However, although smoke-exposed lungs were enriched for infected, highly activated neutrophils, gene signatures of these cells largely reflected an exacerbated form of typical influenza with select unique regulatory features. CONCLUSION: This work provides novel insight into the mechanisms by which cigarette smoke exacerbates influenza infection, unveiling potential therapeutic targets (e.g. excess vRNA accumulation, oedematous CSF3R signalling) for use in this context, and potential limitations for clinical rCSF3 therapy during viral infectious disease.
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Fumar Cigarros , Vírus da Influenza A Subtipo H1N1 , Influenza Humana , Animais , Fumar Cigarros/efeitos adversos , Humanos , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos , NicotianaRESUMO
OBJECTIVE: LDL (low-density lipoprotein) transcytosis across the endothelium is performed by the SR-BI (scavenger receptor class B type 1) receptor and contributes to atherosclerosis. HMGB1 (high mobility group box 1) is a structural protein in the nucleus that is released by cells during inflammation; extracellular HMGB1 has been implicated in advanced disease. Whether intracellular HMGB1 regulates LDL transcytosis through its nuclear functions is unknown. Approach and Results: HMGB1 was depleted by siRNA in human coronary artery endothelial cells, and transcytosis of LDL was measured by total internal reflection fluorescence microscopy. Knockdown of HMGB1 attenuated LDL transcytosis without affecting albumin transcytosis. Loss of HMGB1 resulted in reduction in SR-BI levels and depletion of SREBP2 (sterol regulatory element-binding protein 2)-a transcription factor upstream of SR-BI. The effect of HMGB1 depletion on LDL transcytosis required SR-BI and SREBP2. Overexpression of HMGB1 caused an increase in LDL transcytosis that was unaffected by inhibition of extracellular HMGB1 or depletion of RAGE (receptor for advanced glycation endproducts)-a cell surface receptor for HMGB1. The effect of HMGB1 overexpression on LDL transcytosis was prevented by knockdown of SREBP2. Loss of HMGB1 caused a reduction in the half-life of SREBP2; incubation with LDL caused a significant increase in nuclear localization of HMGB1 that was dependent on SR-BI. Animals lacking endothelial HMGB1 exhibited less acute accumulation of LDL in the aorta 30 minutes after injection and when fed a high-fat diet developed fewer fatty streaks and less atherosclerosis. CONCLUSIONS: Endothelial HMGB1 regulates LDL transcytosis by prolonging the half-life of SREBP2, enhancing SR-BI expression. Translocation of HMGB1 to the nucleus in response to LDL requires SR-BI.
Assuntos
Aterosclerose/metabolismo , Células Endoteliais/metabolismo , Proteína HMGB1/metabolismo , Receptores de LDL/metabolismo , Receptores Depuradores Classe B/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Transcitose , Transporte Ativo do Núcleo Celular , Animais , Aterosclerose/genética , Aterosclerose/patologia , Aterosclerose/prevenção & controle , Células Cultivadas , Modelos Animais de Doenças , Feminino , Proteína HMGB1/deficiência , Proteína HMGB1/genética , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estabilidade Proteica , Receptores de LDL/genética , Receptores Depuradores Classe B/genética , Transdução de Sinais , Proteína de Ligação a Elemento Regulador de Esterol 2/genéticaRESUMO
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.
Assuntos
Receptores de Activinas Tipo II/metabolismo , Caveolina 1/metabolismo , Membrana Celular/metabolismo , Endocitose , Endotélio Vascular/fisiologia , Fator 2 de Diferenciação de Crescimento/metabolismo , Lipoproteínas LDL/metabolismo , Receptores de Activinas Tipo II/genética , Caveolina 1/genética , Células Cultivadas , Endotélio Vascular/citologia , Fator 2 de Diferenciação de Crescimento/genética , Humanos , Fosforilação , Transdução de SinaisRESUMO
Vesicle-mediated transcellular transport or simply "transcytosis" is a cellular process used to shuttle macromolecules such as lipoproteins, antibodies, and albumin from one surface of a polarized cell to the other. This mechanism is in contrast to the transit of small molecules such as anions, cations and amino acids that occur via uptake, diffusion through the cytosol and release and is also distinct from paracellular leak between cells. Importantly, transcytosis has evolved as a process to selectively move macromolecules between 2 neighboring yet unique microenvironments within a multicellular organism. Examples include the movement of lipoproteins out of the circulatory system and into tissues and the delivery of immunoglobulins to mucosal surfaces. Regardless of whether the transport is conducted by endothelial or epithelial cells, the process often involves receptor-mediated uptake of a ligand into an endocytic vesicle, regulated transit of the carrier through the cytoplasm and release of the cargo via an exocytic event. While transcytosis has been examined in detail in epithelial cells, for both historical and technical reasons, the process is less understood in endothelial cells. Here, we spotlight aspects of epithelial transcytosis including recent findings and review the comparative dearth of knowledge regarding the process in endothelial cells highlighting the opportunity for further study.
Assuntos
Células Endoteliais/metabolismo , Transcitose , Vesículas Transportadoras/metabolismo , Animais , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , HumanosRESUMO
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.
Assuntos
Aterosclerose/metabolismo , Caveolina 1/fisiologia , Endotélio Vascular/metabolismo , Lipoproteínas LDL/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Transcitose/fisiologia , Animais , Aterosclerose/patologia , Aterosclerose/prevenção & controle , Células Cultivadas , Cães , Endotélio Vascular/patologia , Ativação Enzimática/fisiologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos TransgênicosRESUMO
The FAO of the UN convened an Expert Working Group meeting to provide recommendations related to protein quality evaluation of Follow-up Formula for Young Children (FUF-YC) and Ready-to-Use Therapeutic Foods (RUTFs). The protein and amino acid (AA) scoring patterns for the target age groups were defined and recommendations provided on the use of currently available protein and indispensable AA digestibility data. For FUF-YC, an age category of 1-2.9 y was identified, and a matching protein requirement of 0.86 g · kg-1 · d-1 with corresponding AA requirements were recommended. For RUTF, the protein requirement recommended was 2.82 g · kg-1 · d-1, to achieve a catch-up weight gain of 10 g · kg-1 · d-1 in children recovering from severe acute malnutrition. The AA requirements were factorially derived based on the adult protein requirement for maintenance and tissue AA composition. A flowchart was proposed for the best available methods to estimate digestibility coefficients (of either protein or AAs), in the following order: human, growing pig, and rat true ileal AA digestibility values. Where this is not possible, fecal protein digestibility values should be used. The Expert Working Group recommends the use of the Protein Digestibility Corrected Amino Acid Score (PDCAAS), with existing protein digestibility values, or the Digestible Indispensable Amino Acid Score provided that individual AA digestibility values are available for protein quality evaluation using the latter score. The Group also recommends the use of ileal digestibility of protein or of AAs for plant-based protein sources, recognizing the possible effects of antinutritional factors and impaired gut function. A PDCAAS score of ≥90% can be considered adequate for these formulations, whereas with a score <90%, the quantity of protein should be increased to meet the requirements. Regardless of the protein quality score, the ability of formulations to support growth in the target population should be evaluated. Future research recommendations are also proposed based on the knowledge gaps identified.
Assuntos
Proteínas Alimentares/normas , Alimento Funcional , Fórmulas Infantis , Animais , Pré-Escolar , Proteínas Alimentares/metabolismo , Digestão , História do Século XXI , Humanos , LactenteRESUMO
Vascular leakage is a characteristic of critical illnesses such as septic shock and acute respiratory distress syndrome. It results in hypotension and tissue edema and contributes to organ dysfunction. It has long been taught that increased vascular permeability is a natural consequence of inflammation; in particular, many clinicians believe that it occurs inevitably during leukocyte recruitment to a site of infection. In fact, abundant research now indicates that vascular leakage and leukocyte emigration do not necessarily occur together in a blood vessel. The molecular mechanisms underpinning these processes-allowing leukocytes to exit the circulation without increasing vascular permeability-are starting to be elucidated and establish vascular leakage as a viable therapeutic target. Several preclinical studies indicate that vascular leakage can be reduced without impairing cytokine production, leukocyte recruitment, and pathogen clearance. The realization that leukocyte traffic and vascular permeability can be regulated separately should spur development of therapies that decrease vascular leakage and tissue edema without compromising the immune response.
Assuntos
Permeabilidade Capilar/fisiologia , Inflamação/fisiopatologia , Síndrome do Desconforto Respiratório/etiologia , Choque Séptico/etiologia , Citocinas/metabolismo , Humanos , Leucócitos/fisiologiaRESUMO
In healthy blood vessels, albumin crosses the endothelium to leave the circulation by transcytosis. However, little is known about the regulation of albumin transcytosis or how it differs in different tissues; its physiological purpose is also unclear. Using total internal reflection fluorescence microscopy, we quantified transcytosis of albumin across primary human microvascular endothelial cells from both lung and skin. We then validated our in vitro findings using a tissue-specific knockout mouse model. We observed that albumin transcytosis was saturable in the skin but not the lung microvascular endothelial cells, implicating a receptor-mediated process. We identified the scavenger receptor CD36 as being both necessary and sufficient for albumin transcytosis across dermal microvascular endothelium, in contrast to the lung where macropinocytosis dominated. Mutations in the apical helical bundle of CD36 prevented albumin internalization by cells. Mice deficient in CD36 specifically in endothelial cells exhibited lower basal permeability to albumin and less basal tissue edema in the skin but not in the lung. Finally, these mice also exhibited a smaller subcutaneous fat layer despite having identical total body weights and circulating fatty acid levels as wild-type animals. In conclusion, CD36 mediates albumin transcytosis in the skin but not the lung. Albumin transcytosis may serve to regulate fatty acid delivery from the circulation to tissues.
Assuntos
Albuminas/metabolismo , Antígenos CD36/metabolismo , Células Endoteliais/metabolismo , Ácidos Graxos/metabolismo , Animais , Antígenos CD36/química , Antígenos CD36/deficiência , Antígenos CD36/genética , Células Cultivadas , Células Endoteliais/citologia , Humanos , Pulmão/irrigação sanguínea , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microvasos/citologia , Microvasos/metabolismo , Mutagênese Sítio-Dirigida , Pinocitose , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Pele/irrigação sanguínea , Gordura Subcutânea/anatomia & histologia , Gordura Subcutânea/metabolismo , Distribuição Tecidual , TranscitoseRESUMO
Objective- The atheroprotective effects of estrogen are independent of circulating lipid levels. Whether estrogen regulates transcytosis of LDL (low-density lipoprotein) across the coronary endothelium is unknown. Approach and Results- Using total internal reflection fluorescence microscopy, we quantified transcytosis of LDL across human coronary artery endothelial cells from multiple donors. LDL transcytosis was significantly higher in cells from men compared with premenopausal women. Estrogen significantly attenuated LDL transcytosis by endothelial cells from male but not female donors; transcytosis of albumin was not affected. Estrogen caused downregulation of endothelial SR-BI (scavenger receptor class B type 1), and overexpression of SR-BI was sufficient to restore LDL transcytosis. Similarly, depletion of SR-BI by siRNA attenuated endothelial LDL transcytosis and prevented any further effect of estrogen. In contrast, treatment with estrogen had no effect on SR-BI expression by liver cells. Inhibition of estrogen receptors α and ß had no effect on estrogen-mediated attenuation of LDL transcytosis. However, estrogen's effect on LDL transcytosis was blocked by depletion of the GPER (G-protein-coupled estrogen receptor). GPER was found to be enriched in endothelial cells compared with hepatocytes and is reported to signal via transactivation of the EGFR (epidermal growth factor receptor); inhibition of EGFR prevented the effect of estrogen on LDL transcytosis and SR-BI mRNA. Last, SR-BI expression was significantly higher in human coronary artery endothelial cells from male compared with premenopausal female donors. Conclusions- Estrogen significantly inhibits LDL transcytosis by downregulating endothelial SR-BI; this effect requires GPER.