Akt is a mediator of artery specification during zebrafish development.

The dorsal aorta (DA) is the first major blood vessel to develop in the embryonic cardiovascular system. Its formation is governed by a coordinated process involving the migration, specification, and arrangement of angioblasts into arterial and venous lineages, a process conserved across species. Although vascular endothelial growth factor a (VEGF-A) is known to drive DA specification and formation, the kinases involved in this process remain ambiguous. Thus, we investigated the role of protein kinase B (Akt) in zebrafish by generating a quadruple mutant (aktΔ/Δ), in which expression and activity of all Akt genes - akt1, -2, -3a and -3b - are strongly decreased. Live imaging of developing aktΔ/Δ DA uncovers early arteriovenous malformations. Single-cell RNA-sequencing analysis of aktΔ/Δ endothelial cells corroborates the impairment of arterial, yet not venous, cell specification. Notably, endothelial specific expression of ligand-independent activation of Notch or constitutively active Akt1 were sufficient to re-establish normal arterial specification in aktΔ/Δ. The Akt loss-of-function mutant unveils that Akt kinase can act upstream of Notch in arterial endothelial cells, and is involved in proper embryonic artery specification. This sheds light on cardiovascular development, revealing a mechanism behind congenital malformations.

TNFα increases the degradation of pyruvate dehydrogenase kinase 4 by the Lon protease to support proinflammatory genes.

The endothelium is a major target of the proinflammatory cytokine, tumor necrosis factor alpha (TNFα). Exposure of endothelial cells (EC) to proinflammatory stimuli leads to an increase in mitochondrial metabolism; however, the function and regulation of elevated mitochondrial metabolism in EC in response to proinflammatory cytokines remain unclear. Studies using high-resolution metabolomics and 13C-glucose and 13C-glutamine labeling flux techniques showed that pyruvate dehydrogenase activity (PDH) and oxidative tricarboxylic acid cycle (TCA) flux are elevated in human umbilical vein ECs in response to overnight (16 h) treatment with TNFα (10 ng/mL). Mechanistic studies indicated that TNFα mediated these metabolic changes via mitochondrial-specific protein degradation of pyruvate dehydrogenase kinase 4 (PDK4, inhibitor of PDH) by the Lon protease via an NF-κB-dependent mechanism. Using RNA sequencing following siRNA-mediated knockdown of the catalytically active subunit of PDH, PDHE1α (PDHA1 gene), we show that PDH flux controls the transcription of approximately one-third of the genes that are up-regulated by TNFα stimulation. Notably, TNFα-induced PDH flux regulates a unique signature of proinflammatory mediators (cytokines and chemokines) but not inducible adhesion molecules. Metabolomics and ChIP sequencing for acetylated modification on lysine 27 of histone 3 (H3K27ac) showed that TNFα-induced PDH flux promotes histone acetylation of specific gene loci via citrate accumulation and ATP-citrate lyase-mediated generation of acetyl CoA. Together, these results uncover a mechanism by which TNFα signaling increases oxidative TCA flux of glucose to support TNFα-induced gene transcription through extramitochondrial acetyl CoA generation and histone acetylation.

Macrophage-Derived 25-Hydroxycholesterol Promotes Vascular Inflammation, Atherogenesis, and Lesion Remodeling.

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, Myh11ERT2CREmT/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.

ZFYVE21 promotes endothelial nitric oxide signaling and vascular barrier function in the kidney during aging.

ZFYVE21 is an ancient, endosome-associated protein that is highly expressed in endothelial cells (ECs) but whose function(s) in vivo are undefined. Here, we identified ZFYVE21 as an essential regulator of vascular barrier function in the aging kidney. ZFYVE21 levels significantly decline in ECs in aged human and mouse kidneys. To investigate attendant effects, we generated EC-specific Zfyve21-/- reporter mice. These knockout mice developed accelerated aging phenotypes including reduced endothelial nitric oxide (ENOS) activity, failure to thrive, and kidney insufficiency. Kidneys from Zfyve21 EC-/- mice showed interstitial edema and glomerular EC injury. ZFYVE21-mediated phenotypes were not programmed developmentally as loss of ZFYVE21 in ECs during adulthood phenocopied its loss prenatally, and a nitric oxide donor normalized kidney function in adult hosts. Using live cell imaging and human kidney organ cultures, we found that in a GTPase Rab5- and protein kinase Akt-dependent manner, ZFYVE21 reduced vesicular levels of inhibitory caveolin-1 and promoted transfer of Golgi-derived ENOS to a perinuclear Rab5+ vesicular population to functionally sustain ENOS activity. Thus, our work defines a ZFYVE21- mediated trafficking mechanism sustaining ENOS activity and demonstrates the relevance of this pathway for maintaining kidney function with aging.

Progressive myoclonus epilepsies-Residual unsolved cases have marked genetic heterogeneity including dolichol-dependent protein glycosylation pathway genes.

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.

SREBP2 regulates the endothelial response to cytokines via direct transcriptional activation of KLF6.

The transcription factor SREBP2 is the main regulator of cholesterol homeostasis and is central to the mechanism of action of lipid-lowering drugs, such as statins, which are responsible for the largest overall reduction in cardiovascular risk and mortality in humans with atherosclerotic disease. Recently, SREBP2 has been implicated in leukocyte innate and adaptive immune responses by upregulation of cholesterol flux or direct transcriptional activation of pro-inflammatory genes. Here, we investigate the role of SREBP2 in endothelial cells (ECs), since ECs are at the interface of circulating lipids with tissues and crucial to the pathogenesis of cardiovascular disease. Loss of SREBF2 inhibits the production of pro-inflammatory chemokines but amplifies type I interferon response genes in response to inflammatory stimulus. Furthermore, SREBP2 regulates chemokine expression not through enhancement of endogenous cholesterol synthesis or lipoprotein uptake but partially through direct transcriptional activation. Chromatin immunoprecipitation sequencing of endogenous SREBP2 reveals that SREBP2 bound to the promoter regions of two nonclassical sterol responsive genes involved in immune modulation, BHLHE40 and KLF6. SREBP2 upregulation of KLF6 was responsible for the downstream amplification of chemokine expression, highlighting a novel relationship between cholesterol homeostasis and inflammatory phenotypes in ECs.

Histone Acetyltransferases p300 and CBP Coordinate Distinct Chromatin Remodeling Programs in Vascular Smooth Muscle Plasticity.

BACKGROUND: Vascular smooth muscle cell (VSMC) phenotypic switching contributes to cardiovascular diseases. Epigenetic regulation is emerging as a key regulatory mechanism, with the methylcytosine dioxygenase TET2 acting as a master regulator of smooth muscle cell phenotype. The histone acetyl-transferases p300 and CREB-binding protein (CBP) are highly homologous and often considered to be interchangeable, and their roles in smooth muscle cell phenotypic regulation are not known. METHODS: We assessed the roles of p300 and CBP in human VSMC with knockdown, in inducible smooth muscle-specific knockout mice (inducible knockout [iKO]; p300iKO or CBPiKO), and in samples of human intimal hyperplasia. RESULTS: P300, CBP, and histone acetylation were differently regulated in VSMCs undergoing phenotypic switching and in vessel remodeling after vascular injury. Medial p300 expression and activity were repressed by injury, but CBP and histone acetylation were induced in neointima. Knockdown experiments revealed opposing effects of p300 and CBP in the VSMC phenotype: p300 promoted contractile protein expression and inhibited migration, but CBP inhibited contractile genes and enhanced migration. p300iKO mice exhibited severe intimal hyperplasia after arterial injury compared with controls, whereas CBPiKO mice were entirely protected. In normal aorta, p300iKO reduced, but CBPiKO enhanced, contractile protein expression and contractility compared with controls. Mechanistically, we found that these histone acetyl-transferases oppositely regulate histone acetylation, DNA hydroxymethylation, and PolII (RNA polymerase II) binding to promoters of differentiation-specific contractile genes. Our data indicate that p300 and TET2 function together, because p300 was required for TET2-dependent hydroxymethylation of contractile promoters, and TET2 was required for p300-dependent acetylation of these loci. TET2 coimmunoprecipitated with p300, and this interaction was enhanced by rapamycin but repressed by platelet-derived growth factor (PDGF) treatment, with p300 promoting TET2 protein stability. CBP did not associate with TET2, but instead facilitated recruitment of histone deacetylases (HDAC2, HDAC5) to contractile protein promoters. Furthermore, CBP inhibited TET2 mRNA levels. Immunostaining of cardiac allograft vasculopathy samples revealed that p300 expression is repressed but CBP is induced in human intimal hyperplasia. CONCLUSIONS: This work reveals that p300 and CBP serve nonredundant and opposing functions in VSMC phenotypic switching and coordinately regulate chromatin modifications through distinct functional interactions with TET2 or HDACs. Targeting specific histone acetyl-transferases may hold therapeutic promise for cardiovascular diseases.

De novo DHDDS variants cause a neurodevelopmental and neurodegenerative disorder with myoclonus.

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.

Structural elucidation of the cis-prenyltransferase NgBR/DHDDS complex reveals insights in regulation of protein glycosylation.

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.

Unbiased proteomics identifies plasminogen activator inhibitor-1 as a negative regulator of endothelial nitric oxide synthase.

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.

Caveolae: The FAQs.

Caveolae are an abundant, but enigmatic, plasma membrane feature of vertebrate cells. In this brief commentary, the authors attempt to answer some key questions related to the formation and function of caveolae based on round-table discussions at the first EMBO Workshop on Caveolae held in France in May 2019.

Defective Flow-Migration Coupling Causes Arteriovenous Malformations in Hereditary Hemorrhagic Telangiectasia.

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.

BMP-9 and LDL crosstalk regulates ALK-1 endocytosis and LDL transcytosis in endothelial cells.

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.

Alcohol-induced Hsp90 acetylation is a novel driver of liver sinusoidal endothelial dysfunction and alcohol-related liver disease.

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.

Cav-1 (Caveolin-1) Deficiency Increases Autophagy in the Endothelium and Attenuates Vascular Inflammation and Atherosclerosis.

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.

Caveolin-1 Regulates Atherogenesis by Attenuating Low-Density Lipoprotein Transcytosis and Vascular Inflammation Independently of Endothelial Nitric Oxide Synthase Activation.

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.

Stimulation of Caveolin-1 Signaling Improves Arteriovenous Fistula Patency.

Objective- Arteriovenous fistulae (AVF) are the most common access created for hemodialysis; however, many AVF fail to mature and require repeated intervention, suggesting a need to improve AVF maturation. Eph-B4 (ephrin type-B receptor 4) is the embryonic venous determinant that is functional in adult veins and can regulate AVF maturation. Cav-1 (caveolin-1) is the major scaffolding protein of caveolae-a distinct microdomain that serves as a mechanosensor at the endothelial cell membrane. We hypothesized that Cav-1 function is critical for Eph-B4-mediated AVF maturation. Approach and Results- In a mouse aortocaval fistula model, both Cav-1 mRNA and protein were increased in the AVF compared with control veins. Cav-1 KO (knockout) mice showed increased fistula wall thickening ( P=0.0005) and outward remodeling ( P<0.0001), with increased eNOS (endothelial NO synthase) activity compared with WT (wild type) mice. Ephrin-B2/Fc inhibited AVF outward remodeling in WT mice but not in Cav-1 KO mice and was maintained in Cav-1 RC (Cav-1 endothelial reconstituted) mice (WT, P=0.0001; Cav-1 KO, P=0.7552; Cav-1 RC, P=0.0002). Cavtratin-a Cav-1 scaffolding domain peptide-decreased AVF wall thickness in WT mice and in Eph-B4 het mice compared with vehicle alone (WT, P=0.0235; Eph-B4 het, P=0.0431); cavtratin also increased AVF patency (day 42) in WT mice ( P=0.0275). Conclusions- Endothelial Cav-1 mediates Eph-B4-mediated AVF maturation. The Eph-B4-Cav-1 axis regulates adaptive remodeling during venous adaptation to the fistula environment. Manipulation of Cav-1 function may be a translational strategy to enhance AVF patency.

Critical role of caveolin-1 in ocular neovascularization and multitargeted antiangiogenic effects of cavtratin via JNK.

Ocular neovascularization is a devastating pathology of numerous ocular diseases and is a major cause of blindness. Caveolin-1 (Cav-1) plays important roles in the vascular system. However, little is known regarding its function and mechanisms in ocular neovascularization. Here, using comprehensive model systems and a cell permeable peptide of Cav-1, cavtratin, we show that Cav-1 is a critical player in ocular neovascularization. The genetic deletion of Cav-1 exacerbated and cavtratin administration inhibited choroidal and retinal neovascularization. Importantly, combined administration of cavtratin and anti-VEGF-A inhibited neovascularization more effectively than monotherapy, suggesting the existence of other pathways inhibited by cavtratin in addition to VEGF-A. Indeed, we found that cavtratin suppressed multiple critical components of pathological angiogenesis, including inflammation, permeability, PDGF-B and endothelial nitric oxide synthase expression (eNOS). Mechanistically, we show that cavtratin inhibits CNV and the survival and migration of microglia and macrophages via JNK. Together, our data demonstrate the unique advantages of cavtratin in antiangiogenic therapy to treat neovascular diseases.

Caveolin-1 regulates lipid droplet metabolism in endothelial cells via autocrine prostacyclin-stimulated, cAMP-mediated lipolysis.

Lipid droplets (LD) are dynamic organelles involved in intracellular lipid metabolism in almost all eukaryotic cells, and LD-associated proteins tightly regulate their dynamics. One LD coat protein is caveolin-1 (Cav-1), an essential component for caveola assembly in highly differentiated cells, including adipocytes, smooth muscle cells, and endothelial cells (EC). However, the role of Cav-1 in LD dynamics is unclear. Here we report that EC lacking Cav-1 exhibit impaired LD formation. The decreased LD formation is due to enhanced lipolysis and not caused by reduced triglyceride synthesis or fatty acid uptake. Mechanistically, the absence of Cav-1 increased cAMP/PKA signaling in EC, as indicated by elevated phosphorylation of hormone-sensitive lipase and increased lipolysis. Unexpectedly, we also observed enhanced autocrine production of prostaglandin I2 (PGI2, also called prostacyclin) in Cav-1 KO EC, and this PGI2 increase appeared to stimulate cAMP/PKA pathways, contributing to the enhanced lipolysis in Cav-1 KO cells. Our results reveal an unanticipated role of Cav-1 in regulating lipolysis in non-adipose tissue, indicating that Cav-1 is required for LD metabolism in EC and that it regulates cAMP-dependent lipolysis in part via the autocrine production of PGI2.

CLOCK phosphorylation by AKT regulates its nuclear accumulation and circadian gene expression in peripheral tissues.

Circadian locomotor output cycles kaput (CLOCK) is a transcription factor that activates transcription of clock-controlled genes by heterodimerizing with BMAL1 and binding to E-box elements on DNA. Although several phosphorylation sites on CLOCK have already been identified, this study characterizes a novel phosphorylation site at serine 845 (Ser-836 in humans). Here, we show that CLOCK is a novel AKT substrate in vitro and in cells, and this phosphorylation site is a negative regulator of CLOCK nuclear localization by acting as a binding site for 14-3-3 proteins. To examine the role of CLOCK phosphorylation in vivo, ClockS845A knockin mice were generated using CRISPR/Cas9 technology. ClockS845A mice are essentially normal with normal central circadian rhythms and hemodynamics. However, examination of core circadian gene expression from peripheral tissues demonstrated that ClockS845A mice have diminished expression of Per2, Reverba, Dbp, and Npas2 in skeletal muscle and Per2, Reverba, Dbp, Per1, Rora, and Npas2 in the liver during the circadian cycle. The reduction in Dbp levels is associated with reduced H3K9ac at E-boxes where CLOCK binds despite no change in total CLOCK levels. Thus, CLOCK phosphorylation by AKT on Ser-845 regulates its nuclear translocation and the expression levels of certain core circadian genes in insulin-sensitive tissues.