Netrin-4 expression by human endothelial cells inhibits endothelial inflammation and senescence.

Netrin-4, recognized in neural and vascular development, is highly expressed by mature endothelial cells. The function of this netrin-4 in vascular biology after development has remained unclear. We found that the expression of netrin-4 is highly regulated in endothelial cells and is important for quiescent healthy endothelium. Netrin-4 expression is upregulated in endothelial cells cultured under laminar flow conditions, while endothelial cells stimulated with tumor necrosis factor alpha resulted in decreased netrin-4 expression. Targeted reduction of netrin-4 in endothelial cells resulted in increased expression of vascular cell adhesion molecule 1 and intercellular adhesion molecule 1. Besides, these endothelial cells were more prone to monocyte adhesion and showed impaired barrier function, measured with electric cell-substrate impedance sensing, as well as in an 'organ-on-a-chip' microfluidic system. Importantly, endothelial cells with reduced levels of netrin-4 showed increased expression of the senescence-associated markers cyclin-dependent kinase inhibitor-1 and -2A, an increased cell size and decreased ability to proliferate. Consistent with the gene expression profile, netrin-4 reduction was accompanied with more senescent associated ß-galactosidase activity, which could be rescued by adding netrin-4 protein. Finally, using human decellularized kidney extracellular matrix scaffolds, we found that pre-treatment of the scaffolds with netrin-4 increased numbers of endothelial cells adhering to the matrix, showing a pro-survival effect of netrin-4. Taken together, netrin-4 acts as an anti-senescence and anti-inflammation factor in endothelial cell function and our results provide insights as to maintain endothelial homeostasis and supporting vascular health.

RIPK1 Expression Associates With Inflammation in Early Atherosclerosis in Humans and Can Be Therapeutically Silenced to Reduce NF-κB Activation and Atherogenesis in Mice.

BACKGROUND: Chronic activation of the innate immune system drives inflammation and contributes directly to atherosclerosis. We previously showed that macrophages in the atherogenic plaque undergo RIPK3 (receptor-interacting serine/threonine-protein kinase 3)-MLKL (mixed lineage kinase domain-like protein)-dependent programmed necroptosis in response to sterile ligands such as oxidized low-density lipoprotein and damage-associated molecular patterns and that necroptosis is active in advanced atherosclerotic plaques. Upstream of the RIPK3-MLKL necroptotic machinery lies RIPK1 (receptor-interacting serine/threonine-protein kinase 1), which acts as a master switch that controls whether the cell undergoes NF-κB (nuclear factor κ-light-chain-enhancer of activated B cells)-dependent inflammation, caspase-dependent apoptosis, or necroptosis in response to extracellular stimuli. We therefore set out to investigate the role of RIPK1 in the development of atherosclerosis, which is driven largely by NF-κB-dependent inflammation at early stages. We hypothesize that, unlike RIPK3 and MLKL, RIPK1 primarily drives NF-κB-dependent inflammation in early atherogenic lesions, and knocking down RIPK1 will reduce inflammatory cell activation and protect against the progression of atherosclerosis. METHODS: We examined expression of RIPK1 protein and mRNA in both human and mouse atherosclerotic lesions, and used loss-of-function approaches in vitro in macrophages and endothelial cells to measure inflammatory responses. We administered weekly injections of RIPK1 antisense oligonucleotides to Apoe-/- mice fed a cholesterol-rich (Western) diet for 8 weeks. RESULTS: We find that RIPK1 expression is abundant in early-stage atherosclerotic lesions in both humans and mice. Treatment with RIPK1 antisense oligonucleotides led to a reduction in aortic sinus and en face lesion areas (47.2% or 58.8% decrease relative to control, P<0.01) and plasma inflammatory cytokines (IL-1α [interleukin 1α], IL-17A [interleukin 17A], P<0.05) in comparison with controls. RIPK1 knockdown in macrophages decreased inflammatory genes (NF-κB, TNFα [tumor necrosis factor α], IL-1α) and in vivo lipopolysaccharide- and atherogenic diet-induced NF-κB activation. In endothelial cells, knockdown of RIPK1 prevented NF-κB translocation to the nucleus in response to TNFα, where accordingly there was a reduction in gene expression of IL1B, E-selectin, and monocyte attachment. CONCLUSIONS: We identify RIPK1 as a central driver of inflammation in atherosclerosis by its ability to activate the NF-κB pathway and promote inflammatory cytokine release. Given the high levels of RIPK1 expression in human atherosclerotic lesions, our study suggests RIPK1 as a future therapeutic target to reduce residual inflammation in patients at high risk of coronary artery disease.

The identification and function of a Netrin-1 mutation in a pedigree with premature atherosclerosis.

BACKGROUND AND AIMS: Neuroimmune guidance cues have been shown to play a role in atherosclerosis, but their exact role in human pathophysiology is largely unknown. In the current study, we investigated the role of a c.1769G > T variant in Netrin-1 in (premature) atherosclerosis. METHODS: To determine the effect of the genetic variation, purified Netrin-1, either wild type (wtNetrin-1) or the patient observed variation (mutNetrin-1), was used for migration, adhesion, endothelial barrier function and bindings assays. Expression of adhesion molecules and transcription proteins was analyzed by RT-PCR, Western blot or ELISA. To further delineate how mutNetrin-1 mediates its effect on cell migration, lenti-viral knockdown of UNC5B or DCC was used. RESULTS: Bindings assays revealed a decreased binding capacity of mutNetrin-1 to the receptors UNC5B, DCC and ß3-integrin and an increased binding capacity to neogenin, heparin and heparan sulfate compared to wtNetrin-1. Exposure of endothelial cells to mutNetrin-1 resulted in enhanced monocyte adhesion and expression of IL-6, CCL2 and ICAM-1 compared to wtNetrin-1. In addition, mutNetrin-1 lacks the inhibitory effect on the NF-κB pathway that is observed for wtNetrin-1. Moreover, the presence of mutNetrin-1 diminished migration of macrophages and smooth muscle cells. Importantly, UNC5B or DCC specific knockdown showed that mutNetrin-1 is unable to act through DCC resulting in enhanced inhibition of migration. CONCLUSIONS: Our data demonstrates that mutNetrin-1 fails to exert anti-inflammatory effects on endothelial cells and more strongly blocks macrophage migration compared to wtNetrin-1, suggesting that the carriers of this genetic molecular variant may well be at risk for premature atherosclerosis.

Disruption of circadian rhythm by alternating light-dark cycles aggravates atherosclerosis development in APOE*3-Leiden.CETP mice.

Disruption of circadian rhythm by means of shift work has been associated with cardiovascular disease in humans. However, causality and underlying mechanisms have not yet been established. In this study, we exposed hyperlipidemic APOE*3-Leiden.CETP mice to either regular light-dark cycles, weekly 6 hours phase advances or delays, or weekly alternating light-dark cycles (12 hours shifts), as a well-established model for shift work. We found that mice exposed to 15 weeks of alternating light-dark cycles displayed a striking increase in atherosclerosis, with an approximately twofold increase in lesion size and severity, while mice exposed to phase advances and delays showed a milder circadian disruption and no significant effect on atherosclerosis development. We observed a higher lesion macrophage content in mice exposed to alternating light-dark cycles without obvious changes in plasma lipids, suggesting involvement of the immune system. Moreover, while no changes in the number or activation status of circulating monocytes and other immune cells were observed, we identified increased markers for inflammation, oxidative stress, and chemoattraction in the vessel wall. Altogether, this is the first study to show that circadian disruption by shifting light-dark cycles directly aggravates atherosclerosis development.

Vascular Semaphorin 7A Upregulation by Disturbed Flow Promotes Atherosclerosis Through Endothelial ß1 Integrin.

OBJECTIVE: Accumulating evidence suggests a role of semaphorins in vascular homeostasis. Here, we investigate the role of Sema7A (semaphorin 7A) in atherosclerosis and its underlying mechanism. APPROACH AND RESULTS: Using genetically engineered Sema7A-/-ApoE-/- mice, we showed that deletion of Sema7A attenuates atherosclerotic plaque formation primarily in the aorta of ApoE-/- mice on a high-fat diet. A higher level of Sema7A in the atheroprone lesser curvature suggests a correlation of Sema7A with disturbed flow. This notion is supported by elevated Sema7A expression in human umbilical venous endothelial cells either subjected to oscillatory shear stress or treated with the PKA (protein kinase A)/CREB (cAMP response element-binding protein) inhibitor H89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide·2HCl hydrate). Further studies using the partial carotid artery ligation model showed that disturbed flow in the left carotid artery of Sema7A+/+ApoE-/- mice promoted the expression of endothelial Sema7A and cell adhesion molecules, leukocyte adhesion, and plaque formation, whereas such changes were attenuated in Sema7A-/-ApoE-/- mice. Further studies showed that blockage of ß1 integrin, a known Sema7A receptor, or inhibition of FAK (focal adhesion kinase), MEK1/2 (mitogen-activated protein kinase kinase 1/2), or NF-κB (nuclear factor-κB) significantly reduced the expression of cell adhesion molecules and THP-1 (human acute monocytic leukemia cell line) monocyte adhesion in Sema7A-overexpressing human umbilical venous endothelial cells. Studies using chimeric mice suggest that vascular, most likely endothelial, Sema7A plays a major role in atherogenesis. CONCLUSIONS: Our findings indicate a significant role of Sema7A in atherosclerosis by mediating endothelial dysfunction in a ß1 integrin-dependent manner.

Silencing of microRNA-132 reduces renal fibrosis by selectively inhibiting myofibroblast proliferation.

Chronic kidney disease is associated with progressive renal fibrosis, where perivascular cells give rise to the majority of α-smooth muscle actin (α-SMA) positive myofibroblasts. Here we sought to identify pericytic miRNAs that could serve as a target to decrease myofibroblast formation. Kidney fibrosis was induced in FoxD1-GC;Z/Red-mice by unilateral ureteral obstruction followed by FACS sorting of dsRed-positive FoxD1-derivative cells and miRNA profiling. MiR-132 selectively increased 21-fold during pericyte-to-myofibroblast formation, whereas miR-132 was only 2.5-fold up in total kidney lysates (both in obstructive and ischemia-reperfusion injury). MiR-132 silencing during obstruction decreased collagen deposition (35%) and tubular apoptosis. Immunohistochemistry, Western blot, and qRT-PCR confirmed a similar decrease in interstitial α-SMA(+) cells. Pathway analysis identified a rate-limiting role for miR-132 in myofibroblast proliferation that was confirmed in vitro. Indeed, antagomir-132-treated mice displayed a reduction in the number of proliferating Ki67(+) interstitial myofibroblasts. Interestingly, this was selective for the interstitial compartment and did not impair the reparative proliferation of tubular epithelial cells, as evidenced by an increase in Ki67(+) epithelial cells, as well as increased phospho-RB1, Cyclin-A and decreased RASA1, p21 levels in kidney lysates. Additional pathway and gene expression analyses suggest miR-132 coordinately regulates genes involved in TGF-ß signaling (Smad2/Smad3), STAT3/ERK pathways, and cell proliferation (Foxo3/p300). Thus, silencing miR-132 counteracts the progression of renal fibrosis by selectively decreasing myofibroblast proliferation and could potentially serve as a novel antifibrotic therapy.

Quaking promotes monocyte differentiation into pro-atherogenic macrophages by controlling pre-mRNA splicing and gene expression.

A hallmark of inflammatory diseases is the excessive recruitment and influx of monocytes to sites of tissue damage and their ensuing differentiation into macrophages. Numerous stimuli are known to induce transcriptional changes associated with macrophage phenotype, but posttranscriptional control of human macrophage differentiation is less well understood. Here we show that expression levels of the RNA-binding protein Quaking (QKI) are low in monocytes and early human atherosclerotic lesions, but are abundant in macrophages of advanced plaques. Depletion of QKI protein impairs monocyte adhesion, migration, differentiation into macrophages and foam cell formation in vitro and in vivo. RNA-seq and microarray analysis of human monocyte and macrophage transcriptomes, including those of a unique QKI haploinsufficient patient, reveal striking changes in QKI-dependent messenger RNA levels and splicing of RNA transcripts. The biological importance of these transcripts and requirement for QKI during differentiation illustrates a central role for QKI in posttranscriptionally guiding macrophage identity and function.

The RNA-binding protein quaking maintains endothelial barrier function and affects VE-cadherin and ß-catenin protein expression.

Proper regulation of endothelial cell-cell contacts is essential for physiological functioning of the endothelium. Interendothelial junctions are actively involved in the control of vascular leakage, leukocyte diapedesis, and the initiation and progression of angiogenesis. We found that the RNA-binding protein quaking is highly expressed by endothelial cells, and that its expression was augmented by prolonged culture under laminar flow and the transcription factor KLF2 binding to the promoter. Moreover, we demonstrated that quaking directly binds to the mRNA of VE-cadherin and ß-catenin and can induce mRNA translation mediated by the 3'UTR of these genes. Reduced quaking levels attenuated VE-cadherin and ß-catenin expression and endothelial barrier function in vitro and resulted in increased bradykinin-induced vascular leakage in vivo. Taken together, we report that quaking is essential in maintaining endothelial barrier function. Our results provide novel insight into the importance of post-transcriptional regulation in controlling vascular integrity.

Hypoxia induces netrin-1 and Unc5b in atherosclerotic plaques: mechanism for macrophage retention and survival.

OBJECTIVE: Hypoxia is intimately linked to atherosclerosis and has become recognized as a primary impetus of inflammation. We recently demonstrated that the neuroimmune guidance cue netrin-1 (Ntn1) inhibits macrophage emigration from atherosclerotic plaques, thereby fostering chronic inflammation. However, the mechanisms governing netrin-1 expression in atherosclerosis are not well understood. In this study, we investigate the role of hypoxia in regulating expression of netrin-1 and its receptor uncoordinated-5-B receptor (Unc5b) in plaque macrophages and its functional consequences on these immune cells. APPROACH AND RESULTS: We show by immunostaining that netrin-1 and Unc5b are expressed in macrophages in hypoxia-rich regions of human and mouse plaques. In vitro, Ntn1 and Unc5b mRNA are upregulated in macrophages treated with oxidized low-density lipoprotein or inducers of oxidative stress (CoCl2, dimethyloxalylglycine, 1% O2). These responses are abrogated by inhibiting hypoxia-inducible transcription factor (HIF)-1α, indicating a causal role for this transcription factor in regulating Ntn1 and Unc5b expression in macrophages. Indeed, using promoter-luciferase reporter genes, we show that Ntn1- and Unc5b-promoter activities are induced by oxidized low-density lipoprotein and require HIF-1α. Correspondingly, J774 macrophages overexpressing active HIF-1α show increased netrin-1 and Unc5b expression and reduced migratory capacity compared with control cells, which was restored by blocking the effects of netrin-1. Finally, we show that netrin-1 protects macrophages from apoptosis under hypoxic conditions in a HIF-1α-dependent manner. CONCLUSIONS: These findings provide a molecular mechanism by which netrin-1 and its receptor Unc5b are expressed in atherosclerotic plaques and implicate hypoxia and HIF-1α-induced netrin-1/Unc5b in sustaining inflammation by inhibiting the emigration and promoting the survival of lesional macrophages.

Endothelial expression of guidance cues in vessel wall homeostasis dysregulation under proatherosclerotic conditions.

OBJECTIVE: Emerging evidence suggests that neuronal guidance cues, typically expressed during development, are involved in both physiological and pathological immune responses. We hypothesized that endothelial expression of such guidance cues may regulate leukocyte trafficking into the vascular wall during atherogenesis. APPROACH AND RESULTS: We demonstrate that members of the netrin, semaphorin, and ephrin family of guidance molecules are differentially regulated under conditions that promote or protect from atherosclerosis. Netrin-1 and semaphorin3A are expressed by coronary artery endothelial cells and potently inhibit chemokine-directed migration of human monocytes. Endothelial expression of these negative guidance cues is downregulated by proatherogenic factors, including oscillatory shear stress and proinflammatory cytokines associated with monocyte entry into the vessel wall. Furthermore, we show using intravital microscopy that inhibition of netrin-1 or semaphorin3A using blocking peptides increases leukocyte adhesion to the endothelium. Unlike netrin-1 and semaphorin3A, the guidance cue ephrinB2 is upregulated under proatherosclerotic flow conditions and functions as a chemoattractant, increasing leukocyte migration in the absence of additional chemokines. CONCLUSIONS: The concurrent regulation of negative and positive guidance cues may facilitate leukocyte infiltration of the endothelium through a balance between chemoattraction and chemorepulsion. These data indicate a previously unappreciated role for axonal guidance cues in maintaining the endothelial barrier and regulating leukocyte trafficking during atherogenesis.

Neuroimmune guidance cue Semaphorin 3E is expressed in atherosclerotic plaques and regulates macrophage retention.

OBJECTIVE: The persistence of myeloid-derived cells in the artery wall is a characteristic of advanced atherosclerotic plaques. However, the mechanisms by which these cells are retained are poorly understood. Semaphorins, a class of neuronal guidance molecules, play a critical role in vascular patterning and development, and recent studies suggest that they may also have immunomodulatory functions. The present study evaluates the expression of Semaphorin 3E (Sema3E) in settings relevant to atherosclerosis and its contribution to macrophage accumulation in plaques. APPROACH AND RESULTS: Immunofluorescence staining of Sema3E, and its receptor PlexinD1, demonstrated their expression in macrophages of advanced atherosclerotic lesions of Apoe(-/-) mice. Notably, in 2 different mouse models of atherosclerosis regression, Sema3E mRNA was highly downregulated in plaque macrophages, coincident with a reduction in plaque macrophage content and an enrichment in markers of reparative M2 macrophages. In vitro, Sema3E mRNA was highly expressed in inflammatory M1 macrophages and in macrophages treated with physiological drivers of plaque progression and inflammation, such as oxidized low-density lipoprotein and hypoxia. To explore mechanistically how Sema3E affects macrophage behavior, we treated macrophages with recombinant protein in the presence/absence of chemokines, including CCL19, a chemokine implicated in the egress of macrophages from atherosclerotic plaques. Sema3E blocked actin polymerization and macrophage migration stimulated by the chemokines, suggesting that it may immobilize these cells in the plaque. CONCLUSIONS: Sema3E is upregulated in macrophages of advanced plaques, is dynamically regulated by multiple atherosclerosis-relevant factors, and acts as a negative regulator of macrophage migration, which may promote macrophage retention and chronic inflammation in vivo.

The neuroimmune guidance cue netrin-1 promotes atherosclerosis by inhibiting the emigration of macrophages from plaques.

Atherosclerotic plaque formation is fueled by the persistence of lipid-laden macrophages in the artery wall. The mechanisms by which these cells become trapped, thereby establishing chronic inflammation, remain unknown. Here we found that netrin-1, a neuroimmune guidance cue, was secreted by macrophages in human and mouse atheroma, where it inactivated the migration of macrophages toward chemokines linked to their egress from plaques. Acting via its receptor, UNC5b, netrin-1 inhibited the migration of macrophages directed by the chemokines CCL2 and CCL19, activation of the actin-remodeling GTPase Rac1 and actin polymerization. Targeted deletion of netrin-1 in macrophages resulted in much less atherosclerosis in mice deficient in the receptor for low-density lipoprotein and promoted the emigration of macrophages from plaques. Thus, netrin-1 promoted atherosclerosis by retaining macrophages in the artery wall. Our results establish a causative role for negative regulators of leukocyte migration in chronic inflammation.

Antagonism of miR-33 in mice promotes reverse cholesterol transport and regression of atherosclerosis.

Plasma HDL levels have a protective role in atherosclerosis, yet clinical therapies to raise HDL levels have remained elusive. Recent advances in the understanding of lipid metabolism have revealed that miR-33, an intronic microRNA located within the SREBF2 gene, suppresses expression of the cholesterol transporter ABC transporter A1 (ABCA1) and lowers HDL levels. Conversely, mechanisms that inhibit miR-33 increase ABCA1 and circulating HDL levels, suggesting that antagonism of miR-33 may be atheroprotective. As the regression of atherosclerosis is clinically desirable, we assessed the impact of miR-33 inhibition in mice deficient for the LDL receptor (Ldlr-/- mice), with established atherosclerotic plaques. Mice treated with anti-miR33 for 4 weeks showed an increase in circulating HDL levels and enhanced reverse cholesterol transport to the plasma, liver, and feces. Consistent with this, anti-miR33-treated mice showed reductions in plaque size and lipid content, increased markers of plaque stability, and decreased inflammatory gene expression. Notably, in addition to raising ABCA1 levels in the liver, anti-miR33 oligonucleotides directly targeted the plaque macrophages, in which they enhanced ABCA1 expression and cholesterol removal. These studies establish that raising HDL levels by anti-miR33 oligonucleotide treatment promotes reverse cholesterol transport and atherosclerosis regression and suggest that it may be a promising strategy to treat atherosclerotic vascular disease.

Endothelial CD81 is a marker of early human atherosclerotic plaques and facilitates monocyte adhesion.

AIMS: In a recent report, we established at the genome-wide level those genes that are specifically upregulated in the endothelium of atherosclerotic plaques in human arteries. As the transcriptome data revealed that mRNA for the tetraspanin family member CD81 is significantly and specifically upregulated in the endothelium overlying early atheroma, we set out to validate these results on the protein level, and investigate the functional consequences of CD81 upregulation. METHODS AND RESULTS: Immunohistochemical analysis in an independent set of donor arteries verified in the endothelium of early human atherosclerotic lesions the enhanced expression of CD81, which appears oxidative stress-dependent. Using lentiviral overexpression and silencing in human umbilical endothelial cells, we established in an in vitro flow adhesion assay that elevated endothelial CD81 is associated with increased monocyte adhesion to non-activated CD81-transduced endothelial cells, approaching the levels normally only attained after tumour necrosis factor alpha stimulation. The CD81 effect was dependent on both intracellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), as it was abolished in the presence of a mixture of anti-ICAM-1 and anti-VCAM-1 antibodies. Flow cytometry revealed that increased CD81 levels did not increase total ICAM-1 and VCAM-1 surface expression. Instead, it concentrated the available adhesion molecules into membrane clusters, as indicated by confocal and electron microscopy. CD81 also colocalized with ICAM-1 and VCAM-1 in the adhesion rings around bound monocytes. CONCLUSION: Endothelial CD81 upregulated in early human atheroma has the potential to play a crucial role in the initial stages of atherosclerotic plaque formation by increasing monocyte adhesion prior to the full-blown inflammatory response.

P-selectin glycoprotein ligand-1 is expressed on endothelial cells and mediates monocyte adhesion to activated endothelium.

OBJECTIVE: The purpose of this study was to investigate the presence and functionality of P-selectin glycoprotein ligand-1 (PSGL-1) on activated endothelial cells (ECs). METHODS AND RESULTS: We show here that PSGL-1 is expressed at the mRNA and protein levels in umbilical vein and microvascular ECs. Furthermore, this endothelial PSGL-1 (ePSGL-1) is functional and mediates adhesion of monocytes or platelet-monocyte complexes (PMCs) to the activated endothelium in a flow model. ePSGL-1 expression was not affected by treating ECs with inflammatory stimuli (tumor necrosis factor alpha, interleukin-1beta, thrombin, or histamine). However, the functional binding capacity of ePSGL-1 to monocytes or P-selectin/Fc chimera significantly increased by stimulation of the ECs with TNFalpha. By means of a siRNA approach to specifically knock-down the genes involved in the glycosylation of PSGL-1 we could show that tumor necrosis factor alpha-induced glycosylation of ePSGL-1 is critical for its binding capacity. CONCLUSION: Our results show that ECs express functional PSGL-1 which mediates tethering and firm adhesion of monocytes and platelets to inflamed endothelium.