Generating human artery and vein cells from pluripotent stem cells highlights the arterial tropism of Nipah and Hendra viruses.

Stem cell research endeavors to generate specific subtypes of classically defined "cell types." Here, we generate >90% pure human artery or vein endothelial cells from pluripotent stem cells within 3-4 days. We specified artery cells by inhibiting vein-specifying signals and vice versa. These cells modeled viral infection of human vasculature by Nipah and Hendra viruses, which are extraordinarily deadly (∼57%-59% fatality rate) and require biosafety-level-4 containment. Generating pure populations of artery and vein cells highlighted that Nipah and Hendra viruses preferentially infected arteries; arteries expressed higher levels of their viral-entry receptor. Virally infected artery cells fused into syncytia containing up to 23 nuclei, which rapidly died. Despite infecting arteries and occupying ∼6%-17% of their transcriptome, Nipah and Hendra largely eluded innate immune detection, minimally eliciting interferon signaling. We thus efficiently generate artery and vein cells, introduce stem-cell-based toolkits for biosafety-level-4 virology, and explore the arterial tropism and cellular effects of Nipah and Hendra viruses.

Atherosclerosis: Recent developments.

Atherosclerosis is an inflammatory disease of the large arteries that is the major cause of cardiovascular disease (CVD) and stroke. Here, we review the current understanding of the molecular, cellular, genetic, and environmental contributions to atherosclerosis, from both individual pathway and systems perspectives. We place an emphasis on recent developments, some of which have yielded unexpected biology, including previously unknown heterogeneity of inflammatory and smooth muscle cells in atherosclerotic lesions, roles for senescence and clonal hematopoiesis, and links to the gut microbiome.

A Cardiovascular Disease-Linked Gut Microbial Metabolite Acts via Adrenergic Receptors.

Using untargeted metabolomics (n = 1,162 subjects), the plasma metabolite (m/z = 265.1188) phenylacetylglutamine (PAGln) was discovered and then shown in an independent cohort (n = 4,000 subjects) to be associated with cardiovascular disease (CVD) and incident major adverse cardiovascular events (myocardial infarction, stroke, or death). A gut microbiota-derived metabolite, PAGln, was shown to enhance platelet activation-related phenotypes and thrombosis potential in whole blood, isolated platelets, and animal models of arterial injury. Functional and genetic engineering studies with human commensals, coupled with microbial colonization of germ-free mice, showed the microbial porA gene facilitates dietary phenylalanine conversion into phenylacetic acid, with subsequent host generation of PAGln and phenylacetylglycine (PAGly) fostering platelet responsiveness and thrombosis potential. Both gain- and loss-of-function studies employing genetic and pharmacological tools reveal PAGln mediates cellular events through G-protein coupled receptors, including α2A, α2B, and ß2-adrenergic receptors. PAGln thus represents a new CVD-promoting gut microbiota-dependent metabolite that signals via adrenergic receptors.

A Watershed Finding for Heart Regeneration.

The adult mammalian heart is minimally regenerative after injury, whereas neonatal hearts fully recover even after major damage. New work from the Red-Horse and Woo labs (Das et al., 2019) shows that collateral artery formation is a key mechanism contributing to successful regeneration in newborn mice and provides insights into how collateral arteries form.

A spatially resolved timeline of the human maternal-fetal interface.

Beginning in the first trimester, fetally derived extravillous trophoblasts (EVTs) invade the uterus and remodel its spiral arteries, transforming them into large, dilated blood vessels. Several mechanisms have been proposed to explain how EVTs coordinate with the maternal decidua to promote a tissue microenvironment conducive to spiral artery remodelling (SAR)1-3. However, it remains a matter of debate regarding which immune and stromal cells participate in these interactions and how this evolves with respect to gestational age. Here we used a multiomics approach, combining the strengths of spatial proteomics and transcriptomics, to construct a spatiotemporal atlas of the human maternal-fetal interface in the first half of pregnancy. We used multiplexed ion beam imaging by time-of-flight and a 37-plex antibody panel to analyse around 500,000 cells and 588 arteries within intact decidua from 66 individuals between 6 and 20 weeks of gestation, integrating this dataset with co-registered transcriptomics profiles. Gestational age substantially influenced the frequency of maternal immune and stromal cells, with tolerogenic subsets expressing CD206, CD163, TIM-3, galectin-9 and IDO-1 becoming increasingly enriched and colocalized at later time points. By contrast, SAR progression preferentially correlated with EVT invasion and was transcriptionally defined by 78 gene ontology pathways exhibiting distinct monotonic and biphasic trends. Last, we developed an integrated model of SAR whereby invasion is accompanied by the upregulation of pro-angiogenic, immunoregulatory EVT programmes that promote interactions with the vascular endothelium while avoiding the activation of maternal immune cells.

Sympathetic and Sensory-Motor Nerves in Peripheral Small Arteries.

Small arteries, which play important roles in controlling blood flow, blood pressure, and capillary pressure, are under nervous influence. Their innervation is predominantly sympathetic and sensory motor in nature, and while some arteries are densely innervated, others are only sparsely so. Innervation of small arteries is a key mechanism in regulating vascular resistance. In the second half of the previous century, the physiology and pharmacology of this innervation were very actively investigated. In the past 10-20 yr, the activity in this field was more limited. With this review we highlight what has been learned during recent years with respect to development of small arteries and their innervation, some aspects of excitation-release coupling, interaction between sympathetic and sensory-motor nerves, cross talk between endothelium and vascular nerves, and some aspects of their role in vascular inflammation and hypertension. We also highlight what remains to be investigated to further increase our understanding of this fundamental aspect of vascular physiology.

Arterial Stiffness: From Basic Primers to Integrative Physiology.

The elastic properties of conductance arteries are one of the most important hemodynamic functions in the body, and data continue to emerge regarding the importance of their dysfunction in vascular aging and a range of cardiovascular diseases. Here, we provide new insight into the integrative physiology of arterial stiffening and its clinical consequence. We also comprehensively review progress made on pathways/molecules that appear today as important basic determinants of arterial stiffness, particularly those mediating the vascular smooth muscle cell (VSMC) contractility, plasticity and stiffness. We focus on membrane and nuclear mechanotransduction, clearance function of the vascular wall, phenotypic switching of VSMCs, immunoinflammatory stimuli and epigenetic mechanisms. Finally, we discuss the most important advances of the latest clinical studies that revisit the classical therapeutic concepts of arterial stiffness and lead to a patient-by-patient strategy according to cardiovascular risk exposure and underlying disease.

Arterialization requires the timely suppression of cell growth.

The formation of arteries is thought to occur by the induction of a highly conserved arterial genetic programme in a subset of vessels that will later experience an increase in oxygenated blood flow1,2. The initial steps of arterial specification require both the VEGF and Notch signalling pathways3-5. Here, we combine inducible genetic mosaics and transcriptomics to modulate and define the function of these signalling pathways in cell proliferation, arteriovenous differentiation and mobilization. We show that endothelial cells with high levels of VEGF or Notch signalling are intrinsically biased to mobilize and form arteries; however, they are not genetically pre-determined, and can also form veins. Mechanistically, we found that increased levels of VEGF and Notch signalling in pre-arterial capillaries suppresses MYC-dependent metabolic and cell-cycle activities, and promotes the incorporation of endothelial cells into arteries. Mosaic lineage-tracing studies showed that endothelial cells that lack the Notch-RBPJ transcriptional activator complex rarely form arteries; however, these cells regained the ability to form arteries when the function of MYC was suppressed. Thus, the development of arteries does not require the direct induction of a Notch-dependent arterial differentiation programme, but instead depends on the timely suppression of endothelial cell-cycle progression and metabolism, a process that precedes arterial mobilization and complete differentiation.

Elastin recoil is driven by the hydrophobic effect.

Elastin is an extracellular matrix material found in all vertebrates. Its reversible elasticity, robustness, and low stiffness are essential for the function of arteries, lungs, and skin. It is among the most resilient elastic materials known: During a human lifetime, arterial elastin undergoes in excess of 2 × 109 stretching/contracting cycles without replacement, and slow oxidative hardening has been identified as a limiting factor on human lifespan. For over 50 y, the mechanism of entropic recoil has been controversial. Herein, we report a combined NMR and thermomechanical study that establishes the hydrophobic effect as the primary driver of elastin function. Water ordering at the solvent:protein interface was observed as a function of stretch using double quantum 2H NMR, and the most extensive thermodynamic analysis performed to date was obtained by measuring elastin length and volume as a function of force and temperature in normal water, heavy water and with cosolvents. When stretched, elastin's heat capacity increases, water is ordered proportional to the degree of stretching, the internal energy decreases, and heat is released in excess of the work performed. These properties show that recoil in elastin under physiological conditions is primarily driven by the hydrophobic effect rather than by configurational entropy as is the case for rubber. Consistent with this conclusion are decreases in the thermodynamic signatures when cosolvents that alter the hydrophobic effect are introduced. We propose that hydrophobic effect-driven recoil, as opposed to a configurational entropy mechanism where hardening from crystallization can occur, is the origin of elastin's unusual resilience.

Insights into hippocampal perfusion using high-resolution, multi-modal 7T MRI.

We present a comprehensive study on the non-invasive measurement of hippocampal perfusion. Using high-resolution 7 tesla arterial spin labeling (ASL) data, we generated robust perfusion maps and observed significant variations in perfusion among hippocampal subfields, with CA1 exhibiting the lowest perfusion levels. Notably, these perfusion differences were robust and already detectable with 50 perfusion-weighted images per subject, acquired in 5 min. To understand the underlying factors, we examined the influence of image quality metrics, various tissue microstructure and morphometric properties, macrovasculature, and cytoarchitecture. We observed higher perfusion in regions located closer to arteries, demonstrating the influence of vascular proximity on hippocampal perfusion. Moreover, ex vivo cytoarchitectonic features based on neuronal density differences appeared to correlate stronger with hippocampal perfusion than morphometric measures like gray matter thickness. These findings emphasize the interplay between microvasculature, macrovasculature, and metabolic demand in shaping hippocampal perfusion. Our study expands the current understanding of hippocampal physiology and its relevance to neurological disorders. By providing in vivo evidence of perfusion differences between hippocampal subfields, our findings have implications for diagnosis and potential therapeutic interventions. In conclusion, our study provides a valuable resource for extensively characterizing hippocampal perfusion.

Building human artery and vein endothelial cells from pluripotent stem cells, and enduring mysteries surrounding arteriovenous development.

Owing to their manifold roles in health and disease, there have been intense efforts to synthetically generate blood vessels in vitro from human pluripotent stem cells (hPSCs). However, there are multiple types of blood vessel, including arteries and veins, which are molecularly and functionally different. How can we specifically generate either arterial or venous endothelial cells (ECs) from hPSCs in vitro? Here, we summarize how arterial or venous ECs arise during embryonic development. VEGF and NOTCH arbitrate the bifurcation of arterial vs. venous ECs in vivo. While manipulating these two signaling pathways biases hPSC differentiation towards arterial and venous identities, efficiently generating these two subtypes of ECs has remained challenging until recently. Numerous questions remain to be fully addressed. What is the complete identity, timing and combination of extracellular signals that specify arterial vs. venous identities? How do these extracellular signals intersect with fluid flow to modulate arteriovenous fate? What is a unified definition for endothelial progenitors or angioblasts, and when do arterial vs. venous potentials segregate? How can we regulate hPSC-derived arterial and venous ECs in vitro, and generate organ-specific ECs? In turn, answers to these questions could avail the production of arterial and venous ECs from hPSCs, accelerating vascular research, tissue engineering, and regenerative medicine.

Transcatheter Arterialization of Deep Veins in Chronic Limb-Threatening Ischemia.

BACKGROUND: Approximately 20% of patients with chronic limb-threatening ischemia have no revascularization options, leading to above-ankle amputation. Transcatheter arterialization of the deep veins is a percutaneous approach that creates an artery-to-vein connection for delivery of oxygenated blood by means of the venous system to the ischemic foot to prevent amputation. METHODS: We conducted a prospective, single-group, multicenter study to evaluate the effect of transcatheter arterialization of the deep veins in patients with nonhealing ulcers and no surgical or endovascular revascularization treatment options. The composite primary end point was amputation-free survival (defined as freedom from above-ankle amputation or death from any cause) at 6 months, as compared with a performance goal of 54%. Secondary end points included limb salvage, wound healing, and technical success of the procedure. RESULTS: We enrolled 105 patients who had chronic limb-threatening ischemia and were of a median age of 70 years (interquartile range, 38 to 89). Of the patients enrolled, 33 (31.4%) were women and 45 (42.8%) were Black, Hispanic, or Latino. Transcatheter arterialization of the deep veins was performed successfully in 104 patients (99.0%). At 6 months, 66.1% of the patients had amputation-free survival. According to Bayesian analysis, the posterior probability that amputation-free survival at 6 months exceeded a performance goal of 54% was 0.993, which exceeded the prespecified threshold of 0.977. Limb salvage (avoidance of above-ankle amputation) was attained in 67 patients (76.0% by Kaplan-Meier analysis). Wounds were completely healed in 16 of 63 patients (25%) and were in the process of healing in 32 of 63 patients (51%). No unanticipated device-related adverse events were reported. CONCLUSIONS: We found that transcatheter arterialization of the deep veins was safe and could be performed successfully in patients with chronic limb-threatening ischemia and no conventional surgical or endovascular revascularization treatment options. (Funded by LimFlow; PROMISE II study ClinicalTrials.gov number, NCT03970538.).

Molecular and Spatial Signatures of Mouse Embryonic Endothelial Cells at Single-Cell Resolution.

BACKGROUND: Mature endothelial cells (ECs) are heterogeneous, with subtypes defined by tissue origin and position within the vascular bed (ie, artery, capillary, vein, and lymphatic). How this heterogeneity is established during the development of the vascular system, especially arteriovenous specification of ECs, remains incompletely characterized. METHODS: We used droplet-based single-cell RNA sequencing and multiplexed error-robust fluorescence in situ hybridization to define EC and EC progenitor subtypes from E9.5, E12.5, and E15.5 mouse embryos. We used trajectory inference to analyze the specification of arterial ECs (aECs) and venous ECs (vECs) from EC progenitors. Network analysis identified candidate transcriptional regulators of arteriovenous differentiation, which we tested by CRISPR (clustered regularly interspaced short palindromic repeats) loss of function in human-induced pluripotent stem cells undergoing directed differentiation to aECs or vECs (human-induced pluripotent stem cell-aECs or human-induced pluripotent stem cell-vECs). RESULTS: From the single-cell transcriptomes of 7682 E9.5 to E15.5 ECs, we identified 19 EC subtypes, including Etv2+Bnip3+ EC progenitors. Spatial transcriptomic analysis of 15 448 ECs provided orthogonal validation of these EC subtypes and established their spatial distribution. Most embryonic ECs were grouped by their vascular-bed types, while ECs from the brain, heart, liver, and lung were grouped by their tissue origins. Arterial (Eln, Dkk2, Vegfc, and Egfl8), venous (Fam174b and Clec14a), and capillary (Kcne3) marker genes were identified. Compared with aECs, embryonic vECs and capillary ECs shared fewer markers than their adult counterparts. Early capillary ECs with venous characteristics functioned as a branch point for differentiation of aEC and vEC lineages. CONCLUSIONS: Our results provide a spatiotemporal map of embryonic EC heterogeneity at single-cell resolution and demonstrate that the diversity of ECs in the embryo arises from both tissue origin and vascular-bed position. Developing aECs and vECs share common venous-featured capillary precursors and are regulated by distinct transcriptional regulatory networks.

Vasodilators mobilize SK3 channels in endothelial cells to produce arterial relaxation.

Endothelial cells (ECs) line the lumen of all blood vessels and regulate functions, including contractility. Physiological stimuli, such as acetylcholine (ACh) and intravascular flow, activate transient receptor potential vanilloid 4 (TRPV4) channels, which stimulate small (SK3)- and intermediate (IK)-conductance Ca2+-activated potassium channels in ECs to produce vasodilation. Whether physiological vasodilators also modulate the surface abundance of these ion channels in ECs to elicit functional responses is unclear. Here, we show that ACh and intravascular flow stimulate rapid anterograde trafficking of an intracellular pool of SK3 channels in ECs of resistance-size arteries, which increases surface SK3 protein more than two-fold. In contrast, ACh and flow do not alter the surface abundance of IK or TRPV4 channels. ACh triggers SK3 channel trafficking by activating TRPV4-mediated Ca2+ influx, which stimulates Rab11A, a Rab GTPase associated with recycling endosomes. Superresolution microscopy data demonstrate that SK3 trafficking specifically increases the size of surface SK3 clusters which overlap with TRPV4 clusters. We also show that Rab11A-dependent trafficking of SK3 channels is an essential contributor to vasodilator-induced SK current activation in ECs and vasorelaxation. In summary, our data demonstrate that vasodilators activate Rab11A, which rapidly delivers an intracellular pool of SK3 channels to the vicinity of surface TRPV4 channels in ECs. This trafficking mechanism increases surface SK3 cluster size, elevates SK3 current density, and produces vasodilation. These data also demonstrate that SK3 and IK channels are differentially regulated by trafficking-dependent and -independent signaling mechanisms in endothelial cells.

Chemokine signaling synchronizes angioblast proliferation and differentiation during pharyngeal arch artery vasculogenesis.

Developmentally, the great vessels of the heart originate from the pharyngeal arch arteries (PAAs). During PAA vasculogenesis, PAA precursors undergo sequential cell fate decisions that are accompanied by proliferative expansion. However, how these two processes are synchronized remains poorly understood. Here, we find that the zebrafish chemokine receptor Cxcr4a is expressed in PAA precursors, and genetic ablation of either cxcr4a or the ligand gene cxcl12b causes PAA stenosis. Cxcr4a is required for the activation of the downstream PI3K/AKT cascade, which promotes not only PAA angioblast proliferation, but also differentiation. AKT has a well-known role in accelerating cell-cycle progression through the activation of cyclin-dependent kinases. Despite this, we demonstrate that AKT phosphorylates Etv2 and Scl, the key regulators of angioblast commitment, on conserved serine residues, thereby protecting them from ubiquitin-mediated proteasomal degradation. Altogether, our study reveals a central role for chemokine signaling in PAA vasculogenesis through orchestrating angioblast proliferation and differentiation.

WASp controls oriented migration of endothelial cells to achieve functional vascular patterning.

Endothelial cell migration and proliferation are essential for the establishment of a hierarchical organization of blood vessels and optimal distribution of blood. However, how these cellular processes are quantitatively coordinated to drive vascular network morphogenesis remains unknown. Here, using the zebrafish vasculature as a model system, we demonstrate that the balanced distribution of endothelial cells, as well as the resulting regularity of vessel calibre, is a result of cell migration from veins towards arteries and cell proliferation in veins. We identify the Wiskott-Aldrich Syndrome protein (WASp) as an important molecular regulator of this process and show that loss of coordinated migration from veins to arteries upon wasb depletion results in aberrant vessel morphology and the formation of persistent arteriovenous shunts. We demonstrate that WASp achieves its function through the coordination of junctional actin assembly and PECAM1 recruitment and provide evidence that this is conserved in humans. Overall, we demonstrate that functional vascular patterning in the zebrafish trunk is established through differential cell migration regulated by junctional actin, and that interruption of differential migration may represent a pathomechanism in vascular malformations.

FOXO1 represses sprouty 2 and sprouty 4 expression to promote arterial specification and vascular remodeling in the mouse yolk sac.

Establishing a functional circulatory system is required for post-implantation development during murine embryogenesis. Previous studies in loss-of-function mouse models showed that FOXO1, a Forkhead family transcription factor, is required for yolk sac (YS) vascular remodeling and survival beyond embryonic day (E) 11. Here, we demonstrate that at E8.25, loss of Foxo1 in Tie2-cre expressing cells resulted in increased sprouty 2 (Spry2) and Spry4 expression, reduced arterial gene expression and reduced Kdr (also known as Vegfr2 and Flk1) transcripts without affecting overall endothelial cell identity, survival or proliferation. Using a Dll4-BAC-nlacZ reporter line, we found that one of the earliest expressed arterial genes, delta like 4, is significantly reduced in Foxo1 mutant YS without being substantially affected in the embryo proper. We show that FOXO1 binds directly to previously identified Spry2 gene regulatory elements (GREs) and newly identified, evolutionarily conserved Spry4 GREs to repress their expression. Furthermore, overexpression of Spry4 in transient transgenic embryos largely recapitulates the reduced expression of arterial genes seen in conditional Foxo1 mutants. Together, these data reveal a novel role for FOXO1 as a key transcriptional repressor regulating both pre-flow arterial specification and subsequent vessel remodeling within the murine YS.

Immunology of Giant Cell Arteritis.

Giant cell arteritis is an autoimmune disease of medium and large arteries, characterized by granulomatous inflammation of the three-layered vessel wall that results in vaso-occlusion, wall dissection, and aneurysm formation. The immunopathogenesis of giant cell arteritis is an accumulative process in which a prolonged asymptomatic period is followed by uncontrolled innate immunity, a breakdown in self-tolerance, the transition of autoimmunity from the periphery into the vessel wall and, eventually, the progressive evolution of vessel wall inflammation. Each of the steps in pathogenesis corresponds to specific immuno-phenotypes that provide mechanistic insights into how the immune system attacks and damages blood vessels. Clinically evident disease begins with inappropriate activation of myeloid cells triggering the release of hepatic acute phase proteins and inducing extravascular manifestations, such as muscle pains and stiffness diagnosed as polymyalgia rheumatica. Loss of self-tolerance in the adaptive immune system is linked to aberrant signaling in the NOTCH pathway, leading to expansion of NOTCH1+CD4+ T cells and the functional decline of NOTCH4+ T regulatory cells (Checkpoint 1). A defect in the endothelial cell barrier of adventitial vasa vasorum networks marks Checkpoint 2; the invasion of monocytes, macrophages and T cells into the arterial wall. Due to the failure of the immuno-inhibitory PD-1 (programmed cell death protein 1)/PD-L1 (programmed cell death ligand 1) pathway, wall-infiltrating immune cells arrive in a permissive tissues microenvironment, where multiple T cell effector lineages thrive, shift toward high glycolytic activity, and support the development of tissue-damaging macrophages, including multinucleated giant cells (Checkpoint 3). Eventually, the vascular lesions are occupied by self-renewing T cells that provide autonomy to the disease process and limit the therapeutic effectiveness of currently used immunosuppressants. The multi-step process deviating protective to pathogenic immunity offers an array of interception points that provide opportunities for the prevention and therapeutic management of this devastating autoimmune disease.

Eradicating Atherosclerotic Events by Targeting Early Subclinical Disease: It Is Time to Retire the Therapeutic Paradigm of Too Much, Too Late.

Recent decades have seen spectacular advances in understanding and managing atherosclerotic cardiovascular disease, but paradoxically, clinical progress has stalled. Residual risk of atherosclerotic cardiovascular disease events is particularly vexing, given recognized lifestyle interventions and powerful modern medications. Why? Atherosclerosis begins early in life, yet clinical trials and mechanistic studies often emphasize terminal, end-stage plaques, meaning on the verge of causing heart attacks and strokes. Thus, current clinical evidence drives us to emphasize aggressive treatments that are delayed until patients already have advanced arterial disease. I call this paradigm "too much, too late." This brief review covers exciting efforts that focus on preventing, or finding and treating, arterial disease before its end-stage. Also included are specific proposals to establish a new evidence base that could justify intensive short-term interventions (induction-phase therapy) to treat subclinical plaques that are early enough perhaps to heal. If we can establish that such plaques are actionable, then broad screening to find them in early midlife individuals would become imperative-and achievable. You have a lump in your coronaries! can motivate patients and clinicians. We must stop thinking of a heart attack as a disease. The real disease is atherosclerosis. In my opinion, an atherosclerotic heart attack is a medical failure. It is a manifestation of longstanding arterial disease that we had allowed to progress to its end-stage, despite knowing that atherosclerosis begins early in life and despite the availability of remarkably safe and highly effective therapies. The field needs a transformational advance to shift the paradigm out of end-stage management and into early interventions that hold the possibility of eradicating the clinical burden of atherosclerotic cardiovascular disease, currently the biggest killer in the world. We urgently need a new evidence base to redirect our main focus from terminal, end-stage atherosclerosis to earlier, and likely reversible, human arterial disease.

SR-B1 drives endothelial cell LDL transcytosis via DOCK4 to promote atherosclerosis.

Atherosclerosis, which underlies life-threatening cardiovascular disorders such as myocardial infarction and stroke1, is initiated by passage of low-density lipoprotein (LDL) cholesterol into the artery wall and its engulfment by macrophages, which leads to foam cell formation and lesion development2,3. It is unclear how circulating LDL enters the artery wall to instigate atherosclerosis. Here we show in mice that scavenger receptor class B type 1 (SR-B1) in endothelial cells mediates the delivery of LDL into arteries and its accumulation by artery wall macrophages, thereby promoting atherosclerosis. LDL particles are colocalized with SR-B1 in endothelial cell intracellular vesicles in vivo, and transcytosis of LDL across endothelial monolayers requires its direct binding to SR-B1 and an eight-amino-acid cytoplasmic domain of the receptor that recruits the guanine nucleotide exchange factor dedicator of cytokinesis 4 (DOCK4)4. DOCK4 promotes internalization of SR-B1 and transport of LDL by coupling the binding of LDL to SR-B1 with activation of RAC1. The expression of SR-B1 and DOCK4 is increased in atherosclerosis-prone regions of the mouse aorta before lesion formation, and in human atherosclerotic arteries when compared with normal arteries. These findings challenge the long-held concept that atherogenesis involves passive movement of LDL across a compromised endothelial barrier. Interventions that inhibit the endothelial delivery of LDL into artery walls may represent a new therapeutic category in the battle against cardiovascular disease.