OCTA quantitative assessment of exercise-induced variations and recovery in retinal microvasculature of healthy subjects with or without masks.

PURPOSE: To investigate the impact of exercise and mask-wearing on retinal microvasculature using optical coherence tomography angiography (OCTA). METHODS: A total of 30 healthy volunteers were enrolled and tasked with physical exercise to reach 75-80 % maximum heart rates. Swept-source OCTA was performed on the macular region and optic nerve head (ONH) in participants with no mask, surgical mask, or N95 mask at quiescent conditions (Step 1) and 0 min, 10 min, 20 min, and 30 min post-exercise (Steps 2-5, respectively). The functional vessel density (VD), including the superficial and deep plex (SP and DP) in the macular area and the superficial plex (SP), nerve fiber plex, and small vessels in the optic nerve head, were measured. RESULTS: Under quiescent conditions, the functional VD of SP and DP exhibited significant reduction with surgical and N95 masks in the foveal area (P < 0.05). In step 2 (immediately after training) with or without masks, functional VD of SP and nerve fiber both showed significant reduction in the inside disc and peripapillary area, small functional VD of nerve fiber in the ONH showed significant reduction in peripapillary area (P < 0.05). These changes had been recovered in Step 5 (30 min post-exercise) in all groups (no-mask, surgical mask and N95 mask groups) (P > 0.05). CONCLUSIONS: Mask-wearing and physical exercise reduce retinal functional VD in macular and ONH areas. The retinal vasoconstriction induced by exercise tends to recover after rest for approximately 30 min. Our research provides insights into mask-wearing and physical exercise's immediate retinal microvasculature effects, hinting at systemic microvascular changes.

Enantioselective Mannich Reaction between Cyclic N-Sulfonyl Ketimines and Isatin-Derived Ketimines.

An enantioselective Mannich reaction with cyclic N-sulfonyl ketimines as the nucleophiles was developed. In the presence of 5 mol % chiral thiourea catalyst C11, the asymmetric Mannich reaction between cyclic N-sulfonyl ketimines and isatin-derived ketimines was achieved in high yields and good-to-excellent enantioselectivities (84-99% yields with 75-99.8% ee). This methodology provided an effective route to construct chiral 3-amino-2-oxindoles containing a cyclic N-sulfonyl ketimine scaffold. The initial biological evaluation of the products in cell-based assays demonstrated that some compounds have excellent antiproliferative activity against human osteosarcoma cells.

Cu-Catalyzed Direct Enantioselective Vinylogous Mannich Reaction between ß,γ-Unsaturated Pyrazoleamides and Ketimines.

A catalytic asymmetric vinylogous Mannich-type reaction between ß,γ-unsaturated amides and ketimines has been developed in excellent regio-, diastereo-, and enantioselectivities. The methodology provides an efficient approach to construct chiral homoallylic amines with a 3-amino-2-oxindole scaffold. Moreover, the transformations of the chiral products, including the removal of the pyrazole group or Boc group, the reduction of the C-C double bond, and Suzuki coupling, have been investigated.

Adjustable Regioselectivity for the Diels-Alder Reactions of Sulfolenodipyrrins upon Molecular Engineering on H-Bonds.

The Diels-Alder reactions of sulfolenodipyrrins prove to be an efficient way to construct aromatic ring-fused dipyrrins. However, adjustable annulation is still hard to achieve. To address this, molecular engineering on the H-bond has been employed. The α-position aryl group-modified sulfolenodipyrrins have been synthesized to react with various dienophiles in Diels-Alder reactions, affording the monoannulation products with different regioselectivity in good yields (45-76%). The remaining sulfolenopyrrole in monoadducts can undergo further fusion in the presence of dienophiles and TEMPO, giving the bisadducts with a lactam subunit in an appropriate yield. According to the crystal structures and theoretical calculations, the intramolecular H-bonds between the α-substituent and the nearby pyrrole confine the conjugation pathway of the dipyrrin core. With respect to the normal NH-sulfolenopyrrole, the imino-type one features low aromaticity, from which SO2 extrusion generates more stable dipyrrin-diene, achieving regioselectivity. In addition, aromatic ring fusion results in red-shifted absorption and emission spectra, and the annulation units regulate the emission intensity. This work shows the versatility of intramolecular H-bonds in regulating the reaction through confinement of the conjugation system.

Pd(II) and Cu(III) Complexes of Multiply Fused Pentaphyrin Isomers with Tunable Structures and NIR Absorption.

Fused porphyrinoids have received increasing interest in light of their extended conjugation and unique coordination behavior. On the basis of our previously reported multiply fused pentaphyrin isomers 1 and 2, a novel isomer 3 has been synthesized in this work. 3 possesses a hexacyclic fused moiety with a nearly coplanar CCNN cavity involving an inverted pyrrole, which is slightly different from the CNNN ones of 1 and 2 involving an N-confused pyrrole. 1-3 possess cavities with three depronatable protons and thus they all can generate Cu(III) complexes. However, only 3Cu is stable under ambient conditions. On the other hand, 3 remains intact upon treatment with Pd(II) ions, while 1 and 2 could undergo structural rearrangement to accommodate Pd(II), affording 1Pd and 2Pd accompanied by the formation of a lactone ring and the addition of a methoxy group, respectively. Compared with the free bases, the complexes show distinct aromaticity and more intense near-infrared (NIR) absorption up to ca. 1600, 1170, and 1500 nm, respectively. The results indicate that the subtle modification of the linking modes between the pyrrolic units in the fused pentaphyrinoids is effective in modulating the coordination behavior for synthesizing complexes with tunable aromaticity and NIR absorption.

Deep Learning Bridged Bioactivity, Structure, and GC-HRMS-Readable Evidence to Decipher Nontarget Toxicants in Sediments.

Identifying causative toxicants in mixtures is critical, but this task is challenging when mixtures contain multiple chemical classes. Effect-based methods are used to complement chemical analyses to identify toxicants, yet conventional bioassays typically rely on an apical and/or single endpoint, providing limited diagnostic potential to guide chemical prioritization. We proposed an event-driven taxonomy framework for mixture risk assessment that relied on high-throughput screening bioassays and toxicant identification integrated by deep learning. In this work, the framework was evaluated using chemical mixtures in sediments eliciting aryl-hydrocarbon receptor activation and oxidative stress response. Mixture prediction using target analysis explained <10% of observed sediment bioactivity. To identify additional contaminants, two deep learning models were developed to predict fingerprints of a pool of bioactive substances (event driver fingerprint, EDFP) and convert these candidates to MS-readable information (event driver ion, EDION) for nontarget analysis. Two libraries with 121 and 118 fingerprints were established, and 247 bioactive compounds were identified at confidence level 2 or 3 in sediment extract using GC-qToF-MS. Among them, 12 toxicants were analytically confirmed using reference standards. Collectively, we present a "bioactivity-signature-toxicant" strategy to deconvolute mixtures and to connect patchy data sets and guide nontarget analysis for diverse chemicals that elicit the same bioactivity.

Ly6C-high monocytes alleviate brain injury in experimental subarachnoid hemorrhage in mice.

BACKGROUND: Subarachnoid hemorrhage (SAH) is an uncommon type of potentially fatal stroke. The pathophysiological mechanisms of brain injury remain unclear, which hinders the development of drugs for SAH. We aimed to investigate the pathophysiological mechanisms of SAH and to elucidate the cellular and molecular biological response to SAH-induced injury. METHODS: A cross-species (human and mouse) multiomics approach combining high-throughput data and bioinformatic analysis was used to explore the key pathophysiological processes and cells involved in SAH-induced brain injury. Patient data were collected from the hospital (n = 712). SAH was established in adult male mice via endovascular perforation, and flow cytometry, a bone marrow chimera model, qPCR, and microglial depletion experiments were conducted to explore the origin and chemotaxis mechanism of the immune cells. To investigate cell effects on SAH prognosis, murine neurological function was evaluated based on a modified Garcia score, pole test, and rotarod test. RESULTS: The bioinformatics analysis confirmed that inflammatory and immune responses were the key pathophysiological processes after SAH. Significant increases in the monocyte levels were observed in both the mouse brains and the peripheral blood of patients after SAH. Ly6C-high monocytes originated in the bone marrow, and the skull bone marrow contribute a higher proportion of these monocytes than neutrophils. The mRNA level of Ccl2 was significantly upregulated after SAH and was greater in CD11b-positive than CD11b-negative cells. Microglial depletion, microglial inhibition, and CCL2 blockade reduced the numbers of Ly6C-high monocytes after SAH. With CCR2 antagonization, the neurological function of the mice exhibited a slow recovery. Three days post-SAH, the monocyte-derived dendritic cell (moDC) population had a higher proportion of TNF-α-positive cells and a lower proportion of IL-10-positive cells than the macrophage population. The ratio of moDCs to macrophages was higher on day 3 than on day 5 post-SAH. CONCLUSIONS: Inflammatory and immune responses are significantly involved in SAH-induced brain injury. Ly6C-high monocytes derived from the bone marrow, including the skull bone marrow, infiltrated into mouse brains via CCL2 secreted from microglia. Moreover, Ly6C-high monocytes alleviated neurological dysfunction after SAH.

Aromatic Ring-Fused Dipyrrins: Programmed [4 + 2]-Cycloaddition Pathway with Regio-selectivity upon Alkylamino-Substitution.

Sulfolenodipyrrins are employed as building blocks to concisely and efficiently construct aromatic rings (e.g., naphthoquinone, anthraquinone, fullerenes, and phthalimide) from fused dipyrrins by programmed [4 + 2]-cycloaddition reactions. Notably, alkylamino-substitution at the α-position not only enhances the reactivity of sulfolenodipyrrins but also results in the regio-selectivity of the cycloaddition reactions. Theoretical calculations in terms of frontier orbitals of dienes, energy of dienes, steric hindrance, and aromaticity have been conducted to understand the reason in depth. Additionally, the fusion of aromatic groups enables bathochromic absorption with up to ∼130 nm for the monoadducts and to ∼200 nm for the bis-adducts. The phthalimide annulation dipyrrin displays red emission, while the other mono- or bis-adducts do not, owing to the presence of typical acceptors such as quinone analogs or fullerene.

[Prediction of quality markers and medicinal value of sea buckthorn leaves based on network pharmacology, content determination, and activity evaluation].

The leaves of sea buckthorn(Hippophae rhamnoides), considered as common food raw materials, have records of medicinal use and diverse pharmacological activities, showing a potential medicinal value. However, the active substances in the sea buckthorn leaves and their mechanisms of action remain unclear. In addition, due to the extensive source and large variety variations, the quality evaluation criteria of sea buckthorn leaves remain to be developed. To solve the problems, this study predicted the main active components, core targets, key pathways, and potential pharmacological effects of sea buckthorn leaves by network pharmacology and molecular docking. Furthermore, ultra-performance liquid chromatography with diode-array detection(UPLC-DAD) was employed to determine the content of active components and establish the chemical fingerprint, on the basis of which the quality markers of sea buckthorn leaves were predicted and then verified by the enzyme activity inhibition method. The results indicated that sea buckthorn leaves had potential therapeutic effects on a variety of digestive tract diseases, metabolic diseases, tumors, and autoimmune diseases, which were consistent with the ancient records and the results of modern pharmacological studies. The core targets of sea buckthorn leaves included PTPN11, AKT1, PIK3R1, ESR1, and SRC, which were mainly involved in the PI3K-AKT, MAPK, and HIF-1 signaling pathways. In conclusion, the active components of sea buckthorn leaves are associated with the rich flavonoids and tannins, among which quercitrin, narcissoside, and ellagic acid can be used as the quality markers of sea buckthorn leaves. The findings provide a reference for the quality control and further development and utilization of sea buckthorn leaves as medicinal materials.

Antiaromatic Sapphyrin Isomer: Transformation into Contracted Porphyrinoids with Variable Aromaticity.

Sapphyrin is a pentapyrrolic expanded porphyrin with a 22π aromatic character. Herein, we report the synthesis of a 20π antiaromatic sapphyrin isomer 1 by oxidative cyclization of a pentapyrrane precursor P5 with a terminal ß-linked pyrrole. The resulting isomer 1, containing a mis-linked bipyrrole unit in the skeleton, exhibits a reactivity for further oxidation due to the distinct antiaromatic electronic structure, affording a fused macrocycle 2, possessing a spiro-carbon-containing [5.6.5.6]-tetracyclic structure. Subsequent treatment with an acid afforded a weakly aromatic pyrrolone-appended N-confused corrole 3, and thermal fusion gave a [5.6.5.7]-tetracyclic-ring-embedded 14π aromatic triphyrin(2.1.1) analog 4. The cyclization at the mis-linked pyrrole moiety of P5 played a crucial role in synthesizing the antiaromatic porphyrinoid susceptible to facile transformation to novel porphyrinoids with variable aromaticity.

High-throughput tandem-microwell assay for ammonia repositions FDA-Approved drugs to inhibit Helicobacter pylori urease.

To date, little attempt has been made to develop new treatments for Helicobacter pylori (H. pylori), although the community is aware of the shortage of treatments for H. pylori. In this study, we developed a 192-tandem-microwell-based high-throughput assay for ammonia that is a known virulence factor of H. pylori and a product of urease. We could identify few drugs, that is, panobinostat, dacinostat, ebselen, captan, and disulfiram, to potently inhibit the activity of ureases from bacterial or plant species. These inhibitors suppress the activity of urease via substrate-competitive or covalent-allosteric mechanism, but all except captan prevent the antibiotic-resistant H. pylori strain from killing human gastric cells, with a more pronounced effect than acetohydroxamic acid, a well-known urease inhibitor and clinically used drug for the treatment of bacterial infection. This study offers several bases for the development of new treatments for urease-containing pathogens and to study the mechanism responsible for the regulation of urease activity.

Grey Wolf Optimizer for Variable Selection in Quantification of Quaternary Edible Blend Oil by Ultraviolet-Visible Spectroscopy.

A novel swarm intelligence algorithm, discretized grey wolf optimizer (GWO), was introduced as a variable selection tool in edible blend oil analysis for the first time. In the approach, positions of wolves were updated and then discretized by logical function. The performance of a wolf pack, the iteration number and the number of wolves were investigated. The partial least squares (PLS) method was used to establish and predict single oil contents in samples. To validate the method, 102 edible blend oil samples containing soybean oil, sunflower oil, peanut oil and sesame oil were measured by an ultraviolet-visible (UV-Vis) spectrophotometer. The results demonstrated that GWO-PLS models can provide best prediction accuracy with least variables compared with full-spectrum PLS, Monte Carlo uninformative variable elimination-PLS (MCUVE-PLS) and randomization test-PLS (RT-PLS). The determination coefficients (R2) of GWO-PLS were all above 0.95. Therefore, the research indicates the feasibility of using discretized GWO for variable selection in rapid determination of quaternary edible blend oil.

Catalytic Desymmetric Dicarbofunctionalization of Unactivated Alkenes.

The construction of multi-stereocenters by a transition metal-catalyzed cross-coupling reaction is a major challenge. The catalytic desymmetric functionalization of unactivated alkenes remains largely unexplored. Herein, we disclose -a desymmetric dicarbofunctionalization of 1,6-dienes via a nickel-catalyzed reductive cross-coupling reaction. The leverage of the underdeveloped chiral 8-Quinox enables the Ni-catalyzed desymmetric carbamoylalkylation of both unactivated mono- and disubstituted alkenes to form pyrrolidinone bearing two nonadjacent stereogenic centers in high enantio- and stereoselectivitives with broad functional-group tolerance. The synthetic application of pyrrolidinones allows the rapid access to complex chiral fused-heterocycles.

Proximity-Dependent Biotinylation to Elucidate the Interactome of TNK2 Nonreceptor Tyrosine Kinase.

Nonreceptor tyrosine kinases (NRTKs) represent an important class of signaling molecules driving diverse cellular pathways. Aberrant expression and hyperphosphorylation of TNK2, an NRTK, have been implicated in multiple cancers. However, the exact proteins and cellular events that mediate phenotypic changes downstream of TNK2 are unclear. Biological systems that employ proximity-dependent biotinylation methods, such as BioID, are being increasingly used to map protein-protein interactions, as they provide increased sensitivity in discovering interaction partners. In this study, we employed stable isotope labeling with amino acids in cell culture and BioID coupled to the biotinylation site identification technology (BioSITe) method that we recently developed to quantitatively explore the interactome of TNK2. By performing a controlled comparative analysis between full-length TNK2 and its truncated counterpart, we were able to not only identify site-level biotinylation of previously well-established TNK2 binders and substrates including NCK1, NCK2, CTTN, and STAT3, but also discover several novel TNK2 interacting partners. We also performed co-immunoprecipitation and immunofluorescence analysis to validate the interaction between TNK2 and CLINT1, a novel TNK2 interacting protein. Overall, this work reveals the power of the BioSITe method coupled to BioID and highlights several molecules that warrant further exploration to assess their functional significance in TNK2-mediated signaling.

Mutation-Specific and Common Phosphotyrosine Signatures of KRAS G12D and G13D Alleles.

KRAS is one of the most frequently mutated genes across all cancer subtypes. Two of the most frequent oncogenic KRAS mutations observed in patients result in glycine to aspartic acid substitution at either codon 12 (G12D) or 13 (G13D). Although the biochemical differences between these two predominant mutations are not fully understood, distinct clinical features of the resulting tumors suggest involvement of disparate signaling mechanisms. When we compared the global phosphotyrosine proteomic profiles of isogenic colorectal cancer cell lines bearing either G12D or G13D KRAS mutation, we observed both shared as well as unique signaling events induced by the two KRAS mutations. Remarkably, while the G12D mutation led to an increase in membrane proximal and adherens junction signaling, the G13D mutation led to activation of signaling molecules such as nonreceptor tyrosine kinases, MAPK kinases, and regulators of metabolic processes. The importance of one of the cell surface molecules, MPZL1, which was found to be hyperphosphorylated in G12D cells, was confirmed by cellular assays as its knockdown led to a decrease in proliferation of G12D but not G13D expressing cells. Overall, our study reveals important signaling differences across two common KRAS mutations and highlights the utility of our approach to systematically dissect subtle differences between related oncogenic mutants and potentially lead to individualized treatments.

Inhibition of P2X4R attenuates white matter injury in mice after intracerebral hemorrhage by regulating microglial phenotypes.

BACKGROUND: White matter injury (WMI) is a major neuropathological event associated with intracerebral hemorrhage (ICH). P2X purinoreceptor 4 (P2X4R) is a member of the P2X purine receptor family, which plays a crucial role in regulating WMI and neuroinflammation in central nervous system (CNS) diseases. Our study investigated the role of P2X4R in the WMI and the inflammatory response in mice, as well as the possible mechanism of action after ICH. METHODS: ICH was induced in mice via collagenase injection. Mice were treated with 5-BDBD and ANA-12 to inhibit P2X4R and tropomyosin-related kinase receptor B (TrkB), respectively. Immunostaining and quantitative polymerase chain reaction (qPCR) were performed to detect microglial phenotypes after the inhibition of P2X4R. Western blots (WB) and immunostaining were used to examine WMI and the underlying molecular mechanisms. Cylinder, corner turn, wire hanging, and forelimb placement tests were conducted to evaluate neurobehavioral function. RESULTS: After ICH, the protein levels of P2X4R were upregulated, especially on day 7 after ICH, and were mainly located in the microglia. The inhibition of P2X4R via 5-BDBD promoted neurofunctional recovery after ICH as well as the transformation of the pro-inflammatory microglia induced by ICH into an anti-inflammatory phenotype, and attenuated ICH-induced WMI. Furthermore, we found that TrkB blockage can reverse the protective effects of WMI as well as neuroprotection after 5-BDBD treatment. This result indicates that P2X4R plays a crucial role in regulating WMI and neuroinflammation and that P2X4R inhibition may benefit patients with ICH. CONCLUSIONS: Our results demonstrated that P2X4R contributes to WMI by polarizing microglia into a pro-inflammatory phenotype after ICH. Furthermore, the inhibition of P2X4R promoted pro-inflammatory microglia polarization into an anti-inflammatory phenotype, enhanced brain-derived neurotrophic factor (BDNF) production, and through the BDNF/TrkB pathway, attenuated WMI and improved neurological function. Therefore, the regulation of P2X4R activation may be beneficial for the reducing of ICH-induced brain injury.

AXL kinase-mediated astrocytic phagocytosis modulates outcomes of traumatic brain injury.

BACKGROUND: Complex changes in the brain microenvironment following traumatic brain injury (TBI) can cause neurological impairments for which there are few efficacious therapeutic interventions. The reactivity of astrocytes is one of the keys to microenvironmental changes, such as neuroinflammation, but its role and the molecular mechanisms that underpin it remain unclear. METHODS: Male C57BL/6J mice were subjected to the controlled cortical impact (CCI) to develop a TBI model. The specific ligand of AXL receptor tyrosine kinase (AXL), recombinant mouse growth arrest-specific 6 (rmGas6) was intracerebroventricularly administered, and selective AXL antagonist R428 was intraperitoneally applied at 30 min post-modeling separately. Post-TBI assessments included neurobehavioral assessments, transmission electron microscopy, immunohistochemistry, and western blotting. Real-time polymerase chain reaction (RT-PCR), siRNA transfection, and flow cytometry were performed for mechanism assessments in primary cultured astrocytes. RESULTS: AXL is upregulated mainly in astrocytes after TBI and promotes astrocytes switching to a phenotype that exhibits the capability of ingesting degenerated neurons or debris. As a result, this astrocytic transformation promotes the limitation of neuroinflammation and recovery of neurological dysfunction. Pharmacological inhibition of AXL in astrocytes significantly decreased astrocytic phagocytosis both in vivo and in primary astrocyte cultures, in contrast to the effect of treatment with the rmGas6. AXL activates the signal transducer and activator of the transcription 1 (STAT1) pathway thereby further upregulating ATP-binding cassette transporter 1 (ABCA1). Moreover, the supernatant from GAS6-depleted BV2 cells induced limited enhancement of astrocytic phagocytosis in vitro. CONCLUSION: Our work establishes the role of AXL in the transformation of astrocytes to a phagocytic phenotype via the AXL/STAT1/ABCA1 pathway which contributes to the separation of healthy brain tissue from injury-induced cell debris, further ameliorating neuroinflammation and neurological impairments after TBI. Collectively, our findings provide a potential therapeutic target for TBI.

Design of a Functional Chromene-Type Kobayashi Precursor: Gram-Scale Total Synthesis of Natural Xanthones by Highly Regioselective Aryne Annulation.

The 2,2-dimethyl-2H-chromene motif is widely found in many bioactive molecules, and is a privileged structure in the pharmaceutical arena. We have developed a concise and regioselective approach to chromenes and chromanes through an aryne-based synthetic strategy. A practical, gram-scale synthetic route to a chromene-type aryne precursor was explored. Subsequently, cyclization under mild conditions afforded tetracyclic xanthone skeletons with excellent regioselectivity. Our approach provides a concise strategy for the gram-scale synthesis of chromene-type xanthones such as 6-deoxyisojacareubin, cylindroxanthone D, staudtiixanthone D, brasilixanthone A and cudracuspixanthone O.

Concise Synthesis of Chromene/Chromane-Type Aryne Precursors and Their Applications.

The gram-scale synthesis of 5,6-, 6,7-, and 7,8-chromene/chromane-type aryne precursors and their applications in regioselective transformation to other functional derivatives is reported. Chromene/chromane-type arynes are generated under mild conditions, which can further undergo [2 + 2], [3 + 2], and [4 + 2] cycloaddition reactions, nucleophilic addition reactions, and σ-insertion reactions to produce structurally novel substituted chromenes and chromanes. The excellent regioselectivity of the reaction is facilitated by the oxygen-containing guiding groups at the ortho-position of the triple bond, which can be removed or switched to other functional groups including alkenyl, aryl, heteroaryl, and arylamino groups.

Endothelial Scaffolding Protein ENH (Enigma Homolog Protein) Promotes PHLPP2 (Pleckstrin Homology Domain and Leucine-Rich Repeat Protein Phosphatase 2)-Mediated Dephosphorylation of AKT1 and eNOS (Endothelial NO Synthase) Promoting Vascular Remodeling.

OBJECTIVE: A decrease in nitric oxide, leading to vascular smooth muscle cell proliferation, is a common pathological feature of vascular proliferative diseases. Nitric oxide synthesis by eNOS (endothelial nitric oxide synthase) is precisely regulated by protein kinases including AKT1. ENH (enigma homolog protein) is a scaffolding protein for multiple protein kinases, but whether it regulates eNOS activation and vascular remodeling remains unknown. Approach and Results: ENH was upregulated in injured mouse arteries and human atherosclerotic plaques and was associated with coronary artery disease. Neointima formation in carotid arteries, induced by ligation or wire injury, was greatly decreased in endothelium-specific ENH-knockout mice. Vascular ligation reduced AKT and eNOS phosphorylation and nitric oxide production in the endothelium of control but not ENH-knockout mice. ENH was found to interact with AKT1 and its phosphatase PHLPP2 (pleckstrin homology domain and leucine-rich repeat protein phosphatase 2). AKT and eNOS activation were prolonged in VEGF (vascular endothelial growth factor)-induced ENH- or PHLPP2-deficient endothelial cells. Inhibitors of either AKT or eNOS effectively restored ligation-induced neointima formation in ENH-knockout mice. Moreover, endothelium-specific PHLPP2-knockout mice displayed reduced ligation-induced neointima formation. Finally, PHLPP2 was increased in the endothelia of human atherosclerotic plaques and blood cells from patients with coronary artery disease. CONCLUSIONS: ENH forms a complex with AKT1 and its phosphatase PHLPP2 to negatively regulate AKT1 activation in the artery endothelium. AKT1 deactivation, a decrease in nitric oxide generation, and subsequent neointima formation induced by vascular injury are mediated by ENH and PHLPP2. ENH and PHLPP2 are thus new proatherosclerotic factors that could be therapeutically targeted.