Oxidation of arachidonic acid generates biologically active derivatives that play a critical role in shaping the inflammatory response. One such derivative, carboxypropylpyrrole (CPP), is a stable end product of arachidonic acid oxidation that has been previously described and detected in inflamed tissues; however, its pathophysiological contribution to inflammation has not been investigated. CPP forms covalent adducts with the extracellular matrix and plasma proteins, altering their biochemical properties and receptor-binding characteristics. Here, we examined how CPP formation impacts neutrophil migration during acute inflammation. Using cell adhesion assays and biolayer interferometry, we identified integrins αMβ2 and αDβ2 as receptors for CPP-modified proteins. Among these, αMβ2 emerged as the primary binding partner on neutrophils, reflecting the low expression of αDβ2 in this cell subset. CPP binding to αMβ2 was mediated by the αM I-domain, required the integrin's high-affinity conformation, and partially overlapped with the classical fibrinogen-binding site. Human and mouse neutrophil swarming assays toward Candida albicans revealed that CPP-modified proteins behave as a unique substrate, unlike classical extracellular matrix protein laminin or inflammatory ligand fibrinogen. While fibrinogen and laminin support cell adhesion and robust migration, CPP-modified proteins promote strong neutrophil adhesion, but inhibit cell migration. CPP deposition within migratory substrates further reduced neutrophil migration in a concentration-dependent manner. Neutrophils isolated from αM-deficient mice confirmed the essential role of αMβ2 in these CPP-mediated effects. These findings suggest that arachidonic acid oxidation products, through selective interaction with αMβ2, regulate neutrophil adhesion and migration and may act as endogenous modulators of the inflammatory response.

High-fat diets are linked to obesity and skin and hair disorders, yet the role of specific lipid classes remains unclear. In this study, we show that cutaneous homeostasis is driven primarily by fatty acid (FA) composition rather than total fat content and is differentially regulated by the gut microbiota. Using omega-3 or omega-6/saturated FA-enriched diets under specific pathogen-free and germ-free conditions, we demonstrate that omega-6/saturated FA feeding promotes weight gain, dermal white adipose tissue expansion, sebaceous gland enlargement, and increased hair follicle proliferation. Systemic and adipose effects occur independently of the microbiota, whereas epithelial and follicular proliferation requires microbial presence. In contrast, omega-3 feeding limits adiposity and dermal white adipose tissue expansion regardless of microbial status but differentially regulates skin compartments in a microbiota-dependent manner. These effects are associated with selective microbiota-dependent regulation of circulating FAs: omega-6 species for omega-6/saturated FA feeding and omega-3 species for an omega-3 diet. Notably, circulating docosahexaenoic acid (22:6, ω-3) levels differ between specific pathogen-free and germ-free conditions in both diet groups, indicating microbiota-mediated control of systemic docosahexaenoic acid bioavailability. Together, these findings identify the gut microbiome as a selective regulator of FA metabolism and define a diet-microbiota-lipid axis that integrates systemic lipid availability with cutaneous homeostasis.

CD36 is a multifunctional receptor widely expressed in immune and nonimmune cells, known for its role in lipid transport and inflammatory signaling. Oxidized phospholipids (oxPLs), a class of prominent lipid oxidation products generated under oxidative stress, bind CD36 with high affinity, contributing to the development of atherogenesis and thrombosis and potentially influencing other CD36-dependent biological events. The molecular basis for the oxPL-CD36 interaction is poorly understood. Here, we used cutting-edge enrichment-mass spectrometry to identify lysine residues of CD36 that directly interact with oxPLs. These residues are located along a putative ligand translocation path─spanning from the apex of the extracellular domain to the entrance, interior, and around the exit of the lipid transport tunnel. Molecular docking revealed two sets of oxPL binding poses: one within a tunnel and the other on a surface loop cluster spanning the top to midsection, including the tallest loop containing oxPL-modified K398/K403. These findings support the selective oxPL binding observed in the LC-MS/MS analysis. Molecular dynamics (MD) simulation demonstrated that the sn-1 chain and headgroup of oxPLs engage distinct CD36 residues through hydrophobic, hydrogen-bonding, and ionic interactions, optimally positioning the reactive sn-2 group for lysine modification. MD and metadynamics simulations further demonstrated oxPL translocation through the tunnel, beginning with sn-1 chain insertion, followed by reorientation at the tunnel midsection, where the sn-2 chain and sn-3 headgroup lead the molecule toward the exit. Together, these studies indicate that CD36 may serve as a transporter of individual oxPL molecules into the cell and outline a translocation pathway, key residues and binding forces involved.

AIMS:In the context of atherosclerosis, macrophages exposed to oxidized low-density lipoproteins (oxLDLs) exhibit cellular abnormalities, specifically in adhesome functions, yet the mechanisms and implications of these adhesive dysfunctions remain largely unexplored. METHODS AND RESULTS:This study reveals a significant depletion of Kindlin3 (K3) or Fermt3, an essential component of the adhesome regulating integrin functions, in macrophages located within atherosclerotic plaques in vivo and following oxLDL exposure in vitro. To examine the effects of K3 deficiency, the study utilized hyperlipidaemic bone marrow chimeras devoid of myeloid Kindlin3 expression. The absence of myeloid K3 increased atherosclerotic plaque burden in the aortas in vivo and enhanced lipid accumulation and lipoprotein uptake in macrophages from Kindlin3-null chimeric mice in vitro. Importantly, re-expression of K3 in macrophages ameliorated these abnormalities. RNA sequencing of bone marrow-derived macrophages (BMDM) from K3-deficient mice revealed extensive deregulation in adhesion-related pathways, echoing changes observed in wild-type cells treated with oxLDL. Notably, there was an increase in Olr1 expression [encoding the lectin-like oxidized LDL receptor-1 (LOX1)], a gene implicated in atherogenesis. The disrupted K3-integrin axis in macrophages led to a significant elevation in the LOX1 receptor, contributing to increased oxLDL uptake and foam cell formation. Inhibition of LOX1 normalized lipid uptake in Kindlin3-null macrophages. A similar proatherogenic phenotype, marked by increased macrophage LOX1 expression and foam cell formation, was observed in myeloid-specific Itgβ1-deficient mice but not in Itgβ2-deficient mice, underscoring the critical role of K3/Itgβ1 interaction. CONCLUSION:This study shows that the loss of Kindlin3 in macrophages upon exposure to oxLDL leads to adhesome dysfunction in atherosclerosis and reveals the pivotal role of Kindlin3 in macrophage function and its contribution to the progression of atherosclerosis, providing valuable insights into the molecular mechanisms that could be targeted for therapeutic interventions.

The etiology of hair loss remains enigmatic, and current remedies remain inadequate. Transcriptome analysis of aging hair follicles uncovered changes in immune pathways, including Toll-like receptors (TLRs). Our findings demonstrate that the maintenance of hair follicle homeostasis and the regeneration capacity after damage depend on TLR2 in hair follicle stem cells (HFSCs). In healthy hair follicles, TLR2 is expressed in a cycle-dependent manner and governs HFSCs activation by countering inhibitory BMP signaling. Hair follicles in aging and obesity exhibit a decrease in both TLR2 and its endogenous ligand carboxyethylpyrrole (CEP), a metabolite of polyunsaturated fatty acids. Administration of CEP stimulates hair regeneration through a TLR2-dependent mechanism. These results establish a novel connection between TLR2-mediated innate immunity and HFSC activation, which is pivotal to hair follicle health and the prevention of hair loss and provide new avenues for therapeutic intervention.

BACKGROUND: Biological sex differences play a vital role in cardiovascular diseases, including atherosclerosis. The endothelium is a critical contributor to cardiovascular pathologies since endothelial cells (ECs) regulate vascular tone, redox balance, and inflammatory reactions. Although EC activation and dysfunction play an essential role in the early and late stages of atherosclerosis development, little is known about sex-dependent differences in EC. METHODS: We used human and mouse aortic EC as well as EC-lineage tracing ( Cdh5 -CreERT2 Rosa-YFP [yellow fluorescence protein]) atherosclerotic Apoe –/– mice to investigate the biological sexual dimorphism of the EC functions in vitro and in vivo. Bioinformatics analyses were performed on male and female mouse aortic EC and human lung and aortic EC. RESULTS: In vitro, female human and mouse aortic ECs showed more apoptosis and higher cellular reactive oxygen species levels than male EC. In addition, female mouse aortic EC had lower mitochondrial membrane potential (ΔΨm), lower TFAM (mitochondrial transcription factor A) levels, and decreased angiogenic potential (tube formation, cell viability, and proliferation) compared with male mouse aortic EC. In vivo, female mice had significantly higher lipid accumulation within the aortas, impaired glucose tolerance, and lower endothelial-mediated vasorelaxation than males. Using the EC-lineage tracing approach, we found that female lesions had significantly lower rates of intraplaque neovascularization and endothelial-to-mesenchymal transition within advanced atherosclerotic lesions but higher incidents of missing EC lumen coverage and higher levels of oxidative products and apoptosis. RNA-seq analyses revealed that both mouse and human female EC had higher expression of genes associated with inflammation and apoptosis and lower expression of genes related to angiogenesis and oxidative phosphorylation than male EC. CONCLUSIONS: Our study delineates critical sex-specific differences in EC relevant to proinflammatory, pro-oxidant, and angiogenic characteristics, which are entirely consistent with a vulnerable phenotype in females. Our results provide a biological basis for sex-specific proatherosclerotic mechanisms.

Akt3 is one of the three members of the serine/threonine cellular processes. We have previously demonstrated that global knockout of Akt3 in mice promotes atherogenesis in a macrophage-dependent manner. Whether enhanced Akt3 kinase activity affects atherogenesis is not known. In this study, we crossed atherosclerosis-prone ApoE-/- mice with a mouse strain that has enhanced Akt3 kinase activity (Akt3nmf350) and assessed atherosclerotic lesion formation and the role of macrophages in atherogenesis. Significant reduction in atherosclerotic lesion area and macrophage accumulation in lesions were observed in ApoE-/-/Akt3nmf350 mice fed a Western-type diet. Experiments using chimeric ApoE-/- mice with either ApoE-/-/Akt3nmf350 bone marrow or ApoE-/- bone marrow cells showed that enhanced Akt3 activity specifically in bone marrow-derived cells is atheroprotective. The atheroprotective effect of Akt3nmf350 was more pronounced in male mice. In line with this result, the release of the pro-inflammatory cytokines IL-6, MCP1, TNF-alpha, and MIP-1 alpha was reduced by macrophages IL-6 and TNF-alpha were also reduced in atherosclerotic lesions of mice in a gender-dependent manner.

Background: Thrombosis is one of the main complications in cancer patients often leading to mortality. However, the mechanisms underlying platelet hyperactivation are poorly understood. Methods: Murine and human platelets were isolated and treated with small extracellular vesicles (sEVs) from various cancer cell lines. The effects of these cancer-sEVs on platelets were evaluated both in vitro and in vivo using various approaches, including the detection of cancer-sEV-specific markers in murine platelets and patient samples, measurement of platelet activation and thrombosis assays. Signaling events induced by cancer-sEVs and leading to platelet activation were identified, and the use of blocking antibodies to prevent thrombosis was demonstrated. Results: We demonstrate that platelets very effectively take up sEVs from aggressive cancer cells. The process of uptake is fast, proceeds effectively in circulation in mice, and is mediated by the abundant sEV membrane protein-CD63. The uptake of cancer-sEVs leads to the accumulation of cancer cell-specific RNA in platelets in vitro and in vivo. The human prostate cancer-sEV-specific RNA marker PCA3 is detected in platelets of ~70% of prostate cancer patients. This was markedly reduced after prostatectomy. In vitro studies showed that platelet uptake of cancer-sEVs induces strong platelet activation in a CD63-RPTPα (receptor-like protein tyrosine phosphatase alpha)-dependent manner. In contrast to physiological agonists ADP and thrombin, cancer-sEVs activate platelets via a noncanonical mechanism. Intravital studies demonstrated accelerated thrombosis both in murine tumor models and in mice that received intravenous injections of cancer-sEVs. The prothrombotic effects of cancer-sEVs were rescued by blocking CD63. Conclusions: Tumors communicate with platelets by means of sEVs, which deliver cancer markers and activate platelets in a CD63-dependent manner leading to thrombosis. This emphasizes the diagnostic and prognostic value of platelet-associated cancer markers and identifies new pathways for intervention.

While platelets are the essential mediators of hemostasis, they are being increasingly recognized for their potential of contributing to host defenses. Here, using immunofluorescent microscopy, western blot, and ELISA, we found that human β-defensin 3 (hBD-3), an important antimicrobial peptide produced by epithelial cells, can be detected in human platelets and megakaryocytes. Flow cytometry and immuno-electron microscopy revealed hBD-3 on the surface of thrombin activated platelets. Moreover, hBD-3 was also found in platelet derived extracellular vesicles (p-EVs), isolated from platelet poor plasma and from platelet supernatants following thrombin stimulation. Incubation of platelets with hBD-3 peptide resulted in modest platelet activation and pre-incubation of platelets with synthetic hBD-3 prior to exposure to thrombin appeared to increase hBD-3 content in platelet lysates as well as in p-EVs, suggesting that hBD-3 can be initially taken up by platelets, perhaps via their open canalicular system. Interestingly, in vitro exposure of primary human endothelial cells to either hBD-3 peptide or purified p-EVs, caused significant endothelial dysfunction as documented by diminished levels of phosphorylated endothelial nitric oxide synthase (eNOS), Krüppel like factor-2 (KLF-2), and elevated relative expression of von Willebrand Factor (vWF). Pre-incubation of platelets with hBD-3 appeared to augment endothelial dysfunction caused by p-EVs. Overall, the current study provides evidence that hBD-3 enriched EVs can be released by activated platelets and may play a role in positive feedback of platelet activation as well as in endothelial dysfunction. Theoretically, these effects could contribute to both cellular recruitment to the endothelium creating a pro-thrombotic vascular microenvironment which serve as a bridge between innate immunity and hemostasis.

Oxidation of polyunsaturated fatty acids contributes to different aspects of the inflammatory response due to the variety of products generated. Specifically, the oxidation of DHA produces the end-product, carboxyethylpyrrole (CEP), which forms a covalent adduct with proteins via an ϵ-amino group of lysines. Previously, we found that CEP formation is dramatically increased in inflamed tissue and CEP-modified albumin and fibrinogen became ligands for αDβ2 (CD11d/CD18) and αMβ2 (CD11b/CD18) integrins. In this study, we evaluated the effect of extracellular matrix (ECM) modification with CEP on the adhesive properties of M1-polarized macrophages, particularly during chronic inflammation. Using digested atherosclerotic lesions and in vitro oxidation assays, we demonstrated the ability of ECM proteins to form adducts with CEP, particularly, DHA oxidation leads to the formation of CEP adducts with collagen IV and laminin, but not with collagen I. Using integrin αDβ2-transfected HEK293 cells, WT and αD−/− mouse M1-polarized macrophages, we revealed that CEP-modified proteins support stronger cell adhesion and spreading when compared with natural ECM ligands such as collagen IV, laminin, and fibrinogen. Integrin αDβ2 is critical for M1 macrophage adhesion to CEP. Based on biolayer interferometry results, the isolated αD I-domain demonstrates markedly higher binding affinity to CEP compared to the “natural” αDβ2 ligand fibrinogen. Finally, the presence of CEP-modified proteins in a 3D fibrin matrix significantly increased M1 macrophage retention. Therefore, CEP modification converts ECM proteins to αDβ2-recognition ligands by changing a positively charged lysine to negatively charged CEP, which increases M1 macrophage adhesion to ECM and promotes macrophage retention during detrimental inflammation, autoimmunity, and chronic inflammation.

OBJECTIVE:It is unclear why adhesion-dependent cells such as epithelium undergo anoikis without anchorage, while adhesion-independent blood cells thrive in suspension. The adhesive machinery of these cells is similar, with the exception of Kindlin orthologs, Kindlin 2 (K2) and Kindlin 3 (K3). Here we address how Kindlins control cell survival and proliferation in anchorage-dependent and independent cells. MATERIAL AND METHODS:To demonstrate the opposite roles of Kindlin's in cell survival we utilized in vivo and in vitro models and K3 and K2 knockdown and knockin cells. We used human lymphocytes from the K3 deficient patients in tumour model, K3 knockout and knockin macrophages and K2 knockout and knockin MEF cells for experiments in under conditions of adhesion and in suspension. RESULTS:Depletion of K3 promotes cell proliferation and survival of anchorage-independent cells regardless of cell attachment. In contrast, the absence of K2 in anchorage-dependent cells accelerates apoptosis and limits proliferation. K3 deficiency promotes human lymphoma growth and survival in vivo. Kindlins' interaction with paxillin, is critical for their differential roles in cell anchorage. While disruption of K2-paxillin binding leads to increased apoptosis, the lack of K3-paxillin binding has an opposite effect in adhesion-independent cells. CONCLUSION:Kindlin ortologs and their interaction to cytoskeletal protein paxillin define the mechanisms of anchorage dependence. Our study identifies the key elements of the cell adhesion machinery in cell survival and tumour metastasis, proposing possible targets for tumour treatment.

As a part of innate immunity, toll-like receptor 2 (TLR2) plays an important function in most defensive responses of the organism, including but not limited to infections. Cutaneous injury, one of the most common challenges for mammals, mobilizes a number of cell types, including epithelial, immune, and vascular cells, for timely tissue repair. However, in contrast to immune cells, little is known about TLR2 function on nonimmune cells during skin regeneration. In this study, we used two tissue-specific conditional Tlr2-knockout mouse lines to address the effects of TLR2 in endothelial and hair follicle stem cells (HFSCs) on cutaneous wound healing. The loss of TLR2 on endothelial cells diminishes their ability to migrate, sprout, and proliferate in response to specific TLR2 ligands and also reduces the secretion of key proangiogenic factors. Lack of TLR2 on endothelial cells prolongs wound healing owing to diminished angiogenesis. TLR2 is expressed in key structures of hair follicles, including HFSCs, secondary hair germ, and dermal papilla. Despite the prominent role of HFSCs in skin regeneration, excision of TLR2 from HFSCs has no effects on their proliferation or wound healing potential. Our study shows that timely tissue regeneration after skin injury is dependent on endothelial TLR2 for robust angiogenesis, whereas HFSC TLR2 is dispensable.

Aims Until recently, the pluripotency factor Octamer (ATGCAAAT)-binding transcriptional factor 4 (OCT4) was believed to be dispensable in adult somatic cells. However, our recent studies provided clear evidence that OCT4 has a critical atheroprotective role in smooth muscle cells. Here, we asked if OCT4 might play a functional role in regulating endothelial cell (EC) phenotypic modulations in atherosclerosis. Methods and results Specifically, we show that EC-specific Oct4 knockout resulted in increased lipid, LGALS3(+) cell accumulation, and altered plaque characteristics consistent with decreased plaque stability. A combination of single-cell RNA sequencing and EC-lineage-tracing studies revealed increased EC activation, endothelial-to-mesenchymal transitions, plaque neovascularization, and mitochondrial dysfunction in the absence of OCT4. Furthermore, we show that the adenosine triphosphate (ATP) transporter, ATP-binding cassette (ABC) transporter G2 (ABCG2), is a direct target of OCT4 in EC and establish for the first time that the OCT4/ABCG2 axis maintains EC metabolic homeostasis by regulating intracellular heme accumulation and related reactive oxygen species production, which, in turn, contributes to atherogenesis. Conclusions These results provide the first direct evidence that OCT4 has a protective metabolic function in EC and identifies vascular OCT4 and its signalling axis as a potential target for novel therapeutics.

CD36 is a multifunctional transmembrane glycoprotein abundantly expressed in several cell types. Recent studies have identified CD36 in circulation (cCD36) in several chronic inflammatory diseases, including type 2 diabetes and chronic kidney disease, and proposed cCD36 to be a biomarker of disease activity. Whether cCD36 is present in hyperlipidemia, a condition characterized by oxidative stress and low-grade inflammation, is not known. In addition, the cellular origin of cCD36 and triggers of CD36 release have not been elucidated. We now demonstrate that plasma cCD36 level is increased in hyperlipidemic ApoE-/- and Ldlr-/- mice. Using several cell-specific CD36 knockout mice, we showed that multiple cell types contribute to cCD36 generation in hyperlipidemic conditions, with a particularly strong contribution from endothelial cells. In vitro studies have demonstrated that oxidized phospholipids, ligands for CD36 (oxPCCD36), which are known to accumulate in circulation in hyperlipidemia, induce a robust release of CD36 from several cell types. In vivo studies have demonstrated CD36 release into the circulation of WT mice in response to tail-vein injection of oxPCCD36. These findings document the presence of cCD36 in hyperlipidemia and identify a link between cCD36 and oxidized phospholipids generated under oxidative stress and low-grade inflammation associated with hyperlipidemia.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease, with poor prognosis and no cure. Substantial evidence implicates inflammation and associated oxidative stress as a potential mechanism for ALS, especially in patients carrying the SOD1 mutation and, therefore, lacking anti-oxidant defense. The brain is particularly vulnerable to oxidation due to the abundance of polyunsaturated fatty acids, such as docosahexaenoic acid (DHA), which can give rise to several oxidized metabolites. Accumulation of a DHA peroxidation product, CarboxyEthylPyrrole (CEP) is dependent on activated inflammatory cells and myeloperoxidase (MPO), and thus marks areas of inflammation-associated oxidative stress. At the same time, generation of an alternative inactive DHA peroxidation product, ethylpyrrole, does not require cell activation and MPO activity. While absent in normal brain tissues, CEP is accumulated in the central nervous system (CNS) of ALS patients, reaching particularly high levels in individuals carrying a SOD1 mutation. ALS brains are characterized by high levels of MPO and lowered anti-oxidant activity (due to the SOD1 mutation), thereby aiding CEP generation and accumulation. Due to DHA oxidation within the membranes, CEP marks cells with the highest oxidative damage. In all ALS cases CEP is present in nearly all astrocytes and microglia, however, only in individuals carrying a SOD1 mutation CEP marks >90% of neurons, thereby emphasizing an importance of CEP accumulation as a potential hallmark of oxidative damage in neurodegenerative diseases.

The accumulation of pro-inflammatory macrophages in the inflamed vascular wall is a critical step in atherogenesis. The mechanism of macrophage retention within the site of inflammation is not understood yet. High adhesion that prevents macrophage migration is one of the potential mechanisms. We previously showed that integrin α D β 2 is upregulated on pro-inflammatory macrophages, promotes macrophage retention, and contributes to atherogenesis. However, we have not identified a key ligand for α D β 2 within the tissue, since α D β 2 does not interact with major ECM proteins, collagens, and laminins. We recently found that during acute inflammation, the oxidation of docosahexaenoic acid (DHA) leads to the generation of end product carboxyethylpyrrole (CEP), which forms an adduct with fibrinogen and albumin via ε-amino group of lysines. Moreover, we revealed that macrophages adhered to CEP-modified albumin in α D β 2 -dependent manner. Now we are testing a hypothesis that DHA oxidation is a universal mechanism during chronic inflammatory diseases that promotes the generation of CEP adducts with different ECM proteins and forms α D β 2 -mediated strong anchorage of macrophages, which is critical for macrophage retention during chronic inflammation. We detected CEP-modified proteins in digested atherosclerotic lesions by western blot. In vitro DHA oxidation leads to the formation of CEP adducts with collagen IV and laminin but not with collagen I. Using α D β 2 -transfected HEK293 cells, WT and α D -/- mouse macrophages, we revealed that CEP-modified proteins support stronger cell adhesion and spreading to compare with natural macrophage ligands. Using site-directed mutagenesis, we generated mutant α D I-domains and α D β 2 -transfected cells with single amino acid substitutions. Applying protein-protein binding and adhesion assays we detected one amino acid within integrin α D , K 246 , which is critical for α D β 2 binding to CEP-modified proteins. In summary, we propose a new mechanism of macrophage retention, which is based on inflammatory modifications of ECM with DHA end-product, CEP. The identification of a binding site for CEP-modified proteins within α D β 2 will help to develop a blocking reagent for the treatment of the inflammatory component of atherosclerosis.

Toll-like receptor 2 (TLR2) is implicated in various pathologies, mainly in terms of its function within innate immune cells. However, TLR2 is also present in endothelial cells. Here, we explored the physiological and pathophysiological roles of endothelial TLR2 signaling. We found that TLR2 was highly abundant in the endothelium within various tissues using TLR2-IRES-EGFP reporter mice and was required for proinflammatory endothelial cell function. Endothelial cells lacking TLR2 exhibited reduced proinflammatory potential at the protein, cell, and tissue levels. Loss of endothelial TLR2 blunted the inflammatory response to both exogenous and endogenous danger signals in endothelial cells in culture and in vivo. Endothelial TLR2 promoted tumor growth, angiogenesis, and protumorigenic immune cell recruitment in a mouse model of prostate cancer. Furthermore, the cell surface localization of P-selectin and the subsequent production of other critical cell adhesion molecules (such as E-selectin, ICAM-1 and VCAM-1) that recruit immune cells required endothelial TLR2. Our findings demonstrate that endothelial cells actively contribute to innate immune pathways and propose that endothelial TLR2 has a pathological role in proinflammatory conditions.

Major myeloid cell functions from adhesion to migration and phagocytosis are mediated by integrin adhesion complexes, also known as adhesome. The presence of a direct integrin binding partner Kindlin-3 is crucial for these functions, and its lack causes severe immunodeficiency in humans. However, how Kindlin-3 is incorporated into the adhesome and how its function is regulated is poorly understood. In this study, using nuclear magnetic resonance spectroscopy, we show that Kindlin-3 directly interacts with paxillin (PXN) and leupaxin (LPXN) via G43/L47 within its F0 domain. Surprisingly, disruption of Kindlin-3-PXN/LPXN interactions in Raw 264.7 macrophages promoted cell spreading and polarization, resulting in upregulation of both general cell motility and directed cell migration, which is in a drastic contrast to the consequences of Kindlin-3 knockout. Moreover, disruption of Kindlin-3-PXN/LPXN binding promoted the transition from mesenchymal to amoeboid mode of movement as well as augmented phagocytosis. Thus, these novel links between Kindlin-3 and key adhesome members PXN/LPXN limit myeloid cell motility and phagocytosis, thereby providing an important immune regulatory mechanism.

Pathological vasculature marks itself for repair by deploying neutrophil extracellular traps Vascular remodeling is essential for building hierarchically structured vascular networks, which in turn support proper organ function (1). The retina is particularly dependent on optimal blood supply, and insufficient or excessive vasculature often leads to blindness. The process of vascular regression reduces blood vessel density, facilitating normalization of vasculature and subsequent tissue repair (2). Regression is either caused by the withdrawal of essential vascular growth factors or by triggering endothelial apoptosis and subsequent pruning of vasculature (3). However, it is unknown how dysfunctional and excessive retinal blood vessels are selected and marked for pruning. On page 934 of this issue, Binet et al. (4) reveal that pathological vasculature in the retina of mice and humans orchestrates its own remodeling by promoting the extrusion of neutrophil extracellular traps (NETs). This mechanism might be applied to other pathologies that require vascular remodeling, such as cancer, pulmonary hypertension, and heart disease (3).

Apolipoprotein A-I (apoA-I) is cross-linked and dysfunctional in human atheroma. Although multiple mechanisms of apoA-I cross-linking have been demonstrated in vitro, the in vivo mechanisms of cross-linking are not well-established. We have recently demonstrated the highly selective and efficient modification of high-density lipoprotein (HDL) apoproteins by endogenous oxidized phospholipids (oxPLs), including ?-ketoalkenal phospholipids. In the current study, we report that ?-ketoalkenal phospholipids effectively cross-link apoproteins in HDL. We further demonstrate that cross-linking impairs the cholesterol efflux mediated by apoA-I or HDL3 in vitro and in vivo. Using LC-MS/MS analysis, we analyzed the pattern of apoprotein cross-linking in isolated human HDL either by synthetic ?-ketoalkenal phospholipids or by oxPLs generated during HDL oxidation in plasma by the physiologically relevant MPO-H2O2-NO2? system. We found that five histidine residues in helices 5?8 of apoA-I are preferably cross-linked by oxPLs, forming stable pyrrole adducts with lysine residues in the helices 3?4 of another apoA-I or in the central domain of apoA-II. We also identified cross-links of apoA-I and apoA-II with two minor HDL apoproteins, apoA-IV and apoE. We detected a similar pattern of apoprotein cross-linking in oxidized murine HDL. We further detected oxPL cross-link adducts of HDL apoproteins in plasma and aorta of hyperlipidemic LDLR?/? mice, including cross-link adducts of apoA-I His-165?apoA-I Lys-93, apoA-I His-154?apoA-I Lys-105, apoA-I His-154?apoA-IV Lys-149, and apoA-II Lys-30?apoE His-227. These findings suggest an important mechanism that contributes to the loss of HDL's atheroprotective function in vivo.