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The first heteroditopic [2]rotaxane host systems capable of strong and selective binding of lithium chloride ion-pair species are described. Importantly, a cooperative 'switch on' mechanism was found to operate, in which complexation of a lithium metal cation enhances the halide anion affinity of the rotaxanes via a combination of favourable proximal electrostatic and preorganised allosteric effects. The mechanically bonded rotaxane host design features a macrocycle component possessing a 2,6-dialkoxy pyridyl cation binding motif and an isophthalamide anion binding group, as well as an axle component functionalised with either a halogen bonding (XB) iodotriazole or hydrogen bonding (HB) prototriazole moiety. Extensive quantitative 1H NMR titration studies in CD3CN/CDCl3 solvent mixtures determined enhanced ion-pair binding affinities for lithium halides over the corresponding sodium or potassium halide salts, with the axle prototriazole-containing HB rotaxane in particular demonstrating a marked selectivity for lithium chloride. Solid-state X-ray crystallographic studies and computational DFT investigations provide evidence for a [2]rotaxane host axle-separated ion-pair binding mode, in which complementary cation and anion binding motifs from both the macrocycle and axle components act convergently to recognise each of the charged guest species.
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The synthesis and ion-pair binding properties of a heteroditopic [2]catenane receptor exhibiting highly potent and selective recognition of sodium halide salts are described. The receptor design consists of a bidentate halogen bonding donor motif for anion binding, as well as a di(ethylene glycol)-derived cation binding pocket which dramatically enhances metal cation affinity over previously reported homo[2]catenane analogues. 1H NMR cation, anion and ion-pair binding studies reveal significant positive cooperativity between the cation and anion binding events in which cation pre-complexation to the catenane subsequently 'switches-on' anion binding. Notably, the heteroditopic catenane displayed impressive selectivity for sodium halide recognition over the corresponding potassium halides. We further demonstrate that the catenane is capable of extracting solid alkali metal salts into organic media. Crucially, the observed solution phase binding selectivity for sodium halides translates to superior functional extraction capabilities of these salts relative to potassium halides, overcoming the comparatively higher lattice enthalpies NaX > KX dictated by the smaller alkali metal sodium cation. This is further exemplified in competitive solid-liquid experiments which revealed the exclusive extraction of sodium halide salts from solid mixtures of sodium and potassium halide salts.
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The first example of a heteroditopic receptor capable of cooperative recognition and extraction of ammonium salt (NH4X) ion-pairs is described. Consisting of a bidentate 3,5-bis-tellurotriazole chalcogen bond donor binding cleft, the appendage of benzo-15-crown-5 (B15C5) substituents to the tellurium centres facilitates binding of the ammonium cation via a co-facial bis-B15C5 sandwich complex, which serves to switch on chalcogen bonding-mediated anion binding potency. Extensive quantitative ion-pair recognition 1H NMR titration studies in CD3CN/CDCl3 (1 : 1, v/v) solvent media reveal impressive ion-pair binding affinities towards a variety of ammonium halide, nitrate and thiocyanate salts, with the heteroditopic receptor displaying notable ammonium halide salt selectivity. The prodigious solution phase NH4X recognition also translates to efficient solid-liquid and liquid-liquid extraction capabilities.
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The first example of a [2]rotaxane shuttle capable of selective optical sensing of chloride anions over other halides is reported. The rotaxane was synthesised via a chloride ion template-directed cyclisation of an isophthalamide macrocycle around a multi-station axle containing peripheral naphthalene diimide (NDI) stations and a halogen bonding (XB) bis(iodotriazole) based station. Proton NMR studies indicate the macrocycle resides preferentially at the NDI stations in the free rotaxane, where it is stabilised by aromatic donor-acceptor charge transfer interactions between the axle NDI and macrocycle hydroquinone moieties. Addition of chloride ions in an aqueous-acetone solvent mixture induces macrocycle translocation to the XB anion binding station to facilitate the formation of convergent XBâ â â Cl- and hydrogen bonding HBâ â â Cl- interactions, which is accompanied by a reduction of the charge-transfer absorption band. Importantly, little to no optical response was induced by addition of bromide or iodide to the rotaxane, indicative of the size discriminative steric inaccessibility of the interlocked cavity to the larger halides, demonstrating the potential of using the mechanical bond effect as a potent strategy and tool in chloride-selective chemo-sensing applications in aqueous containing solvent environments.
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Cell-free RNAs and extracellular vesicles (EVs) are valuable biomarkers in liquid biopsies, but they are prone to preanalytical variabilities such as nonstandardized centrifugation or ex vivo blood degradation. Herein, we report a high-throughput and label-free inertial microfluidic device (ExoArc) for isolation of platelet-free plasma from blood for RNA and EV analysis. Unlike conventional inertial microfluidic devices widely used for cell sorting, a submicrometer size cutoff (500 nm) was achieved which completely removed all leukocytes, RBCs, platelets, and cellular debris based on differential lateral migration induced by Dean vortices. The single-step operation also reduced platelet-associated miRNAs (â¼2-fold) compared to centrifugation. We clinically validated ExoArc for plasma miRNA profiling (39 samples) and identified a 7-miRNA panel that detects non-small cell lung cancer with â¼90% sensitivity. ExoArc was also coupled with size exclusion chromatography (SEC) to isolate EVs within 50 min with â¼10-fold higher yield than ultracentrifugation. As a proof-of-concept for EV-based transcriptomics analysis, we performed miRNA analysis in healthy and type 2 diabetes mellitus (T2DM) subjects (n = 3 per group) by coupling ExoArc and ExoArc+SEC with quantitative polymerase chain reaction (RT-qPCR) assay. Among 293 miRNAs detected, plasmas and EVs showed distinct differentially expressed miRNAs in T2DM subjects. We further demonstrated automated in-line EV sorting from low volume culture media for continuous EV monitoring. Overall, the developed ExoArc offers a convenient centrifugation-free workflow to automate plasma and EV isolation for point-of-care diagnostics and quality control in EV manufacturing.
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Carcinoma Pulmonar de Células não Pequenas , Diabetes Mellitus Tipo 2 , Vesículas Extracelulares , Neoplasias Pulmonares , MicroRNAs , Humanos , MicroRNAs/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Microfluídica , Neoplasias Pulmonares/metabolismo , Vesículas Extracelulares/metabolismoRESUMO
Urinary tract infection (UTI) diagnosis based on urine culture for bacteriuria analysis is time-consuming and often leads to wastage of hospital resources due to false-positive UTI cases. Direct cellular phenotyping (e.g., RBCs, neutrophils, epithelial cells) of urine samples remains a technical challenge as low cell concentrations, and urine characteristics (conductivities, pH, microbes) can affect the accuracy of cell measurements. In this work, we report a microfluidic inertial-impedance cytometry technique for label-free rapid (<5 min) neutrophil sorting and impedance profiling from urine directly. Based on size-based inertial focusing effects, neutrophils are isolated, concentrated, and resuspended in saline (buffer exchange) to improve consistency in impedance-based single-cell analysis. We first observed that both urine pH and the presence of bacteria can affect neutrophil high-frequency impedance measurements possibly due to changes in nucleus morphology as neutrophils undergo NETosis and phagocytosis, respectively. As a proof-of-concept for clinical testing, we report for the first time, rapid UTI testing based on multiparametric impedance profiling of putative neutrophils (electrical size, membrane properties, and distribution) in urine samples from non-UTI (n = 20) and UTI patients (n = 20). A significant increase in cell count was observed in UTI samples, and biophysical parameters were used to develop a UTI classifier with an area under the receiver operating characteristic curve of 0.84. Overall, the developed platform facilitates rapid culture-free urine screening which can be further developed to assess disease severity in UTI and other urologic diseases based on neutrophil electrical signatures.
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Bacteriúria , Infecções Urinárias , Humanos , Impedância Elétrica , Microfluídica , Infecções Urinárias/diagnóstico , Infecções Urinárias/microbiologia , Infecções Urinárias/urina , Bacteriúria/diagnóstico , Bacteriúria/urina , Urinálise/métodosRESUMO
Blood tests are considered as standard clinical procedures to screen for markers of diseases and health conditions. However, the complex cellular background (>99.9% RBCs) and biomolecular composition often pose significant technical challenges for accurate blood analysis. An emerging approach for point-of-care blood diagnostics is utilizing "label-free" microfluidic technologies that rely on intrinsic cell properties for blood fractionation and disease detection without any antibody binding. A growing body of clinical evidence has also reported that cellular dysfunction and their biophysical phenotypes are complementary to standard hematoanalyzer analysis (complete blood count) and can provide a more comprehensive health profiling. In this review, we will summarize recent advances in microfluidic label-free separation of different blood cell components including circulating tumor cells, leukocytes, platelets and nanoscale extracellular vesicles. Label-free single cell analysis of intrinsic cell morphology, spectrochemical properties, dielectric parameters and biophysical characteristics as novel blood-based biomarkers will also be presented. Next, we will highlight research efforts that combine label-free microfluidics with machine learning approaches to enhance detection sensitivity and specificity in clinical studies, as well as innovative microfluidic solutions which are capable of fully integrated and label-free blood cell sorting and analysis. Lastly, we will envisage the current challenges and future outlook of label-free microfluidics platforms for high throughput multi-dimensional blood cell analysis to identify non-traditional circulating biomarkers for clinical diagnostics.
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Técnicas Analíticas Microfluídicas , Microfluídica , Microfluídica/métodos , Separação Celular , Leucócitos , Testes Hematológicos , BiomarcadoresRESUMO
The first examples of halogen bonding (XB) heteroditopic homo[2]catenanes were prepared by discrete Na+ template-directed assembly of oligo(ethylene glycol) units derived from XB donor-containing macrocycles and acyclic bis-azide precursors, followed by a CuI -mediated azide-alkyne cycloaddition macrocyclisation reaction. Extensive 1 Hâ NMR spectroscopic studies show the [2]catenane hosts exhibit positive cooperative ion-pair recognition behaviour, wherein XB-mediated halide recognition is enhanced by alkali metal cation pre-complexation. Notably, subtle changes in the catenanes' oligo(ethylene glycol) chain length dramatically alters their ion-binding affinity, stoichiometry, complexation mode, and conformational dynamics. Solution-phase and single-crystal X-ray diffraction studies provide evidence for competing host-separated and direct-contact ion-pair binding modes. We further demonstrate the [2]catenanes are capable of extracting solid alkali-metal halide salts into organic media.
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Vascular stenosis caused by atherosclerosis instigates activation and aggregation of platelets, eventually resulting in thrombus formation. Although antiplatelet drugs are commonly used to inhibit platelet activation and aggregation, they unfortunately cannot prevent recurrent thrombotic events in patients with atherosclerosis. This is partially due to the limited understanding of the efficacy of antiplatelet drugs in the complex hemodynamic environment of vascular stenosis. Conventional methods for evaluating the efficacy of antiplatelet drugs under stenosis either fail to simulate the hemodynamic environment of vascular stenosis characterized by high shear stress and recirculatory flow or lack spatial resolution in their analytical techniques to statistically identify and characterize platelet aggregates. Here we propose and experimentally demonstrate a method comprising an in vitro 3D stenosis microfluidic chip and an optical time-stretch quantitative phase imaging system for studying the efficacy of antiplatelet drugs under stenosis. Our method simulates the atherogenic flow environment of vascular stenosis while enabling high-resolution and statistical analysis of platelet aggregates. Using our method, we distinguished the efficacy of three antiplatelet drugs, acetylsalicylic acid (ASA), cangrelor, and eptifibatide, for inhibiting platelet aggregation induced by stenosis. Specifically, ASA failed to inhibit stenosis-induced platelet aggregation, while eptifibatide and cangrelor showed high and moderate efficacy, respectively. Furthermore, we demonstrated that the drugs tested also differed in their efficacy for inhibiting platelet aggregation synergistically induced by stenosis and agonists (e.g., adenosine diphosphate, and collagen). Taken together, our method is an effective tool for investigating the efficacy of antiplatelet drugs under vascular stenosis, which could assist the development of optimal pharmacologic strategies for patients with atherosclerosis.
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Aterosclerose , Trombose , Humanos , Inibidores da Agregação Plaquetária/farmacologia , Eptifibatida/farmacologia , Constrição Patológica , Plaquetas , Aspirina/farmacologia , Aterosclerose/diagnóstico por imagem , Aterosclerose/tratamento farmacológico , Dispositivos Lab-On-A-ChipRESUMO
Exceptionally strong halogen bonding (XB) donor-chloride interactions are exploited for the chloride anion template synthesis of neutral XB [2]rotaxane host systems which contain perfluoroaryl-functionalised axle components, including a remarkably potent novel 4,6-dinitro-1,3-bis-iodotriazole motif. Halide anion recognition properties in aqueous-organic media, determined via extensive 1 H NMR halide anion titration experiments, reveal the rotaxane host systems exhibit dramatically enhanced affinities for hydrophilic Cl- and Br- , but conversely diminished affinities for hydrophobic I- , relative to their non-interlocked axle counterparts. Crucially, this mechanical bond effect induces a binding selectivity which directly opposes Hofmeister bias. Free-energy analysis of this mechanical bond enhancement demonstrates anion recognition by neutral XB interlocked host systems as a rare and general strategy to engineer anti-Hofmeister bias anion selectivity in synthetic receptor design.
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A fluorescent, chiral coordination polymer (CP) with a novel topology has been synthesised using a dipyridyl ligand derived from 1,1'-bi-2-naphthol (BINOL). Enantioselectivity ratios up to 2.61 were obtained in fluorescence sensing studies with chiral analytes.
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Polímeros , Ligantes , Espectrometria de Fluorescência , EstereoisomerismoRESUMO
The intrinsic biophysical states of neutrophils are associated with immune dysfunctions in diseases. While advanced image-based biophysical flow cytometers can probe cell deformability at high throughput, it is nontrivial to couple different sensing modalities (e.g., electrical) to measure other critical cell attributes including cell viability and membrane integrity. Herein, an "optics-free" impedance-deformability cytometer for multiparametric single cell mechanophenotyping is reported. The microfluidic platform integrates hydrodynamic cell pinching, and multifrequency impedance quantification of cell size, deformability, and membrane impedance (indicative of cell viability and activation). A newly-defined "electrical deformability index" is validated by numerical simulations, and shows strong correlations with the optical cell deformability index of HL-60 experimentally. Human neutrophils treated with various biochemical stimul are further profiled, and distinct differences in multimodal impedance signatures and UMAP analysis are observed. Overall, the integrated cytometer enables label-free cell profiling at throughput of >1000 cells min-1 without any antibodies labeling to facilitate clinical diagnostics.
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Técnicas Analíticas Microfluídicas , Microfluídica , Impedância Elétrica , Citometria de Fluxo , Células HL-60 , Humanos , NeutrófilosRESUMO
BACKGROUND: Polypoidal choroidal vasculopathy (PCV), a subtype of age-related macular degeneration (AMD), is a global leading cause of vision loss in older populations. Distinct from typical AMD, PCV is characterized by polyp-like dilatation of blood vessels and turbulent blood flow in the choroid of the eye. Gold standard anti-vascular endothelial growth factor (anti-VEGF) therapy often fails to regress polypoidal lesions in patients. Current animal models have also been hampered by their inability to recapitulate such vascular lesions. These underscore the need to identify VEGF-independent pathways in PCV pathogenesis. RESULTS: We cultivated blood outgrowth endothelial cells (BOECs) from PCV patients and normal controls to serve as our experimental disease models. When BOECs were exposed to heterogeneous flow, single-cell transcriptomic analysis revealed that PCV BOECs preferentially adopted migratory-angiogenic cell state, while normal BOECs undertook proinflammatory cell state. PCV BOECs also had a repressed protective response to flow stress by demonstrating lower mitochondrial functions. We uncovered that elevated hyaluronidase-1 in PCV BOECs led to increased degradation of hyaluronan, a major component of glycocalyx that interfaces between flow stress and vascular endothelium. Notably, knockdown of hyaluronidase-1 in PCV BOEC improved mechanosensitivity, as demonstrated by a significant 1.5-fold upregulation of Krüppel-like factor 2 (KLF2) expression, a flow-responsive transcription factor. Activation of KLF2 might in turn modulate PCV BOEC migration. Barrier permeability due to glycocalyx impairment in PCV BOECs was also reversed by hyaluronidase-1 knockdown. Correspondingly, hyaluronidase-1 was detected in PCV patient vitreous humor and plasma samples. CONCLUSIONS: Hyaluronidase-1 inhibition could be a potential therapeutic modality in preserving glycocalyx integrity and endothelial stability in ocular diseases with vascular origin.
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Hialuronoglucosaminidase , Degeneração Macular , Idoso , Corioide/irrigação sanguínea , Corioide/patologia , Células Endoteliais , Angiofluoresceinografia , Glicocálix/patologia , Humanos , Hialuronoglucosaminidase/genética , Hialuronoglucosaminidase/uso terapêutico , Degeneração Macular/tratamento farmacológico , Degeneração Macular/patologiaRESUMO
Extracellular vesicles (EVs) are recognized as next generation diagnostic biomarkers due to their disease-specific biomolecular cargoes and importance in cell-cell communications. A major bottleneck in EV sample preparation is the inefficient and laborious isolation of nanoscale EVs (≈50-200 nm) from endogenous proteins in biological samples. Herein, a unique microfluidic platform is reported for EV-protein fractionation based on the principle of size exclusion chromatography (SEC). Using a novel rapid (≈20 min) replica molding technique, a fritless microfluidic SEC device (µSEC) is fabricated using thiol-ene polymer (UV glue NOA81, Young's modulus ≈1 GPa) for high pressure (up to 6 bar) sample processing. Controlled on-chip nanoliter sample plug injection (600 nL) using a modified T-junction injector is first demonstrated with rapid flow switching response time (<1.5 s). Device performance is validated using fluorescent nanoparticles (50 nm), albumin, and breast cancer cells (MCF-7)-derived EVs. As a proof-of-concept for clinical applications, EVs are directly isolated from undiluted human platelet-poor plasma using µSEC and show distinct elution profiles between EVs and proteins based on nanoparticle particle analysis (NTA), Western blot and flow cytometry analysis. Overall, the optically transparent µSEC can be readily automated and integrated with EV detection assays for EVs manufacturing and clinical diagnostics.
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Vesículas Extracelulares , Microfluídica , Proteínas Sanguíneas/metabolismo , Cromatografia em Gel , Vesículas Extracelulares/metabolismo , Humanos , PlasmaRESUMO
Incorporation of extracellular matrix (ECM) and hydrogel in microfluidic 3D cell culture platforms is important to create a physiological microenvironment for cell morphogenesis and to establish 3D co-culture models by hydrogel compartmentalization. Here, we describe a simple and scalable ECM patterning method for microfluidic cell cultures by achieving hydrogel confinement due to the geometrical expansion of channel heights (stepped height features) and capillary burst valve (CBV) effects. We first demonstrate a sequential "pillar-free" hydrogel patterning to form adjacent hydrogel lanes in enclosed microfluidic devices, which can be further multiplexed with one to two stepped height features. Next, we developed a novel "spheroid-in-gel" culture device that integrates (1) an on-chip hanging drop spheroid culture and (2) a single "press-on" hydrogel confinement step for rapid ECM patterning in an open-channel microarray format. The initial formation of breast cancer (MCF-7) spheroids was achieved by hanging a drop culture on a patterned polydimethylsiloxane (PDMS) substrate. Single spheroids were then directly encapsulated on-chip in individual hydrogel islands at the same positions, thus, eliminating any manual spheroid handling and transferring steps. As a proof-of-concept to perform a spheroid co-culture, endothelial cell layer (HUVEC) was formed surrounding the spheroid-containing ECM region for drug testing studies. Overall, this developed stepped height-based hydrogel patterning method is simple to use in either enclosed microchannels or open surfaces and can be readily adapted for in-gel cultures of larger 3D cellular spheroids or microtissues.
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Hidrogéis , Microfluídica , Técnicas de Cultura de Células em Três Dimensões , Esferoides CelularesRESUMO
Elevated serum concentrations of leucine-rich α-2-glycoprotein (LRG1) have been reported in patients with inflammatory, autoimmune, and cardiovascular diseases. This study aims to investigate the role of LRG1 in endothelial activation. LRG1 in endothelial cells (ECs) of arteries and serum of patients with critical limb ischemia (CLI) was assessed by immunohistochemistry and ELISA, respectively. LRG1 expression in sheared and tumor necrosis factor-α (TNF-α)-treated ECs was analyzed. The mechanistic role of LRG1 in endothelial activation was studied in vitro. Plasma of 37-week-old Lrg1 -/- mice was used to investigate causality between LRG1 and tumor necrosis factor receptor 1 (TNFR1) shedding. LRG1 was highly expressed in ECs of stenotic but not normal arteries. LRG1 concentrations in serum of patients with CLI were elevated compared to healthy controls. LRG1 expression was shear dependent. It could be induced by TNF-α, and the induction of its expression was mediated by NF-κB activation. LRG1 inhibited TNF-α-induced activation of NF-κB signaling, expression of VCAM-1 and ICAM-1, and monocyte capture, firm adhesion, and transendothelial migration. Mechanistically, LRG1 exerted its function by causing the shedding of TNFR1 via the ALK5-SMAD2 pathway and the subsequent activation of ADAM10. Consistent with this mechanism, LRG1 and sTNFR1 concentrations were correlated in the serum of CLI patients. Causality between LRG1 and TNFR1 shedding was established by showing that Lrg1 -/- mice had lower plasma sTNFR1 concentrations than wild type mice. Our results demonstrate a novel role for LRG1 in endothelial activation and its potential therapeutic role in inflammatory diseases should be investigated further.
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The ubiquity of anions in biological and environmental systems has motivated the development of many novel anion receptors and sensors over the past two decades. Optical anion sensors, which undergo a spectral change in response to anion binding, are particularly desirable due to the technical simplicity and fast response time of such systems. A myriad of macrocyclic host molecules have been shown to be effective anion receptors and present a promising platform for elaboration into optical anion sensors by incorporation of an appropriate fluorogenic or chromogenic group. The enhanced anion binding properties of the three-dimensional binding cavities in mechanically interlocked host molecules have also been exploited to design anion sensors that exhibit remarkable selectivity and sensitivity. This review summarises recent progress in the development of optical anion sensors based on macrocyclic and interlocked hosts. The major classes of macrocyclic receptors possessing neutral, cationic and metal-based binding motifs are examined, followed by a survey of optically-responsive interlocked anion hosts.
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Extracellular vesicles (EVs) are key mediators of communication among cells, and clinical utilities of EVs-based biomarkers remain limited due to difficulties in isolating EVs from whole blood reliably. We report a novel inertial-based microfluidic platform for direct isolation of nanoscale EVs (exosomes, 50 to 200 nm) and medium-sized EVs (microvesicles, 200 nm to 1 µm) from blood with high efficiency (three-fold increase in EV yield compared to ultracentrifugation). In a pilot clinical study of healthy (n = 5) and type 2 diabetes mellitus (T2DM, n = 9) subjects, we detected higher EV levels in T2DM patients (P < 0.05), and identified a subset of "high-risk" T2DM subjects with abnormally high (â¼10-fold to 50-fold) amounts of platelet (CD41a+) or leukocyte-derived (CD45+) EVs. Our in vitro endothelial cell assay further revealed that EVs from "high-risk" T2DM subjects induced significantly higher vascular inflammation (ICAM-1 expression) (P < 0.05) as compared to healthy and non-"high-risk" T2DM subjects, reflecting a pro-inflammatory phenotype. Overall, the EV isolation tool is scalable, and requires less manual labour, cost and processing time. This enables further development of EV-based diagnostics, whereby a combined immunological and functional phenotyping strategy can potentially be used for rapid vascular risk stratification in T2DM.
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Diabetes Mellitus Tipo 2 , Exossomos , Vesículas Extracelulares , Biomarcadores , Células Endoteliais , HumanosRESUMO
Chirality is a key aspect of amino acids and is essential for life. Here, a chiral metal-organic framework, S-Mg2dobpdc, is used to determine the chirality of three BOC protected amino acids (alanine, valine and proline) by 13C solid-state NMR with chemical shift differences of up to 1.3 ppm observed between enantiomers. The chiral sensitivity persists upon in situ deprotection of the amino acids by thermolysis of the BOC group.
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Aminoácidos/química , Magnésio/química , Estruturas Metalorgânicas/química , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , EstereoisomerismoRESUMO
Delayed wound healing is commonly associated with diabetes. It may lead to amputation and death if not treated in a timely fashion. Limited treatments are available partially due to the poor understanding of the complex disease pathophysiology. Here, we investigated the role of leucine-rich α-2-glycoprotein 1 (LRG1) in normal and diabetic wound healing. First, our data showed that LRG1 was significantly increased at the inflammation stage of murine wound healing, and bone marrow-derived cells served as a major source of LRG1. LRG1 deletion causes impaired immune cell infiltration, reepithelialization, and angiogenesis. As a consequence, there is a significant delay in wound closure. On the other hand, LRG1 was markedly induced in diabetic wounds in both humans and mice. LRG1-deficient mice were resistant to diabetes-induced delay in wound repair. We further demonstrated that this could be explained by the mitigation of increased neutrophil extracellular traps (NETs) in diabetic wounds. Mechanistically, LRG1 mediates NETosis in an Akt-dependent manner through TGFß type I receptor kinase ALK5. Taken together, our studies demonstrated that LRG1 derived from bone marrow cells is required for normal wound healing, revealing a physiological role for this glycoprotein, but that excess LRG1 expression in diabetes is pathogenic and contributes to chronic wound formation.