We isolated and characterized a panel of EDIII-specific monoclonal antibodies (mAbs) and identified several that potently inhibit POWV infection and engage distinct epitopes within the lateral ridge and C-C′ loop of the EDIII. By creating a subunit-based nanoparticle immunogen with vaccine potential that elicits antibodies with protective activity against POWV infection, our findings enhance our understanding of the molecular determinants of antibody-mediated neutralization of TBFVs.Powassan virus (POWV) is an emerging tick borne flavivirus (TBFV) that causes severe neuroinvasive disease. Currently, there are no approved treatments or vaccines to combat POWV infection. Here, we generated and characterized a nanoparticle immunogen displaying domain III (EDIII) of the POWV E glycoprotein. Immunization with POWV EDIII presented on nanoparticles resulted in significantly higher serum neutralizing titers against POWV than immunization with monomeric POWV EDIII. Furthermore, passive transfer of EDIII-reactive sera protected against POWV challenge  Powassan virus (POWV) is an emerging tick-borne flavivirus that circulates in North America. Infection can cause severe neuro-invasive disease, and currently there is no treatment or vaccine against POWV. We generated a novel POWV immunogen that displays the EDIII of the envelope glycoprotein on the surface of a self-assembling protein nanoparticle. We found that immunization of the EDIII-displaying nanoparticle in mice elicited potently neutralizing and protective antibodies. We further characterized EDIII-specific neutralizing mAbs and mapped their epitopes to specific regions within the POWV EDIII. Our study sheds light on determinants of antibody-mediated neutralization and protection against POWV and other TBFVs. It also informs EDIII-based vaccine and antiviral efforts to combat POWV infection. Powassan virus (POWV) is an emerging tick-borne flavivirus (TBFV) that causes neurological disease in humans –4. The fIxodes cookei and Ixodes scapularis, respectively  or  samples were expressed in cells grown in M9 minimal media supplemented with 15NH4Cl and/or [U-13C] D-glucose. Cells were lysed by sonication in 50 mM Tris pH 8.0, 250 mM NaCl, and MBP-POWV fusion proteins were purified by Ni NTA chromatography and confirmed by SDS-PAGE. Proteolytic cleavage with TEV was done overnight (1:50 w/w), and cleavage products were dialyzed in low salt buffer  before anion exchange with MonoQ 10/100 GL.POWV, TBEV, and LGTV  EDIII sequences (residues 303–402) were codon optimized for Aquifex aeolicus lumazine synthase -POWV EDIII in 20 mM phosphate, 5 mM NaCl, pH 6.2 with trimethylsilyl propionate  in 90% H2O/10% D2O in 3 mm tube or ~1 mM -POWV EDIII in 20 mM phosphate, 50 mM NaCl, pH 6.2 with TSP in 90% H2O/10% D2O in 3 mm tube or ~1 mM -POWV EDIII in 20 mM phosphate, 50mM NaCl, pH 6.2 with TSP in 100% D2O in 3 mm tube.All NMR data were collected at 25°C on Bruker Avance III 600 MHz, 700 MHz or 800 MHz spectrometers equipped with 5 mm cryogenically cooled triple resonance probes. NMR samples consisted of ~500 μM [1H[15N] HSQC, 1H[13C] HSQC, HNHA, HNCA, HN(CO)CA, HNCO, HN(CA)CO, HNCACB, HN(CO)CACB, (H)CC(CO)NH-tocsy, H(CCCO)NH-tocsy, 1H[15N] TOCSY-HSQC and HC(C)H-TOCSY. Distance restraints were derived from 3D 15N- and 13C-edited NOESY-HSQC spectra with a mixing time of 120 ms. All 3D experiments were acquired as non-uniform sampled (NUS) experiments. All NMR data sets were processed on NMRbox  POWV EDIII and complex with m158.25 Fab at a 1:1.1 stoichiometry.Sequence-specific resonance assignments were obtained using standard triple resonance NMR spectroscopy  includinn NMRbox  with nmrn NMRbox  using SMn NMRbox . 1H chemerencing . 200 strerencing , and eneerencing  in expliS1 Fig50 values were determined using non-linear regression analysis. One-way ANOVA with Tukey’s multiple comparison test was done. .(A) IgG subclass profiles of POWV EDIII and LS-POWV EDIII antisera bound to POWV EDIII by ELISA. (B) ELISA reactivity of POWV EDIII and LS-POWV EDIII antisera to monomeric and multivalent POWV EDIII antigen. (C) Cross-reactivity of POWV EDIII and LS-POWV EDIII antisera to related flavivirus EDIIIs. ELISA reactivity of antisera to POWV, TBEV, LGTV, and DENV2 recombinant EDIII was determined. For A-C, groups shown are POWV EDIII monomer (n = 5) and LS-POWV-EDIII nanoparticle (n = 5) at day 56. Full curve ELISAs were performed twice independently in triplicate wells (mean ± SD) and EC(EPS)Click here for additional data file.S2 FigAlignment of amino acid sequences of structural polyproteins used for immunogen and reporter virus constructs. EDIII sequence is highlight as indicated. Alignment was generated using ESPript 3.0(EPS)Click here for additional data file.S3 FigELISA reactivity of mAbs B2 and B4 against POWV-I RVPs. ELISAs were performed twice independently in triplicate wells (mean ± SD).(EPS)Click here for additional data file.S4 Fig+, CD8+, F4/80+, and Gr-1+ cells were excluded and CD19+ IgG+ POWV EDIII+ B cells were collected in individual wells.Representative flow cytometric gating of mouse splenocytes is shown. Cells were filtered for size and granularity. CD3(EPS)Click here for additional data file.S5 FigTwo-phase binding of neutralizing mAbs to POWV EDIII vs. POWV-80 by BLI. Neutralizing mAbs m61.65, m158.25, m158.26 and m158.36 were first bound to a sensor loaded with POWV EDIII and then POWV-80 was added. Representative data from two independent experiments are shown.(EPS)Click here for additional data file.S6 FigFull curve ELISAs against POWV, TBEV, LFTV, and DENV2 EDIIIs were performed twice independently in triplicate wells (mean ± SD).(EPS)Click here for additional data file.S7 FigA) SDS-PAGE of purified MBP-POWV EDIII mutants. (B) ELISA reactivity of MBP-POWV EDIII mutants to mAb POWV-55. Full binding curves were determined by eight-point serial dilution of POWV-55 against each mutant. Experiment was performed twice independently in triplicate wells.((EPS)Click here for additional data file.S8 FigFull curve ELISAs against the indicated EDIII mutants were performed twice independently in triplicate wells (mean ± SD).(EPS)Click here for additional data file.S9 FigResidue labels for POWV EDIII assigned peaks are shown.(EPS)Click here for additional data file.S10 FigBinding of Fab m158.25 to POWV EDIII. A representative data set from two independent experiments is shown.(EPS)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Following the publication of the original article , the texfromThe institutional review board of Chia-yi Chang Gung Memorial Hospital approved this retrospective study (100-4178B) and (201900447B0C601). Consent to participate was not applicable.toThe institutional review board of Chia-yi Chang Gung Memorial Hospital (IRB number: 100-4178B0C504 and 201900447B0C601) and Kaohsiung Veterans General Hospital (IRB number: VGHKS18-CT6-04) were approved this retrospective study. Consent to participate was not applicable.The original article has been corrected.


O-GlcNAcylation, oxidation, S-nitrosylation, ubiquitination, and proteolytic cleavage, regulate its activity and ATG8 processing, thus modulating its autophagic activity. We reviewed recent advances in our understanding of the effect of post-translational modification on the regulation, activity, and function of ATG4, the main protease that controls autophagy.Autophagy plays a key role in eliminating and recycling cellular components in response to stress, including starvation. Dysregulation of autophagy is observed in various diseases, including neurodegenerative diseases, cancer, and diabetes. Autophagy is tightly regulated by autophagy-related (ATG) proteins. Autophagy-related 4 (ATG4) is the sole cysteine protease, and four homologs (ATG4A–D) have been identified in mammals. These proteins have two domains: catalytic and short fingers. ATG4 facilitates autophagy by promoting autophagosome maturation through reversible lipidation and delipidation of seven autophagy-related 8 (ATG8) homologs, including microtubule-associated protein 1-light chain 3 (LC3) and GABA type A receptor-associated protein (GABARAP). Each ATG4 homolog shows a preference for a specific ATG8 homolog. Post-translational modifications of ATG4, including phosphorylation/dephosphorylation,  Macroautophagy (autophagy) is a highly conserved catabolic process that maintains cellular homeostasis . AutophaAutophagy-related 8 (ATG8) is a ubiquitin-like protein that is widely used as an autophagosome marker ,7. In maO-GlcNAcylation, and redox modifications [Post-translational protein modifications such as adding covalent functional groups, proteolytically cleaving regulatory subunits, or degrading proteins increase the functional diversity of the proteome. Autophagy is modulated by post-translational modifications, including phosphorylation, ications . SeveralAutophagy-related 4 (ATG4) is the only ATG that displays cysteine protease activity . Four ATDysfunctional autophagy is associated with several pathological conditions ,13. ATG4O-GlcNAcylation, oxidation, S-nitrosylation, ubiquitination, and proteolytic cleavage, regulate its proteolytic activity and ATG8 processing, leading to the modulation of autophagy under various conditions /MST4 . Proteom31RKYS34) for AKT1 at its N-terminus and identified the Ser-34 residue of ATG4B as a target in HCC cells. ATG4B phosphorylation by AKT1 had little effect on basal autophagic flux, although it enhanced the Warburg effect in HCC cells. Cells expressing wild-type ATG4B or a phospho-defective ATG4B mutant (S34A) with AKT1 had different levels of L-lactate, the main product of the Warburg effect. L-lactate was produced in wild-type ATG4B-expressing cells in greater quantities than in phospho-defective ATG4B cells. Overexpression of ATG4B phosphorylated on Ser-34 by AKT1 results in the inhibition of mitochondrial function via a decrease in F1F0-ATP synthase activity and an increase in mitochondrial reactive oxygen species (ROS). Therefore, phosphorylation of ATG4B at residue Ser-34 plays a noncanonical role under pathological conditions by regulating metabolic reprogramming [ATG4B in hepatocellular carcinoma (HCC) cells is also phosphorylated by AKT Serin/Threonine Kinase 1 (AKT1) . The AKTgramming .AKT1 directly phosphorylates ATG4B at Ser-34. However, whether this phosphorylation increases or decreases ATG4B activity remains unknown. Pengo et al. showed that AKT2 is a novel gene that activates ATG4B . High-thThe Ser-34 residue in ATG4B can be phosphorylated by phosphofructokinase 1 platelet isoform (PFKP), a rate-limiting canonical metabolic enzyme in the glycolytic pathway . High-fiIn yeast, Atg1 kinase is an Atg protein that participates in phagophore formation. At least seven putative Atg1 phosphorylation sequences are conserved in Atg4. Atg4 is an Atg1 substrate and was phosphorylated at Ser-307 in an in vitro assay. The phosphorylation of Atg4 at Ser-307 inhibits interactions with Atg8 and cleavage activity, particularly at sites of autophagosome biogenesis. The Atg8-PE pool is critical for autophagosome formation and is partially protected by Atg4′s phosphorylation by Atg1. However, newly synthesized Atg8 is constitutively processed by Atg4 .ULK1 also mediates ATG4B phosphorylation in mammalian cells . A proteThus, several kinases bind to and phosphorylate ATG4B; however, the effects of phosphorylation on ATG4B activity are not fully understood. These findings point to the complex regulation of ATG4B activity by kinases and indicate that such phospho-regulation may be dependent on different kinases under different stress conditions .Dephosphorylation is the removal of phosphoryl groups by phosphatases. Dephosphorylation and phosphorylation activate and deactivate proteins by detaching or attaching phosphate esters and anhydrides, respectively. Thus, phosphorylation–dephosphorylation cycles are common post-translational modifications. This process involves the reversible addition of phosphoryl groups to amine moieties on serine/threonine and tyrosine residues. Atg1/ULK1 negatively regulates ATG4B activity in yeast and mammalian cells ,57. An uO-linked-N-acetylglucosaminylation (O-GlcNAcylation) is a glycosylation process that attaches a monosaccharide, O-GlcNAc, to serine or threonine residues [O-GlcNAcylation and phosphorylation. O-GlcNAcylation influences various cellular processes, including nutrient sensing, metabolism, and transcription [O-GlcNAcylation is regulated by O-GlcNAc transferase (OGT) and is reversed by O-GlcNAcase (OGA) [O-GlcNAcylation is associated with several illnesses, including neurodegenerative diseases, diabetes, and cancer, all of which are also associated with autophagy disorders [O-GlcNAcylation plays a role in sensing nutrients and stress conditions under which autophagy is initiated [O-GlcNAcylation of ATG4B was elevated following treatment with PugNAc, as well as under low-glucose conditions. A luciferase assay showed that O-GlcNAcylation increased LC3 cleavage activity. Specific target residues for ATG4B O-GlcNAcylation have not been identified, although mass spectrometry analyses showed several possible target residues, suggesting that ATG4B is O-GlcNAcylated at multiple residues following autophagic activation [residues ,62, suggcription . O-GlcNAse (OGA) ,63. Dysrnitiated ,65. Treanitiated . Bimolectivation 2O2, are generated in a partial class III PI3K-dependent manner [2O2 at a cysteine residue near the ATG4 proteolytic residue. Of the 12 cysteine residues in ATG4A, Cys-81 is an oxidative target for the redox regulation of ATG4A [2O2 treatment and restored by N-acetyl-L-cysteine treatment in an in vitro ATG4B proteolytic assay, suggesting that ATG4B is reversibly redox-regulated [ROS are signaling molecules involved in growth, differentiation, adhesion, and autophagy. Both free radicals and ROS induce oxidative post-translational modification (Ox-PTM), including protein hydroperoxides and hydroxylation of aromatic groups and aliphatic amino acid chains . Ox-PTM t manner . Variousof ATG4A . ATG4B iof ATG4A . ATG4B aegulated . To idenegulated .S-nitrosylation is a PTM that reversibly adds a nitric oxide (NO) group to cysteine residues to generate S-nitrosothiols (SNOs) in response to redox-mediated reactions ,68. S-niCaenorhabditis elegans increased the levels of LLG-1/LC3:GFP puncta. Infection with group A Streptococcus was lower in RNF5-/- mice because autophagosome formation increased and mediated the clearance of bacteria by RNF5-/- macrophages. Overall, ATG4B is negatively regulated by RNF5 in a ubiquitin-dependent manner [Ubiquitination is an enzymatic post-translational modification in which a small ubiquitin protein (8.5 kDa) is attached to substrate proteins. Ubiquitination affects proteins in various ways, including degradation in the proteasome system, alterations in cellular location-affecting activity, and promotion or prevention of protein interactions ,70. Ubiqt manner .63K, a canonical caspase sequence. This sequence is also conserved in ATG4C, but not in ATG4A or ATG4B. Caspase-3 can cleave ATG4D, but not a ATG4D DEVA63K mutant. Furthermore, caspase-3-dependent cleavage of ATG4D enhances delipidation and the activity of its main substrate, GABARAP-L1. The caspase-cleavage-mimicking mutant, ΔN63 Atg4D, further enhanced GAPARAP-L1 delipidation in cells, while ATG4D depletion decreased GAPARAP-L1 processing and autophagosome formation. The overexpression of cleaved ATG4D localized to mitochondria affects the mitochondrial structure and reduces the mitochondrial cristae. This process results in increased ROS production and mitochondrial clearance (mitophagy) [Proteolytic cleavage is an irreversible process mediated by proteases . Autophatophagy) ,21. Noneautophagy plays an essential role in maintaining homeostasis, dysregulation of ATG4 is associated with multiple human diseases, including cancer. Understanding the complex post-translational modifications of ATG4 will facilitate the development of a therapeutic strategy for autophagy-associated diseases.Our understanding of autophagy has improved in recent years. Autophagic regulation is critical for maintaining cellular homeostasis under various stress conditions. Many ATG proteins are regulated through post-translational modifications, affecting their ability to modulate autophagy under various stress conditions. ATG4 plays a prominent role in autophagy regulation because it participates in the cleavage of both pro-ATG8 and ATG8-II, two essential steps in autophagosome formation and recycling. However, ATG4 is regulated via multiple modifications. Elucidating how these different modifications interact and how regulation varies in different tissues and under different physiological or pathological conditions is required. As


The broad spectrum of curcumin's beneficial properties has encouraged medicinal researchers to investigate its therapeutic efficacy against diverse diseases. The clinical potential of curcumin is, however limited due to its poor pharmacodynamic/pharmacokinetic properties . 3,5-Bis(ylidene)-4-piperidone scaffolds are considered a curcumin mimic that exhibit diverse bio-properties. The current review provides a brief overview of these mimics and highlights biological activities relevant to drug development. 3,5-Diyliden-4-piperidone scaffold are considered as curcumin mimic exhibiting diverse bio-properties. The discoverer of artemisinin was granted the Nobel Prize in 2015 in recognition of contribution to medicine.1 Curcumin 2 is another famous example of a natural compound with clinical potential. Curcumin was extracted from roots/rhizomes of Curcuma longa and has earned a high reputation among medicinal chemists due to its usage in Ayurvedic medicine, as a food additive and as a dye in many Asian countries. Historically, it was first described over 4000 years ago in ancient India where its usage was associated with religious practice.2Natural products remain one of the main resources for a variety of diverse human needs. Many natural compounds are used directly as drugs or have inspired the development of potent biologically active agents for clinically use. Artemisinin 1  is a cla3 Antioxidant properties determined by the DPPH , ABTS , ROO˙ (TRAP) and O2˙ (NET) assays have also shown a free radical scavenging effect of curcumin.4 Antiproliferative properties of curcumin against many human cancer cell lines  have been reported.4,5 In addition curcumin has been shown to display anti-malarial properties against Plasmodium falciparum.6The broad spectrum of curcumin's beneficial properties has encouraged medicinal researchers into its therapeutic efficacy against diverse diseases. It has been reported to possess anti-inflammatory properties and its sodium salt derivative modulates iNOS and COX-2 (cyclooxygenase-2) gene expression in cultured RAW 264.7 cells.in vitro results obtained in pre-clinical studies, but poor in vivo activities. Thus far curcumin has only been approved as a dietary supplement.1Although an enormous amount of research effort has been invested in bringing curcumin and its analogs towards clinical use, thus far no curcumin based product has been approved for use. Over 120 clinical trials on curcumin have failed and consumed federal funds equivalent to 150 million dollars as stated in NIH reports from the last 25 years. This is in part attributed to the promising The next section highlights some of the important findings of the biological properties of curcumin reported within the last decades with focus on the promising bio-properties of curcumin and its analogs and the potential in identifying more effective hits/leads.2.7 pH instability,8 poor absorption in circulation, rapid elimination9 and photochemical degradation .10It has been reported that the major clinical limitation of curcumin is due to poor pharmacological properties such as poor pharmacodynamics/pharmacokinetics, low solubility,et al. reported that the production of the prostaglandin E2 (PGE2) was (57–62%) decreased in blood samples after 1 h oral administration in patients . Mild diarrhea and discernible toxicity was detected at doses of 0.5–3.6 g daily administration up to four months. Curcumin and its conjugates were detected in plasma and urine and also noticed in patient feces. The entire study concluded that doses of 3.6 g curcumin are recommended for more systemic pharmacological antitumor studies. Additionally, low oral bioavailability  is supported probably due to intestinal metabolism. The observed bio-properties of curcumin (anti-inflammatory and anticancer) can be attributed to its antioxidant capacity in neutral and acidic pH.11–13The bioavailability of curcumin has been intensively studied. In a phase I clinical trial, Sharma in vivo) utilizing high performance liquid chromatography. Disappearance of curcumin was noticed from the rat's plasma within one hour of dosing (i.v. 40 mg kg−1). However, it has been detected in plasma upon p.o. 400 mg kg−1 administration suggesting that the gastrointestinal tract is more exposed to the unmetabolized curcumin compared to other tissues.14Another study investigated the bioavailability of curcumin in rats (−1) with piperine (20 mg kg−1) in rats, with a significant reduction in the half time of clearance. Similar observations were noticed in human volunteers with slight differences. Time to reach curcumin maximum serum concentration was shorter (earlier) in humans than rats, presumably due to physiological differences. Also, the elimination (clearance) time was shorter in humans than rats.15Low solubility is one of the major limiting parameters for curcumin's therapeutic use. Different approaches have been considered to overcome this problem and improve bioavailability. Piperine  has beenp-hydroxybenzoic acid and l-tartaric acid)   in diffe17 Curcumin encapsulation in liposomes modified with DDAB (didecyldimethylammonium bromide) was studied using cervical cancer cell lines . It was observed that the uptake of DDAB liposomes was better than the non-modified ones but more toxic. Additionally, curcumin was released at a faster rate from cationic DDAB liposomes presumably due to the decrease in interaction of the lipid chains as a result of cationic charges.18Another study mentioned the possibility of water solubility enhancement through nano-vehicles with curcumin encapsulated in liposomes, exosomes, dendrimers and micelles.19 Other studies also adopted bioavailability enhancement of curcumin via combination with cyclodextrin,20 conjugation with biopolymers21 or composite nanoparticles.22Sodium salt curcumin diacetate  was also3.9Many efforts have been directed towards designing novel bio-active agents of enhanced potency and better bioavailability to overcome the drawbacks of curcumin. Manipulation of curcumin chemical structure is usually focused on the aryl rings, carbonyl groups, active methylene or the carbon linker . Most of- Modification of the main curcumin skeletal entities.- Conjugation with other moieties.- Curcumin mimics.4.via alkylation of the appropriate aldehyde with chloroacetic acid in the presence of NaOH followed by reaction with glycine ethyl ester hydrochloride. Reaction with 2,4-pentandione followed by hydrolysis with methanolic NaOH afforded the targeted agents 3. The synthesized water-soluble agents displayed enhanced antiproliferative properties (MTT assay) against HeLa  cells (IC50 = 0.5 μM for both the synthesized agents) relative to curcumin (IC50 = 4.33 μM) with induction of p53 activity, p21 expression and mediated apoptosis. The p53 is the tumor suppressor protein capable for induction of genes controlling cell cycle and apoptosis. It usually arrests the cell cycle at G2/M phase/transition affecting cyclin dependent kinase (Cdc2) necessary for mitosis.23Curcumin connected to amino acid sodium salts 3 were reported as water-soluble agents. ter-butyloxycarbonyl) protected analogs  and S. typhi, P. aeruginosa (Gram-negative) bacteria with potency higher than norfloxacin and ciprofloxacin .24A series of curcumin–amino acid conjugates 4 were synthesized through reaction of curcumin 2 with the corresponding protected amino acid (benzyloxycarbonyl “Cbz” and fluorenylmethyloxycarbonyl “Fmoc”) in the presence of EDAC [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide] and DMAP (4-dimethylaminopyridine) at −5 to 0 °C. The unprotected curcumin-amino acid conjugates 5 were obtained from the Boc  condensation of the appropriate aldehyde with acetone were found to be potent inhibitors (MTT assay) on a variety of human pancreatic cancers  and apoptosis inducers relative to curcumin26 .2 complex with the appropriate aldehyde under nitrogen atmosphere in ethyl acetate in presence of n-butylamine (3CO)3C6H2; 3,4,5-(H3CO)3C6H2; 3,4-(H3CO)2C6H3; 3-(H3CO)-4-(H3COO)C6H3] showed notable antiproliferative properties through NCI  screening program (NCI-60) against a variety of human cancer cell lines (SRB technique).27Curcuminoid-difluoroborons 9 were synthesized through the reaction of acetyl acetone–BFtylamine . Some of28Multi-component Biginelli reaction of curcumin with furochromone carbaldehyde 10 and amines, hydrazines, hydroxylamine hydrochloride, urea or thiourea afforded the corresponding furochromone-containing heterocycles 11–14 . All the2Cl2 was used for Boc group removal -3-hydroxy-1-(2-hydroxyphenyl)-3-phenylprop-2-ene-1-one 16 (mimicking curcumin) (31The antiproliferation properties of (urcumin)  was obseE,4E)-1,5-Bis-penta-1,4-dien-3-one  is the most potent analog synthesized against the breast cancer cell lines tested .32Bis(arylidene)monocarbonyl compounds 17–19 were synthesized as modified curcuminoid analogs comprising one ketonic group conjugated with two olefinic groups  through E,4E)-1-aryl-5-(hydroxyphenyl)penta-1,4-dien-3-ones with 3--5,7-dimethoxy-2--4H-chromen-4-one in DMF containing K2CO3 -3-oxo-5-(pyridin-2-yl)penta-1,4-dien-1-yl)phenoxy)propoxy)-2--4H-chromen-4-one, showed the most promise against gastric cancer cell lines  relative to doxorubicin (standard reference) with inhibitory properties against TrxR (thioredoxin reductase) observed.33A series of curcumin mimics conjugated with chromen-4-one analogs 20 were synthesized through reaction of (1ng K2CO3 . Some of5.5.1.E)-2-fluorobenzylidene)piperidin-4-one 21 -4-piperidones 22 with alkylaminomethyl substituent  revealedN-Acryloyl-3,5-bis(ylidene)-4-piperidones 23 were synthesized through acid catalyzed condensation (AcOH/HCl gas) of 4-piperidone hydrate hydrochloride with the appropriate aldehyde followed by reaction with acryloyl chloride in CH2Cl2 containing TEA (triethylamine) at 0 °C. However, 3,5-bis(ylidene)-1-[N-(aryl)maleamoyl]-4-piperidones 24 were obtained through reaction of maleamic acid  and the corresponding 3,5-bis(ylidene)-4-piperidones in THF (tetrahydrofuran) containing ethyl chloroformate and TEA  and in vivo (mice) testing. Some of the synthesized agents showed potent inhibitory properties of topoisomerase IIα. This enzyme facilitates DNA replication by preventing the buildup of supercoils during replication fork progression. Inhibitors of this enzyme result in the generation of multiple DNA strand breaks, arrest cell division and eventually lead to apoptosis. As such numerous topoisomerase inhibitors are currently used in clinics as chemotherapy for numerous malignancies. The synthesized agents also revealed antioxidant properties.36 and TEA . Most ofN-arylsulfonyl-3,5-bis(arylidene)-4-piperidones 25 were synthesized through base-catalyzed arylsulfonation  of the corresponding N-unsubstituted piperidones -4-piperidones 26–29 were also reported  with antd]pyrimidines 30 were synthesized through reaction of the corresponding fluoro-containing N-arylsulfonyl-3,5-bis(arylidene)-4-piperidones with guanidine hydrochloride in ethanolic KOH  and A431 (skin/squamous) cancer cell lines were exhibited by 1--3,5-bis(ylidene)-4-piperidinones 31  relativevia reaction of isocyanate with the appropriate N-unsubstituted 3,5-diylidene-4-piperidine in DMF in the presence of TEA propionyl]-4-piperidones 33 and their thio-analogs 34 were synthesized through reaction of the acyl chloride with the appropriate 3,5-bis(ylidene)-4-piperidone in CHectively . Antipro43The piperidone-salicylate conjugates 37 were synthesized through dehydrohalogentation of acetylsalicylic acid chloride with the appropriate piperidone in DMF containing TEA as basic catalyst . Potent E)-arylidene]-1-[3-(4-methylpiperazin-1-yl)alkyl]piperidin-4-ones 38 were prepared through acylation of the appropriate 3,5-bis(ylidene)-4-piperidones followed by dehydrohalogenation via reaction with N-methylpiperazine  -4-piperidones connected with 1,2,3-triazolyl heterocycle bearing phosphonate group 39 were obtained t2O·BF3) . Promisivia reaction of α-amino(aryl)methyl phosphonates  with aromatic aldehyde in the presence of LiClO4/Et3N (Lewis acid) -4-piperidones attached to diethyl[(aryl)methyl]phosphonate moiety 40 were synthesized is acid) . NoticeaH-indole-3,2′-pyrrolidine-3′,3′′-piperidines] 41 were synthesized through dipolar cycloaddition of the appropriate 3,5-bis(ylidene)-4-piperidones with azomethine ylide (which obtained in situ through reaction of sarcosine with isatins) (47–49A series of dispiro[3isatins) . The ste50Dispiro-analog bearing a 1-[(4-morpholinyl)methylene] group 42 was also synthesized through azomethine dipolar cycloaddition reaction with the appropriate piperidone . Quantum5.2.51,52Alzheimer's disease is a neurodegenerative disease comprising the main cause of dementia particular in elder people. One of the most common methods for treatment is the elevation of acetylcholine levels in the brain. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are two enzymes that exist in the central nervous system capable of hydrolyzing acetylcholine (neurotransmitter at cholinergic synapses). For this reason compounds with inhibitory properties against these enzymes are valuable for controlling progress of this disease. A series of 3,5-bis(ylidene)-4-piperidones 43  showed pd]pyrimidines 44 obtained through reaction of 3,5-bis(ylidene)-4-piperidones with thiourea in presence of NaOEt. Reaction of 44 with phenacyl bromides afforded the formation of pyridothiazolopyrimidines 45. Alternatively, 45 could be obtained directly through domino reaction of the appropriate piperidone, thiourea and phenacyl bromide in ionic liquid (1-butyl-3-methylimidazolium bromide “[bmim]Br”) under microwave irradiation -N-substituted-4-piperidones 46 with azomethine ylides formed from condensation of the appropriate isatin and sarcosine in presence of ionic liquid “[bmim]Br” (56Spiropyrrolidines 47 were synthesized bmim]Br” . Promisil-proline) with N-acryloyl-3,5-bis(ylidene)-4-piperidones 23 afforded the corresponding spiro-heterocycles 48via reaction with the acryloyl linkage rather than the exocyclic ylidene olefinic linkage (57Cycloaddition reaction of azomethine ylide (formed from condensation of 5-chloroisatin and  linkage . Stereocl-proline) with N-acryloyl-3,5-bis(ylidene)-4-piperidones 23 in refluxing MeOH in equimolar values afforded the mono-spiro-heterocycles 49 in a similar manner to that of the aforementioned formation of 48. However, reaction of azomethine ylide in double folds amount  to that of piperidones 23, the bis-spiro-heterocycles 50 were obtained due to double cycloaddition reactions with both acryloyl and ylidene linkages -4-piperidones 23 afforded the mono-spiro-pyrrolidines 51 due to cycloaddition reaction with the exocyclic ylidene linkage. However, reaction of the azomethine ylide with 23 in 2 : 1 molar value equivalent afforded the bis-spiro-pyrrolidines 52. The difference in observations of this reaction to that mentioned in l-proline). Additionally, bis-spiro-pyrrolidines 52 were obtained from mono-spiro-pyrrolidines 51 by reaction with another mol equivalent of the azomethine ylide -4-piperidones 23 with azomethine ylide generated from phenylglycine and isatin  in ionic liquid “[bmim]Br” medium afforded the mono-spiro-pyrrolidines 53 due to cycloaddition reaction with the exocyclic ylidene linkage. However, reaction of the azomethine ylide with 23 in 2 : 1 molar value equivalent afforded the bis-spiro-pyrrolidines 54. Reaction of 53 with another mol equivalent of azomethine ylide also afforded the bis-spiro-pyrrolidines 54  with 3,5-bis(ylidene)-4-piperidine afforded the dispiropyrrolidines 55 -1-(cyclopropyl)-4-piperidone 58  with various aldehydes   and TNF-α (tumor necrosis factor). The 3,5-bis-4-piperidones 57 were synthesized through acid-catalyzed condensation [HCl(gas)/AcOH] of 4-piperidones with 3-allyl-4-hydroxybenzaldehyde (56). Meanwhile, 3-aryliden-4-piperidone 59 was obtained OH/EtOH) . Piperidin vivo carrageenin-induced paw oedema of rats . Some of the tested agents revealed higher efficacy than that of indomethacin  after 3.5 h of administration.63Anti-inflammatory properties of 3,5-bis(ylidene)-4-piperidones 60  were sup642-{3,5-[Bis(ylidene)-4-oxopiperidin-1-yl]}-2-oxoethylmorpholine-4-carbodithioates 61 where synthesized through reaction of sodium morpholine-4-carbodithioate with the appropriate 1-chloroacetyl-3,5-bis(ylidene)-4-piperidones in methanol–water (1 : 1) at 60 °C . Some of65Dispiro-heterocyles 62 were synthesized by the azomethine ylide [3 + 2]-cycloaddition reaction with 3,5-bis(ylidene)-4-piperidones in refluxing EtOH . Single 5.4.Mycobacterium sp.) have been identified causing TB. Many drugs have been discovered for treating patients with TB but due to the side effects and drug resistant TB strains, novel therapeutical agents are still needed.66Infectious diseases are still one of the main human global health problems. Tuberculosis (TB) is one of the top ten human life threatening globally. It is the second cause of mortality after HIV/AIDS due to single infectious pathogen. Various pathogenic agents (M. tuberculosis H37Rv). Some of them revealed properties in the rat liver mitochondria respiration with swelling in mitochondria67 -4-piperidones 63 were identified with noticeable antimycobacterial properties  cycloaddition to 64 in refluxing methanol yielded the corresponding spiro-heterocycles 65–67. Single crystal X-ray studies supported the synthesized structures. Antimycobacterial properties were observed by the synthesized spiro-heterocycles 65–67 against gmatis68 .5.5.Candida albicans ATCC 10231. Some of the synthesized agents showed potent inhibitory properties relative to fluconazole (standard reference).69Dispiropyrrolidines 68 were synthesized through multi-component domino azomethine ylide (formed from condensation of 2-amino-3-phenylpropanoic acid and isatin) dipolar cycloaddition reaction with the appropriate 3,5-bis(arylidene)-4-piperidones in ionic liquid “[bmim]Br” . Antifun5.6.Plasmodium sp. which is a protozoan organism transmitted to humans due to mosquito bites. Although many drugs are known to combat malaria emerging drug resistance means that new effective agents are still needed.70Malaria is one of the most widely distributed infectious diseases in tropical and subtropical regions. N-methyl-4-piperidones 69 against chloroquine-sensitive Pf3D7, chloroquine-resistant PfINDO, and artemisinin-resistant PfMRA-1240 strains71 -trains71 .N-acyl-3,5-bis(ylidene)-4-piperidones 70 were synthesized via dehydrohalogenation of the unsubstituted piperidone with the corresponding acid chloride  strains which also subjected to Plasmodium berghei revealing higher efficacy than chloroquine and mefloquine (standard reference drugs).72A series of chloride . Some of5.7.2CO3 methylene]-4-piperidones 71 were synthesized through base catalyzed condensation (piperidine in refluxing toluene) of 1-ethoxycarbonyl-4-piperidine with 3-indolecarboxaldehyde followed by alkylation with various alkyl halides in refluxing DMF/K2CO3 . Lipase 6.in vivo. 3,5-Bis(ylidene)-4-piperidone scaffolds are considered a curcumin mimic with diverse promising bio-properties. The distinguished biological observations of curcumin mimics can be considered for optimizing high potent hits/leads accessible in drug discovery program. Many articles dealing with the biological properties of this scaffold have appeared. It has been also intensively utilized for construction of diverse potentially bioactive heterocycles.Curcumin is an important natural compound with broad spectrum biological properties. Limitation of clinical application of curcumin is mainly due to its poor bio-availability There is no conflict to declare.


N-acetyl-L-cysteine (NAC) as a model drug with low transferability to the spinal cord and determined the physicochemical properties of NAC/PEG-PCL-Tat. After intranasal administration of NAC/PEG-PCL-Tat, we measured the survival duration of superoxide dismutase-1 G93A mutant transgenic mice (G93A mice), widely used in ALS studies, and quantitatively analyzed the tissue distribution of NAC/PEG-PCL-Tat in ddY mice. The mean particle size and zeta potential of NAC/PEG-PCL-Tat were 294 nm and + 9.29 mV, respectively. Treatment with repeated intranasal administration of NAC/PEG-PCL-Tat considerably prolonged the median survival of G93A mice by 11.5 days compared with that of untreated G93A mice. Moreover, the highest distribution after a single administration of NAC/PEG-PCL-Tat was measured in the spinal cord. These results suggest that intranasal administration combined with PEG-PCL-Tat might represent a useful DDS for ALS therapeutics.Intranasal administration is a promising route for direct drug delivery to the brain; its combination with nanocarriers enhances delivery. We have previously shown that intranasal administration combined with PEG-PCL-Tat (a nanocarrier) efficiently delivers drugs to the brain and exhibits excellent therapeutic efficacy against brain diseases. We aimed to clarify whether intranasal administration combined with PEG-PCL-Tat represents a useful drug delivery system (DDS) for amyotrophic lateral sclerosis (ALS) pharmacotherapy. We used  SOD1G93A) are widely used as mouse models for mechanistic and therapeutic studies of ALS. Several hypotheses for ALS pathogenesis include the involvement of oxidative stress, mitochondrial dysfunction, excitotoxicity, neuroinflammation, and protein aggregation  N-Acetyl-L-cysteine  was purchased from American Radiolabeled Chemicals Inc. . All other reagents used in this study were commercial products without further purification.Methoxy poly(ethylene glycol)-block-poly(ε-caprolactone)  was purchased from Sigma-Aldrich Co. , and Tat peptide (GRKKRRQRRRG) was purchased from BEX Co., Ltd. . NAC was purchased from Sigma-Aldrich Co.  ethanesulfonic acid (HEPES) buffer (pH 7.4), respectively. NAC was dissolved in 10 mM HEPES buffer (pH 7.4) to prepare a stock solution at a concentration of 100 mg/mL. The NAC solution (20 or 100 mg/mL) was mixed by adding, in equal volumes, the PEG-PCL-Tat solution (5 or 25 mg/mL). The resulting solution was incubated at room temperature (25 °C) for 30 min to obtain 0.2 mg NAC/PEG-PCL-Tat (0.2NAC/PEG-PCL-Tat) or 1 mg NAC/PEG-PCL-Tat (NAC/PEG-PCL-Tat) as a concentration of 10 mg/mL or 50 mg/mL of NAC solution. In 0.2NAC/PEG-PCL-Tat and NAC/PEG-PCL-Tat solutions, the weight ratio of PEG-PCL-Tat (1.5 or 7.5 mg) to NAC (6 or 30 mg) was 1:4. The PEG-PCL-Tat solution (12.5 mg/mL) without NAC was prepared by diluting the PEG-PCL-Tat solution (25 mg/mL) in 10 mM HEPES buffer (pH 7.4). The NAC containing PEG-PCL-Tat solution was used in the experiment immediately after the preparation.The mean particle size, polydispersity index (PDI), and zeta potential of the PEG-PCL-Tat, 0.2NAC/PEG-PCL-Tat, and NAC/PEG-PCL-Tat solutions were measured after diluting them to an appropriate concentration range via the Zetasizer Ultra .All efforts were made to minimize the number of animals used and their distress. Animal experiments were carried out after obtaining approval from the Nihon University Animal Care and Use Committee  . Animals were housed under controlled conditions  and with ad libitum access to feed and water.Transgenic mice of the B6SJL-TgN (SOD1-G93A) 1Gur strain were used as an ALS mouse model, purchased from the Jackson Laboratory . The hemizygous G93A mice were obtained by mating male G93A mice with wild-type (WT) females. Mice were genotyped by polymerase chain reaction (PCR) using genomic DNA isolated from blood as previously described . Only maFour-week-old ddY mice  were obtained from Japan SLC, Inc.  for the pharmacokinetic investigation of the intranasal administration. Mice weighing 25–35 g were used for the experiments after being pre-fed as previously reported .Intranasal administration was performed as previously described ,33. In b2. The median survival duration is the time period taken for 50% of G93A mice to die. The mean survival time is the average amount of days that G93A mice reached the endpoint. In the group that received intranasal administration (Groups 3–6) of For survival analyses, a total of 108 male G93A mice were randomly assigned into six groups as shown in 14C]-NAC solution, which contained unlabeled-NAC equivalent to that of group 3, was prepared by adding an equal volume of 10 mM HEPES buffer (pH 7.4) into the NAC solution (25 µL). The [14C]-NAC/PEG-PCL-Tat solution (10 µCi/mL), containing 1 mg NAC, was prepared by adding the NAC solution as described above dropwise into the PEG-PCL-Tat solution and further mixed and incubated at room temperature (25 °C) for 30 min. Mice were administered either 20 µL of [14C]-NAC or [14C]-NAC/PEG-PCL-Tat solution, each containing 1 mg of NAC.NAC was dissolved in 10 mM HEPES buffer (pH 7.4) to prepare the stock solution (200 mg/mL). For radioactive tracer experiments, the [14C]-NAC or [14C]-NAC/PEG-PCL-Tat solution, each containing unlabeled-NAC (1 mg). Plasma samples (20 µL) were obtained through centrifugation .Blood samples (50 µL) were collected from the tail vein of ddY mice at 0, 3, 15, 30, 60, and 90 min after an intranasal administration of [14C]-NAC or [14C]-NAC/PEG-PCL-Tat solution to ddY mice. CSF was slowly withdrawn (approximately 20 µL) from the cisterna magna through a needle (30 G) connected to a cannula. Plasma samples (approximately 0.5 mL) were obtained through blood centrifugation , collected from the heart. Tissues  were obtained after systemic perfusion with phosphate-buffered saline (pH 7.4) using a peristaltic pump until the circulating fluid was decolored. Subsequently, each tissue sample was weighed (wet weight) and prepared as previously described [CSF and tissue samples were obtained at 60 min after an intranasal administration of [escribed .14C] in plasma, CSF, or tissues was measured using a liquid scintillation counter , as per our previous report [max) was determined as the highest value among the calculated values of %ID/mL plasma. The area under the plasma concentration–time curve (AUC0–90) was calculated by the linear trapezoidal method using the [14C]-NAC radioactivity in the plasma to the last time point (t = 90 min).The radioactivity of -NAC/PEG-PCL-Tat to ddY mice. The post-administration Cmax of [14C]-NAC/PEG-PCL-Tat was 1.81 ± 0.7% ID/mL plasma, showing a trend slightly higher than that measured following the administration of [14C]-NAC without PEG-PCL-Tat. In the [14C]-NAC/PEG-PCL-Tat group, the concentration in plasma trended higher than that measured in the [14C]-NAC group at early time points after the intranasal administration (at 10 and 13 min from administration commencement). In the elimination phase, 30 to 90 min after the intranasal administration (40 to 100 min after the start of the administration), the plasma concentrations of [14C]-NAC/PEG-PCL-Tat group showed no difference in concentration compared with that of [14C]-NAC group and both plasma concentrations decreased at a similar pace over time . The AUC0–90 in the [14C]-NAC/PEG-PCL-Tat group was approximately 75% ID/mL plasma min and was not significantly different from that of the [14C]-NAC group . In the [14C]-NAC/PEG-PCL-Tat group, the [14C]-NAC amount distributed within the spinal cord was 1.5- and 2.1-fold higher than that measured in the brain and CSF, respectively  when combined with a PEG-PCL-Tat nanocarrier. After a single intranasal administration to healthy ddY mice, the [


PTK7 is a catalytically defective receptor protein tyrosine kinase. We previously demonstrated that PTK7 enhances angiogenesis by interacting with KDR, a vascular endothelial growth factor (VEGF) receptor important for angiogenesis, and activating it through oligomerization. To control angiogenesis by inhibiting PTK7 function, we developed anti-PTK7 monoclonal antibodies (mAbs). The selected PTK7 mAbs reduced VEGF-induced angiogenic phenotypes of endothelial cells and angiogenesis ex vivo and in vivo. The PTK7 mAbs also inhibited VEGF-induced KDR activation in endothelial cells and its downstream signaling and PTK7–KDR interaction. Our results show that the PTK7 mAbs inhibit angiogenesis by blocking PTK7 function. Therefore, PTK7 mAbs could be applied as therapeutics to control angiogenesis-associated diseases such as metastatic cancers.PTK7, a catalytically defective receptor protein tyrosine kinase, promotes angiogenesis by activating KDR through direct interaction and induction of KDR oligomerization. This study developed anti-PTK7 monoclonal antibodies (mAbs) to regulate angiogenesis by inhibiting PTK7 function. The effect of anti-PTK7 mAbs on vascular endothelial growth factor (VEGF)-induced angiogenic phenotypes in human umbilical vascular endothelial cells (HUVECs) was examined. Analysis of mAb binding with PTK7 deletion mutants revealed that mAb-43 and mAb-52 recognize immunoglobulin (Ig) domain 2 of PTK7, whereas mAb-32 and mAb-50 recognize Ig domains 6–7. Anti-PTK7 mAbs inhibited VEGF-induced adhesion and wound healing in HUVECs. mAb-32, mAb-43, and mAb-52 dose-dependently mitigated VEGF-induced migration and invasion in HUVECs without exerting cytotoxic effects. Additionally, mAb-32, mAb-43, and mAb-52 inhibited capillary-like tube formation in HUVECs, and mAb-32 and mAb-43 suppressed angiogenesis ex vivo (aortic ring assay) and in vivo (Matrigel plug assay). Furthermore, mAb-32 and mAb-43 downregulated VEGF-induced KDR activation and downstream signaling and inhibited PTK7–KDR interaction in PTK7-overexpressing and KDR-overexpressing HEK293 cells. Thus, anti-PTK7 mAbs inhibit angiogenic phenotypes by blocking PTK7–KDR interaction. These findings indicate that anti-PTK7 mAbs that neutralize PTK7 function can alleviate impaired angiogenesis-associated pathological conditions, such as cancer metastasis. Receptor protein tyrosine kinases (RPTKs), which are active tyrosine kinases, play an important role in signal transduction involved in cellular processes, such as proliferation, differentiation, and metabolism . Of the PTK7 comprises an extracellular region with seven immunoglobulin (Ig)-like loops, a transmembrane domain, and a cytosolic region containing a defective tyrosine kinase catalytic domain . PreviouAngiogenesis, which is the process of formation of new blood vessels, involves protease production for extracellular matrix degradation, chemotactic migration and proliferation of endothelial cells, and capillary-like tube formation induced by angiogenic factors, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor  . As the PTK7 inhibits VEGF-induced migration, invasion, tube formation, and angiogenesis in vivo [Treatment with high concentrations of soluble PTK7  as a decoy receptor or a knockdown of  in vivo . PTK7 pr in vivo . Additio in vivo . In peri in vivo . TherefoTo develop an agent that inhibits PTK7 function with in vivo stability, anti-PTK7 monoclonal antibodies (mAbs) were produced in this study. The ability of anti-PTK7 mAbs to neutralize PTK7-dependent angiogenic phenotypes was examined. The effects of anti-PTK7 mAbs on VEGF-induced adhesion, migration, invasion, and tube formation in human umbilical vein endothelial cells (HUVECs) in vitro and angiogenesis ex vivo and in vivo were analyzed. Additionally, the effects of anti-PTK7 mAbs on the VEGF-induced signaling pathway and the PTK7–KDR interaction were examined. The findings of this study indicate that anti-PTK7 mAbs inhibit angiogenesis and that they have potential therapeutic applications for impaired angiogenesis-associated diseases.2, and 95% air.Human embryonic kidney 293 (HEK293) cells were obtained from Korean Cell Line Bank  and cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% bovine serum , 100 U/mL penicillin, and 100 μg/mL streptomycin. HUVECs were purchased from Zenbio  and cultured in Medium 199 (Gibco) supplemented with 20% fetal bovine serum , 5 U/mL heparin , and 3 ng/mL human bFGF . HUVECs were used at passages 5–10 for the experiments. All cells were cultured at 37 °C, 5% COAntibodies were purchased from the following vendors: Santa Cruz Biotechnology , anti-phospho-ERK (sc-7383) and anti-FAK (sc-557) antibodies; Cell Signaling Technology , anti-phospho-KDR , anti-KDR (2479S), anti-phospho-Src family Tyr416; 2101S), anti-Src (2109S), anti-phospho-JNK , and anti-JNK (9252S) antibodies; Merck Millipore , anti-phospho-FAK antibodies ; Bioss , anti-ERK2 antibodies (bms-52068R); Sigma-Aldrich, anti-FLAG-M2 antibodies (F1804); BioLegend , anti-HA antibodies (902302); Qiagen , anti-penta-His antibodies; AbClone , anti-GAPDH antibodies (abc2003); KOMA Biotech , horseradish peroxidase-conjugated goat anti-mouse-IgG and anti-rabbit-IgG antibodies. Rabbit anti-PTK7 anti-serum has been previously described [01S, antiEcoRI and XbaI and ligated into the pcDNA3.1 vector that was digested with EcoRI and XbaI. The pcDNA3.1-hPTK7-Ig1-4-His construct encoding human PTK7-Ig1-4-His was generated using DpnI-mediated deletion mutagenesis with the primer pair Ig1-4-His-F and Ig1-4-His-R  has been previously described . The pcD-4-His-R  and the ectively , and the2HPO4, 2.7 mM KCl, and 2 mM KH2PO4; pH 7.4) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 mM ethylenediaminetetraacetic acid (EDTA) and dialyzed against PBS. His-tagged proteins were purified using Ni2+-NTA agarose .The expression constructs for His-tagged sPTK7 and sPTK7 domains were transfected into HEK293 cells using the calcium phosphate method, following a previously reported protocol . To seleMouse anti-PTK7 hybridoma cell lines were established using purified human sPTK7 as the antigen . Anti-PTK7 mAbs were purified from ascites obtained via intraperitoneal injection of hybridomas into mice (AbClone).2+-NTA agarose resins . Protein-bound resins were washed twice with PBS containing 0.1% Tween 20. Pulled-down proteins were resuspended in sodium dodecyl sulfate (SDS) sample buffer and subjected to Western blotting.Purified sPTK7-His and its deletion domains were incubated with anti-PTK7 mAbs at a 1:1 molar ratio for 2 h at 4 °C and pulled down with NiWestern blotting was performed as described previously . Briefly4 cells/100 μL) was pre-treated with anti-PTK7 mAbs (10 μg/mL) or human sPTK7 (4 μg/mL) for 30 min at 25 °C and loaded into gelatin-coated 96-well plates. Then, the cells were incubated with 10 ng/mL human VEGF (KOMA Biotech) for 1 h. Next, the cells were fixed with 3.7% paraformaldehyde in PBS and stained with 0.005% crystal violet. The stained cells were lysed with 1% SDS. The absorbance of the mixture at 600 nm was measured.HUVECs were starved for 6 h in M199 medium containing 1% FBS and the cells were resuspended in the same medium. The cell suspension (1 × 10The monolayer of HUVECs grown in 12-well plates was starved in the M199 medium containing 1% FBS for 6 h. A scratch was introduced into the monolayer using a micropipette tip. The cells were washed to remove debris and pre-treated with anti-PTK7 mAbs (10 μg/mL) or human sPTK7 (4 μg/mL) in M199 medium with 1% FBS. Next, the cells were incubated with 10 ng/mL human VEGF for 14 h and observed under a light microscope.HUVECs were starved in the M199 medium containing 1% FBS for 6 h. Chemotactic migration and invasion assays were performed as described previously  with min5 cells/well of a 24-well plate) were pre-treated with anti-PTK7 mAbs (10 μg/mL) or sPTK7 (4 μg/mL) for 30 min and loaded on a 24-well plate whose wells were pre-coated with 300 μL of growth-factor-reduced Matrigel . The cells were then incubated with a final 20 ng/mL human VEGF at 37 °C for 16 h and analyzed using light microscopy. The number and length of capillary-like tubes were quantified using ImageJ angiogenesis analyzer [Capillary-like tube formation assays were performed as previously described . Brieflyanalyzer .The aortic ring assay was performed as described previously . The thoThe Matrigel plug assay was performed as described previously . Briefly3VO4 and 5 mM NaF. Cell lysates were resuspended in SDS sample buffer and subjected to Western blotting. Protein bands from Western blots were quantified with ImageJ.Subconfluent HUVECs were starved in the M199 medium supplemented with 1% FBS for 6 h. The cells were pre-incubated with anti-PTK7 mAbs (10 μg/mL) or sPTK7 (4 μg/mL) for 30 min. Next, the cells were stimulated with 10 ng/mL human VEGF for 2 min to analyze receptor phosphorylation, 1 h to analyze the phosphorylation of FAK ,32, or 1The lentiviral transfer vector pHRST-hPTK7-FLAG-IRES-eGFP encoding human PTK7 with a C-terminal FLAG tag has been previously described . Lentivi3VO4, and protease inhibitor cocktail III . The lysates were incubated with mouse anti-FLAG M2 antibodies (Sigma-Aldrich) for 2 h. The protein-bound resins were then washed with NP-40 lysis buffer. Pulled-down proteins were resuspended in SDS sample buffer and subjected to Western blotting.Subconfluent HEK293 cells co-expressing PTK7-FLAG and KDR-HA were incubated with anti-PTK7 mAbs (10 μg/mL) or sPTK7 (4 μg/mL) for 2 h. Cells were lysed with NP-40 lysis buffer (50 mM Tris–HCl (pH 7.4), 150 mM NaCl, and 1% NP-40) containing 5 mM NaF, 1 mM Nat-test. All data obtained from at least three independent experiments are expressed as mean ± standard deviation. Differences were considered significant at p < 0.05.The means between the two groups were compared using Student’s To analyze the PTK7-binding domains of anti-PTK7 mAbs, a pull-down assay was performed using anti-PTK7 mAbs. The following deletion mutants of the extracellular region of PTK7  were used for the analysis: PTK7-Ig1-5-His, PTK7-Ig1-4-His, PTK7-Ig1-3-His, PTK7-Ig2-4-His, and PTK7-Ig3-4-His A. mAb-32The effect of anti-PTK7 mAbs on angiogenic phenotypes  in HUVECs was analyzed. As high concentrations of sPTK7 inhibit angiogenic phenotypes ,24, sPTKThe effect of anti-PTK7 mAbs on angiogenesis in vitro was analyzed using the capillary-like tube formation assay. VEGF (20 ng/mL) induced capillary-like tube formation in HUVECs cultured on Matrigel. mAb-32, mAb-43, mAb-52 (10 μg/mL), and sPTK7 (4 μg/mL) significantly decreased the VEGF-induced tube formation to 55.2% ± 9.3%, 49.4% ± 3.8%, 49.4% ± 1.3%, and 45.2% ± 5.0% based on tube numbers, and to 54.7% ± 10.8%, 51.4% ± 8.5%, 66.9% ± 11.4%, and 48.9% ± 6.5% based on tube length, respectively .The effect of anti-PTK7 mAbs on ex vivo angiogenesis was evaluated using the mouse aortic ring assay. Treatment with 20 ng/mL VEGF induced sprouting and outgrowth of endothelial cells from the aortas. The endothelial cell outgrowth in the mAb-32-, mAb-43- (10 μg/mL), and sPTK7 (4 μg/mL)-treated groups was inhibited to 29.9% ± 3.7%, 21.6% ± 6.3%, and 21.7% ± 11.1%, respectively .To examine the effect of anti-PTK7 mAbs on angiogenesis in vivo, a Matrigel plug assay was performed. Treatment with mouse VEGF resulted in plugs with a dark red color, indicating induction of angiogenesis. Co-treatment with 3 μg/mL mAb-32 or mAb-43 and VEGF resulted in plugs with an orange or pale red color. In contrast, co-treatment with 10 μg/mL of mAb-32 or mAb-43 and VEGF resulted in plugs with a white or yellow color A. The deAngiogenesis is mediated by various signaling pathways, including ERK and JNK signaling pathways involved in cell proliferation and differentiation and FAK and Src signaling pathways involved in cell adhesion and migration ,36. TherNext, the effect of anti-PTK7 mAbs on PTK7–KDR interaction was examined. PTK7–KDR interaction in HEK293 cells expressing PTK7 and KDR was analyzed by co-precipitation of KDR-HA with PTK7-FLAG after treatment with mAb-32 or mAb-43. Previously, we had reported that sPTK7 (4 μg/mL) decreased the PTK7–KDR interaction by competing with PTK7 ,24. CompPTK7 knockdown inhibits the angiogenic functions of endothelial cells and angiogenesis in vivo [As PTK7 is reported to be involved in oncogenesis and angiogenesis, it is a potential therapeutic target for cancer and vascular diseases. However, a small-molecule tyrosine kinase inhibitor targeting PTK7 cannot be designed as PTK7 is a catalytically defective RPTK. To suppresses the oncogenic function of ERBB3 , which is another catalytically defective RPTK, an anti-ERBB3 antibody was developed and is currently undergoing clinical trials as an anti-cancer drug . Previou in vivo . Additio in vivo . As the To determine the PTK7 subdomains to which the selected anti-PTK7 mAbs  bind, this study constructed deletion mutants of PTK7 and evaluated their abilities to bind to mAbs. mAb-43 and mAb-52 recognized the PTK7 Ig2 domain, whereas mAb-32 and mAb-50 recognized the PTK7 Ig6–7 domains.Angiogenesis involves adhesion, migration, and invasion following chemotaxis of endothelial tip cells . PreviouIt was known that endothelial cells can differentiate into a tubular structure, mimicking a capillary, on Matrigel in vitro . HoweverDana and colleagues reported that PTK7 knockdown decreased VEGF-induced phosphorylation of FLT-1 but not KDR, in mouse endothelial MS1 cells, and that PTK7 interacted with FLT-1 but not with KDR or FLT-4 . In contTo examine the mechanism underlying mAb-mediated inhibition of KDR activation, the binding of PTK7 to KDR was examined in the presence of anti-PTK7 mAbs. mAb-32 and mAb-43 inhibited the interaction between PTK7 and KDR. Previously, we had demonstrated that PTK7 promotes KDR activation by inducing KDR oligomerization . AdditioTumorigenesis can be inhibited by blocking angiogenesis. Several anti-angiogenic drugs that specifically target the VEGF signaling pathway have been developed , and theWhereas endothelial cells have decreased somatic mutation burdens, tumor endothelial cells exhibit increased genetic instability owing to angiogenic factors, cytokines, and growth factors secreted into the tumor microenvironment ,55. TherIn addition to its role in KDR activation, PTK7 enhances FGFR1 activation in the absence of ligands or the presence of aFGF or bFGF . TherefoIn addition to anti-angiogenic drugs, including mAbs and tyrosine kinase inhibitors, antibody–drug conjugates (ADCs) that target tumor vasculature have been reported ,64. PF-0PTK7, a catalytically defective RPTK, promotes angiogenesis by activating catalytically active RPTKs, such as KDR and FGFR1. To regulate angiogenesis by blocking PTK7 function, this study developed anti-PTK7 mAbs and analyzed their effects on angiogenesis. The selected anti-PTK7 mAbs inhibited VEGF-induced angiogenic phenotypes, including endothelial cell adhesion, migration, invasion, and tube formation, as well as angiogenesis ex vivo and in vivo. Additionally, anti-PTK7 mAbs downregulated the VEGF-induced activation of KDR and its downstream signaling proteins and inhibited the interaction of PTK7 with KDR. These results suggest that anti-PTK7 neutralizing mAbs have potential applications for the treatment of aberrant angiogenesis-associated diseases, such as cancer metastasis.


We have recently shown that VEGF165b inhibits ischemic angiogenesis by blocking VEGFR1, not VEGFR2 activation. Here we wanted to determine whether VEGF165b inhibition using a monoclonal isoform-specific antibody against VEGF165b vs. control, improved perfusion recovery in preclinical PAD models that have impaired VEGFR2-NO signaling, including (1) type-2 diabetic model, (2) endothelial Nitric oxide synthase-knock out mice, and (3) Myoglobin transgenic mice that have impaired NO bioavailability. In all PAD models, VEGF165b inhibition vs. control enhanced perfusion recovery, increased microvascular density in the ischemic limb, and activated VEGFR1-STAT3 signaling. In vitro, VEGF165b inhibition vs. control enhanced a VEGFR1-dependent endothelial survival/proliferation and angiogenic capacity. These data demonstrate that VEGF165b inhibition induces VEGFR1-STAT3 activation, which does not require increased NO to induce therapeutic angiogenesis in PAD. These results may have implications for advancing therapies for patients with PAD where the VEGFR2-eNOS-NO pathway is impaired.Nitric oxide (NO) is the critical regulator of VEGFR2-induced angiogenesis. Neither VEGF-A over-expression nor L-Arginine (NO-precursor) supplementation has been effective in helping patients with Peripheral Artery Disease (PAD) in clinical trials. One incompletely studied reason may be due to the presence of the less characterized anti-angiogenic VEGF-A (VEGF Atherosclerotic occlusions that develop in the lower extremities can result in a moderate to severe decrease in blood flow, resulting in tissue ischemia, which causes Peripheral Artery Disease (PAD). PAD is a large contributor to the global cardiovascular disease burden with >200 million patients worldwide . Approxi165b isoform production in ischemic muscle over pro-angiogenic VEGF165a isoform [165b isoforms occur by alternative splicing in the 8th exon of VEGF-A. These isoforms are considered to be ‘inactive competitive inhibitors’ of VEGFR2 signaling that act by competing with pro-angiogenic VEGF-A isoforms for binding sites on VEGFR2 [165a, VEGF165b is a weak agonist of VEGFR2 that can induce VEGFR2-phosphorylation, but not downstream signaling, needed to elicit an angiogenic response. As such VEGF165b was thought to be an ‘inactive blocker’ of VEGF-A-induced VEGFR2 signaling [165b–VEGFR1 interactions in regulating endothelial function in cardiovascular pathologies [165b inhibits ischemic angiogenesis by decreasing VEGFR1 activation [165b–VEGFR1 interactions in regulating microvascular hydraulic conductivity in rat mesenteric vessels.One of the major mechanisms by which VEGF-A regulates angiogenesis is by activating VEGFR2-AKT-ERK-eNOS signaling, which produces NO to induce angiogenesis. However, despite its ability to induce a strong pro-angiogenic phenotype in vitro and in vivo PAD models, none of the VEGF-A clinical trials or NO supplementation by L-arginine showed any clinical benefit in PAD ,5,6,7. A isoform ,9. The an VEGFR2 . Previouignaling ,12,13. Lhologies . We havetivation  showed t165b binding to VEGFR2 correlated with increased VEGFR2 activation, whereas increased VEGF165b binding to VEGFR1 correlated with decreased VEGFR1 activation vs. non-PAD controls, and (2) in in vitro competition studies, exogenous VEGF165b-induced VEGFR2 activation almost to the same extent as VEGF165a in ECs and (3) VEGF165b inhibition-induced VEGFR1 activation without changes in VEGFR2 activation [165b treatment did not increase the angiogenic potential of normal or ischemic endothelial cells [165b using an isoform-specific monoclonal antibody against VEGF165b isoform-induced angiogenesis by activating VEGFR1 signaling [165b induces VEGFR2 activation in pulmonary arterial endothelial (PAE) cells and Chinese Hamster Ovarian (CHO) cells that express VEGFR2, respectively [165b in ovarian cancer cell lines, including SKOV3 and OVCAR3, inhibits VEGFR2 signaling. However, the role of VEGF165b in regulating VEGFR1 vs. VEGFR2 signaling in ischemic endothelial cells is not clear.The rationale of this study is based on our data that (1) in human PAD muscle biopsies increased VEGFtivation . Despiteal cells . Whereasignaling ,15. Consignaling  and Cateignaling  also shoectively ,13. On t165b inhibition has the ability to induce ischemic angiogenesis even when VEGFR2-eNOS-NO signaling is impaired e.g., diabetic-PAD [Based on these data, we questioned whether VEGFR1 activation achieved by VEGFetic-PAD ,18. We uetic-PAD ,18, (2) etic-PAD ), and  in iHuman Umbilical Vein endothelial cells (HUVECs) were purchased and cultured in an all-in-one complete endothelial growth medium that contains 5 mM Glucose (Cat No.: 211-500) from Cell Applications Inc. . HUVECs were purchased from Cell Applications and cultured in HUVEC growth medium . C57BL/6165a (Cat No.: 293-VE) and recombinant human/murine VEGF165b (Cat No.: 3045-VE) were purchased from R&D . Recombinant mouse VEGF165a (Cat No.: 200-34) was purchased from Shenandoah Biotechnology . STATTIC (Cat No.: 27-981-0) was purchased from Tocris Biosciences . BSA-Palmitate Saturated Fatty Acid Complex  was purchased from Cayman Chemicals .Recombinant human VEGFNon-targeting (Cat No.: D-001206-13-20) and VEGFR1 siRNA (Cat No.: M-003136-03-0005) were purchased from Horizon Discovery . RNAiMax (Cat No.: 13778150) was purchased from ThermoFisher Scientific . HUVECs were transfected with 150 nM control or VEGFR1 siRNA using RNAiMAX for 48 h according to the manufacturer’s instructions.165b (Cat No.: MAB3045), used in Western blotting as well as to block VEGF165b in vitro and in vivo, was purchased from R&D, Minneapolis, MN, USA. PlGF antibody  was a kind gift from Dr. Yao Liang Tang, Vascular Biology Center, Augusta University.Antibodies against pVEGFR1 , pVEGFR2 , and VEGFR2 (Cat No.: SAB4501643) were purchased from Sigma-Aldrich, St. Louis, MO, USA; Flt1  and VEGF-A (Cat No.: SC-7269) were purchased from Santa Cruz Biotechnology Inc., Dallas, TX, USA; VEGFR1 (Cat No.: PA5-16493) and CD31 (Cat No.: MA3105) purchased from ThermoFisher Scientific, Grand Island, NY, USA; VEGFR2 (Cat No.: 55B11), pAKT (Cat No.: 4060), AKT (Cat No.: 4691), pERK1/2 (Cat No.: 4370), ERK1/2 (Cat No.: 9102), pSTAT3 (Cat No.: 9145), and STAT3 (Cat No.: 12640) were purchased for Cell Signaling and Technology, Danvers, MA, USA; eNOS (Cat No.: 610299) was purchased from BD Pharmingen, San Jose, CA, USA; peNOS (Cat No.: 66127) from Abcam, Cambridge, MA, USA; VEGF-B (Cat No.: MAB751) and VEGF165a (50 ng/mL) or VEGF165b (50 ng/mL) for 10 min under normal growth conditions. HUVECs were challenged with hypoxia serum starvation for 24 h followed by treatment with VEGF165a (50 ng/mL) or VEGF165b (50 ng/mL) for 10 min.Confluent endothelial cells were treated with VEGFCells were incubated in an endothelial starvation medium purchased from Cell applications Inc. that contained 5 mM Glucose (Cat No.: 209-250) and were subjected to hypoxia (2% O2) for 24 h ,15.Confluent ECs were treated with varying concentrations of STATTIC ranging from 0.1 μM to 1.0 μM for 24 h under normal growth conditions or HSS. Control cells received the same volume of DMSO as 1.0 μM STATTIC.Confluent ECs were treated with 500 μM BSA-Palmitate under HSS conditions for 24 h. Control cells received the same amount of BSA matching the amount of BSA-Palmitate.As an in vitro model for diabetic-PAD, HUVECs were incubated with 500 μM BSA-Palmitate in endothelial starvation medium and challenged with 2% hypoxia for 24 h. HUVECs treated with 500 μM BSA-Palmitate in a normal growth medium under normal growth conditions were used as a model for diabetes. Cells treated with an equal volume of BSA matching BSA-Palmitate treatment were used as a control in these experiments.Endothelial cells post-treatments were trypsinized and the cell number was quantified using Corning CytoSmart cell counter. An equal number of cells  were plated on growth factor reduced matrigel  to avoid any interference from VEGF-A present in the matrigel. Capillary-like tubes formed on the matrigel were photographed at 1 h, 2 h, and 3 h at the center concave, and the number of nodes was quantified by at least 2 people who were blinded to the treatments using NIH Image J Angiogenesis Analyzer ,22.Endothelial cells were plated in 96 well plates at equal density. Post-treatment, cells were incubated with CCK9 at 1–2 h according to the manufacturer’s instructions. Post incubation, the plates were colorimetrically read at 450 nM using a BioTek Synergy LX Multi-Mode Plate Reader.All animal experiments were approved by the Augusta University IACUC and conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. C57BL/6 mice on 60 kCal (high-fat diet) for 4 months were purchased from Jackson laboratories (DIO C57BL/6J Mice). Only males on a high-fat diet were used in T2D-PAD studies. Fasting glucose levels in these mice on a high-fat diet were measured by performing a glucose tolerance test using One Touch Ultra Glucometer and Strips according to our previous publications ,24. The HLI was performed as described previously ,16. A coThe extent of necrosis was scored as follows: Grade I—involving only toes, Grade II—extending to dorsum pedis, Grade III—extending to crus, and Grade IV—extending to the thigh or complete necrosis ,15.Gastrocnemius muscle tissues were fixed in 4% paraformaldehyde overnight at 4 °C, followed by RT for another 24 h. Later, tissues were incubated in 30% sucrose for ~24 h (until the tissues sink to the bottom) and embedded in OCT for cryosectioning. Thick cryosections of 5 µM were processed for immunostaining 12–14. Briefly, sections were washed in PBS twice followed by antigen retrieval in citrate buffer , blocked in 5% goat serum for 1 h, followed by incubation in CD31 antibody overnight at 4 °C. Later, sections were washed (PBS) and incubated in secondary antibodies conjugated to AlexaFluor-555  for 2 h at RT. Sections were washed (PBS) and mounted using Prolong gold anti-fade mounting media . Immunofluorescent images were obtained using an EVOS-M5000 fluorescent microscope.® TUNEL Alexa Fluor® 488 Imaging Assay (Cat No.: C10245) purchased from ThermoFisher Scientific, Grand Island, NY, USA, according to the manufacturer’s instructions, followed by immunostaining with CD31 antibody as described above. Four random images per section were photographed on EVOS M5000 fluorescent microscope and quantified as an average of TUNEL+ ECs per image between treatment groups [TUNEL assay was performed using Click-iTt groups .At least 50 μgm of tissue or 20 μgm of cell lysates were resolved on SDS-PAGE and transferred onto nitrocellulose membrane for Western blotting as previously described, using the iBright Imaging System ,15,22.~1 µg of the antibody was incubated in 50 μgm of protein lysates overnight at 4 °C in an end-to-end mixer. Antibody–antigen complexes were isolated by adding Goat-Anti-Rabbit (Cat No.: 21356) or Goat-Anti-Mouse (Cat No.: 21354) conjugated magnetic beads  for 2–3 h at room temperature. Antibody–antigen complexes were separated by boiling in sample buffer  for 5–10 min, resolved in SDS-PAGE and Western blotted, as previously described ,15.t-test was used to compare 2 specific groups. One-way ANOVA with Bonferroni select pair comparison or Dunnett’s post-tests were used to compare more than 2 groups. Repeated measures ANOVA was used for time-course laser doppler perfusion recovery measurements. A nonparametric Mann–Whitney test was used for necrosis scores. Statistical tests used to determine the significance of a specific experiment accompanied each figure legend. p < 0.05 is considered significant for all experiments. Data are presented as Mean ± SEM for all experiments.GraphPad Prism 9 was used to analyze the statistical significance of the data and generate graphical representations. An unpaired 165b activates VEGFR2 signaling in normal and hypoxia serum starved ECs in vitro.VEGF Hypoxia is a known inducer of VEGF-A [165b levels in HUVECs [165b is ~3X higher than the pro-angiogenic VEGF-A levels [165a is decreased, and the fraction of anti-angiogenic VEGF165b isoform is increased in HSS HUVECs vs. normoxic controls.f VEGF-A ,26,27. Tf VEGF-A ,28,29,30f VEGF-A ,32,33,34165b-induced VEGFR2-phosphorylation results in the activation of downstream AKT-ERK-eNOS signaling similar to VEGF165a in confluent ECs under normoxia or challenged with HSS . The immunoblots of normal HUVECs treated with recombinant-VEGF165b or recombinant-VEGF165a showed a significant increase in pVEGFR2Y1175 , pAKT , pERK , and peNOS  activation almost to the same extent vs. untreated controls , pAKT (~2-fold), and pERK activation to the same extent in HSS-HUVECs vs. untreated controls  with isotype-matched IgG or VEGF165b-Ab (10 μg/mL [165b-Ab showed no significant difference in pVEGFR2Y1175 activation but significantly decreased pAKT (~1.5-fold), and pERK (~1.3-fold) activation (165b-Ab showed a significant decrease in pVEGFR2 Y1175 (~1.8-fold), pAKT (~3.6-fold) and pERK (~3.9-fold) activation vs. IgG . HSStivation A. HSS-MV vs. IgG B. No sig vs. IgG A,B.165b inhibition in regulating VEGFR2 signaling in vivo, we performed immunoblot analysis of day 3 ischemic muscle samples treated with IgG or VEGF165b-Ab . Immunoblot analysis showed that VEGF165b-Ab significantly decreased pAKT (~5.1-fold) and pERK (~2.9-fold) activation with no changes in peNOS activation vs. IgG  and pSTAT3  activation vs. IgG  vs. normal chow-fed mice skeletal muscle samples . No significant difference in pVEGFR1Y1333 activation was observed between HFD-ischemic samples vs. normal chow ischemic samples  was injected intramuscularly into HFD mouse gastrocnemius (two non-overlapping sites) and tibialis anterior (one site) immediately after HLI (d0) and days 3, 7, 14, and 21 post-HLI. VEGFR1 pull-down at day 3 post HLI with IgG or VEGF165b-Ab-treated samples showed a significant increase in total VEGF-A bound to VEGFR1 indicating that VEGF165b binding to VEGFR1 was reduced by VEGF165b-Ab treatment  in HFD-ischemic muscle vs. IgG  activation was observed in VEGF165b-Ab-treated HFD-ischemic muscle vs. IgG  CD31+ (Red) apoptotic ECs  PAD mice that have impaired NO bioavailabilityVEGF. Excessive myoglobin expression in Mg-Tg mice skeletal muscle functions as a nitric oxide sink and impairs perfusion recovery [165b-Ab could induce perfusion recovery despite the lack of the capacity to increase NO bioavailability. Immunoblot analysis to determine relative changes in VEGF165b vs. total VEGF-A at day 3 post-HLI, (a time point where the perfusion recovery between WT and Mg-Tg mice is comparable) showed no significant difference in the ratio of total VEGF-A to VEGF165b between WT vs. Mg-Tg ischemic muscle , which showed a significant decrease in Mg-Tg ischemic muscle vs. WT, correlating with decreased VEGFR1 levels  vs. IgG  have a strong association with Type-2 diabetes patients [165b-VEGFR1 signaling in regulating angiogenesis in a pathologically relevant in vitro model with impaired VEGFR2 signaling.patients ,40 and ipatients ,42,43. B165b inhibition can rescue the PA-induced angiogenic inhibition. Matrigel analysis showed a significant decrease in the angiogenic capacity in normal  vs. IgG-treated PA-HSS-HUVECs  and microvascular density in diabetic mouse models, including the high fat-high sucrose diet model and Ob-Ob mice model [165b inhibition induces both arteriogenesis and angiogenesis in ischemic diabetic muscle to enhance perfusion recovery. The ability of VEGFR1-STAT3 signaling to induce angiogenesis and perfusion recovery in the T2D-HLI model presented a critical role of VEGFR1-STAT3 signaling to induce therapeutic angiogenesis in PAD.In mice models, and in humans, diabetes further impairs arteriogenesis and angiogenesis in PAD muscle compared with PAD muscle alone ,23. Attec muscle . Howeverce model . Taken t165b, an alternatively spliced VEGF-A isoform blocks VEGF-A-induced angiogenesis by inhibiting VEGFR2 activation. On the contrary VEGF165b has been shown to be a weak activator of VEGFR2 albeit under normal growth conditions [165b induces VEGFR2 and downstream AKT-ERK-eNOS signaling to a similar extent as VEGF165a both under normal growth conditions, and when challenged with HSS, an in vitro model used to simulate the PAD muscle that is deprived of oxygen and growth factors [165b is lower vs. VEGF165a indicating that VEGF165b inhibits angiogenesis independent of VEGFR2 in ischemic vasculature. Accordingly, VEGF165b inhibition decreased VEGFR2 signaling in HSS-HUVECs, indicating that VEGF165b is a potent activator of VEGFR2. A schematic of these data is presented in Previous reports have shown that VEGFnditions ,13,33,35 factors ,15. Desp165b inhibition in activating VEGFR1 in T2D-PAD vasculature, we developed an in vitro model of T2D-PAD. Diabetes is one of the most important risk factors for amputation in PAD patients [165b in the context of this in vitro T2D-PAD model was able to rescue impaired angiogenesis. More importantly, VEGF165b inhibition induced VEGFR1 activation without affecting VEGFR2 activation or downstream signaling. To confirm the role of VEGFpatients . Microvapatients . Hence, patients ,49. Multpatients . Furtherpatients  have sugpatients . Taking 165b inhibition-induced ERK activation without changes in AKT activation in eNOS-KO ischemic muscle, VEGF165b inhibition in Mg-Tg ischemic muscle-inhibited AKT activation with no changes in ERK suggesting distinct VEGFR1 signaling events are activated post-VEGF165b inhibition across these mouse models.To confirm whether VEGFR1 activation can induce therapeutic angiogenesis and perfusion independent of NO, we used eNOS-KO mice  and Myog165b, a selective and potent inhibitor of VEGFR1. The ability of VEGFR1 activation to enhance perfusion even when eNOS-NO signaling is impaired demonstrates the translational potential of targeting VEGF165b to induce VEGFR1-mediated perfusion relief in PAD, where targeting VEGFR2/NO signaling has failed.Amputations due to PAD in addition to other cardiovascular risk factors increase the acute mortality rate to ~30% and decrease the 5-year prognostic survival rate to less than 30%, clearly indicating that there is a great need for identifying new therapeutic strategies to treat PAD ,3,46. Ou165b activates VEGFR2-AKT-ERK-eNOS-NO signaling in ischemic vasculature with no pro-angiogenic effects.Anti-angiogenic VEGF165b allowed VEGFR1-STAT3 activation to induce angiogenesis and promote perfusion recovery in preclinical PAD models, where VEGFR2-eNOS-NO signaling is impaired.Antibody-mediated depletion of VEGF165b in a Type-2 diabetic-PAD model induced VEGFR1-STAT3 signaling activation to promote perfusion recoveryInhibiting VEGF165b in eNOS-KO mice induced VEGFR1-STAT3 signaling to decrease necrosis incidence and severity in preclinical PADBlocking VEGF165b promoted therapeutic angiogenesis where eNOS-NO signaling is impaired, demonstrating the translational potential of inducing VEGFR1-mediated perfusion relief in PAD, where targeting VEGFR2/NO signaling has failed.Targeting VEGF