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Catabolic degradation of endothelial VEGFA via autophagy

  • Thomas Neill
    Correspondence
    To whom correspondence may be addressed: 1020 Locust St., Suite 336G Jefferson Alumni Hall, Thomas Jefferson University, Philadelphia, PA 19107. Tel.: 215-503-6146;
    Affiliations
    Department of Pathology, Anatomy, and Cell Biology, and the Cancer Cell Biology and Signaling Program, Sidney Kimmel Medical College at Thomas Jefferson University, Philadelphia, Pennsylvania 19107
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  • Carolyn G. Chen
    Affiliations
    Department of Pathology, Anatomy, and Cell Biology, and the Cancer Cell Biology and Signaling Program, Sidney Kimmel Medical College at Thomas Jefferson University, Philadelphia, Pennsylvania 19107
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  • Simone Buraschi
    Affiliations
    Department of Pathology, Anatomy, and Cell Biology, and the Cancer Cell Biology and Signaling Program, Sidney Kimmel Medical College at Thomas Jefferson University, Philadelphia, Pennsylvania 19107
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  • Renato V. Iozzo
    Correspondence
    To whom correspondence may be addressed: 1020 Locust St., Suite 336 Jefferson Alumni Hall, Thomas Jefferson University, Philadelphia, PA 19107. Tel.: 215-503-2208; Fax: 215-923-7969;
    Affiliations
    Department of Pathology, Anatomy, and Cell Biology, and the Cancer Cell Biology and Signaling Program, Sidney Kimmel Medical College at Thomas Jefferson University, Philadelphia, Pennsylvania 19107
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Open AccessPublished:March 24, 2020DOI:https://doi.org/10.1074/jbc.RA120.012593
      Extracellular matrix-evoked angiostasis and autophagy within the tumor microenvironment represent two critical, but unconnected, functions of the small leucine-rich proteoglycan, decorin. Acting as a partial agonist of vascular endothelial growth factor 2 (VEGFR2), soluble decorin signals via the energy sensing protein, AMP-activated protein kinase (AMPK), in the autophagic degradation of intracellular vascular endothelial growth factor A (VEGFA). Here, we discovered that soluble decorin evokes intracellular catabolism of endothelial VEGFA that is mechanistically independent of mTOR, but requires an autophagic regulator, paternally expressed gene 3 (PEG3). We found that administration of autophagic inhibitors such as chloroquine or bafilomycin A1, or depletion of autophagy-related 5 (ATG5), results in accumulation of intracellular VEGFA, indicating that VEGFA is a basal autophagic substrate. Mechanistically, decorin increased the VEGFA clearance rate by augmenting autophagic flux, a process that required RAB24 member RAS oncogene family (RAB24), a small GTPase that facilitates the disposal of autophagic compartments. We validated these findings by demonstrating the physiological relevance of this process in vivo. Mice starved for 48 h exhibited a sharp decrease in overall cardiac and aortic VEGFA that could be blocked by systemic chloroquine treatment. Thus, our findings reveal a unified mechanism for the metabolic control of endothelial VEGFA for autophagic clearance in response to decorin and canonical pro-autophagic stimuli. We posit that the VEGFR2/AMPK/PEG3 axis integrates the anti-angiogenic and pro-autophagic bioactivities of decorin as the molecular basis for tumorigenic suppression. These results support future therapeutic use of decorin as a next-generation protein therapy to combat cancer.

      Introduction

      Decorin, an archetypical member of the small leucine-rich proteoglycan gene family (
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      Proteoglycan form and function: a comprehensive nomenclature of proteoglycans.
      ,
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      • Vallet S.D.
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      Decorin interacting network: A comprehensive analysis of decorin-binding partners and their versatile functions.
      ), acts as a partial agonist for a diverse set of cell surface receptor-tyrosine kinases (RTKs)
      The abbreviations used are: RTK
      receptor tyrosine kinase
      AICAR
      N1-(β-d-ribofuranosyl)-5-aminoimidazole-4 carboxamide
      AMPKα
      5′-AMP-activated protein kinase catalytic subunit α
      ATG
      autophagy regulated genes
      HAEC
      human aortic endothelial cells
      HIF-1α
      hypoxia-inducible factor 1 subunit α
      HUVEC
      Human umbilical vein endothelial cells
      LC3
      microtubule associated light chain 3
      p62/SQSTM1
      sequestome 1
      VEGFA
      vascular endothelial growth factor-A
      VEGFR2
      vascular endothelial growth factor receptor 2
      PEG3
      paternally expressed gene 3
      CQ
      chloroquine
      PAER2
      porcine aortic endothelial cells overexpressing VEGFR2
      DIC
      differential interference contrast
      EGFR
      epidermal growth factor receptor
      mTOR
      mechanistic target of rapamycin
      GAPDH
      glyceraldehyde-3-phosphate dehydrogenase
      HA
      hemagglutinin
      STAT
      signal transducers and activators of transcription
      DAPI
      4′,6-diamidino-2-phenylindole
      ANOVA
      analysis of variance.
      that directly regulate a surfeit of intracellular pathways via outside-in signaling (
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      Altered proteoglycan gene expression and the tumor stroma.
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      Small leucine-rich proteoglycans orchestrate receptor crosstalk during inflammation.
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      Biologically active decorin is a monomer in solution.
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      Decoding the matrix: Instructive roles of proteoglycan receptors.
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      Proteoglycans regulate autophagy via outside-in signaling: an emerging new concept.
      ,
      • Iozzo R.V.
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      Extracellular matrix: the driving force of mammalian diseases.
      ). Classically, decorin evokes anti-oncogenic and angiostatic responses (
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      ,
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      Decorin as a multivalent therapeutic agent against cancer.
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      An oncosuppressive role for decorin.
      ) in vitro (
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      • Neill T.
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      Decorin antagonizes Met receptor activity and downregulates β-catenin and Myc levels.
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      Decorin antagonizes the angiogenic network: concurrent inhibition of Met, hypoxia inducible factor-1α and vascular endothelial growth factor A and induction of thrombospondin-1 and TIMP3.
      ) and in vivo (
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      The systemic delivery of an oncolytic adenovirus expressing decorin inhibits bone metastasis in a mouse model of human prostate cancer.
      ,
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      • Hu Z.
      • Wang C.H.
      • Xiao X.
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      • Guise T.
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      • Seth P.
      Systemic delivery of an oncolytic adenovirus expressing decorin for the treatment of breast cancer bone metastases.
      ,
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      • Owens R.T.
      • Iniguez L.A.
      • Purkins G.
      • Vadigepalli R.
      • Evans B.
      • Schaefer L.
      • Peiper S.C.
      • Wang Z.X.
      • Iozzo R.V.
      Decorin protein core affects the global gene expression profile of the tumor microenvironment in a triple-negative orthotopic breast carcinoma xenograft model.
      ) via proteolytic degradation of oncogenes such as β-catenin, Myc (
      • Buraschi S.
      • Pal N.
      • Tyler-Rubinstein N.
      • Owens R.T.
      • Neill T.
      • Iozzo R.V.
      Decorin antagonizes Met receptor activity and downregulates β-catenin and Myc levels.
      ), and HIF-1α (
      • Neill T.
      • Painter H.
      • Buraschi S.
      • Owens R.T.
      • Lisanti M.P.
      • Schaefer L.
      • Iozzo R.V.
      Decorin antagonizes the angiogenic network: concurrent inhibition of Met, hypoxia inducible factor-1α and vascular endothelial growth factor A and induction of thrombospondin-1 and TIMP3.
      ), whereas concurrently inducing tumor suppressors, i.e. p21WAF1 (
      • De Luca A.
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      • Baldi A.
      • Giordano A.
      • Iozzo R.V.
      Decorin-induced growth suppression is associated with upregulation of p21, an inhibitor of cyclin-dependent kinases.
      ). Decorin is unique among onco-suppressive agents (
      • Neill T.
      • Schaefer L.
      • Iozzo R.V.
      An oncosuppressive role for decorin.
      ) as it selectively and simultaneously binds RTKs expressed in the stroma such as VEGFR2 (
      • Buraschi S.
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      • Goyal A.
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      • Owens R.T.
      • Schaefer L.
      • Torres A.
      • Iozzo R.V.
      Decorin causes autophagy in endothelial cells via Peg3.
      ) and those expressed by the tumor parenchyma such as EGFR and/or Met (
      • Iozzo R.V.
      • Moscatello D.K.
      • McQuillan D.J.
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      Decorin is a biological ligand for the epidermal growth factor receptor.
      ,
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      • Nyström A.
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      Decorin is a novel antagonistic ligand of the Met receptor.
      ). Decorin-deficient mice are prone to tumorigenesis when either p53 is missing (
      • Iozzo R.V.
      • Chakrani F.
      • Perrotti D.
      • McQuillan D.J.
      • Skorski T.
      • Calabretta B.
      • Eichstetter I.
      Cooperative action of germline mutations in decorin and p53 accelerates lymphoma tumorigenesis.
      ) or when subjected to a Western high-fat diet (
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      • Dockendorff A.
      • Bancroft L.
      • Gallagher L.
      • Guzman G.
      • Iozzo R.V.
      • Augenlicht L.H.
      • Yang W.
      Genetic deficiency of decorin causes intestinal tumor formation through disruption of intestinal cell maturation.
      ,
      • Bi X.
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      • Qian Z.
      • Yang G.R.
      • Gou Y.
      • Guzman G.
      • Kajdacsy-Balla A.
      • Iozzo R.V.
      • Yang W.
      Decorin-mediated inhibition of colorectal cancer growth and migration is associated with E-cadherin in vitro and in mice.
      ).
      Mechanistically, decorin reduces intracellular, cell-associated, and secreted vascular endothelial growth factor A (VEGFA) in a noncanonical manner and evokes the expression and rapid secretion of thrombospondin-1 downstream of VEGFR2 or Met (
      • Neill T.
      • Painter H.
      • Buraschi S.
      • Owens R.T.
      • Lisanti M.P.
      • Schaefer L.
      • Iozzo R.V.
      Decorin antagonizes the angiogenic network: concurrent inhibition of Met, hypoxia inducible factor-1α and vascular endothelial growth factor A and induction of thrombospondin-1 and TIMP3.
      ,
      • Grant D.S.
      • Yenisey C.
      • Rose R.W.
      • Tootell M.
      • Santra M.
      • Iozzo R.V.
      Decorin suppresses tumor cell-mediated angiogenesis.
      ,
      • Neill T.
      • Jones H.R.
      • Crane-Smith Z.
      • Owens R.T.
      • Schaefer L.
      • Iozzo R.V.
      Decorin induces rapid secretion of thrombospondin-1 in basal breast carcinoma cells via inhibition of Ras homolog gene family, member A/Rho-associated coiled-coil containing protein kinase 1.
      ,
      • Torres A.
      • Gubbiotti M.A.
      • Iozzo R.V.
      Decorin-inducible Peg3 evokes beclin 1-mediated autophagy and thrombospondin 1-mediated angiostasis.
      ,
      • Gubbiotti M.A.
      • Buraschi S.
      • Kapoor A.
      • Iozzo R.V.
      Proteoglycan signaling in tumor angiogenesis and endothelial cell autophagy.
      ). The protracted suppression of potent pro-angiogenic factors constitutes a key hallmark of decorin as a soluble tumor repressor and emphasizes its eponym as a guardian from the matrix (
      • Gubbiotti M.A.
      • Vallet S.D.
      • Ricard-Blum S.
      • Iozzo R.V.
      Decorin interacting network: A comprehensive analysis of decorin-binding partners and their versatile functions.
      ,
      • Järveläinen H.
      • Puolakkainen P.
      • Pakkanen S.
      • Brown E.L.
      • Höök M.
      • Iozzo R.V.
      • Sage H.
      • Wight T.N.
      A role for decorin in cutaneous wound healing and angiogenesis.
      ,
      • Goldoni S.
      • Seidler D.G.
      • Heath J.
      • Fassan M.
      • Baffa R.
      • Thakur M.L.
      • Owens R.T.
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      An anti-metastatic role for decorin in breast cancer.
      ,
      • Neill T.
      • Schaefer L.
      • Iozzo R.V.
      Decorin, a guardian from the matrix.
      ,
      • Mongiat M.
      • Buraschi S.
      • Andreuzzi E.
      • Neill T.
      • Iozzo R.V.
      Extracellular matrix: the gatekeeper of tumor angiogenesis.
      ). Decorin has been linked to metabolic reprogramming via regulation of cardiac O-β-N-acetylglycosylation (
      • Gubbiotti M.A.
      • Seifert E.
      • Rodeck U.
      • Hoek J.B.
      • Iozzo R.V.
      Metabolic reprogramming of murine cardiomyocytes during autophagy requires the extracellular nutrient sensor decorin.
      ,
      • Vigetti D.
      • Caon I.
      • Passi A.
      A nutrient sentinel stands guard outside the cell.
      ).
      Using high-resolution transcriptomics to identify decorin-regulated genes in vivo, we discovered that paternally expressed gene 3 (Peg3) was specifically induced within the Mus musculus stroma in triple-negative breast carcinoma orthotopic tumor xenografts treated with systemic delivery of decorin (
      • Buraschi S.
      • Neill T.
      • Owens R.T.
      • Iniguez L.A.
      • Purkins G.
      • Vadigepalli R.
      • Evans B.
      • Schaefer L.
      • Peiper S.C.
      • Wang Z.X.
      • Iozzo R.V.
      Decorin protein core affects the global gene expression profile of the tumor microenvironment in a triple-negative orthotopic breast carcinoma xenograft model.
      ). Peg3 is a genomically-imprinted Krüpple-like zinc finger transcription factor possessing inherent tumor suppressor properties (
      • Kohda T.
      • Asai A.
      • Kuroiwa Y.
      • Kobayashi S.
      • Aisaka K.
      • Nagashima G.
      • Yoshida M.C.
      • Kondo Y.
      • Kagiyama N.
      • Kirino T.
      • Kaneko-Ishino T.
      • Ishino F.
      Tumour suppressor activity of human imprinted gene PEG3 in a glioma cell line.
      ,
      • Nye M.D.
      • Hoyo C.
      • Huang Z.
      • Vidal A.C.
      • Wang F.
      • Overcash F.
      • Smith J.S.
      • Vasquez B.
      • Hernandez B.
      • Swai B.
      • Oneko O.
      • Mlay P.
      • Obure J.
      • Gammon M.D.
      • Bartlett J.A.
      • Murphy S.K.
      Association between methylation of paternally expressed gene 3 (PEG3), cervical intraepithelial neoplasia and invasive cervical cancer.
      ). Peg3 is commonly lost due to biallelic promoter hypermethylation (
      • Dowdy S.C.
      • Gostout B.S.
      • Shridhar V.
      • Wu X.
      • Smith D.I.
      • Podratz K.C.
      • Jiang S.-W.
      Biallelic methylation and silencing of paternally expressed gene 3 (PEG3) in gynecologic cancer cell lines.
      ,
      • Maegawa S.
      • Yoshioka H.
      • Itaba N.
      • Kubota N.
      • Nishihara S.
      • Shirayoshi Y.
      • Nanba E.
      • Oshimura M.
      Epigenetic silencing of PEG3 gene expression in human glioma cell lines.
      ) or loss of heterozygosity (
      • Feng W.
      • Marquez R.T.
      • Lu Z.
      • Liu J.
      • Lu K.H.
      • Issa J.-P.
      • Fishman D.M.
      • Yu Y.
      • Bast Jr., R.C.
      Imprinted tumor suppressor genes ARHI and PEG3 are the most frequently down-regulated in human ovarian cancers by loss of heterozygosity and promoter methylation.
      ). Querying the ONCOMINE database (
      • Buraschi S.
      • Neill T.
      • Owens R.T.
      • Iniguez L.A.
      • Purkins G.
      • Vadigepalli R.
      • Evans B.
      • Schaefer L.
      • Peiper S.C.
      • Wang Z.X.
      • Iozzo R.V.
      Decorin protein core affects the global gene expression profile of the tumor microenvironment in a triple-negative orthotopic breast carcinoma xenograft model.
      ), we found that Peg3 expression is substantially and significantly reduced in invasive ductal breast carcinoma (
      • Karnoub A.E.
      • Dash A.B.
      • Vo A.P.
      • Sullivan A.
      • Brooks M.W.
      • Bell G.W.
      • Richardson A.L.
      • Polyak K.
      • Tubo R.
      • Weinberg R.A.
      Mesenchymal stem cells within tumor stroma promote breast cancer metastasis.
      ) and ductal breast carcinoma (
      • Richardson A.L.
      • Wang Z.C.
      • De Nicolo A.
      • Lu X.
      • Brown M.
      • Miron A.
      • Liao X.
      • Iglehart J.D.
      • Livingston D.M.
      • Ganesan S.
      X chromosomal abnormalities in basal-like human breast cancer.
      ). Due to the functional similarities of Peg3 for the noncanonical disruption of Wnt/β-catenin signaling in a glycogen synthase kinase 3β-independent manner (
      • Jiang X.
      • Yu Y.
      • Yang H.W.
      • Agar N.Y.
      • Frado L.
      • Johnson M.D.
      The imprinted gene PEG3 inhibits Wnt signaling and regulates glioma growth.
      ), which is strikingly akin to the activity of decorin (
      • Buraschi S.
      • Pal N.
      • Tyler-Rubinstein N.
      • Owens R.T.
      • Neill T.
      • Iozzo R.V.
      Decorin antagonizes Met receptor activity and downregulates β-catenin and Myc levels.
      ), Peg3 emerged as a prime candidate for further study.
      We discovered that Peg3 co-localizes and physically interacts with the autophagic regulators LC3 and Beclin 1 (
      • Levine B.
      • Kroemer G.
      Biological functions of autophagy genes: a disease perspective.
      ) in response to decorin and canonical autophagic stimuli in endothelial cells (
      • Buraschi S.
      • Neill T.
      • Goyal A.
      • Poluzzi C.
      • Smythies J.
      • Owens R.T.
      • Schaefer L.
      • Torres A.
      • Iozzo R.V.
      Decorin causes autophagy in endothelial cells via Peg3.
      ). Peg3 is necessary and sufficient to maintain and evoke BECN1, MAP1LC3A, and TFEB expression (
      • Buraschi S.
      • Neill T.
      • Goyal A.
      • Poluzzi C.
      • Smythies J.
      • Owens R.T.
      • Schaefer L.
      • Torres A.
      • Iozzo R.V.
      Decorin causes autophagy in endothelial cells via Peg3.
      ,
      • Torres A.
      • Gubbiotti M.A.
      • Iozzo R.V.
      Decorin-inducible Peg3 evokes beclin 1-mediated autophagy and thrombospondin 1-mediated angiostasis.
      ,
      • Poluzzi C.
      • Casulli J.
      • Goyal A.
      • Mercer T.J.
      • Neill T.
      • Iozzo R.V.
      Endorepellin evokes autophagy in endothelial cells.
      ,
      • Neill T.
      • Sharpe C.
      • Owens R.T.
      • Iozzo R.V.
      Decorin-evoked paternally expressed gene 3 (PEG3) is an upstream regulator of the transcription factor EB (TFEB) in endothelial cell autophagy.
      ), and drives LC3-positive autophagosome formation and autophagic flux (
      • Torres A.
      • Gubbiotti M.A.
      • Iozzo R.V.
      Decorin-inducible Peg3 evokes beclin 1-mediated autophagy and thrombospondin 1-mediated angiostasis.
      ). Moreover, we found that Peg3 is required for thrombospondin-1 expression and secretion (
      • Torres A.
      • Gubbiotti M.A.
      • Iozzo R.V.
      Decorin-inducible Peg3 evokes beclin 1-mediated autophagy and thrombospondin 1-mediated angiostasis.
      ). Thus, Peg3 may act as a nexus to parse anti-angiogenic and pro-autophagic signals for the concerted regulation of decorin-evoked angiogenesis and autophagy.
      Decorin actively signals via the VEGFR2 tyrosine kinase to evoke autophagy (
      • Buraschi S.
      • Neill T.
      • Goyal A.
      • Poluzzi C.
      • Smythies J.
      • Owens R.T.
      • Schaefer L.
      • Torres A.
      • Iozzo R.V.
      Decorin causes autophagy in endothelial cells via Peg3.
      ,
      • Neill T.
      • Torres A.
      • Buraschi S.
      • Iozzo R.V.
      Decorin has an appetite for endothelial cell autophagy.
      ). Decorin differentially modulates AMPK and mTOR phosphorylation, critical rheostats for maintaining metabolic homeostasis (
      • Goyal A.
      • Neill T.
      • Owens R.T.
      • Schaefer L.
      • Iozzo R.V.
      Decorin activates AMPK, an energy sensor kinase, to induce autophagy in endothelial cells.
      ,
      • Yang Z.
      • Klionsky D.J.
      Mammalian autophagy: core molecular machinery and signaling regulation.
      ,
      • Alers S.
      • Löffler A.S.
      • Wesselborg S.
      • Stork B.
      Role of AMPK-mTOR-Ulk1/2 in the regulation of autophagy: crosstalk, shortcuts, and feedbacks.
      ,
      • Kim J.
      • Kundu M.
      • Viollet B.
      • Guan K.-L.
      AMPK and mTOR regulate autophagy through direct phopshorylation of Ulk1.
      ), to favor pro-autophagic outcomes. Indeed, AMPK and mTOR subsume opposing roles in the governance of autophagic induction. AMPK is required for autophagic initiation via ULK1/2 phosphorylation (
      • Kim J.
      • Kundu M.
      • Viollet B.
      • Guan K.-L.
      AMPK and mTOR regulate autophagy through direct phopshorylation of Ulk1.
      ,
      • Kim J.
      • Guan K.L.
      AMPK connects energy stress to PIK3C3/VPS34 regulation.
      ,
      • Lee J.W.
      • Park S.
      • Takahashi Y.
      • Wang H.-G.
      The association of AMPK with ULK1 regulates autophagy.
      ,
      • Kuhajda F.P.
      AMP-activated protein kinase and human cancer: cancer metabolism revisited.
      ) and mTOR for autophagic inhibition and termination (
      • Rosenbluth J.M.
      • Pietenpol J.A.
      mTOR regulates autophagy-associated genes downstream of p73.
      ,
      • Yu L.
      • McPhee C.K.
      • Zheng L.
      • Mardones G.A.
      • Rong Y.
      • Peng J.
      • Mi N.
      • Zhao Y.
      • Liu Z.
      • Wan F.
      • Hailey D.W.
      • Oorschot V.
      • Klumperman J.
      • Baehrecke E.H.
      • Lenardo M.J.
      Termination of autophagy and reformation of lysosomes regulated by mTOR.
      ). We found prolonged phosphorylation of the AMPKα catalytic subunit at Thr172 denoting activation with a concurrent suppression of phosphorylated mTOR at Ser2448 and its downstream effectors (Akt and p70S6K) in endothelial cells for up to 48 h (
      • Goyal A.
      • Neill T.
      • Owens R.T.
      • Schaefer L.
      • Iozzo R.V.
      Decorin activates AMPK, an energy sensor kinase, to induce autophagy in endothelial cells.
      ). Importantly, decorin-evoked endothelial cell autophagy occurs under nutrient-rich conditions, thereby designating decorin as a noncanonical stimulus for evoking excessive autophagy.
      Our understanding of the molecular mechanisms of VEGFs regulating vascular homeostasis and angiogenesis has dramatically expanded in the past two decades (
      • Apte R.S.
      • Chen D.S.
      • Ferrara N.
      VEGF in signaling and disease: beyond discovery and development.
      ,
      • Ferrara N.
      • Adamis A.P.
      Ten years of anti-vascular endothelial growth factor therapy.
      ). Given that decorin suppresses VEGFA with the concurrent induction of autophagy via VEGFR2/AMPKα/Peg3 in endothelial cells (
      • Buraschi S.
      • Neill T.
      • Goyal A.
      • Poluzzi C.
      • Smythies J.
      • Owens R.T.
      • Schaefer L.
      • Torres A.
      • Iozzo R.V.
      Decorin causes autophagy in endothelial cells via Peg3.
      ,
      • Torres A.
      • Gubbiotti M.A.
      • Iozzo R.V.
      Decorin-inducible Peg3 evokes beclin 1-mediated autophagy and thrombospondin 1-mediated angiostasis.
      ,
      • Neill T.
      • Sharpe C.
      • Owens R.T.
      • Iozzo R.V.
      Decorin-evoked paternally expressed gene 3 (PEG3) is an upstream regulator of the transcription factor EB (TFEB) in endothelial cell autophagy.
      ,
      • Goyal A.
      • Neill T.
      • Owens R.T.
      • Schaefer L.
      • Iozzo R.V.
      Decorin activates AMPK, an energy sensor kinase, to induce autophagy in endothelial cells.
      ), we hypothesized these pathways could converge to catabolize intracellular VEGFA by autophagy as the mechanism for the onco-suppressive effects of decorin. In this study, we found that decorin evokes the protracted catabolism of intracellular VEGFA via endothelial cell autophagy in a VEGFR2/AMPK/Peg3-dependent manner as the functional apparatus to achieve angiostasis.

      Results

      Intracellular VEGFA protein levels are controlled by AMPK

      We found via confocal imaging that decorin reduced intracellular VEGFA (Fig. 1A) in primary endothelial cells (HUVEC) and human aortic endothelial cells immortalized by stable expression of the human catalytic subunit of telomerase (TeloHAEC). VEGFA suppression proceeded in a VEGFR2-dependent manner as inhibition with the small molecule inhibitor, SU5416 (
      • Mendel D.B.
      • Schreck R.E.
      • West DC.
      • Li. G
      • Strawn L.M.
      • Tanciongco S.S.
      • Vasile S.
      • Shawver L.K.
      • Cherrington J.M.
      The angiogenesis inhibitor SU5416 has long-lasting effects on vascular endothelial growth factor receptor phopshorylation and function.
      ,
      • Fong T.A.
      • Shawver L.K.
      • Sun L.
      • Tang C.
      • App H.
      • Powell T.J.
      • Kim Y.H.
      • Schreck R.
      • Wang X.
      • Risau W.
      • Ullrich A.
      • Hirth K.P.
      • McMahon G.
      SU5416 is a potent and selective inhibitor of the vascular endothelial growth factor receptor (Flk-1/KDR) that inhibits tyrosine kinase catalysis, tumor vascularization, and growth of multiple tumor types.
      ,
      • Ban H.S.
      • Uno M.
      • Nakamura H.
      Suppression of hypoxia-induced HIF-1α accumulation by VEGFR inhibitors: different profiles of AAL993, versus SU5416 and KRN633.
      ) significantly abrogated the effect of decorin (Fig. 1A). We previously found that exogenous decorin evokes endothelial cell autophagy by activating the master energy sensor kinase, AMPK (
      • Kim J.
      • Guan K.L.
      AMPK connects energy stress to PIK3C3/VPS34 regulation.
      ,
      • Liang J.
      • Mills G.B.
      AMPK: a contextual oncogene or tumor suppressor?.
      ,
      • Garcia D.
      • Shaw R.J.
      AMPK: mechanisms of cellular energy sensing and restoration of metabolic balance.
      ) by augmenting the phosphorylation of the α-catalytic subunit at Thr172 (
      • Goyal A.
      • Neill T.
      • Owens R.T.
      • Schaefer L.
      • Iozzo R.V.
      Decorin activates AMPK, an energy sensor kinase, to induce autophagy in endothelial cells.
      ). We discovered that treatment with Compound C (Dorsomorphin), a reversible and selective AMPKα inhibitor (
      • Kim J.
      • Kundu M.
      • Viollet B.
      • Guan K.-L.
      AMPK and mTOR regulate autophagy through direct phopshorylation of Ulk1.
      ), potently attenuated VEGFA suppression (Fig. 1A), underscoring a role for the VEGFR2/AMPK axis.
      Figure thumbnail gr1
      Figure 1Intracellular VEGFA and Peg3 are governed by AMPK in endothelial cells. A, gallery of confocal images in HUVEC (top) or TeloHAEC (bottom) treated in combination with decorin (200 nm) ± SU5416 (1 μm) or Compound C (30 μm) and immunostained for VEGFA (green) for 6 h. Nuclei (blue) were visualized with DAPI. Bar ∼10 μm. B, representative immunoblot of HUVEC treated with AICAR (500 μm) at the indicated time points. C–E, quantification of targets as depicted in B. F and G, semi-quantification of immunofluorescence signal intensity for VEGFA (F) or Peg3 (G) normalized to total cell number in HUVEC treated as in B. H and I, gene expression of VEGFA (H) or PEG3 (I) in HUVEC as treated in B. GAPDH served as the loading control in B. DMSO served as vehicle for all experiments to account for SU5146, Compound C, and AICAR. Data are reflective of several (n = 3–4) independent biological replicates. Data are expressed as mean ± S.E.
      Next, we used AICAR, an analog of adenosine monophosphate that stimulates AMPK activity (
      • Giri S.
      • Nath N.
      • Smith B.
      • Viollet B.
      • Singh A.K.
      • Singh I.
      5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside inhibits proinflammatory response in glial cells: a possible role of AMP-activated protein kinase.
      ), to assess whether AMPK alone was capable of modulating VEGFA. We verified that AICAR potently activated AMPK as shown by increasing levels of P-Thr172 (Fig. 1B), that peaked at 4 h (Fig. 2C). This is in stark contrast to treating with vehicle only (DMSO), which has no effect on P-AMPKα or VEGFA levels (Fig. S1, A and B). We found that turning on AMPK via AICAR was sufficient to increase Peg3 (Fig. 1, B and D), a master autophagic regulator induced by decorin (
      • Buraschi S.
      • Neill T.
      • Goyal A.
      • Poluzzi C.
      • Smythies J.
      • Owens R.T.
      • Schaefer L.
      • Torres A.
      • Iozzo R.V.
      Decorin causes autophagy in endothelial cells via Peg3.
      ,
      • Torres A.
      • Gubbiotti M.A.
      • Iozzo R.V.
      Decorin-inducible Peg3 evokes beclin 1-mediated autophagy and thrombospondin 1-mediated angiostasis.
      ,
      • Neill T.
      • Sharpe C.
      • Owens R.T.
      • Iozzo R.V.
      Decorin-evoked paternally expressed gene 3 (PEG3) is an upstream regulator of the transcription factor EB (TFEB) in endothelial cell autophagy.
      ), and simultaneously decrease intracellular VEGFA with a t½ of 2.6 h (Fig. 1, B and E). Notably, immunofluorescence analysis showed that AICAR treatment caused a similar induction of Peg3 (Fig. 1F) and concurrent VEGFA suppression (Fig. 1G) with a comparable half-life (t½ = 3.2 h). We then assessed the effect of AMPK on PEG3 and VEGFA mRNA expression. AICAR dynamically increased PEG3 mRNA peaking at ∼2.5 h and then declining to basal levels (Fig. 1H). Importantly, AICAR concomitantly decreased VEGFA mRNA (Fig. 1I). However, the half-life of VEGFA mRNA was nearly 5 h (t½ = 4.9 h), significantly longer (p = 0.0014) than VEGFA protein (cf. Fig. 1, B and E).
      Figure thumbnail gr2
      Figure 2AMPK kinase activity is required for VEGFA and Peg3 modulation. A, representative immunoblots of HUVEC treated with AICAR (500 μm) ± Compound C (30 μm) for 6 h. B–F, quantification of targets as immunostained in A. GAPDH served as the loading control for immunoblot in A. Data are reflective of three independent biological replicates. Data are expressed as arbitrary units (A.U.) on a dot density plot. DMSO served as vehicle for the control condition. Statistics were calculated via one-way ANOVA.
      We sought to better define a role of the AMPK kinase for the differential regulation of Peg3 and VEGFA by treating HUVEC with AICAR, either individually or in combination with Compound C. We validated AICAR function by immunoblotting for P-Thr172 and found increased phosphorylation (Fig. 2, A and B). Treatment with or without Compound C did not alter basal or AICAR-induced P-Thr172 (Fig. 2, A and B). These data are congruent with the established mechanism of AMPKα activation via canonical phosphorylation by the upstream LKB1/MO25α/STRAD1 trimeric kinase complex (
      • Alers S.
      • Löffler A.S.
      • Wesselborg S.
      • Stork B.
      Role of AMPK-mTOR-Ulk1/2 in the regulation of autophagy: crosstalk, shortcuts, and feedbacks.
      ). Importantly, Compound C significantly inhibited AICAR-mediated induction of Peg3 (p = 0.002, Fig. 2, A and C) and corresponding VEGFA suppression (p = 0.006, Fig. 2, A and D). We confirmed these biochemical findings using quantitative immunofluorescence under identical experimental conditions and found that Compound C markedly prevented AICAR-evoked induction of Peg3 (p = 0.0006, Fig. 2E) or reduction of VEGFA (p = 0.008, Fig. 2F).
      Collectively, these data support a dynamic and reciprocal role of AMPK in inducing Peg3 and simultaneously decreasing VEGFA in endothelial cells. Moreover, activating AMPK alone with AICAR to mimic a low cellular energy state in an otherwise enveloping nutrient-rich environment appears equivalent to the decorin-dependent suppression of cellular VEGFA.

      Endothelial VEGFA suppression is mTOR independent

      We have previously shown that decorin turns off mTOR signaling in a protracted fashion by attenuating phosphorylation at Ser2448 downstream of VEGFR2 and Akt, resulting in decreased activation of its effectors such as p70S6K (
      • Goyal A.
      • Neill T.
      • Owens R.T.
      • Schaefer L.
      • Iozzo R.V.
      Decorin activates AMPK, an energy sensor kinase, to induce autophagy in endothelial cells.
      ).
      Thus, we tested whether VEGFA suppression depended on mTOR inhibition. To this end, we used Torin 1, a highly-specific ATP competitive inhibitor of mTORC1 (
      • Francipane M.G.
      • Lagasse E.
      Selective targeting of human colon cancer stem-like cells by the mTOR inhibitor Torin-1.
      ,
      • Chen C.
      • Kapoor A.
      • Iozzo R.V.
      Methods for monitoring matrix-induced autophagy.
      ). We assessed either p62/SQSTM1 (p62) or LC3 protein levels, two established autophagic markers regulated by mTOR inhibition (
      • Mizushima N.
      Methods for monitoring autophagy using GFP-LC3 transgenic mice.
      ). We found that Torin 1 dynamically modulated p62 in a dose-dependent manner in HUVEC (Fig. 3, A and B) and PAER2 (Fig. 3, C and D). Intriguingly, the responses were different between both models, with a maximal induction of p62 in HUVEC occurring with as little as ∼1 nm Torin 1, then decreasing at subsequently higher doses (Fig. 3, A and B). In PAER2, which have been immortalized by the stable integration and expression of VEGFR2, progressively increasing the amount of Torin 1 significantly decreased p62/SQSTM1 (Fig. 3, C and D). This was not a result of increased cell death as the internal loading control (GAPDH) remained equal (Fig. 3, C and D) and total cell numbers did not waver at the highest dose (500 nm) administered (data not shown). However, despite positive modulation of p62 as a surrogate for autophagy, we found no change of VEGFA with increasing amounts of Torin 1 in HUVEC (Fig. 3, A and B) or PAER2 (Fig. 3, C and D).
      Figure thumbnail gr3
      Figure 3Endothelial VEGFA suppression is mTOR independent. A and B, representative immunoblots of increasing Torin 1 concentrations, as indicated, for 6 h in HUVEC (A) with corresponding quantification (B). C and D, identical experiment performed in PAER2 as in A and B. E and F, representative immunoblots of Torin 1 (20 nm) at the indicated time points in HUVEC (E) with quantifications (F). G and H, identical experiment performed in PAER2 as in E and F. GAPDH served as the loading control in all immunoblots (A, C, E, and G). In E–H, DMSO served as the vehicle for the 0-h control time point. All data are reflective of four independent biological replicates. Data are expressed as mean ± S.E.
      We next investigated the temporal effect of Torin 1 on VEGFA. We used 20 nm Torin 1 as we previously found this to be most effective dosage for autophagic induction in our system (
      • Goyal A.
      • Gubbiotti M.A.
      • Chery D.R.
      • Han L.
      • Iozzo R.V.
      Endorepellin-evoked autophagy contributes to angiostasis.
      ) and LC3 as a positive control for mTOR inhibition because p62 levels are only subtly modulated under this concentration of Torin 1 (Fig. 3, A and C). Similar time-dependent administration of Torin 1 induced dynamic LC3-II formation, the bioactive form of LC3, in HUVEC that peaked at 1 h (Fig. 3, E and F). In PAER2, LC3-II levels declined with Torin 1 treatment over time (Fig. 3, G and H). However, similar to the dose-response, VEGFA was not responsive to Torin 1 over time despite induction of autophagy in either HUVEC (Fig. 3, E and F) or PAER2 (Fig. 3, G and H).
      We complemented Torin 1 inhibition with INK128 (Sapanisertib), which more potently attenuates mTORC1/2 and has a longer half-life than Torin 1 (
      • Liu Q.
      • Thoreen C.
      • Wang J.
      • Sabatini D.
      • Gray N.S.
      mTOR mediated anti-cancer drug discovery.
      ,
      • Hsieh A.C.
      • Liu Y.
      • Edlind M.P.
      • Ingolia N.T.
      • Janes M.R.
      • Sher A.
      • Shi E.Y.
      • Stumpf C.R.
      • Christensen C.
      • Bonham M.J.
      • Wang S.
      • Ren P.
      • Martin M.
      • Jessen K.
      • Feldman M.E.
      • et al.
      The translational landscape of mTOR signalling steers cancer initiation and metastasis.
      ). Under comparable experimental conditions, we found a similar trend in HUVEC where p62 exhibited a dynamic induction, peaking at 0.1 nm followed by a decline to baseline at higher concentrations (Fig. S2, A and C). In PAER2, INK128 caused a progressive decline in p62 levels from baseline, akin to the effect of Torin 1, as INK128 concentrations increased (Fig. S2, B and D). Importantly, using this more potent inhibitor of the mTOR complexes, VEGFA levels were unperturbed in both endothelial cell types (Fig. S2, A–D).
      Collectively, these data demonstrate an mTOR-independent role of VEGFA modulation in endothelial cells utilizing pharmacological agents that mimic low-energy conditions, despite lively regulation of established autophagic markers. Thus, bioenergetics-mediated regulation of intracellular VEGFA proceeds in an AMPK-dependent and mTOR-independent mechanism in endothelial cells.

      VEGFA is sensitive to nutrient deprivation

      As VEGFA is untouched by mTORC1/2 inhibition as a mimic of the nutrient replete state, we performed nutrient deprivation in PAER2 cells using Earle's balanced salt solution (
      • Klionsky D.J.
      • Abdelmohsen K.
      • Abe A.
      • Abedin M.J.
      • Abeliovich H.
      • Acevedo A.A.
      • Adachi H.
      • Adams C.M.
      • Adams P.D.
      • Adeli K.
      • Adhihetty P.J.
      • Adler S.G.
      • Agam G.
      • Agarwal R.
      • Aghi M.K.
      • et al.
      Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition).
      ) and evaluated VEGFA over time. After confirming starvation-induced autophagy by increased P-AMPKα at Thr172 (data not shown), we showed a concurrent decrease in p62 (Fig. S2, E and F). Importantly, we found a significant decrease in VEGFA, nearly mirroring the profile of p62 over this same time frame (Fig. S2, E and F).
      To evaluate the generality of the above results, we assessed VEGFA regulation by pro-autophagic stimuli (AICAR, Torin 1, and HBSS) in two other cell types, namely, human primary fibroblasts and NIH-3T3 immortalized mouse fibroblasts. Surprisingly, in response to AICAR, p62 levels did not appreciably change in either cell type (Fig. S3, A, C, B, and E). In contrast, AICAR significantly reduced VEGFA in human and mouse fibroblasts (Fig. S3, A, D, B, and F), similar to endothelial cells. Treatment with Torin 1 reduced p62 (Fig. S3, A, C, B, and E) without a concomitant decrease in VEGFA levels (Fig. S3, A, D, B, and F), akin to HUVEC and PAER2. Nutrient deprivation via HBSS suppressed p62 levels (Fig. S3, A, C, B, and E), whereas concurrently decreasing VEGFA (Fig. S3, A, D, B, and F). Collectively, our findings highlight a general sensitivity of cellular VEGFA to pro-autophagic agents that is not restricted to endothelia, further reinforcing the independence of mTOR in this pathway.

      Pro-autophagic stimuli evokes co-localization of VEGFA with LC3 positive autophagosomes

      We evaluated whether VEGFA would localize to autophagosomes using confocal imaging. We discovered that even basal levels of VEGFA co-localized with LC3-positive autophagosomes (Fig. 4, A and B). However, 6 h treatment with decorin, AICAR, or nutrient deprivation caused a marked increase in the number and size of the VEGFA/LC3 dually-positive autophagosomes (Fig. 4, C, E, and G) as did the signal intensity ascertained by semi-quantitative line scanning profiles for each stimulus (Fig. 4, D, F, and H).
      Figure thumbnail gr4
      Figure 4Pro-autophagic stimuli evokes co-localization of VEGFA with LC3 positive autophagosomes. A, C, E, and G, gallery of confocal laser microscopy images treated with vehicle (DMSO) (A), decorin (200 nm) (C), AICAR (500 μm) (E), or HBSS (G) for 6 h and immunostained for VEGFA (green) and LC3 (red). Nuclei (blue) were visualized with DAPI. Bar ∼10 μm. B, D, F, and H, line scanning profiles for each condition in the top set of panels. Confocal imaging is representative of three independent biological replicates, each assaying ∼10 fields per condition/biological replicate with comparable outcomes.
      Next, we investigated the formation of VEGFA/LC3 dually-positive autophagosomes using differential interference contrast (DIC) microscopy on HUVEC challenged with decorin, AICAR, or HBSS. We immunostained the cells with antibodies against either LC3/VEGFA (Fig. 5, A–D) or Beclin 1/VEGFA (Fig. 5, E–H) and found that all three stimuli significantly promoted the formation of dually-positive VEGFA/LC3 (Fig. 5, B–D; Fig. S4A) or VEGFA/Beclin-1 (Fig. 5, F–H; Fig. S4B) autophagosomes over basal conditions (Fig. 5, A and B; Fig. S4, A and B). Moreover, we found discrete VEGFA/LC3 puncta within large autophagosomes following decorin, AICAR, or HBSS (Fig. 5, B–D, white arrows) that were absent under basal conditions. Next, we evaluated VEGFA/p62 autophagosomal positivity and found that decorin significantly increases the number of VEGFA/p62 dually-positive autophagosomes as well (Fig. S4, C and D). Mechanistically, we found that the formation of VEGFA/LC3-positive autophagosomes in HUVEC was suppressed by treatment with the VEGFR2 kinase inhibitor SU5416 (p < 0.001, Fig. 5, I and J). We validated the immunofluorescence by omitting the primary or secondary antibodies in the presence or absence of BafA1 (Fig. S5). BafA1 served as a positive control for LC3. Treating with BafA1 recapitulated the augmented co-localization of VEGFA with LC3 (cf. Figure 4, Figure 5; Fig. S5, A and B) over basal conditions. Omitting either the primary (Fig. S5, C and D) or secondary (Fig. S5, E and F) antibodies annulled the immunofluorescence signal, even in the presence of BafA1. These data reinforce the robustness of our immunofluorescence data.
      Figure thumbnail gr5
      Figure 5Differential interference contrast microscopy of VEGFA-positive autophagosomes. A–D, gallery of DIC microscopy images in HUVEC treated with pro-autophagic stimuli (vehicle, DMSO; decorin, 200 nm; AICAR, 500 μm) as listed for 6 h and immunostained with VEGFA (green) or LC3 (red). E–H, identical experiment in HUVEC immunostained with VEGFA (green) or Beclin 1 (red). Nuclei (blue) were visualized with DAPI. Bar ∼10 μm. I, representative DIC images of HUVEC treated with decorin (200 nm) ± SU5416 (1 μm) for 6 h and immunostained with VEGFA (green) or LC3 (red). J, quantification of the number of autophagosomes per cell in HUVEC as depicted in I. The graph is representative of four independent biological replicates where ∼40 cells per field were counted per biological replicate. Statistics were calculated via one-way ANOVA (***, p < 0.001).
      Collectively, these imaging data posit VEGFA as an autophagic substrate that is present in LC3- or p62-positive autophagosomes under basal conditions. The incorporation of VEGFA into autophagosomes was substantially augmented in response to decorin/VEGFR2 signaling, as well as conventional pro-autophagic cues.

      Peg3 drives VEGFA clearance via autophagy

      As AMPK activation up-regulates Peg3 (cf. Fig. 1, B and C), and Peg3 drives autophagosome formation (
      • Torres A.
      • Gubbiotti M.A.
      • Iozzo R.V.
      Decorin-inducible Peg3 evokes beclin 1-mediated autophagy and thrombospondin 1-mediated angiostasis.
      ), we postulated that Peg3 could be directly involved in regulating VEGFA clearance via autophagy. Structurally, Peg3 is composed of an N-terminal SCAN domain required for protein–protein interactions and an elongated C terminus of 12 C2H2 Krüpple-like zinc fingers interspersed with proline-rich regions potentially used for DNA binding and transcriptional regulation (Fig. 6A) (
      • Kohda T.
      • Asai A.
      • Kuroiwa Y.
      • Kobayashi S.
      • Aisaka K.
      • Nagashima G.
      • Yoshida M.C.
      • Kondo Y.
      • Kagiyama N.
      • Kirino T.
      • Kaneko-Ishino T.
      • Ishino F.
      Tumour suppressor activity of human imprinted gene PEG3 in a glioma cell line.
      ,
      • Kuroiwa Y.
      • Kaneko-Ishino T.
      • Kagitani F.
      • Kohda T.
      • Li L.-L.
      • Tada M.
      • Suzuki R.
      • Yokoyama M.
      • Shiroishi T.
      • Wakana S.
      • Barton S.C.
      • Ishino F.
      • Surani M.A.
      Peg3 imprinted gene on proximal chromosome 7 encodes for a zinc finger protein.
      ,
      • Edelstein L.C.
      • Collins T.
      The SCAN domain family of zinc finger transcription factors.
      ,
      • Rimsa V.
      • Eadsforth T.C.
      • Hunter W.N.
      Structure of the SCAN domain of human paternally expressed gene 3 protein.
      ). We transiently transfected PAER2 cells with empty vector (pcDNA3.1), HA-PEG3, or HA-SCAN (Fig. 6A) and verified their correct expression by immunoblotting using anti-HA antibodies (Fig. 6B). Importantly, and in contrast to either empty vector or SCAN-domain, overexpression of full-length Peg3 drove the accumulation of VEGFA into LC3-positive autophagosomes (Fig. 6, C and D).
      Figure thumbnail gr6
      Figure 6Peg3 drives VEGFA clearance via autophagy. A, schematic depiction of Peg3 architecture and engineered constructs. B, representative immunoblot validating expression of HA-tagged full-length Peg3, or HA-SCAN. WB, Western blot. C, immunofluorescence images of PAER2 transfected with equimolar amounts of empty vector, full-length Peg3, or SCAN domain 48 h post-transfection. D, quantification of autophagosomes per cell in PAER2 as depicted in C. Graph represents four independent biological replicates where ∼50 cells per field were counted. E, confocal images of HUVEC transfected with siScr or siPeg3 (100 pm each) in combination with decorin (200 nm) for 6 h. F, calculation of Pearson's coefficient of colocalization for the degree of overlap between VEGFA and LC3 as demonstrated in E. In all images, immunostaining was performed for VEGFA (green) or LC3 (red). Nuclei (blue) were visualized with DAPI. Bar ∼10 μm. GAPDH served as loading control in B. For Pearson's coefficient in F, ∼10 fields per experimental condition over three biological replicates were utilized. Statistics were calculated via one-way ANOVA. N.S., not significant (***, p < 0.001).
      Next, we performed loss-of-function experiments to determine the role of Peg3 in promoting VEGFA-positive autophagosomes in response to decorin. We verified Peg3 knockdown via RNAi and found a significant (p = 0.002) depletion (Fig. S4E). Corroborating the gain-of-function experiments above, we discovered that transient depletion of Peg3 abrogated the ability of decorin to drive VEGFA into LC3-positive autophagosomes in HUVEC (Fig. 6E) vis à vis vehicles. Quantitation of the Pearson's coefficient of colocalization as a readout for dual VEGFA/LC3 positivity supported (Fig. 6F) our confocal imaging that Peg3 is required for decorin-mediated VEGFA/LC3 autophagosome formation in endothelial cells. Collectively, these data delineate a critical role for Peg3 as a necessary and sufficient autophagic regulator to clear VEGFA by autophagy in response to pro-autophagic stimuli.

      Interfering with basal autophagic flux leads to an accumulation of intracellular VEGFA

      We observed that VEGFA co-localized under basal conditions with LC3 (cf. Figure 4, Figure 5), Beclin 1 (cf. Fig. 5), and p62 (cf. Fig. S4), suggesting that VEGFA could be directly degraded via autophagy. We pharmacologically blocked autophagic flux using bafilomycin A1 (BafA1 hereafter), a universally established method to assay autophagic processes (
      • Moulis M.
      • Vindis C.
      Methods for measuring autophagy in mice.
      ,
      • Galluzzi L.
      • Bravo-San Pedro J.M.
      • Levine B.
      • Green D.R.
      • Kroemer G.
      Pharmacological modulation of autophagy: therapeutic potential and persisting obstacles.
      ,
      • Gurney M.A.
      • Huang C.
      • Ramil J.M.
      • Ravindran N.
      • Andres A.M.
      • Sin J.
      • Linton P.J.
      • Gottlieb R.A.
      Measuring cardiac autophagic flux in vitro and in vivo.
      ). Mechanistically, BafA1 inhibits the vacuolar H+-ATPase to preclude mature autophagosomes from fusing with lysosomes, thereby giving a metric on the substrates undergoing autophagic degradation. Using PAER2 cells, we performed a BafA1 dose response. Our positive control, proteolytic conversion of LC3-I to LC3-II, which amasses following autophagic inhibition (
      • Galluzzi L.
      • Bravo-San Pedro J.M.
      • Levine B.
      • Green D.R.
      • Kroemer G.
      Pharmacological modulation of autophagy: therapeutic potential and persisting obstacles.
      ,
      • Martinez J.
      • Almendinger J.
      • Oberst A.
      • Ness R.
      • Dillon C.P.
      • Fitzgerald P.
      • Hengartner M.O.
      • Green D.R.
      Microtubule-associated protein 1 light chain 3α (LC3)-associated phagocytosis is required for the efficient clearance of dead cells.
      ,
      • Mizushima N.
      • Yoshimori T.
      • Levine B.
      Methods in mammalian autophagy research.
      ), increased in a concentration-dependent manner with as little as 1 nm BafA1 (Fig. 7, A and B). Concurrent with LC3-II induction, we found that VEGFA increased proportionally (Fig. 7, A and B). Notably, we found nearly identical results in HUVEC (Fig. 7, C and D).
      Figure thumbnail gr7
      Figure 7Interfering with basal autophagic flux leads to an accumulation of intracellular VEGFA. A, representative immunoblots of PAER2 treated with increasing concentrations of bafilomycin A1 (BafA1) for 6 h. B, quantification of targets as in A. C and D, immunoblot of HUVEC ± BafA1 (500 nm) (C) and quantification (D). E and F, immunoblot of HUVEC ± chloroquine (30 μm) for 6 h (E) and quantification (F). G and H, immunoblot depicting HUVEC following the transient transfection of siScr or siATG5 (100 pm each) (G) and corresponding quantification of targets (H) as in G. GAPDH served as the loading control for immunoblots depicted in A, C, E, and G. Data are reflective of four independent biological replicates. Data are expressed as arbitrary units (A.U.) on a dot density plot. Statistics were calculated via two-tailed Student's t test for D, F, and H.
      Next, we used a different autophagic inhibitor, chloroquine, to assay the sensitivity of VEGFA to basal autophagy. Chloroquine (CQ), a lysosomotropic agent, has previously been thought to prevent lysosomal acidification as a mechanism of action (
      • Moore B.R.
      • Page-Sharp M.
      • Stoney J.R.
      • Ilett K.F.
      • Jago J.D.
      • Batty K.T.
      Pharmacokinetics, pharmacodynamics, and allometric scaling of chloroquine in a murine malaria model.
      ). However, recently it has been shown that CQ inhibits autophagic flux by decreasing autophagosome-lysosome fusion (
      • Mauthe M.
      • Orhon I.
      • Rocchi C.
      • Zhou X.
      • Luhr M.
      • Hijlkema K.J.
      • Coppes R.P.
      • Engedal N.
      • Mari M.
      • Reggiori F.
      Chloroquine inhibits autophagic flux by decreasing autophagosome-lysosome fusion.
      ). Evaluating HUVEC with CQ, we found that LC3-II increased (Fig. 7, E and F), validating CQ function, concomitantly with an accumulation of VEGFA (Fig. 7, E and F). Treating PAER2 cells with CQ resulted in a similar pattern wherein p62 and VEGFA both increased (Fig. S6, A and B) in response to autophagic inhibition.
      Next, we complemented the pharmacological approach with RNAi-mediated silencing of ATG5, an E1-like activating protease that processes LC3 for lipidation with phosphatidylethanolamine (
      • Singh R.
      • Kaushik S.
      • Wang Y.
      • Xiang Y.
      • Novak I.
      • Komatsu M.
      • Tanaka K.
      • Cuervo A.M.
      • Czaja M.J.
      Autophagy regulates lipid metabolism.
      ). Depleting ATG5 potently abrogates autophagosomal maturation leading to a cessation of autophagy for flux analyses (
      • Katayama H.
      • Kogure T.
      • Mizushima N.
      • Yoshimori T.
      • Miyawaki A.
      A sensitive and quantitative technique for detecting autophagic events based on lysosomal delivery.
      ,
      • Nishida Y.
      • Arakawa S.
      • Fujitani K.
      • Yamaguchi H.
      • Mizuta T.
      • Kanaseki T.
      • Komatsu M.
      • Otsu K.
      • Tsujimoto Y.
      • Shimizu S.
      Discovery of Atg5/Atg7-independent alternative macroautophagy.
      ). Loss of ATG5 results in a build-up of LC3-I, the unprocessed form of LC3 (
      • Hara T.
      • Nakamura K.
      • Matsui M.
      • Yamamoto A.
      • Nakahara Y.
      • Suzuki-Migishima R.
      • Yokoyama M.
      • Mishima K.
      • Saito I.
      • Okano H.
      • Mizushima N.
      Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice.
      ). We verified our siRNA and found a significant knockdown of ATG5 in HUVEC (Fig. 7, G and H). ATG5 loss led to a marked increase in LC3-I (Fig. 7, G and H), thus functionally validating ATG5 silencing. Analogous to our findings, genetically silencing a critical enzyme for autophagosome maturation, we discovered a significant (p < 0.001) increase in intracellular VEGFA with respect to our nontargeting vehicle (Fig. 7, G,H).
      To determine the generality of our findings, we applied a similar strategy to nonendothelial systems. We evaluated NIH3T3 mouse fibroblasts with either BafA1 (Fig. S6, C and D) or CQ (Fig. S6, E and F), with LC3-II conversion as a positive control, and recapitulated the results insofar as VEGFA is degraded by basal autophagic flux. Finally, we evaluated highly-malignant MDA-MB-231 triple-negative breast carcinoma cells by treating with BafA1. We found increased LC3-II with a build-up of VEGFA (Fig. S6, G and H). Collectively, these pharmacological and genetic data indicate that VEGFA is degraded by basal autophagy in a diverse number of cellular systems and types.

      Decorin evokes intracellular degradation of VEGFA via autophagy and RAB24

      A hallmark of decorin bioactivity is the dual property of being anti-angiogenic (
      • Neill T.
      • Schaefer L.
      • Iozzo R.V.
      An oncosuppressive role for decorin.
      ,
      • Neill T.
      • Painter H.
      • Buraschi S.
      • Owens R.T.
      • Lisanti M.P.
      • Schaefer L.
      • Iozzo R.V.
      Decorin antagonizes the angiogenic network: concurrent inhibition of Met, hypoxia inducible factor-1α and vascular endothelial growth factor A and induction of thrombospondin-1 and TIMP3.
      ,
      • Neill T.
      • Jones H.R.
      • Crane-Smith Z.
      • Owens R.T.
      • Schaefer L.
      • Iozzo R.V.
      Decorin induces rapid secretion of thrombospondin-1 in basal breast carcinoma cells via inhibition of Ras homolog gene family, member A/Rho-associated coiled-coil containing protein kinase 1.
      ,
      • Neill T.
      • Schaefer L.
      • Iozzo R.V.
      Decorin, a guardian from the matrix.
      ,
      • Karamanos N.K.
      • Piperigkou Z.
      • Theocharis A.D.
      • Watanabe H.
      • Franchi M.
      • Baud S.
      • Brézillon S.
      • Götté M.
      • Passi A.
      • Vigetti D.
      • Ricard-Blum S.
      • Sanderson R.D.
      • Neill T.
      • Iozzo R.V.
      Proteoglycan chemical diversity drives multifunctional cell regulation and therapeutics.
      ,
      • Karamanos N.K.
      • Theocharis A.D.
      • Neill T.
      • Iozzo R.V.
      Matrix modeling and remodeling: a biological interplay regulating tissue homeostasis and diseases.
      ) and pro-autophagic (
      • Buraschi S.
      • Neill T.
      • Owens R.T.
      • Iniguez L.A.
      • Purkins G.
      • Vadigepalli R.
      • Evans B.
      • Schaefer L.
      • Peiper S.C.
      • Wang Z.X.
      • Iozzo R.V.
      Decorin protein core affects the global gene expression profile of the tumor microenvironment in a triple-negative orthotopic breast carcinoma xenograft model.
      ,
      • Buraschi S.
      • Neill T.
      • Goyal A.
      • Poluzzi C.
      • Smythies J.
      • Owens R.T.
      • Schaefer L.
      • Torres A.
      • Iozzo R.V.
      Decorin causes autophagy in endothelial cells via Peg3.
      ,
      • Torres A.
      • Gubbiotti M.A.
      • Iozzo R.V.
      Decorin-inducible Peg3 evokes beclin 1-mediated autophagy and thrombospondin 1-mediated angiostasis.
      ,
      • Neill T.
      • Sharpe C.
      • Owens R.T.
      • Iozzo R.V.
      Decorin-evoked paternally expressed gene 3 (PEG3) is an upstream regulator of the transcription factor EB (TFEB) in endothelial cell autophagy.
      ,
      • Neill T.
      • Torres A.
      • Buraschi S.
      • Iozzo R.V.
      Decorin has an appetite for endothelial cell autophagy.
      ,
      • Goyal A.
      • Neill T.
      • Owens R.T.
      • Schaefer L.
      • Iozzo R.V.
      Decorin activates AMPK, an energy sensor kinase, to induce autophagy in endothelial cells.
      ). Moreover, the Dcn gene itself is an autophagic-inducible gene (
      • Gubbiotti M.A.
      • Neill T.
      • Frey H.
      • Schaefer L.
      • Iozzo R.V.
      Decorin is an autophagy-inducible proteoglycan and is required for proper in vivo autophagy.
      ). We postulated that decorin-mediated autophagy is the mechanism by which intracellular VEGFA is reduced.
      We investigated this hypothesis by performing autophagic flux assays in PAER2 and found that decorin significantly suppressed VEGFA levels (Fig. 8, A and B). Treatment with BafA1 alone increased both LC3-II and VEGFA above basal conditions (Fig. 8, A and B), further reinforcing our above findings (cf. Fig. 7, A and B) that VEGFA is degraded by basal autophagy. Combinatorial treatment of decorin and BafA1 increased LC3-II levels beyond the levels expected of basal autophagy (Fig. 8, A and B), corroborating the concept that decorin evokes excessive autophagy by increasing autophagic flux. Critically, VEGFA was no longer suppressed by decorin following autophagic inhibition with BafA1. Intracellular VEGFA levels appeared modestly increased above basal levels with the combinatorial treatment indicating that decorin enhances the rate of VEGFA catabolism (Fig. 8, A and B). These data indicate that decorin clears intracellular VEGFA by inducing endothelial cell autophagy.
      Figure thumbnail gr8
      Figure 8Decorin evokes intracellular degradation of VEGFA via autophagy and RAB24. A, representative immunoblots of PAER2 cells treated with decorin (200 nm) ± BafA1 (500 nm) for 6 h. B, quantification of targets from A. C, representative immunoblots of PAER2 transiently transfected with siScr or siRAB24 (100 pm each) ± decorin (200 nm) for 6 h. D, quantification of targets from C. GAPDH served as loading control for immunoblots depicted in A and C. Data are reflective of four independent biological replicates and expressed as arbitrary units (A.U.) on a dot density plot. Statistics were calculated via one-way ANOVA (**, p < 0.01; ***, p < 0.001).
      Next, we investigated the small GTPase RAB24, a gene induced by decorin via NanoString analysis.
      T. Neill, C. G. Chen, S. Buraschi, and R. V. Iozzo, unpublished dataset.
      RAB24 has recently been implicated in the autophagic pathway (
      • Ylä-Anttila P.
      • Eskelinen E.L.
      Roles for RAB24 in autophagy and disease.
      ). RAB24 facilitates autophagic clearance of components undergoing basal autophagy, but it does not aid in autophagosomal formation, as depletion of RAB24 results in a significant increase in late autophagic compartments (
      • Ylä-Anttila P.
      • Mikkonen E.
      • Happonen K.E.
      • Holland P.
      • Ueno T.
      • Simonsen A.
      • Eskelinen E.L.
      RAB24 facilitates clearance of autophagic compartments during basal conditions.
      ). Therefore, as VEGFA is degraded by autophagy and decorin promotes increased autophagic flux of VEGFA and lysosomal biogenesis via TFEB (
      • Neill T.
      • Sharpe C.
      • Owens R.T.
      • Iozzo R.V.
      Decorin-evoked paternally expressed gene 3 (PEG3) is an upstream regulator of the transcription factor EB (TFEB) in endothelial cell autophagy.
      ), we evaluated the role of RAB24. We silenced RAB24 by RNAi and verified its depletion (Fig. 8, C and D). We noted that decorin induced RAB24 by ∼1.9-fold (p = 0.014) (Fig. 8, C and D). Moreover, we found that LC3-I was significantly increased following loss of RAB24 indicating a role for RAB24 in autophagosome maturation with no significant changes seen in p62 (Fig. 8, C and D). Decorin reduced VEGFA in PAER2 cells and this effect was abrogated following RAB24 silencing, recapitulating the findings from the flux assays (Fig. 8, C and D). Moreover, loss of RAB24 alone increased VEGFA levels, corroborating the role of RAB24 in basal autophagic regulation and strengthening the shuttling of VEGFA in the autophagic pathway for clearance (Fig. 8, C and D).
      Taken together, these data illustrate that decorin promotes VEGFA catabolism by autophagic clearance via RAB24 in endothelial cells. This mechanism serves as a nexus by which decorin integrates and exerts its powerful anti-angiogenic and pro-autophagic properties.

      VEGFA is reduced in vivo by protracted fasting

      To determine whether our in vitro results could also occur in vivo, we fasted WT C57BL/6 mice for 48 h. We found that VEGFA levels in both heart and aorta of fasted mice were significantly reduced compared with those fed ad libitum (Fig. 9, A–D). As a positive control for starvation-induced autophagic induction, we tested LC3-II levels and found a marked increase in both cardiac and aortic tissues (Fig. 9, A–D). To provide definitive proof for the in vivo autophagic catabolism of VEGFA, we starved the mice for 48 h and treated them with CQ (50 mg/kg) for the last 4 h. We found a significant increase in VEGFA in cardiac and aortic tissues relative to starved mice alone (Fig. 9, E–G). As a negative control, we evaluated β-catenin, a critical regulator of cell behavior in response to Wnt signals (
      • Clevers H.
      Wnt/β-catenin signaling in development and disease.
      ) and canonical decorin target (
      • Buraschi S.
      • Pal N.
      • Tyler-Rubinstein N.
      • Owens R.T.
      • Neill T.
      • Iozzo R.V.
      Decorin antagonizes Met receptor activity and downregulates β-catenin and Myc levels.
      ). We found no significant changes in β-catenin levels in either the heart (p = 0.456) or aorta (p = 0.433) following in vivo autophagy inhibition (Fig. 9, E and F). Collectively, these results demonstrate that VEGFA is regulated in a nutrient-dependent manner in parenchymal tissues in vivo. These findings greatly reinforce our in vitro data whereby VEGFA is governed by prevailing nutrient conditions and by in vivo autophagic flux.
      Figure thumbnail gr9
      Figure 9VEGFA is reduced in vivo by protracted fasting. A, representative immunoblot of cardiac tissue from WT C57BL/6 mice following 48 h starvation. B, quantification of immunoblot targets as in A. C, representative immunoblot of aortic tissue from WT C57BL/6 mice following 48 h starvation. D, quantification of immunoblot targets as in C. E and F, representation immunoblot of in vivo autophagic flux in cardiac (E) or aortic (F) tissue following a 48-h starvation period with concurrent systemic administration of chloroquine (50 mg/kg) for the last 4 h. G, quantification of VEGFA from the tissues as shown in E and F. GAPDH served as a loading control for immunoblots depicted in A, C, E, and F. Data are reflective of four independent biological replicates and expressed as arbitrary units (A.U.) on a dot density plot. Statistics were calculated via two-tailed Student's t test.

      Discussion

      Autophagic modulation by soluble extracellular matrix components is rapidly emerging as a common, conserved, and fundamental cellular mechanism intricately intertwined in health and disease outcomes (
      • Gubbiotti M.A.
      • Iozzo R.V.
      Proteoglycans regulate autophagy via outside-in signaling: an emerging new concept.
      ,
      • Neill T.
      • Schaefer L.
      • Iozzo R.V.
      Instructive roles of extracellular matrix on autophagy.
      ,
      • Douglass S.
      • Goyal A.
      • Iozzo R.V.
      The role of perlecan and endorepellin in the control of tumor angiogenesis and endothelial cell autophagy.
      ). In this study, we functionally unified the anti-angiogenic and pro-autophagic properties of decorin downstream of VEGFR2, AMPK, and Peg3 signaling in endothelial cells and demonstrated that intracellular VEGFA is catabolized via autophagy (Fig. 10).
      Figure thumbnail gr10
      Figure 10Schematic depiction of decorin-evoked catabolism of VEGFA via endothelial cell autophagy. Please refer to the text for additional details. Star-shaped symbols denote lysosomal acid hydrolases.
      Stimulation with the AMP-mimetic AICAR phenocopies the effect of decorin. Our data support a reciprocal role of AMPK that requires the inherent kinase activity of the catalytic α-subunit, to induce Peg3 and simultaneously decrease VEGFA in endothelial cells at the protein level. Activating AMPK alone is biologically equivalent to decorin-dependent suppression of intracellular VEGFA, indicating that decorin and AMPK are in the same signaling pathway, presumably downstream of decorin/VEGFR2 interactions (Fig. 10). The precise connection between VEGFR2 and phosphorylated AMPK remains elusive. Considering the noncanonical activation of AMPK under replete nutrient conditions (
      • Goyal A.
      • Neill T.
      • Owens R.T.
      • Schaefer L.
      • Iozzo R.V.
      Decorin activates AMPK, an energy sensor kinase, to induce autophagy in endothelial cells.
      ) and cytosolic Ca2+ mobilization by decorin (
      • Patel S.
      • Santra M.
      • McQuillan D.J.
      • Iozzo R.V.
      • Thomas A.P.
      Decorin activates the epidermal growth factor receptor and elevates cytosolic Ca2+ in A431 cells.
      ), it is feasible that decorin is engaging the nucleotide-independent pathway to activate AMPK via CAMKK2 (
      • Garcia D.
      • Shaw R.J.
      AMPK: mechanisms of cellular energy sensing and restoration of metabolic balance.
      ) downstream of VEGFR2.
      Decorin differentially modulates signaling to favor autophagic induction by attenuating mTOR at P-Ser2448 and inducing AMPKα at P-Thr172. It is known that mTOR can evoke VEGFA expression via p70S6K1 activation and STAT3 nuclear translocation to the VEGFA promoter (
      • Yang F.
      • Zhang W.
      • Li D.
      • Zhan Q.
      Gadd45a suppresses tumor angiogenesis via inhibition of the mTOR/STAT3 protein pathway.
      ,
      • Dodd K.M.
      • Yang J.
      • Shen M.H.
      • Sampson J.R.
      • Tee A.R.
      mTORC1 drives HIF-1α and VEGF-A signalling via multiple mechanisms involving 4E-BP1, S6K1 and STAT3.
      ). Here, we demonstrate an mTOR-independent role of VEGFA suppression in endothelial cells utilizing pharmacological agents that inhibit the mTOR kinase and mimic low-energy conditions (
      • Guertin D.A.
      • Sabatini D.M.
      The pharmacology of mTOR inhibition.
      ), despite the lively regulation of LC3-I/-II and/or p62 (Fig. 10). Thus, bioenergetics-mediated regulation of intracellular VEGFA proceeds in an AMPK-dependent and mTOR-independent mechanism in endothelial cells and fibroblasts. It is possible that decorin initiates autophagy solely via AMPK and ULK1/2 (
      • Alers S.
      • Löffler A.S.
      • Wesselborg S.
      • Stork B.
      Role of AMPK-mTOR-Ulk1/2 in the regulation of autophagy: crosstalk, shortcuts, and feedbacks.
      ,
      • Kim J.
      • Kundu M.
      • Viollet B.
      • Guan K.-L.
      AMPK and mTOR regulate autophagy through direct phopshorylation of Ulk1.
      ,
      • Jung C.H.
      • Jun C.B.
      • Ro S.H.
      • Kim Y.M.
      • Otto N.M.
      • Cao J.
      • Kundu M.
      • Kim D.H.
      ULK-Atg13-FIP200 complexes mediate mTOR signaling to the autophagy machinery.
      ) as decorin requires the class III phosphatidylinositol 3-kinase or Vps34 and Beclin 1-positive complexes (
      • Buraschi S.
      • Neill T.
      • Goyal A.
      • Poluzzi C.
      • Smythies J.
      • Owens R.T.
      • Schaefer L.
      • Torres A.
      • Iozzo R.V.
      Decorin causes autophagy in endothelial cells via Peg3.
      ,
      • Stjepanovic G.
      • Baskaran S.
      • Lin M.G.
      • Hurley J.H.
      Vps34 kinase domain dynamics regulate the autophagic PI 3-kinase complex.
      ) that are heavily involved in the early stages of autophagic progression (
      • Russell R.C.
      • Tian Y.
      • Yuan H.
      • Park H.W.
      • Chang Y.Y.
      • Kim J.
      • Kim H.
      • Neufeld T.P.
      • Dillin A.
      • Guan K.L.
      ULK1 induces autophagy by phosphorylating Beclin-1 and activating VPS34 lipid kinase.
      ).
      We found that conventional autophagic stimuli trigger VEGFA incorporation into LC3- and Beclin 1-positive autophagosomes to a similar magnitude and frequency to that of decorin. This process occurs downstream of VEGFR2 as tyrosine kinase inhibition prevents autophagosomal incorporation, highlighting an active role of this RTK for the autophagic degradation of VEGFA in response to anti-angiogenic signals originating from the extracellular matrix. Decorin transduces signals through VEGFR2 that engage and augment catabolic hubs for the degradation of powerful angiokines residing within the cytosol. Interestingly, the overall levels of VEGFA are sensitive to the ambient nutrient conditions as nutrient deprivation in multiple cellular models representing diverse tissues, such as endothelium, stromal, and triple negative breast cancer all resulted in a significant decrease of VEGFA.
      Mechanistically, the mobilization of VEGFA into LC3-positive autophagosomes requires the putative tumor suppressor gene, Peg3 (Fig. 10). Transient depletion or overexpression of full-length Peg3 is necessary and sufficient for the formation of VEGFA/LC3-positive structures, respectively, whereas genetically ablating the C-terminal zinc finger domains abrogates this process. Peg3 is capable of driving endothelial BECN1 gene expression, a critical effector necessary for autophagosomes biogenesis (
      • Kang R.
      • Zeh H.J.
      • Lotze M.T.
      • Tang D.
      The beclin 1 network regulates autophagy and apoptosis.
      ,
      • Liang X.H.
      • Jackson S.
      • Seaman M.
      • Brown K.
      • Kempkes B.
      • Hibshoosh H.
      • Levine B.
      Induction of autophagy and inhibition of tumorigenesis by beclin 1.
      ,
      • Yue Z.
      • Jin S.
      • Yang C.
      • Levine A.J.
      • Heintz N.
      Beclin 1, an authophagy gene essential for early embryonic development, is a haploinsufficient tumor suppressor.
      ), and LC3-mediated autophagic flux (
      • Torres A.
      • Gubbiotti M.A.
      • Iozzo R.V.
      Decorin-inducible Peg3 evokes beclin 1-mediated autophagy and thrombospondin 1-mediated angiostasis.
      ). Truncating the zinc finger domains may result in a loss of BECN1 expression culminating in decreased autophagic flux, thereby manifesting as reduced VEGFA/LC3 autophagosomes. Alternately, loss of the zinc fingers may abrogate the scaffolding function of Peg3, as its incorporation into Beclin 1-positive protein complexes (
      • Buraschi S.
      • Neill T.
      • Goyal A.
      • Poluzzi C.
      • Smythies J.
      • Owens R.T.
      • Schaefer L.
      • Torres A.
      • Iozzo R.V.
      Decorin causes autophagy in endothelial cells via Peg3.
      ) may be impaired resulting in dampened autophagosome formation. The exact molecular partners that shuttle VEGFA into the autophagosome remain unidentified. Bioinformatics analyses of the various VEGFA isoforms did not identify a canonical LC3-interacting region that would enable specific autophagosomal targeting; therefore, we cannot exclude the possibility that VEGFA is merely a constituent of the larger cytoplasmic milieu subject to bulk degradation by macroautophagy.
      Intracellular VEGFA is subject to autophagic flux as pharmacological inhibition with BafA1 or CQ, lysosomotropic agents that block autophagosomal/lysosomal fusion, results in a considerable accumulation of VEGFA under basal conditions (Fig. 10). These findings were recapitulated by depleting ATG5, a critical protease for LC3 maturation (
      • Hara T.
      • Nakamura K.
      • Matsui M.
      • Yamamoto A.
      • Nakahara Y.
      • Suzuki-Migishima R.
      • Yokoyama M.
      • Mishima K.
      • Saito I.
      • Okano H.
      • Mizushima N.
      Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice.
      ). Importantly, decorin augments VEGFA flux, suggesting that this is a primary pathway for intracellular control of VEGFA. Mechanistically, decorin-evoked catabolism of VEGFA depends on RAB24, a small GTPase recently implicated in autophagic progression, particularly of substrates undergoing basal autophagy (
      • Ylä-Anttila P.
      • Eskelinen E.L.
      Roles for RAB24 in autophagy and disease.
      ,
      • Ylä-Anttila P.
      • Mikkonen E.
      • Happonen K.E.
      • Holland P.
      • Ueno T.
      • Simonsen A.
      • Eskelinen E.L.
      RAB24 facilitates clearance of autophagic compartments during basal conditions.
      ). However, whether the control of RAB24 is under the auspices of the VEGFR2/AMPK/Peg3 signaling axis remains to be investigated (Fig. 10).
      We validated our findings in vivo by demonstrating that starvation decreased VEGFA content in cardiac and aortic tissues, suggesting physiological relevance for the metabolic control over VEGFA. The starvation-mediated reduction of cardiac and aortic VEGFA was blocked by systemically administrating CQ, providing evidence for the in vivo role of autophagy in VEGFA catabolism. The precise reservoir of VEGFA that is being depleted, whether intracellular, cell-surface associated, extracellular matrix, and/or blood, is currently unknown.
      Therapeutic targeting of VEGFA with biological agents, such as anti-VEGFA therapies using bevacizumab and ranibizumab, have revolutionized the treatment of metastatic colorectal cancer and severe ophthalmological disorders (
      • Apte R.S.
      • Chen D.S.
      • Ferrara N.
      VEGF in signaling and disease: beyond discovery and development.
      ,
      • Ferrara N.
      • Adamis A.P.
      Ten years of anti-vascular endothelial growth factor therapy.
      ). Mechanistically, these agents function by inhibiting VEGFA thereby preventing rampant neovascularization (
      • Ferrara N.
      • Adamis A.P.
      Ten years of anti-vascular endothelial growth factor therapy.
      ). Considering the effect of decorin in suppressing VEGFA via autophagic degradation, we recognize the potential clinical implications of decorin administered as an adjuvant that specifically targets the RTK-rich environment of the growing cancer (
      • Buraschi S.
      • Pal N.
      • Tyler-Rubinstein N.
      • Owens R.T.
      • Neill T.
      • Iozzo R.V.
      Decorin antagonizes Met receptor activity and downregulates β-catenin and Myc levels.
      ). Moreover, our general finding of VEGFA sensitivity to starvation and subsequent accumulation with autophagic inhibitors transcends proteoglycan-driven endothelial cell autophagy. Starvation-evoked VEGFA clearance may augment the clinical benefits of caloric restriction as a potent modality to curb breast tumorigenesis by enhancing immunotherapies (
      • Orillion A.
      • Damayanti N.P.
      • Shen L.
      • Adelaiye-Ogala R.
      • Affronti H.
      • Elbanna M.
      • Chintala S.
      • Ciesielski M.
      • Fontana L.
      • Kao C.
      • Elzey B.D.
      • Ratliff T.L.
      • Nelson D.E.
      • Smiraglia D.
      • Abrams S.I.
      • Pili R.
      Dietary protein restriction reprograms tumor-associated macrophages and enhances immunotherapy.
      ), radiation efficacy (
      • Saleh A.D.
      • Simone B.A.
      • Palazzo J.
      • Savage J.E.
      • Sano Y.
      • Dan T.
      • Jin L.
      • Champ C.E.
      • Zhao S.
      • Lim M.
      • Sotgia F.
      • Camphausen K.
      • Pestell R.G.
      • Mitchell J.B.
      • Lisanti M.P.
      • Simone N.L.
      Caloric restriction augments radiation efficacy in breast cancer.
      ), and alleviating the metastatic burden (
      • Jin L.
      • Lim M.
      • Zhao S.
      • Sano Y.
      • Simone B.A.
      • Savage J.E.
      • Wickstrom E.
      • Camphausen K.
      • Pestell R.G.
      • Simone N.L.
      The metastatic potential of triple-negative breast cancer is decreased via caloric restriction-mediated reduction of the miR-17∼92 cluster.
      ,
      • Simone B.A.
      • Dan T.
      • Palagani A.
      • Jin L.
      • Han S.Y.
      • Wright C.
      • Savage J.E.
      • Gitman R.
      • Lim M.K.
      • Palazzo J.
      • Mehta M.P.
      • Simone N.L.
      Caloric restriction coupled with radiation decreases metastatic burden in triple negative breast cancer.
      ).
      Collectively, we have integrated two critical properties of soluble decorin operating downstream of receptor binding for its putative tumor repressor properties. At the nexus of this network is Peg3, which acts as a molecular conduit for coordinated angiostasis and autophagic induction in response to decorin and perhaps other autophagic stimuli derived from extracellular matrix interactions (
      • Garantziotis S.
      • Savani R.C.
      Hyaluronan biology: a complex balancing act of structure, function, location and context.
      ). These data further reinforce the candidacy of decorin as a next-generation anti-angiogenic protein therapy (
      • Ricard-Blum S.
      • Vallet S.D.
      Fragments generated upon extracellular matrix remodeling: biological regulators and potential drugs.
      ) in the ongoing fight against cancer.

      Experimental procedures

      Cells, chemicals, and general reagents

      Primary HUVEC were obtained from Lifeline Cell Technology, grown in basal media supplemented with the VascuLife EnGS LifeFactors Kit, also from Lifeline Cell Technology, and used within the first five passages. Porcine aortic endothelial cells overexpressing VEGFR2 (PAER2) were described previously (
      • Goyal A.
      • Pal N.
      • Concannon M.
      • Paul M.
      • Doran M.
      • Poluzzi C.
      • Sekiguchi K.
      • Whitelock J.M.
      • Neill T.
      • Iozzo R.V.
      Endorepellin, the angiostatic module of perlecan, interacts with both the α2β1 integrin and vascular endothelial growth factor receptor 2 (VEGFR2).
      ,
      • Goyal A.
      • Poluzzi C.
      • Willis C.D.
      • Smythies J.
      • Shellard A.
      • Neill T.
      • Iozzo R.V.
      Endorepellin affects angiogenesis by antagonizing diverse VEGFR2-evoked signaling pathways: transcriptional repression of HIF-1α and VEGFA and concurrent inhibition of NFAT1 activation.
      ). Human aortic endothelial cells immortalized by stable expression of the human catalytic subunit of telomerase (TeloHAEC), immortalized NIH-3T3 murine fibroblasts, and human MDA-MB-231 triple-negative breast carcinoma cells were procured from American Type Cell Culture. Cells were grown at 37 °C in a 5% CO2 atmosphere in Dulbecco's modified Eagle's medium containing 4.5 g/liter of glucose, l-glutamine, and sodium pyruvate from Life Technologies and supplemented with 5% fetal bovine serum from ThermoFisher Scientific and 100 units/ml of penicillin/streptomycin from Life Technologies. Dimethyl sulfoxide (DMSO) and Hanks' balanced salt solution also came from ThermoFisher Scientific, whereas Earle's balanced salt solution was from HyClone. Rabbit monoclonal antibodies against GAPDH, HA, AMPKα, and P-AMPKα were obtained from Cell Signaling Technologies. The rabbit polyclonal antibody against RAB24 was from Santa Cruz Biotechnology. Rabbit polyclonal antibodies against p62 and LC3B were purchased from Sigma. Mouse monoclonal antibodies against VEGFA and ATG5 were purchased from Santa Cruz Biotechnology. A second mouse mAb against VEGFA was purchased from Abcam. The rabbit anti-Beclin 1 and horseradish peroxidase-conjugated goat anti-rabbit and donkey anti-mouse secondary antibodies were obtained from EMD Millipore. A custom rabbit polyclonal antibody against the N-terminal human SCAN domain of Peg3 (amino acids 164–177) was generated by GenScript. Compound C, AICAR, SU5416, bafilomycin A1, and chloroquine were purchased from Sigma. Torin 1 was purchased from Tocris Biosciences. INK128 was acquired from Cayman Chemicals. DMSO was used as vehicle where appropriate for all experiments. All primary antibodies were used at 1:1,000 diluted in 1% BSA/TBST except for GAPDH, which was used at 1:10,000 and VEGFA (from Santa Cruz Biotechnology) was used at 1:500. For immunofluorescence, primary antibodies were used at 1:200 in 1% BSA in PBS. Secondary antibodies for chemiluminescence were used at 1:5,000 in the same buffer as above. The SuperSignal West Pico Enhanced Chemiluminescence substrate was purchased from ThermoFisher Scientific (USA). Purification and validation of human recombinant decorin free of any co-purifying contaminants can be found elsewhere (
      • Buraschi S.
      • Neill T.
      • Owens R.T.
      • Iniguez L.A.
      • Purkins G.
      • Vadigepalli R.
      • Evans B.
      • Schaefer L.
      • Peiper S.C.
      • Wang Z.X.
      • Iozzo R.V.
      Decorin protein core affects the global gene expression profile of the tumor microenvironment in a triple-negative orthotopic breast carcinoma xenograft model.
      ).

      Quantitative real-time PCR and analysis

      Briefly, quantitative PCR was carried out on subconfluent 6-well-plates seeded with ∼2 × 105 of HUVEC cells, treated per experimental conditions, and lysed in 1 ml of TRIzol reagent (Life Technologies). RNA isolation was carried out with Direct-zol RNA Miniprep Kit as per the manufacturer's protocol (Zymo Research). Total RNA (1 μg) was annealed with oligo(dT) primers, and cDNA was synthesized using SuperScript Reverse Transcriptase III (Life Technologies). Gene-specific primer sets for Homo sapiens PEG3 and VEGFA were designed and validated. Gene expression analysis was performed on a Roche LightCycler 480-II and calculated with the comparative ΔΔCt method. A full description can be found in Ref.
      • Buraschi S.
      • Neill T.
      • Goyal A.
      • Poluzzi C.
      • Smythies J.
      • Owens R.T.
      • Schaefer L.
      • Torres A.
      • Iozzo R.V.
      Decorin causes autophagy in endothelial cells via Peg3.
      .

      Transient DNA expression and RNAi-mediated silencing

      We transiently transfected PAER2 with empty vector (pcDNA3.1) or plasmids encoding HA-Peg3 or HA-SCAN using Lipofectamine 2000 (Life Technologies) in Opti-MEM reduced serum media (Gibco). Expression was verified by immunoblot using target-specific antibodies. A full description of the DNA transfection protocol has been described elsewhere (
      • Neill T.
      • Torres A.
      • Buraschi S.
      • Owens R.T.
      • Hoek J.B.
      • Baffa R.
      • Iozzo R.V.
      Decorin induces mitophagy in breast carcinoma cells via peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and mitostatin.
      ). Cells were transiently transfected using Lipofectamine RNAiMAX (Life Technologies) mixed with siRNA against Peg3, ATG5, and RAB24 that were purchased from Santa Cruz Biotechnology. The siRNA used represents a validated mixture of 3–5 targeting oligonucleotides for each gene of interest. Scrambled siRNA (sc-37007, Santa Cruz Biotechnology) served as a control for all siRNA experiments presented herein. The full protocol is described in Ref.
      • Poluzzi C.
      • Casulli J.
      • Goyal A.
      • Mercer T.J.
      • Neill T.
      • Iozzo R.V.
      Endorepellin evokes autophagy in endothelial cells.
      .

      Immunofluorescence, confocal laser microscopy, line scanning, and Pearson's coefficient of colocalization

      Typically, ∼5 × 104 HUVEC or PAER2 were plated on 0.2% gelatin-coated 4-well-chamber slides (Nunc, ThermoFischer Scientific) and grown to full confluence in their respective growth media at 37 °C. Cells were treated as per the experimental conditions contained herein. Slides were incubated with conjugated secondary antibodies such as: goat anti-rabbit IgG Alexa Fluor® 488 and goat anti-mouse IgG Alexa Fluor® 564 (Life Technologies). Nuclei were visualized with DAPI (Vector Laboratories). Immunofluorescence (
      • Rudnicka L.
      • Varga J.
      • Christiano A.M.
      • Iozzo R.V.
      • Jimenez S.A.
      • Uitto J.
      Elevated expression of type VII collagen in the skin of patients with systemic sclerosis.
      ) and differential interference contract images were acquired with a ×63, 1.3 oil-immersion objective installed on a LEICA DM5500B microscope with the Leica Application suite, advanced fluorescence version 1.8 software from Leica Microsystems, Inc. Confocal analyses were carried out utilizing a ×63, 1.3 oil-immersion objective of a Zeiss LSM-780 confocal laser-scanning microscope with Zen Imaging Software. To determine colocalization of proteins, Z-stack series were acquired maintaining the same number of slices (n = 30). All images were then analyzed in ImageJ (NIH) and Photoshop CS6 (Adobe Systems). Line scanning plots were generated using SigmaPlot software (Systat Software). A full description can be found elsewhere (
      • Buraschi S.
      • Neill T.
      • Goyal A.
      • Poluzzi C.
      • Smythies J.
      • Owens R.T.
      • Schaefer L.
      • Torres A.
      • Iozzo R.V.
      Decorin causes autophagy in endothelial cells via Peg3.
      ).

      Animal experiments

      All animal experiments contained herein were performed as per the Guide for Care and Use of Laboratory Animals and the Institutional Animal Care and Use Committee of Thomas Jefferson University. C57BL/6 mice were purchased from Jackson Laboratories. Both male and female mice and of various ages ranging from 4 weeks to 6 months were used. Fasting experiments involved withholding food for 48 h, but water was allowed ad libitum. Chloroquine (50 mg/kg in sterile dH2O) was administered by intraperitoneal injection during the last 4 h of the starvation period. After animals were euthanized, organs were removed and immediately snap-frozen in liquid nitrogen. Cardiac and aortic tissues were homogenized, solubilized in T-PER Tissue Protein Extraction Buffer from ThermoFischer Scientific, and resolved by SDS-PAGE.

      Statistical analysis

      Immunoblots were quantified by scanning densitometry using Scion Image software (NIH). Graphs were generated using Sigma Stat 3.10. Experiments with three or more comparison groups were subjected to one-way ANOVA followed by a Bonferroni post hoc test using the Systat Package of SigmaPlot 13.0. Differences were considered significant at two sided p < 0.05.

      Data availability

      All data presented here are contained within the manuscript.

      Author contributions

      T. N., C. G. C., and R. V. I. conceptualization; T. N., C. G. C., S. B., and R. V. I. data curation; T. N., C. G. C., S. B., and R. V. I. formal analysis; T. N. and R. V. I. supervision; T. N. and R. V. I. funding acquisition; T. N., C. G. C., S. B., and R. V. I. investigation; T. N., C. G. C., and R. V. I. writing-original draft; T. N. and R. V. I. project administration.

      Acknowledgments

      We thank L. Claesson-Welsh for providing the porcine aortic endothelial cells overexpressing VEGFR2, C. Sharp and S. Ward for their scientific contributions during the early stages of this project, M. Johnson for providing the full-length HA-tagged Peg3, and A. Torres for the HA-tagged Peg3 SCAN domain.

      Supplementary Material

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