title: CDKN2B Regulates TGFβ Signaling and Smooth Muscle Cell Investment of Hypoxic Neovessels.
abstract: BACKGROUND
  Genetic variation at the chromosome 9p21 cardiovascular risk locus has been associated with peripheral artery disease, but its mechanism remains unknown.
  
  
  OBJECTIVE
  To determine whether this association is secondary to an increase in atherosclerosis, or it is the result of a separate angiogenesis-related mechanism.
  
  
  RESULTS
  Quantitative evaluation of human vascular samples revealed that carriers of the 9p21 risk allele possess a significantly higher burden of immature intraplaque microvessels than carriers of the ancestral allele, irrespective of lesion size or patient comorbidity. To determine whether aberrant angiogenesis also occurs under nonatherosclerotic conditions, we performed femoral artery ligation surgery in mice lacking the 9p21 candidate gene, Cdkn2b. These animals developed advanced hindlimb ischemia and digital autoamputation, secondary to a defect in the capacity of the Cdkn2b-deficient smooth muscle cell to support the developing neovessel. Microarray studies identified impaired transforming growth factor β (TGFβ) signaling in cultured cyclin-dependent kinase inhibitor 2B (CDKN2B)-deficient cells, as well as TGFβ1 upregulation in the vasculature of 9p21 risk allele carriers. Molecular signaling studies indicated that loss of CDKN2B impairs the expression of the inhibitory factor, SMAD-7, which promotes downstream TGFβ activation. Ultimately, this manifests in the upregulation of a poorly studied effector molecule, TGFβ1-induced-1, which is a TGFβ-rheostat known to have antagonistic effects on the endothelial cell and smooth muscle cell. Dual knockdown studies confirmed the reversibility of the proposed mechanism, in vitro.
  
  
  CONCLUSIONS
  These results suggest that loss of CDKN2B may not only promote cardiovascular disease through the development of atherosclerosis but may also impair TGFβ signaling and hypoxic neovessel maturation.
authors.affiliation: From the Departments of Surgery (V.N., K.P.D., J.Y., S.X., Y.K., D.D., K.T.N., J.D., N.J.L.), Medicine (J.M.S., N.J.L.), and Pathology (A.J.C.), Stanford University School of Medicine, CA; Department of Medicine, McGill University, Montreal, Canada (S.D.); Departments of Molecular Medicine and Surgery (L.P., U.H.) and Medicine (L.M.), Karolinska Institute, Stockholm, Sweden; and CVPath Institute, Gaithersburg, MD (L.G., X.Z., F.D.K., R.V., H.R.D.).
authors.forename: Vivek
authors.initials: V
authors.lastname: Nanda
authors.affiliation: From the Departments of Surgery (V.N., K.P.D., J.Y., S.X., Y.K., D.D., K.T.N., J.D., N.J.L.), Medicine (J.M.S., N.J.L.), and Pathology (A.J.C.), Stanford University School of Medicine, CA; Department of Medicine, McGill University, Montreal, Canada (S.D.); Departments of Molecular Medicine and Surgery (L.P., U.H.) and Medicine (L.M.), Karolinska Institute, Stockholm, Sweden; and CVPath Institute, Gaithersburg, MD (L.G., X.Z., F.D.K., R.V., H.R.D.).
authors.forename: Kelly P
authors.initials: KP
authors.lastname: Downing
authors.affiliation: From the Departments of Surgery (V.N., K.P.D., J.Y., S.X., Y.K., D.D., K.T.N., J.D., N.J.L.), Medicine (J.M.S., N.J.L.), and Pathology (A.J.C.), Stanford University School of Medicine, CA; Department of Medicine, McGill University, Montreal, Canada (S.D.); Departments of Molecular Medicine and Surgery (L.P., U.H.) and Medicine (L.M.), Karolinska Institute, Stockholm, Sweden; and CVPath Institute, Gaithersburg, MD (L.G., X.Z., F.D.K., R.V., H.R.D.).
authors.forename: Jianqin
authors.initials: J
authors.lastname: Ye
authors.affiliation: From the Departments of Surgery (V.N., K.P.D., J.Y., S.X., Y.K., D.D., K.T.N., J.D., N.J.L.), Medicine (J.M.S., N.J.L.), and Pathology (A.J.C.), Stanford University School of Medicine, CA; Department of Medicine, McGill University, Montreal, Canada (S.D.); Departments of Molecular Medicine and Surgery (L.P., U.H.) and Medicine (L.M.), Karolinska Institute, Stockholm, Sweden; and CVPath Institute, Gaithersburg, MD (L.G., X.Z., F.D.K., R.V., H.R.D.).
authors.forename: Sophia
authors.initials: S
authors.lastname: Xiao
authors.affiliation: From the Departments of Surgery (V.N., K.P.D., J.Y., S.X., Y.K., D.D., K.T.N., J.D., N.J.L.), Medicine (J.M.S., N.J.L.), and Pathology (A.J.C.), Stanford University School of Medicine, CA; Department of Medicine, McGill University, Montreal, Canada (S.D.); Departments of Molecular Medicine and Surgery (L.P., U.H.) and Medicine (L.M.), Karolinska Institute, Stockholm, Sweden; and CVPath Institute, Gaithersburg, MD (L.G., X.Z., F.D.K., R.V., H.R.D.).
authors.forename: Yoko
authors.initials: Y
authors.lastname: Kojima
authors.affiliation: From the Departments of Surgery (V.N., K.P.D., J.Y., S.X., Y.K., D.D., K.T.N., J.D., N.J.L.), Medicine (J.M.S., N.J.L.), and Pathology (A.J.C.), Stanford University School of Medicine, CA; Department of Medicine, McGill University, Montreal, Canada (S.D.); Departments of Molecular Medicine and Surgery (L.P., U.H.) and Medicine (L.M.), Karolinska Institute, Stockholm, Sweden; and CVPath Institute, Gaithersburg, MD (L.G., X.Z., F.D.K., R.V., H.R.D.).
authors.forename: Joshua M
authors.initials: JM
authors.lastname: Spin
authors.affiliation: From the Departments of Surgery (V.N., K.P.D., J.Y., S.X., Y.K., D.D., K.T.N., J.D., N.J.L.), Medicine (J.M.S., N.J.L.), and Pathology (A.J.C.), Stanford University School of Medicine, CA; Department of Medicine, McGill University, Montreal, Canada (S.D.); Departments of Molecular Medicine and Surgery (L.P., U.H.) and Medicine (L.M.), Karolinska Institute, Stockholm, Sweden; and CVPath Institute, Gaithersburg, MD (L.G., X.Z., F.D.K., R.V., H.R.D.).
authors.forename: Daniel
authors.initials: D
authors.lastname: DiRenzo
authors.affiliation: From the Departments of Surgery (V.N., K.P.D., J.Y., S.X., Y.K., D.D., K.T.N., J.D., N.J.L.), Medicine (J.M.S., N.J.L.), and Pathology (A.J.C.), Stanford University School of Medicine, CA; Department of Medicine, McGill University, Montreal, Canada (S.D.); Departments of Molecular Medicine and Surgery (L.P., U.H.) and Medicine (L.M.), Karolinska Institute, Stockholm, Sweden; and CVPath Institute, Gaithersburg, MD (L.G., X.Z., F.D.K., R.V., H.R.D.).
authors.forename: Kevin T
authors.initials: KT
authors.lastname: Nead
authors.affiliation: From the Departments of Surgery (V.N., K.P.D., J.Y., S.X., Y.K., D.D., K.T.N., J.D., N.J.L.), Medicine (J.M.S., N.J.L.), and Pathology (A.J.C.), Stanford University School of Medicine, CA; Department of Medicine, McGill University, Montreal, Canada (S.D.); Departments of Molecular Medicine and Surgery (L.P., U.H.) and Medicine (L.M.), Karolinska Institute, Stockholm, Sweden; and CVPath Institute, Gaithersburg, MD (L.G., X.Z., F.D.K., R.V., H.R.D.).
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Connolly
authors.affiliation: From the Departments of Surgery (V.N., K.P.D., J.Y., S.X., Y.K., D.D., K.T.N., J.D., N.J.L.), Medicine (J.M.S., N.J.L.), and Pathology (A.J.C.), Stanford University School of Medicine, CA; Department of Medicine, McGill University, Montreal, Canada (S.D.); Departments of Molecular Medicine and Surgery (L.P., U.H.) and Medicine (L.M.), Karolinska Institute, Stockholm, Sweden; and CVPath Institute, Gaithersburg, MD (L.G., X.Z., F.D.K., R.V., H.R.D.).
authors.forename: Sonny
authors.initials: S
authors.lastname: Dandona
authors.affiliation: From the Departments of Surgery (V.N., K.P.D., J.Y., S.X., Y.K., D.D., K.T.N., J.D., N.J.L.), Medicine (J.M.S., N.J.L.), and Pathology (A.J.C.), Stanford University School of Medicine, CA; Department of Medicine, McGill University, Montreal, Canada (S.D.); Departments of Molecular Medicine and Surgery (L.P., U.H.) and Medicine (L.M.), Karolinska Institute, Stockholm, Sweden; and CVPath Institute, Gaithersburg, MD (L.G., X.Z., F.D.K., R.V., H.R.D.).
authors.forename: Ljubica
authors.initials: L
authors.lastname: Perisic
authors.affiliation: From the Departments of Surgery (V.N., K.P.D., J.Y., S.X., Y.K., D.D., K.T.N., J.D., N.J.L.), Medicine (J.M.S., N.J.L.), and Pathology (A.J.C.), Stanford University School of Medicine, CA; Department of Medicine, McGill University, Montreal, Canada (S.D.); Departments of Molecular Medicine and Surgery (L.P., U.H.) and Medicine (L.M.), Karolinska Institute, Stockholm, Sweden; and CVPath Institute, Gaithersburg, MD (L.G., X.Z., F.D.K., R.V., H.R.D.).
authors.forename: Ulf
authors.initials: U
authors.lastname: Hedin
authors.affiliation: From the Departments of Surgery (V.N., K.P.D., J.Y., S.X., Y.K., D.D., K.T.N., J.D., N.J.L.), Medicine (J.M.S., N.J.L.), and Pathology (A.J.C.), Stanford University School of Medicine, CA; Department of Medicine, McGill University, Montreal, Canada (S.D.); Departments of Molecular Medicine and Surgery (L.P., U.H.) and Medicine (L.M.), Karolinska Institute, Stockholm, Sweden; and CVPath Institute, Gaithersburg, MD (L.G., X.Z., F.D.K., R.V., H.R.D.).
authors.forename: Lars
authors.initials: L
authors.lastname: Maegdefessel
authors.affiliation: From the Departments of Surgery (V.N., K.P.D., J.Y., S.X., Y.K., D.D., K.T.N., J.D., N.J.L.), Medicine (J.M.S., N.J.L.), and Pathology (A.J.C.), Stanford University School of Medicine, CA; Department of Medicine, McGill University, Montreal, Canada (S.D.); Departments of Molecular Medicine and Surgery (L.P., U.H.) and Medicine (L.M.), Karolinska Institute, Stockholm, Sweden; and CVPath Institute, Gaithersburg, MD (L.G., X.Z., F.D.K., R.V., H.R.D.).
authors.forename: Jessie
authors.initials: J
authors.lastname: Dalman
authors.affiliation: From the Departments of Surgery (V.N., K.P.D., J.Y., S.X., Y.K., D.D., K.T.N., J.D., N.J.L.), Medicine (J.M.S., N.J.L.), and Pathology (A.J.C.), Stanford University School of Medicine, CA; Department of Medicine, McGill University, Montreal, Canada (S.D.); Departments of Molecular Medicine and Surgery (L.P., U.H.) and Medicine (L.M.), Karolinska Institute, Stockholm, Sweden; and CVPath Institute, Gaithersburg, MD (L.G., X.Z., F.D.K., R.V., H.R.D.).
authors.forename: Liang
authors.initials: L
authors.lastname: Guo
authors.affiliation: From the Departments of Surgery (V.N., K.P.D., J.Y., S.X., Y.K., D.D., K.T.N., J.D., N.J.L.), Medicine (J.M.S., N.J.L.), and Pathology (A.J.C.), Stanford University School of Medicine, CA; Department of Medicine, McGill University, Montreal, Canada (S.D.); Departments of Molecular Medicine and Surgery (L.P., U.H.) and Medicine (L.M.), Karolinska Institute, Stockholm, Sweden; and CVPath Institute, Gaithersburg, MD (L.G., X.Z., F.D.K., R.V., H.R.D.).
authors.forename: XiaoQing
authors.initials: X
authors.lastname: Zhao
authors.affiliation: From the Departments of Surgery (V.N., K.P.D., J.Y., S.X., Y.K., D.D., K.T.N., J.D., N.J.L.), Medicine (J.M.S., N.J.L.), and Pathology (A.J.C.), Stanford University School of Medicine, CA; Department of Medicine, McGill University, Montreal, Canada (S.D.); Departments of Molecular Medicine and Surgery (L.P., U.H.) and Medicine (L.M.), Karolinska Institute, Stockholm, Sweden; and CVPath Institute, Gaithersburg, MD (L.G., X.Z., F.D.K., R.V., H.R.D.).
authors.forename: Frank D
authors.initials: FD
authors.lastname: Kolodgie
authors.affiliation: From the Departments of Surgery (V.N., K.P.D., J.Y., S.X., Y.K., D.D., K.T.N., J.D., N.J.L.), Medicine (J.M.S., N.J.L.), and Pathology (A.J.C.), Stanford University School of Medicine, CA; Department of Medicine, McGill University, Montreal, Canada (S.D.); Departments of Molecular Medicine and Surgery (L.P., U.H.) and Medicine (L.M.), Karolinska Institute, Stockholm, Sweden; and CVPath Institute, Gaithersburg, MD (L.G., X.Z., F.D.K., R.V., H.R.D.).
authors.forename: Renu
authors.initials: R
authors.lastname: Virmani
authors.affiliation: From the Departments of Surgery (V.N., K.P.D., J.Y., S.X., Y.K., D.D., K.T.N., J.D., N.J.L.), Medicine (J.M.S., N.J.L.), and Pathology (A.J.C.), Stanford University School of Medicine, CA; Department of Medicine, McGill University, Montreal, Canada (S.D.); Departments of Molecular Medicine and Surgery (L.P., U.H.) and Medicine (L.M.), Karolinska Institute, Stockholm, Sweden; and CVPath Institute, Gaithersburg, MD (L.G., X.Z., F.D.K., R.V., H.R.D.).
authors.forename: Harry R
authors.initials: HR
authors.lastname: Davis
authors.affiliation: From the Departments of Surgery (V.N., K.P.D., J.Y., S.X., Y.K., D.D., K.T.N., J.D., N.J.L.), Medicine (J.M.S., N.J.L.), and Pathology (A.J.C.), Stanford University School of Medicine, CA; Department of Medicine, McGill University, Montreal, Canada (S.D.); Departments of Molecular Medicine and Surgery (L.P., U.H.) and Medicine (L.M.), Karolinska Institute, Stockholm, Sweden; and CVPath Institute, Gaithersburg, MD (L.G., X.Z., F.D.K., R.V., H.R.D.). nleeper@stanford.edu.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Leeper
mesh_terms: D000818:Animals; D050197:Atherosclerosis / Q000201:enzymology* / Q000235:genetics / Q000401:mortality / Q000473:pathology; D002339:Carotid Arteries / Q000201:enzymology / Q000473:pathology; D015687:Cell Hypoxia; D002478:Cells, Cultured; D002899:Chromosomes, Human, Pair 9; D003331:Coronary Vessels / Q000201:enzymology / Q000473:pathology; D050762:Cyclin-Dependent Kinase Inhibitor p15 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D004195:Disease Models, Animal; D005260:Female; D020022:Genetic Predisposition to Disease; D006614:Hindlimb; D006801:Humans; D008297:Male; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D018482:Muscle, Skeletal / Q000098:blood supply*; D009131:Muscle, Smooth, Vascular / Q000201:enzymology* / Q000503:physiopathology; D032389:Myocytes, Smooth Muscle / Q000201:enzymology*; D009389:Neovascularization, Pathologic; D018919:Neovascularization, Physiologic; D010641:Phenotype; D034622:RNA Interference; D015398:Signal Transduction; D051906:Smad7 Protein / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D053773:Transforming Growth Factor beta1 / Q000235:genetics / Q000378:metabolism*
chemical_list: C493966:CDKN2B protein, human; C493968:Cdkn2b protein, mouse; D050762:Cyclin-Dependent Kinase Inhibitor p15; D051906:Smad7 Protein; C497807:Smad7 protein, mouse; C508960:TGFB1 protein, human; C508961:Tgfb1 protein, mouse; D053773:Transforming Growth Factor beta1
keywords: atherosclerosis; cyclin-dependent kinase inhibitor p15; genetic variation; pathologic angiogenesis; peripheral artery disease; smooth muscle cells