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Thromb Haemost 1999; 81(05): 808-814
DOI: 10.1055/s-0037-1614574
Rapid Communication
Schattauer GmbH

Increased Expression of Protease Activated Receptor-2 (PAR-2) in Balloon-Injured Rat Carotid Artery

Bruce P. Damiano
1   From Drug Discovery, The R. W. Johnson Pharmaceutical Research Institute, Spring House, Pennsylvania, USA
,
Michael R. D’Andrea
1   From Drug Discovery, The R. W. Johnson Pharmaceutical Research Institute, Spring House, Pennsylvania, USA
,
Lawrence de Garavilla
1   From Drug Discovery, The R. W. Johnson Pharmaceutical Research Institute, Spring House, Pennsylvania, USA
,
Wai-man Cheung
1   From Drug Discovery, The R. W. Johnson Pharmaceutical Research Institute, Spring House, Pennsylvania, USA
,
Patricia Andrade-Gordon
1   From Drug Discovery, The R. W. Johnson Pharmaceutical Research Institute, Spring House, Pennsylvania, USA
› Author Affiliations
Further Information

Publication History

Received 24 November 1998

Accepted after revision 08 February 1999

Publication Date:
09 December 2017 (online)

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Summary

Protease-induced cell signaling is mediated by specific receptors such as the emerging family of protease activated receptors (PARs). Since proteases are involved in various aspects of vascular injury, we assessed expression of PAR-2, a protease-activated receptor closely related to the thrombin receptor (PAR-1) but activated by an unknown protease, in vascular injury. Rat carotids were subjected to balloon-catheter injury and perfusion fixed at 1, 3, 7 or 14 days after injury. Sections of injured and normal carotid arteries were immunohisto-chemically labeled with a polyclonal antibody raised against the N-terminal residues 37-53 of human PAR-2. Sections were also labeled with antibodies to factor VIII-related antigen, smooth muscle actin and a proliferating cell nuclear antigen (PCNA). In normal vessels, PAR-2 labeling was diffuse and patchy in medial smooth muscle and endothelium. At one and three days after injury, before appearance of neointima, PAR-2 labeling increased in cells adjacent to damaged or necrotic smooth muscle cells. In addition, proliferating adventitial myofibroblasts labeled strongly for PAR-2. At 7 and 14 days after injury, the media and neointima of injured vessels had increased PAR-2 labeling which was most intense at the luminal edge of the neointima. Double immunohistochemical labeling confirmed the greatest expression of PAR-2 in areas with the greatest density of PCNA-positive cells. In addition, PAR-2 mRNA localization using in situ hybridization paralleled PAR-2 expression. The data suggest an upregulation of PAR-2 in response to vascular injury which is associated with medial smooth muscle damage, proliferating adventitial myofibroblasts and smooth muscle cells of the neointima, particularly those at the proliferating luminal edge of the neointima. Possible functional consequences of this receptor upregulation and its role in the response to vascular injury remain to be determined.

 

  • References

  • 1 Mullins DE, Rohrlich ST. The role of proteinases in cellular invasiveness.. Biochim Biophys Acta 1983; 695: 177-214.
    PubMedGoogle Scholar
  • 2 Hollenberg MD. Protease-mediated signalling: new paradigms for cell regulation and drug development.. Trends Pharmacol Sci 1996; 17: 3-6.
    PubMedGoogle Scholar
  • 3 Vu T-KH, Hung DT, Wheaton I V, Coughlin SR. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation.. Cell 1991; 64: 1057-68.
    CrossrefPubMedGoogle Scholar
  • 4 Rasmussen UB, Vouret-Craviari V, Jallat S, Schlesinger Y, Pagès G, Pavirani A, Lecocq J-P, Pouysségur J, Van Obberghen-Schilling E. cDNA cloning and expression of a hamster alpha-thrombin receptor coupled to Ca 2+ mobilization.. FEBS Letters 1991; 288: 123-8.
    CrossrefPubMedGoogle Scholar
  • 5 Nystedt S, Emilsson IE, Wahlestedt C, Sundelin J. Molecular cloning of a potential proteinase activated receptor.. Proc Natl Acad Sci 1994; 91: 9208-12.
    CrossrefPubMedGoogle Scholar
  • 6 Nystedt S, Emilsson K, Larsson AK, Strombeck B, Sundelin J. Molecular cloning and functional expression of the gene encoding the human pro-teinase-activated receptor 2.. Eur J Biochem 1995; 232: 84-9.
    CrossrefPubMedGoogle Scholar
  • 7 Saifeddine M, Alani B, Cheng CH, Wang L, Hollenberg MD. Rat protein-ase-activated receptor-2 (PAR-2): cDNA sequence and activity of receptor-derived peptides in gastric and vascular tissue.. Br J Pharmacol 1996; 118: 521-30.
    CrossrefPubMedGoogle Scholar
  • 8 Mirza H, Yatsula V, Bahou WF. The proteinase activated receptor-2 (PAR-2) mediates mitogenic responses in human vascular endothelial cells – molecular characterization and evidence for functional coupling to the thrombin receptor.. J Clin Inves 1996; 97: 1705-14.
    CrossrefPubMedGoogle Scholar
  • 9 Nystedt S, Ramakrishnan V, Sundelin J.. The proteinase-activated receptor 2 is induced by inflammatory mediators in human endothelial cells. Comparison with the thrombin receptor.. J Biol Chem 1996; 271:: 14910-5.
    CrossrefPubMedGoogle Scholar
  • 10 Storck J, Kuesters B, Vahland M, Morys-Wortmann C, Zimmermann ER. Trypsin induced von Willebrand factor release from human endothelial cells is mediated by PAR-2 activation.. Thromb Res 1996; 84: 463-73.
    CrossrefPubMedGoogle Scholar
  • 11 Magazine I H, King JM, Srivastava KD. Protease activated receptors modulate aortic vascular tone.. Int J Cardiol 1996; 53: S75-S80.
    CrossrefPubMedGoogle Scholar
  • 12 Alani B, Saifeddine M, Hollenberg MD. Detection of functional receptors for the proteinase-activated receptor-2-activating polypeptide, SLIGRLNH2, in rat vascular and gastric smooth muscle.. Can J Physiol Pharmacol 1995; 73: 1203-7.
    CrossrefPubMedGoogle Scholar
  • 13 Bono F, Lamarche I, Herbert JM. Induction of vascular smooth muscle cell growth by selective activation of the proteinase activated receptor-2 (PAR-2).. Biochem Biophys Res Commun 1997; 241: 762-4.
    CrossrefPubMedGoogle Scholar
  • 14 Molino M, Barnathan ES, Numerof R, Clark J, Dreyer M, Cumashi A, Hoxie JA, Schechter N, Woolkalis M, Brass LF. Interactions of mast cell tryptase with thrombin receptors and PAR-2.. J Biol Chem 1997; 272: 4043-9.
    CrossrefPubMedGoogle Scholar
  • 15 D’Andrea MR, Derian CK, Leturcq D, Baker SM, Brunmark A, Ling P, Darrow AL, Santulli RJ, Brass LF, Andrade-Gordon P. Characterization of protease-activated receptor-2 immunoreactivity in normal human tissues.. J Histochem Cytochem 1998; 46: 157-64.
    CrossrefPubMedGoogle Scholar
  • 16 Kong W, McConalogue K, Khitin LM, Hollenberg MD, Payan DG, Bohm SK, Bunnett NW. Luminal trypsin may regulate enterocytes through proteinase-activated receptor 2.. Proc Natl Acad Sci USA 1997; 94: 8884-9.
    CrossrefPubMedGoogle Scholar
  • 17 Koshikawa N, Nagashima Y, Miyagi Y, Mizushima H, Yanoma S, Yasumitsu H, Miyazaki K.. Expression of trypsin in vascular endothelial cells.. FEBS Lett 1997; 409:: 442-8.
    CrossrefPubMedGoogle Scholar
  • 18 Jeziorska M, Mccollum C, Woolley DE.. Mast cell distribution, activation, and phenotype in atherosclerotic lesions of human carotid arteries.. J Pathol. 1997; 182:: 115-22.
    CrossrefPubMedGoogle Scholar
  • 19 Kaartinen M, Penttila A, Kovanen PT.. Mast cells of two types differing in neutral protease composition in the human aortic intima. Demonstration of tryptase- and tryptase/chymase-containing mast cells in normal intimas, fatty streaks, and the shoulder region of atheromas.. Arterioscler Thromb 1994; 14:: 966-72.
    CrossrefPubMedGoogle Scholar
  • 20 Clowes AW, Reidy MA, Clowes MM.. Mechanisms of stenosis after arterial injury.. Lab Invest 1983; 49:: 208-15.
    PubMedGoogle Scholar
  • 21 Bahou WF, Schmidt VA. Molecular characterization of proteolytically activated receptors: insights from the thrombin receptor and proteinase activated receptor-2 (PAR-2) genes.. Platelets 1996; 7: 253-60.
    CrossrefPubMedGoogle Scholar
  • 22 Molino M, Raghunath PN, Kuo A, Ahuja M, Hoxie JA, Brass LF, Barna-than ES.. Differential expression of functional protease-activated receptor-2 (PAR-2) in human vascular smooth muscle cells.. Arterioscler, Thromb, Vasc Biol 1998; 18:: 825-32.
    CrossrefPubMedGoogle Scholar
  • 23 Zeymer U, Fishbein MC, Forrester JS, Cercek B.. Proliferating cell nuclear antigen immunohistochemistry in rat aorta after balloon denudation.. Am J Pathol 1992; 141:: 685-90.
    PubMedGoogle Scholar
  • 24 Clowes AW, Reidy MA, Clowes MM. Kinetics of cellular proliferation after arterial injury. I. Smooth muscle growth in the absence of endothelium.. Lab Invest. 1983; 49: 327-33.
    PubMedGoogle Scholar
  • 25 Shi Y, Pieniek M, Fard A, BJ O, Mannion J, Zalewski A. Adventitial remodeling after coronary arterial injury.. Circ 1996; 93: 340-8.
    CrossrefPubMedGoogle Scholar
  • 26 Andersen H, Maeng M, Thorwest M, Falk E. Remodeling rather than neo-intimal formation explains luminal narrowing after deep vessel wall injury: insights from a porcine coronary (re)stenosis model.. Circ 1996; 93: 1716-24.
    CrossrefPubMedGoogle Scholar
  • 27 Scott N, Cipolla G, Ross C, Dunn B, Martin F, Simonet L, Wilcox J. Identification of a potential role for the adventitia in vascular lesion formation after balloon overstretch injury of porcine coronary arteries.. Circ 1996; 93: 2178-87.
    CrossrefPubMedGoogle Scholar
  • 28 Shi Y, BJ O, Fard A, Mannion J, Wang D, Zalewski A.. Adventitial myo-fibroblasts contribute to neointimal formation in injured porcine coronary arteries.. Circ 1996; 94:: 1655-64.
    CrossrefPubMedGoogle Scholar
  • 29 Wilcox JN, Rodriguez J, Subramanian R, Ollerenshaw J, Zhong CZ, Hayzer DJ, Horaist C, Hanson SR, Lumsden A, Salam TA, Kelly AB, Harker LA, Runge M. Characterization of thrombin receptor expression during vascular lesion formation.. Circ Res 1994; 75: 1029-38.
    CrossrefPubMedGoogle Scholar
  • 30 Nelken NA, Soifer SJ, O"Keefe J, Vu T-KH, Charo IF, Coughlin SR. Thrombin receptor expression in normal and atherosclerotic human arteries.. J Clin Invest 1992; 90: 1614-21.
    CrossrefPubMedGoogle Scholar
  • 31 Dechend R, Mo X, Schulz W, Gross M, Praus M, Dietz R, Gulba DC. Thrombin receptor and urokinase-type plasminogen activator are colocalized in vascular smooth muscle cells derived from human carotid atherosclerotic plaques.. Fibrinolysis 1996; 10: 41-5.
    PubMedGoogle Scholar
  • 32 Walters TK, Gorog DA, Wood RFM. Thrombin Generation Following Arterial Injury Is a Critical Initiating Event in the Pathogenesis of the Proliferative Stages of the Atherosclerotic Process.. J Vasc Res 1994; 31: 173-7.
    CrossrefPubMedGoogle Scholar
  • 33 Barry WL, Gimple LW, Humphries JE, Powers ER, Mccoy KW, Sanders JM, Owens GK, Sarembock IJ.. Arterial thrombin activity after angioplasty in an atherosclerotic rabbit model: time course and effect of hirudin.. Circ 1996; 94:: 88-93.
    CrossrefPubMedGoogle Scholar
  • 34 Abendschein DR, Recchia D, Meng YY, Oltrona L, Wickline SA, Eisen-berg PR. Inhibition of thrombin attenuates stenosis after arterial injury in minipigs.. J Amer Coll Cardiol 1996; 28: 1849-55.
    CrossrefPubMedGoogle Scholar
  • 35 Stouffer GA, Schmedtje JF, Gulba D, Huber K, Bode C, Aaron J, Runge MS.. Restenosis following percutaneous revascularization – the potential role of thrombin and the thrombin receptor.. Ann Hematol 1996; 73:: S39-S41.
    CrossrefPubMedGoogle Scholar
  • 36 Corvera CU, Dery O, Mcconalogue K, Bohm SK, Khitin LM, Caughey GH, Payan DG, Bunnett NW.. Mast cell tryptase regulates rat colonic myocytes through proteinase-activated receptor 2.. J Clin Invest 1997; 100:: 1383-93.
    CrossrefPubMedGoogle Scholar
  • 37 Fox MT, Harriott P, Walker B, Stone SR.. Identification of potential activators of proteinase-activated receptor-2.. FEBS Lett 1997; 417:: 267-9.
    CrossrefPubMedGoogle Scholar
  • 38 Mirza H, Schmidt VA, Derian CK, Jesty J, Bahou WF.. Mitogenic responses mediated through the proteinase-activated receptor-2 are induced by expressed forms of mast cell α- or α-tryptases.. Blood 1997; 90:: 3914-22.
    PubMedGoogle Scholar
  • 39 Holm J, Hansson GK. Cellular and immunologic features of carotid artery disease in man and experimental animal models.. Eur J Vasc Surg 1990; 4: 49-55.
    CrossrefPubMedGoogle Scholar