A novel cell permeant peptide inhibitor of MAPKAP kinase II inhibits intimal hyperplasia in a human saphenous vein organ culture model

J Vasc Surg. 2010 Dec;52(6):1596-607. doi: 10.1016/j.jvs.2010.06.168. Epub 2010 Sep 22.

Abstract

Objective: The present study was aimed at developing a new cell-permeant peptide inhibitor (MK2i) of the kinase that phosphorylates and activates heat-shock protein (HSP)27 (MAPKAP kinase II), and evaluating the ability of this peptide to inhibit HSP27 phosphorylation and intimal thickening.

Methods: The ability of MK2i to reduce HSP27 phosphorylation and cell migration was evaluated in A7R5 cells stimulated with arsenite or lysophosphatidic acid. Stable isotopic labeling using amino acids in cell culture, in combination with liquid chromatography mass spectrometry, was used to characterize the effect of MK2i on global protein expression in fibroblasts. The effect of MK2i on intimal thickening and connective tissue growth factor expression was evaluated in human saphenous vein (HSV) rings maintained with 30% fetal bovine serum for 14 days by light microscopy and immunoblotting.

Results: Pretreatment of cells with MK2i (10 μM) prior to arsenite or lysophosphatidic acid stimulation decreased phosphorylation of HSP27 (36% ± 9% and 33% ± 10%, respectively) compared with control (not pretreated) cells. MK2i also inhibited A7R5 migration, and downregulated the transforming growth factor-induced expression of collagen and fibronectin in keloid cells, two major matrix proteins involved in the development of intimal hyperplasia. Treatment of HSV segments with MK2i enhanced relaxation, reduced HSP27 phosphorylation (40% ± 17%), connective tissue growth factor expression (17% ± 5%), and intimal thickness (48.2% ± 10.5%) compared with untreated segments. On the other hand, treatment with a recombinant fusion protein containing a cell-permeant peptide attached to the HSP27 sequence increased intimal thickness of HSV segments by 48% ± 14%.

Conclusion: Our results suggest that HSP27 may play a role in the development of processes leading to intimal hyperplasia in HSV, and reduction of HSP27 phosphorylation by MK2i may be a potential strategy to inhibit the development of intimal hyperplasia in HSV to prevent the autologous vascular graft failure.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Aorta / cytology
  • Arsenites / pharmacology
  • Cell Movement / drug effects
  • Cells, Cultured
  • Collagen / metabolism
  • Connective Tissue Growth Factor / metabolism
  • Enzyme Inhibitors / pharmacology
  • Fibronectins / metabolism
  • HSP27 Heat-Shock Proteins / metabolism
  • Heat-Shock Proteins
  • Humans
  • Hyperplasia
  • Intracellular Signaling Peptides and Proteins / antagonists & inhibitors*
  • Intracellular Signaling Peptides and Proteins / pharmacology
  • Lysophospholipids / pharmacology
  • Microscopy, Confocal
  • Molecular Chaperones
  • Muscle, Smooth, Vascular / cytology
  • Peptides / pharmacology*
  • Phosphorylation / drug effects
  • Protein Array Analysis
  • Protein-Serine-Threonine Kinases / antagonists & inhibitors*
  • Protein-Serine-Threonine Kinases / pharmacology
  • Rats
  • Saphenous Vein / cytology*
  • Sodium Compounds / pharmacology
  • Tissue Culture Techniques
  • Tunica Intima / drug effects
  • Tunica Intima / pathology*
  • p38 Mitogen-Activated Protein Kinases / antagonists & inhibitors

Substances

  • Arsenites
  • Enzyme Inhibitors
  • Fibronectins
  • HSP27 Heat-Shock Proteins
  • HSPB1 protein, human
  • Heat-Shock Proteins
  • Intracellular Signaling Peptides and Proteins
  • Lysophospholipids
  • MK2i peptide
  • Molecular Chaperones
  • Peptides
  • Sodium Compounds
  • Connective Tissue Growth Factor
  • sodium arsenite
  • Collagen
  • MAP-kinase-activated kinase 2
  • Protein-Serine-Threonine Kinases
  • p38 Mitogen-Activated Protein Kinases
  • lysophosphatidic acid