Abstract
Increased force generation and smooth muscle remodeling follow the implantation of saphenous vein as an arterial bypass graft. Previously, we characterized and mapped 129 proteins in human saphenous vein medial smooth muscle using two-dimensional (2-D) PAGE and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Here, we focus on actin filament remodeling in response to simulated arterial flow. Human saphenous vein was exposed to simulated venous or arterial flow for 90 min in vitro, and the contractile medial smooth muscle was dissected out and subjected to 2-D gel electrophoresis using a non-linear immobilized pH 3-10 gradient in the first dimension. Proteins were analyzed quantitatively using PDQuest 2-D software. The actin polymerization inhibitor cytochalasin B (1 microm) prevented increases in force generation after 90 min of simulated arterial flow. At this time point, there were several consistent changes in actin filament-associated protein expression (seven paired vein samples). The heat shock protein HSP27, identified as a three-spot charge train, showed a 1.6-fold increase in abundance (p = 0.01), but with reduced representation of the phosphorylated Ser(82) and Ser(15)Ser(82) isoforms (p = 0.018). The abundance of actin-capping protein alpha2 subunit CapZ had decreased 3-fold, p = 0.04. A 19-kDa proteolytic fragment of actin increased 2-fold, p = 0.04. For the four-spot charge train of gelsolin, there was reduced representation of the more acidic isoforms, p = 0.022. The abundance of other proteins associated with actin filaments, including cofilin and destrin, remained unchanged after arterial flow. Actin filament remodeling with differential expression and/or post-translational modification of proteins involved in capping the barbed end of actin filaments, HSP27 and CapZ, is an early response of contractile saphenous vein smooth muscle cells to hemodynamic stress. The observed changes would favor the generation of contractile stress fibers.
Highlights
Increased force generation and smooth muscle remodeling follow the implantation of saphenous vein as an arterial bypass graft
We previously have described the changes in force generation when intact vein is exposed to arterial hemodynamics in vitro [4] and characterized and mapped protein expression in Human saphenous vein (HSV) medial smooth muscle using two-dimensional (2-D) PAGE and a combination of peptide mass fingerprinting by matrixassisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (MS) and partial amino acid sequencing by nanospray tandem MS [6]
The apparent increase in EC50 for phenylephrine after arterial flow in the presence of cytochalasin B was not significant. These results indicated that actin polymerization was necessary to support the increased force generation after 90 min of simulated arterial flow
Summary
Emma McGregor‡§, Lee Kempster‡, Robin Wait¶, Martin Gosling‡, Michael J. We considered that to progress our understanding of the changes in protein expression and/or post-translational modifications associated with the increased force generation of HSV smooth muscle in response to arterial hemodynamics, the proteomic analysis of media dissected from intact vein had considerable advantages. We previously have described the changes in force generation when intact vein is exposed to arterial hemodynamics in vitro [4] and characterized and mapped protein expression in HSV medial smooth muscle using two-dimensional (2-D) PAGE and a combination of peptide mass fingerprinting by matrixassisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (MS) and partial amino acid sequencing by nanospray tandem MS [6]. We provide evidence for early actin filament remodeling, detailing quantitative changes in smooth muscle protein expression, and post-translational modifications following exposure of HSV to arterial hemodynamics
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