Transduction of peptide analogs of the small heat shock–related protein HSP20 inhibits intimal hyperplasia

Tessier, Deron J.; Komalavilas, Padmini; Liu, Bo; Kent, K. Craig; Thresher, Jeffrey S.; Dreiza, Catherine M.; Panitch, Alyssa; Joshi, Lokesh; Furnish, Elizabeth J.; Stone, William M.; Fowl, Richard J.; Brophy, Colleen M.

doi:10.1016/j.jvs.2004.03.028

Cited by 35 publications

(42 citation statements)

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“…The observation that treatment of serum-starved cultured cells with TAT-pHSP20 resulted in the destruction of actin stress fibers is congruent with previously published studies on heat shock proteins and the actin cytoskeleton [8][9][10][33][34][35]. In general, the presence of stress fibers is associated with a proliferative response whereas the absence of stress fibers is associated with a quiescent phenotype [36].…”

Section: Discussionsupporting

confidence: 90%

“…Peptide mimetics are advantageous in determining full-length protein activity as they have fewer domains available for binding/interaction, are usually easier to synthesize in large quantities, and less susceptible to proteolytic activity. A synthesized HSP20 analog, PTD-pHSP20 composed of an optimized protein transduction domain, fused to sequences encoding the active portion (phosphorylation site, serine 16 of HSP20) [6] has been shown to relax a wide array of smooth muscles including bovine carotid artery, porcine coronary artery, human saphenous vein and human umbilical artery [8,10,12,13]. To compare the magnitude of the physiological responses of the entire recombinant molecule to the peptide mimetics, rings of rat aorta contracted with 5-HT (0.5 μM) displayed steady contractions that were sustained for long periods of time (> 20 min), whereas treatment of aorta rings with increasing amounts of PTD-pHSP20 (100 μM, 500 μM and 1 mM respectively) led to a dosedependent decrease in contraction that was maximal at a PTD-pHSP20 concentration of 1 mM (data not shown).…”

Section: Physiological Activity Of Tat-phsp20 Analogsmentioning

confidence: 99%

“…Like other members of the heat shock protein family, HSP20 requires phosphorylation at a serine residue (S16) to become functionally active [5,6]. HSP20 is a specific substrate protein of PKA and PKG and accumulating evidence suggests HSP20 has several physiological and biochemical roles including controlling muscle tone, regulating cell motility, modulating actin filament dynamics and protection against intimal hyperplasia and ischemia/reperfusion injury [7][8][9][10][11][12][13][14]. Although the molecular mechanisms of action for HSP20 have not been determined fully, it is an actinassociated protein and we have shown that HSP20 peptide treatment leads to loss of actin stress fibers and disruption of focal adhesion complexes [9][10][11].…”

Section: Introductionmentioning

confidence: 99%

“…PTDs, first discovered independently by the Green and Frankel laboratories, are positively charged amino acid sequences facilitating the entry of proteins into cells through macropinocytotic mechanisms [15][16][17]. Our laboratory has successfully implemented the use of these sequences to transduce different proteins and peptides into numerous tissues and cells [8][9][10][11][12][13][14].…”

Section: Introductionmentioning

confidence: 99%

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Phosphorylation and activation of a transducible recombinant form of human HSP20 in Escherichia coli

Flynn

Smoke

Furnish

et al. 2007

Protein Expression and Purification

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Protein based cellular therapeutics have been limited by getting the molecules into cells and the fact that many proteins require post-translational modifications for activation. Protein transduction domains (PTDs), including that from the HIV TAT protein (TAT), are small arginine rich peptides that carry molecules across the cell membrane. We have shown that the heat shock-related protein, HSP20 is a downstream mediator of cyclic nucleotide-dependent relaxation of vascular smooth muscle and is activated by phosphorylation. In this study, we co-expressed in E. coli the cDNAs encoding the catalytic subunit of protein kinase G and a TAT-HSP20 fusion protein composed of the TAT PTD (-YGRKKRRQRRR-) fused to the N-terminus of human HSP20. Immunoblot and HPLC-ESI-MS/MS analysis of the purified TAT-HSP20 demonstrated that it was phosphorylated at serine 40 (equivalent to serine 16 in wild-type human HSP20). This phosphorylated TAT-HSP20 was physiologically active in intact smooth muscles in that it inhibited 5-hydroxytryptamine-induced contractions by 57% ± 4.5. The recombinant phosphorylated protein also led to changes in actin cytoskeletal morphology in 3T3 cells. These results delineate strategies for the expression and activation of therapeutic molecules for intracellular protein based therapeutics.

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Section: Discussionsupporting

confidence: 90%

Section: Physiological Activity Of Tat-phsp20 Analogsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Phosphorylation and activation of a transducible recombinant form of human HSP20 in Escherichia coli

Flynn

Smoke

Furnish

et al. 2007

Protein Expression and Purification

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“…P20 peptide has been demonstrated to mimic the biological effect of the entire HSP20 molecule, inducing relaxation in bovine carotid artery (21), porcine coronary artery (26), human saphenous vein (27), human umbilical artery (28), rat aorta (29), bovine ASM (14), and canine ASM (15). In National Institutes of Health 3T3 cells and human ASM (HASM) cells, treatment with the P20 peptide led to depolymerization of actin, resulting in disruption of actin stress fibers (14,15,24).…”

mentioning

confidence: 99%

Heat Shock–Related Protein 20 Peptide Decreases Human Airway Constriction Downstream of β₂-Adrenergic Receptor

Banathy¹,

Cheung‐Flynn²,

Goleniewska³

et al. 2016

Am J Respir Cell Mol Biol

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Severe bronchospasm refractory to b-agonists is a challenging aspect of asthma therapy, and novel therapeutics are needed. b-agonist-induced airway smooth muscle (ASM) relaxation is associated with increases in the phosphorylation of the small heat shock-related protein (HSP) 20. We hypothesized that a transducible phosphopeptide mimetic of HSP20 (P20 peptide) causes relaxation of human ASM (HASM) by interacting with target(s) downstream of the b 2 -adrenergic receptor (b 2 AR) pathway. The effect of the P20 peptide on ASM contractility was determined in human and porcine ASM using a muscle bath. The effect of the P20 peptide on filamentous actin dynamics and migration was examined in intact porcine ASM and cultured primary HASM cells. The efficacy of the P20 peptide in vivo on airway hyperresponsiveness (AHR) was determined in an ovalbumin (OVA) sensitization and challenge murine model of allergic airway inflammation. P20 peptide caused dose-dependent relaxation of carbachol-precontracted ASM and blocked carbachol-induced contraction. The b 2 AR inhibitor, (6)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol hydrochloride (ICI 118,551), abrogated isoproterenol but not P20 peptide-mediated relaxation. The P20 peptide decreased filamentous actin levels in intact ASM, disrupted stress fibers, and inhibited platelet-derived growth factor-induced migration of HASM cells. The P20 peptide treatment reduced methacholine-induced AHR in OVA mice without affecting the inflammatory response. These results suggest that the P20 peptide decreased airway constriction and disrupted stress fibers through regulation of the actin cytoskeleton downstream of b 2 AR. Thus, the P20 peptide may be a potential therapeutic for asthma refractory to b-agonists.Keywords: asthma; airway smooth muscle relaxation; ovalbumin mouse model; peptide therapeutic; filamentous actin Clinical RelevanceInhaled b-agonists are the mainstay of asthma therapy. However, regular use of these drugs has detrimental effects in some patients with asthma, owing to desensitization and/or genetic polymorphism of the b 2 -adrenergic receptor. This article demonstrates that a cell-permeant phosphomimetic peptide of heat shock-related protein 20, P20 peptide, reduced airway hypercontractility by regulating the actin cytoskeleton via mechanism(s) downstream of the adrenergic receptor pathway. Furthermore, inhaled delivery of P20 peptide attenuated methacholine-induced airway hyperresponsiveness in a mouse model of allergic airway inflammation that mimics human asthma. Thus, P20 peptide may be a potential therapeutic for asthma refractory to b-agonists.

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Pressure Control During Preparation of Saphenous Veins

Eagle

Brophy

et al. 2014

JAMA Surg

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Importance Long-term patency of human saphenous veins (HSVs) used as autologous conduits for coronary artery bypass grafting (CABG) procedures remains limited because of vein graft failure (VGF). Vein graft failure has been reported to be as high as 45% at 12 to 18 months after surgery and leads to additional surgery, myocardial infarction, recurrent angina, and death. Preparation of HSVs before implantation leads to conduit injury, which may promote VGF. Objectives To investigate whether pressure distension during vein graft preparation leads to endothelial injury and intimal thickening and whether limiting intraluminal pressure during pressure distension by using a pressure release valve (PRV) preserves endothelial function and prevents neointima thickening. Design, Setting, and Participants Segments of HSVs were collected in a university hospital from 13 patients undergoing CABG procedures immediately after harvest (unmanipulated [UM]), after pressure distension (after distension [AD]), and after typical intraoperative surgical graft preparation (after manipulation [AM]). Porcine saphenous veins (PSVs) from 7 healthy research animals were subjected to manual pressure distension with or without an in-line PRV that prevents pressures of 140 mm Hg or greater. Endothelial function of the HSVs and PSVs was determined in a muscle bath, endothelial integrity was assessed, and intimal thickening in PSVs was evaluated after 14 days in organ culture. Main outcomes and measures Endothelial function was measured in force, converted to stress, and defined as the percentage relaxation of maximal phenylephrine-induced contraction. Endothelial integrity was assessed by immunohistologic examination. Neointimal thickness was measured by histomorphometric analysis. Results Pressure distension of HSVs led to decreased mean (SEM) endothelial-dependent relaxation (5.3% [2.3%] for AD patients vs 13.7% [2.5%] for UM patients; P < .05) and denudation. In the AM group, the function of the conduits was further decreased (−3.2% [3.2%]; P < .05). Distension of the PSVs led to reduced endothelial-dependent relaxation (7.6% [4.4%] vs 61.9% [10.2%] in the control group; P < .05), denudation, and enhanced intimal thickening (15.0 [1.4] μm vs 2.2 [0.8] μm in the control group; P < .05). Distension with the PRV preserved endothelial-dependent relaxation (50.3% [9.6%]; P = .32 vs control), prevented denudation, and reduced intimal thickening (3.4 [0.8] μm; P = .56 vs controls) in PSVs. Conclusions and Relevance Use of a PRV during graft preparation limits intraluminal pressure generated by manual distension, preserves endothelial integrity, and reduces intimal hyperplasia. Integration of this simple device may contribute to improved long-term vein graft patency.

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Transduction of peptide analogs of the small heat shock–related protein HSP20 inhibits intimal hyperplasia

Cited by 35 publications

References 42 publications

Phosphorylation and activation of a transducible recombinant form of human HSP20 in Escherichia coli

Phosphorylation and activation of a transducible recombinant form of human HSP20 in Escherichia coli

Heat Shock–Related Protein 20 Peptide Decreases Human Airway Constriction Downstream of β₂-Adrenergic Receptor

Pressure Control During Preparation of Saphenous Veins

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Transduction of peptide analogs of the small heat shock–related protein HSP20 inhibits intimal hyperplasia

Cited by 35 publications

References 42 publications

Phosphorylation and activation of a transducible recombinant form of human HSP20 in Escherichia coli

Phosphorylation and activation of a transducible recombinant form of human HSP20 in Escherichia coli

Heat Shock–Related Protein 20 Peptide Decreases Human Airway Constriction Downstream of β2-Adrenergic Receptor

Pressure Control During Preparation of Saphenous Veins

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Heat Shock–Related Protein 20 Peptide Decreases Human Airway Constriction Downstream of β₂-Adrenergic Receptor