Transduction of peptide analogs of the small heat shock–related protein HSP20 inhibits intimal hyperplasia Deron J Tessier, MD, Padmini Komalavilas,

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Transduction of peptide analogs of the small heat shock–related protein HSP20 inhibits intimal hyperplasia Deron J Tessier, MD, Padmini Komalavilas, PhD, Bo Liu, PhD, Craig K Kent, MD, Jeffrey S Thresher, MS, Catherine M Dreiza, BS, Alyssa Panitch, PhD, Lokesh Joshi, PhD, Elizabeth Furnish, PhD, William Stone, MD, Richard Fowl, MD, Colleen M Brophy, MD Journal of Vascular Surgery Volume 40, Issue 1, Pages 106-114 (July 2004) DOI: 10.1016/j.jvs.2004.03.028

Fig 1 Transducible HSP20 phosphopeptide analogs relaxes human saphenous vein. A, Rings of saphenous vein were suspended in a muscle bath and were precontracted with norepinephrine (10−7 mol/L) for 10 minutes. Cumulative increasing doses (0.1 - 1 mmol/L) of the PTD-pHSP20, (PTD-P20) or PTD-scHSP20 peptides (PTD-ScP20) were then added. The percentage of relaxation was calculated as the force of the maximal norepinephrine contraction minus the force after the addition of the peptide divided by the force of the maximal norepinephrine contraction × 100 (n= 3, *P < .05 compared to PTD-scHSP20). B, Rings of saphenous vein were pretreated with cumulative doses (1-10 mmol/L) of PTD-pHSP20 peptide in 7.5% dextran gel for 10 min and then challenged with norepinephrine (10−7 mol/L). C, Histologic representation of transduction of FITC-labeled peptides into saphenous vein. Representative strips were treated with 100 μmol/L PTD-FITC-pHSP20 peptide for 30 minutes at 37°C then fixed in 4% paraformaldehyde, paraffin embedded, and 6-μm sections were mounted on polylysine slides and examined under fluorescence microscopy. Journal of Vascular Surgery 2004 40, 106-114DOI: (10.1016/j.jvs.2004.03.028)

Fig 2 Localization of FITC-PTD-phospho HSP20 peptide analogs. Swiss albino 3T3 cells were treated with 25μmol/L FITC-PTD-pHSP20 peptides for 30 minutes at 37°C as described in the methods section and analyzed by confocal microscopy. A representative optical section taken from a confocal projection image, sectioned at 10-um intervals is depicted here. Non-peptide–treated control cells (A). Cells treated with FITC-PTD-pHSP20 peptide analogs (B). FITC-PTD-pHSP20 peptides localization was diffuse throughout cytoplasm. There was also punctate staining consistent with a vesicular distribution (arrow), along with nuclear staining (arrow). Scale bar 200 μm. Journal of Vascular Surgery 2004 40, 106-114DOI: (10.1016/j.jvs.2004.03.028)

Fig 3 Phospho HSP20 peptides inhibit migration of smooth muscle cells. A10 cells in the upper transwell chamber were treated with either 0.5% FBS alone or in combination with PTD-scHSP20 or PTD-pHSP20. Migration against a serum gradient was determined by placing 10% FBS into the lower chamber. No serum gradient (0.5% FBS) was used for the control. Four separate experiments were performed. No significant difference was found between control cells and PTD-pHSP20 + FBS-treated cells (#P > .05). PTD-pHSP20 significantly inhibited migration over both FBS and PTD-scHSP20 + FBS-treated cells (*P < .05). Journal of Vascular Surgery 2004 40, 106-114DOI: (10.1016/j.jvs.2004.03.028)

Fig 4 Phospho HSP20 peptides inhibit intimal hyperplasia in saphenous vein. A, Six human saphenous veins were cultured for 14 days in media alone (control) or media containing either PTD-scHSP20 or PTD-pHSP20. Prior to culture several rings from each vein were fixed in formalin (preculture). PTD-pHSP20 maintained intimal thickness compared to preculture (#P > .05) and significantly inhibited intimal hyperplasia compared to the control and PTD-scHSP20-treated veins (*P < .05). B, Representative segments of control and peptide-treated human saphenous vein stained with Verhoeff-van Gieson stain. The thickness of the internal elastic lamina (IEL) is denoted by a black line on the left of each image and is approximately 50 μm in the control image. C, The intima to media ratio (I/M) was calculated [intimal area/(intimal area + medial area)] for each vein segment. The control and PTD-scHSP20 treated veins demonstrated a significant increase in the I/M ratio compared to PTD-pHSP20 (*P < .05). PTD-pHSP20 maintained the I/M ratio compared to pre-culture (#P > .05). Journal of Vascular Surgery 2004 40, 106-114DOI: (10.1016/j.jvs.2004.03.028)