Inhibition of intimal thickening after vascular injury with a cocktail of vascular endothelial growth factor and cyclic Arg-Gly-Asp peptide Yue Li, Lucinda.

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Presentation transcript:

Inhibition of intimal thickening after vascular injury with a cocktail of vascular endothelial growth factor and cyclic Arg-Gly-Asp peptide Yue Li, Lucinda S. McRobb, Levon M. Khachigian International Journal of Cardiology Volume 220, Pages 185-191 (October 2016) DOI: 10.1016/j.ijcard.2016.06.300 Copyright © 2016 Elsevier Ireland Ltd Terms and Conditions

Fig. 1 Divergent effects of cocktail on EC and SMC proliferation. Serum-arrested EC and SMC were treated with cocktail (VEGF-A/-D 50ng/ml and cRGD 200nM) in the presence of serum and cell proliferation monitored by xCELLigence (Roche) over time. Cell index represents a quantitative measure of cell growth. No addition denotes that no cocktail was added. Data represent the mean±SEM of the means of 4–5 independent experiments. P values are indicated in the figure. International Journal of Cardiology 2016 220, 185-191DOI: (10.1016/j.ijcard.2016.06.300) Copyright © 2016 Elsevier Ireland Ltd Terms and Conditions

Fig. 2 Cocktail inhibits Egr-1 expression in SMC. SMCs were exposed to cocktail (VEGF-A/-D 50ng/ml plus cRGD 200nM) with or without the NOS inhibitor L-NAME (100μM) for 0.5h and Egr-1 expression was assessed by Western blot analysis. Egr-1 and ß-actin antibodies recognize 75 and 42kDa proteins, respectively. The blot was stripped and reprobed. SF denotes serum-free medium. NT denotes no treatment. Data is representative of at least 2 independent experiments. International Journal of Cardiology 2016 220, 185-191DOI: (10.1016/j.ijcard.2016.06.300) Copyright © 2016 Elsevier Ireland Ltd Terms and Conditions

Fig. 3 Cocktail inhibits neointima formation in the rat carotid balloon injury model. The left carotid arteries of Sprague–Dawley rats were subjected to balloon injury followed by local treatment with Pluronic gel with vehicle (Pluronic gel only, n=6 animals) or cocktail at low concentration (LC: VEGF-A/-D at 100ng/ml, cRGD at 400nM; n=4) or high concentration (HC: VEGF-A/-D at 2500ng/ml, cRGD at 10μM; n=6), with or without subcutaneous injections of L-NAME (3mg/kg/day; n=4). A L-NAME alone treatment group with injury but no cocktail was also included (n=6). Neointima formation was examined in segments 2 and 3 after 14days. (A) Representative photomicrographs (100× magnification) of Verhoeff van Gieson (elastin) stained carotid arteries 14days after injury and treatment. (B) Intimal, medial and luminal areas were quantified using Image-Pro Plus software and mean±SEM determined. The I/M ratio is also indicated. (n=4–6). P values are indicated in the figure. International Journal of Cardiology 2016 220, 185-191DOI: (10.1016/j.ijcard.2016.06.300) Copyright © 2016 Elsevier Ireland Ltd Terms and Conditions

Fig. 4 Cocktail increases re-endothelialization and NOS3 expression, and decreases Egr-1 expression in injured rat carotid arteries. (A) Re-endothelialization was examined by immunohistochemical staining for CD31 in fixed and embedded entire rat carotid artery sections in segments 2 and 3 taken 14days after balloon injury and treatment with high concentration cocktail (HC: VEGF-A/-D at 2500ng/ml, cRGD at 10μM; n=6), with or without subcutaneous injections of L-NAME (3mg/kg/day; n=4–6). NOS3 expression (B) and Egr-1 (C) expression were similarly examined at the 14day time point. Protein labeling was visualized with DAB. CD31 and NOS3 stained sections were counterstained with hematoxylin. No counter staining was implemented with Egr-1 stained sections where Egr-1 positive cells in entire sections were counted. Open arrows indicate lack of endothelial staining. Closed arrows indicate positive staining. IEL denotes internal elastic lamina. Integrated Optical Density (IOD) of positive staining was determined using software Image-Pro Plus (Cybernetics), n=4–6. (IOD=Area×Density (mean)). All photomicrographs were taken at 400× magnification. Plotted data represents mean±SEM. P values are indicated in the figure. International Journal of Cardiology 2016 220, 185-191DOI: (10.1016/j.ijcard.2016.06.300) Copyright © 2016 Elsevier Ireland Ltd Terms and Conditions