Fig 1.
R1R2 decreases the binding between fibronectin (FN) and collagen.
4 nM of FN was incubated with the indicated amount of R1R2 or scrambled peptide at 4°C overnight. The mixture of FN and R1R2 or scrambled peptide was added into collagen or BSA coated wells. Bound FN was detected with an anti-FN antibody followed by HRP-conjugated secondary antibody, and ABTS as described in Methods. The absorbance at 405nm was quantitated.
Fig 2.
The collagen homologous peptide, R1R2, decreases vascular remodeling in the carotid artery.
(A) Representative photomicrographs of the left carotid artery 14 days after ligation. Lumen (L), neointima (I) and media (M) in ligated vessels are shown. C indicates blood clot. Bar, 200 μm. Morphometric analyses of the lumen (B), intima+media (C), the external elastic lamina compartment (EEL; D), and intima/media ratio (E) 7 and 14 days after ligation. * indicates p<0.05, ** p< 0.01 and *** p< 0.001.
Table 1.
Morphometric analysis of vascular remodeling in experimental groups.
Fig 3.
R1R2 decreases collagen content in the ligated vessel.
(A) Immunohistochemistry (IHC) for collagen type I 14 days post-surgery. Bar, 100 μm. (B) Quantification of immunostaining intensity of collagen I in the intima-media area 7 (Sham: n = 6, Scrambled: n = 6, R1R2: n = 6) and 14 days (Sham: n = 6, Scrambled: n = 6, R1R2: n = 5) after the ligation. (C) Collagen content was biochemically measured by hydroxyproline assay in the carotid artery 14 days after ligation. n = 3 for each experimental group. * indicates p<0.05, ** p< 0.01 and *** p< 0.001.
Fig 4.
R1R2 does not attenuate FN content in the left carotid artery.
(A) IHC for FN 14 days post-surgery. Bar, 100 μm. (B) Quantification of IHC intensity of FN in the intima-media area 7 (Sham: n = 6, Scrambled: n = 6, R1R2: n = 6) and 14 days (Sham: n = 7, Scrambled: n = 7, R1R2: n = 7) after ligating the vessel. * indicates p<0.05, ** p< 0.01 and *** p< 0.001.
Fig 5.
R1R2 maintains the contractile phenotype in smooth muscle cells.
(A) Sections of the left carotid artery from mice 7 days post-surgery were immunostained for SM myosin heavy chain (SM-MHC) and SM α-actin (SMAA) and Ki-67. Arrows indicate Ki-67 (+) cells. Bar, 50 μm. (B) Quantification of IHC intensity of SM-MHC in the intima-media area (Sham: n = 6, Scrambled: n = 8, R1R2: n = 7). (C) Quantitative analysis of immunostaining intensities of SMAA in the intima-media area 7 days post-ligation (Sham: n = 5, Scrambled: n = 6, R1R2: n = 5). (D) Proliferation index (Ki-67 (+) cells/total cells) in the intima and media of the vessel wall (Sham: n = 6, Scrambled: n = 5, R1R2: n = 5). * indicates p<0.05, *** p< 0.001.
Fig 6.
R1R2 reduces SMC proliferation and migration.
(A) A7r5 SMC growth study over 96 hours. After serum starvation and treatment with R1R2 or scrambled peptides for 36 hours, cells were stimulated with 10% FBS over 96 hours. Cells were counted at the indicated time in the figure (n = 5). (B) A7r5 SMCs were pretreated with R1R2 or scramble peptide for 36 hours. SMCs underwent a scratch wound injury and were then stimulated with 10 ng/ml PDGF-BB for 30 hours. Representative images from the scratch wound assays are shown. (C) Quantitation of SMC migration in the scratch wound healing assay was performed by subtracting the cell-free area 30 hours after PDGF-BB stimulation from the cell-free area in the beginning (n = 5). * indicates p<0.05 and ** p< 0.01.
Fig 7.
R1R2 decreases inflammatory cell accumulation and VCAM-1 and ICAM-1 levels.
(A) Photographs showing representative immunostaining of CD-45, VCAM-1 and ICAM-1 in the left carotid artery from the animals subjected to ligation 7 days after the surgery. Arrows indicate CD45 (+) leukocytes. Bar, 50 μm. (B-D) Percentage of the area which is CD45 (+) (Sham: n = 7, Scrambled: n = 6, R1R2: n = 5) (B), VCAM-1 (+) (Sham: n = 7, Scrambled: n = 9, R1R2: n = 7) (C) or ICAM-1 (+) (Sham: n = 5, Scrambled: n = 8, R1R2: n = 7) (D) were assessed in the intima-media of the vessel. (E) Western blot analysis of ICAM-1 and VCAM-1 expression in ligated carotid artery at 7 days. Equal protein loading was confirmed with GAPDH. * indicates p<0.05 and ** p<0.01.
Fig 8.
R1R2 decreases TNF-α-induced monocyte U937 cell adhesion to HUVECs and transendothelial migration and reduces ICAM-1 and VCAM-1 levels.
(A) HUVECs were pretreated with R1R2 or scramble peptide before treatment with TNF-α (10ng/ml) for 6 hours in the continued presence of R1R2 or scrambled peptide. Calcein-AM labeled U937 monocyte adhesion to TNF-α HUVECs was quantitated by fluorescence intensity. Microscopic images showing U937 monocytes adhering to HUVECs as assessed by in vitro adhesion assay. (B) Calcein-AM-labeled U937 monocytes transmigrated through TNF-α-treated HUVECs. (C) Western blot analysis of ICAM-1 and VCAM-1 expression in TNF-α-treated HUVECs. * indicates p<0.05 and ** p< 0.01.