R1R2 decreases TNF-α-induced monocyte U937 cell adhesion to HUVECs and transendothelial migration and reduces ICAM-1 and VCAM-1 levels.

<p>(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 <i>p</i><0.05 and ** <i>p</i>< 0.01.</p

R1R2 peptide ameliorates pulmonary fibrosis in mice through fibrocyte migration and differentiation

<div><p>Circulating fibrocytes play a key role in the pathogenesis of pulmonary fibrosis. Fibrocytes are bone marrow-derived leukocytes, which enter the lungs in response to their chemoattractant CXCL12 and differentiate into fibroblasts or myofibroblasts, leading to excess deposition of the collagen-rich extracellular matrix. Matrix metalloproteinase (MMP)-9 and MMP-2, secreted by fibrocytes, degrade the subendothelial basement membrane and promote fibrocyte influx into the lungs. Here, we demonstrate that R1R2, a novel peptide derived from the bacterial adhesin SFS, attenuates pulmonary fibrosis by preventing the differentiation of fibrocytes into myofibroblasts and by reducing the invasion of fibrocytes through basement membrane-like proteins. Moreover, our findings reveal dual regulation of R1R2 on MMP-9 through reduced enzymatic activity on gelatin and increased cleavage of CXCL12. These data suggest that R1R2 has potent anti-fibrotic effects against pulmonary fibrosis.</p></div

R1R2 decreases the binding between fibronectin (FN) and collagen.

<p>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.</p

R1R2 maintains the contractile phenotype in smooth muscle cells.

<p>(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.</p

R1R2 reduces SMC proliferation and migration.

<p>(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.</p

R1R2 decreases collagen content in the ligated vessel.

<p>(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 *** <i>p</i>< 0.001.</p

The collagen homologous peptide, R1R2, decreases vascular remodeling in the carotid artery.

<p>(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 <i>p</i><0.05, ** <i>p</i>< 0.01 and *** <i>p</i>< 0.001.</p

R1R2 does not attenuate FN content in the left carotid artery.

<p>(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 *** <i>p</i>< 0.001.</p

R1R2 decreases inflammatory cell accumulation and VCAM-1 and ICAM-1 levels.

<p>(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.</p

Morphometric analysis of vascular remodeling in experimental groups.

<p>Morphometric analysis of vascular remodeling in experimental groups.</p