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Collagen inhibitory peptide R1R2 mediates vascular remodeling by decreasing inflammation and smooth muscle cell activation.

Lee TH, Sottile J, Chiang HY - PLoS ONE (2015)

Bottom Line: Morphometric analysis demonstrated that R1R2 reduced intima-media thickening compared to the control groups.This decrease was accompanied by decreased VCAM-1 and ICAM-1 expression.Our in vitro studies revealed that R1R2 attenuated SMC proliferation and migration, and also decreased monocyte adhesion and transendothelial migration through endothelial cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan.

ABSTRACT
The extracellular matrix (ECM) is a major constituent of the vessel wall. In addition to providing a structural scaffold, the ECM controls numerous cellular functions in both physiologic and pathologic settings. Vascular remodeling occurs after injury and is characterized by endothelial cell activation, inflammatory cell infiltration, phenotypic modulation of smooth muscle cells (SMCs), and augmented deposition of collagen-rich ECM. R1R2, a peptide derived from the bacterial adhesin SFS, with sequence homology to collagen, is known to inhibit collagen type I deposition in vitro by inhibiting the binding of fibronectin to collagen. However, the inhibitory effects of R1R2 during vascular remodeling have not been explored. We periadventitially delivered R1R2 to carotid arteries using pluronic gel in a vascular remodeling mouse model induced by blood flow cessation, and evaluated its effects on intima-media thickening, ECM deposition, SMC activation, and inflammatory cell infiltration. Morphometric analysis demonstrated that R1R2 reduced intima-media thickening compared to the control groups. R1R2 treatment also decreased collagen type I deposition in the vessel wall, and maintained SMC in the contractile phenotype. Interestingly, R1R2 dramatically reduced inflammatory cell infiltration into the vessel by ∼ 78%. This decrease was accompanied by decreased VCAM-1 and ICAM-1 expression. Our in vitro studies revealed that R1R2 attenuated SMC proliferation and migration, and also decreased monocyte adhesion and transendothelial migration through endothelial cells. Together, these data suggest that R1R2 attenuates vascular remodeling responses by decreasing inflammation and by modulating SMC proliferation and migration, and suggest that the R1R2 peptide may have therapeutic potential in treating occlusive vascular diseases.

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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.
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pone.0117356.g005: 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.

Mentions: Leukocyte infiltration through the activated endothelium and phenotypic modulation of vascular smooth muscle cells are two hallmarks of vascular remodeling [41–43]. To further define the mechanism by which R1R2 regulates vascular remodeling, we investigated the effect of R1R2 on phenotypic modulation and leukocyte infiltration in the vessel wall. To assess the effect of R1R2 on smooth muscle phenotype, we immunostained sections of vessels 7 days post-ligation with the smooth muscle cell differentiation markers, myosin heavy chain (SM-MHC) and smooth muscle α-actin (SMAA) (Fig. 5A, 5B, and 5C). IHC analysis demonstrated that vessels treated with scrambled peptide showed significantly decreased SM-MHC (0.407 ± 0.0135, n = 8 vs. 0.717 ± 0.021, n = 6) and SMAA staining in the intima-media area (0.176 ± 0.004, n = 6 vs. 0.232 ± 0.011, n = 5). In contrast, vessels treated with R1R2 maintained the same high SM-MHC (0.541 ± 0.025, n = 7 vs. 0.407 ± 0.014, n = 8) and SMAA staining as the sham group (0.214 ± 0.004, n = 5 vs. 0.232 ± 0.011, n = 5). We also evaluated smooth muscle cell proliferation in the intima and media by performing IHC with Ki-67 (Fig. 5D). R1R2 treatment inhibited cell proliferation in the ligated artery compared to the scrambled peptide treatment (11.20 ± 0.62%, n = 5 vs. 28.77 ± 1.94%, n = 5).


Collagen inhibitory peptide R1R2 mediates vascular remodeling by decreasing inflammation and smooth muscle cell activation.

Lee TH, Sottile J, Chiang HY - PLoS ONE (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4326127&req=5

pone.0117356.g005: 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.
Mentions: Leukocyte infiltration through the activated endothelium and phenotypic modulation of vascular smooth muscle cells are two hallmarks of vascular remodeling [41–43]. To further define the mechanism by which R1R2 regulates vascular remodeling, we investigated the effect of R1R2 on phenotypic modulation and leukocyte infiltration in the vessel wall. To assess the effect of R1R2 on smooth muscle phenotype, we immunostained sections of vessels 7 days post-ligation with the smooth muscle cell differentiation markers, myosin heavy chain (SM-MHC) and smooth muscle α-actin (SMAA) (Fig. 5A, 5B, and 5C). IHC analysis demonstrated that vessels treated with scrambled peptide showed significantly decreased SM-MHC (0.407 ± 0.0135, n = 8 vs. 0.717 ± 0.021, n = 6) and SMAA staining in the intima-media area (0.176 ± 0.004, n = 6 vs. 0.232 ± 0.011, n = 5). In contrast, vessels treated with R1R2 maintained the same high SM-MHC (0.541 ± 0.025, n = 7 vs. 0.407 ± 0.014, n = 8) and SMAA staining as the sham group (0.214 ± 0.004, n = 5 vs. 0.232 ± 0.011, n = 5). We also evaluated smooth muscle cell proliferation in the intima and media by performing IHC with Ki-67 (Fig. 5D). R1R2 treatment inhibited cell proliferation in the ligated artery compared to the scrambled peptide treatment (11.20 ± 0.62%, n = 5 vs. 28.77 ± 1.94%, n = 5).

Bottom Line: Morphometric analysis demonstrated that R1R2 reduced intima-media thickening compared to the control groups.This decrease was accompanied by decreased VCAM-1 and ICAM-1 expression.Our in vitro studies revealed that R1R2 attenuated SMC proliferation and migration, and also decreased monocyte adhesion and transendothelial migration through endothelial cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan.

ABSTRACT
The extracellular matrix (ECM) is a major constituent of the vessel wall. In addition to providing a structural scaffold, the ECM controls numerous cellular functions in both physiologic and pathologic settings. Vascular remodeling occurs after injury and is characterized by endothelial cell activation, inflammatory cell infiltration, phenotypic modulation of smooth muscle cells (SMCs), and augmented deposition of collagen-rich ECM. R1R2, a peptide derived from the bacterial adhesin SFS, with sequence homology to collagen, is known to inhibit collagen type I deposition in vitro by inhibiting the binding of fibronectin to collagen. However, the inhibitory effects of R1R2 during vascular remodeling have not been explored. We periadventitially delivered R1R2 to carotid arteries using pluronic gel in a vascular remodeling mouse model induced by blood flow cessation, and evaluated its effects on intima-media thickening, ECM deposition, SMC activation, and inflammatory cell infiltration. Morphometric analysis demonstrated that R1R2 reduced intima-media thickening compared to the control groups. R1R2 treatment also decreased collagen type I deposition in the vessel wall, and maintained SMC in the contractile phenotype. Interestingly, R1R2 dramatically reduced inflammatory cell infiltration into the vessel by ∼ 78%. This decrease was accompanied by decreased VCAM-1 and ICAM-1 expression. Our in vitro studies revealed that R1R2 attenuated SMC proliferation and migration, and also decreased monocyte adhesion and transendothelial migration through endothelial cells. Together, these data suggest that R1R2 attenuates vascular remodeling responses by decreasing inflammation and by modulating SMC proliferation and migration, and suggest that the R1R2 peptide may have therapeutic potential in treating occlusive vascular diseases.

Show MeSH
Related in: MedlinePlus