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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 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.
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pone.0117356.g008: 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.

Mentions: In injured vessels, there is increased expression of cell adhesion molecules in the activated endothelium, leading to enhanced leukocyte recruitment and transendothelial infiltration [47,48]. Our study demonstrates that there is a reduction in leukocyte infiltration in the vessel wall in R1R2-treated animals compared to scrambled peptide-treated mice (Fig. 7). To determine whether R1R2 modulates leukocyte recruitment to activated endothelial cells during inflammation, we assessed monocyte adhesion to HUVECs and transendothelial migration, which both involve interaction with cell adhesion molecules such as ICAM-1 and VCAM-1. In the adhesion assay, HUVECs treated with 10 ng/ml TNF-α for 16 hours were incubated with calcein-AM stained monocyte U937 cells. As shown in Fig. 8A, TNF-α-induced monocyte U937 adhesion to HUVECs was significantly blunted by the R1R2 peptide (197697 ± 34184 vs. 259093 ± 14720). In addition, R1R2 significantly reduced monocyte transendothelial migration in comparison to control peptide (Fig. 8B, 65633 ± 20412 vs. 110055 ± 42517). To rule out the possibility that the inhibitory effect of R1R2 on U937 adhesion and transmigration was due to a decrease in endothelial cell number, we assayed the effect of R1R2 on cell proliferation in comparison with control peptide-treated cells. No significant decrease was observed between the R1R2-treated HUVECs and the control group at the start of the adhesion assay (data not shown). Consistent with our in vivo data (Fig. 7), the expression levels of ICAM-1 and VCAM-1 on HUVECs were significantly decreased with R1R2 treatment (Fig. 8C and 8D). Interestingly, the inhibitory effect of R1R2 on ICAM-1 expression was transient and detected earlier than the effect on VCAM-1 expression after TNF-α stimulation (Fig. 8D). This result is supported by a previous report showing that VCAM-1 expression is induced by ICAM-1 activation or clustering [49]. Taken together, these data show that R1R2 reduces monocyte adhesion and infiltration, as well as the expression of cell adhesion molecules by endothelial cells.


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 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.
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pone.0117356.g008: 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.
Mentions: In injured vessels, there is increased expression of cell adhesion molecules in the activated endothelium, leading to enhanced leukocyte recruitment and transendothelial infiltration [47,48]. Our study demonstrates that there is a reduction in leukocyte infiltration in the vessel wall in R1R2-treated animals compared to scrambled peptide-treated mice (Fig. 7). To determine whether R1R2 modulates leukocyte recruitment to activated endothelial cells during inflammation, we assessed monocyte adhesion to HUVECs and transendothelial migration, which both involve interaction with cell adhesion molecules such as ICAM-1 and VCAM-1. In the adhesion assay, HUVECs treated with 10 ng/ml TNF-α for 16 hours were incubated with calcein-AM stained monocyte U937 cells. As shown in Fig. 8A, TNF-α-induced monocyte U937 adhesion to HUVECs was significantly blunted by the R1R2 peptide (197697 ± 34184 vs. 259093 ± 14720). In addition, R1R2 significantly reduced monocyte transendothelial migration in comparison to control peptide (Fig. 8B, 65633 ± 20412 vs. 110055 ± 42517). To rule out the possibility that the inhibitory effect of R1R2 on U937 adhesion and transmigration was due to a decrease in endothelial cell number, we assayed the effect of R1R2 on cell proliferation in comparison with control peptide-treated cells. No significant decrease was observed between the R1R2-treated HUVECs and the control group at the start of the adhesion assay (data not shown). Consistent with our in vivo data (Fig. 7), the expression levels of ICAM-1 and VCAM-1 on HUVECs were significantly decreased with R1R2 treatment (Fig. 8C and 8D). Interestingly, the inhibitory effect of R1R2 on ICAM-1 expression was transient and detected earlier than the effect on VCAM-1 expression after TNF-α stimulation (Fig. 8D). This result is supported by a previous report showing that VCAM-1 expression is induced by ICAM-1 activation or clustering [49]. Taken together, these data show that R1R2 reduces monocyte adhesion and infiltration, as well as the expression of cell adhesion molecules by endothelial cells.

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