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Vascular remodeling in response to hemodynamic alterations is a physiological process that requires coordinated signaling between endothelial, inflammatory and vascular smooth muscle cells (VSMCs). Extensive experimental and clinical studies have indicated the critical role of the Ras homolog gene family, member A/Rho-associated kinase (ROCK) signaling pathway in the pathogenesis of cardiovascular disease, where ROCK activation has been demonstrated to promote inflammation and remodeling through inducing the expression of proinflammatory cytokines and adhesion molecules in endothelial cells and VSMCs. However, the role of ROCK in flow-induced vascular remodeling has not been fully defined. The current study aimed to investigate the effect of the ROCK signaling pathway in flow-induced vascular remodeling by comparing the responses to partial carotid artery ligation in mice treated with fasudil (a ROCK inhibitor) and untreated mice. Intima-media thickness and neointima formation were evaluated by morphology. VSMC proliferation and inflammation of the vessel wall were assessed by immunohistochemistry. In addition, the expression levels of ROCK and the downstream effectors of ROCK, myosin light chain (MLC) and phosphorylated-MLC (p-MLC), were quantified by western blot analysis. Following a reduction in blood flow, ROCK1 and p-MLC expression increased in the untreated left common carotid arteries (LCA). Fasudil-treated mice developed a significantly smaller intima-media thickness compared with the untreated mice. Quantitative immunohistochemistry of the fasudil-treated LCA indicated that there was a reduction in proliferation when compared with untreated vessels. There were fewer CD45+ cells observed in the fasudil-treated LCA compared with the untreated LCA. In conclusion, the expression of ROCK was enhanced in flow-induced carotid artery remodeling and ROCK inhibition as a result of fasudil treatment may attenuate flow-induced carotid artery remodeling.
Vascular remodeling of the carotid arteries, characterized by carotid artery intima-media thickening (IMT), may be key in the pathogenesis of various cardiovascular diseases (
A total of 85 male mice (weight, 20–25 g; age, 8-weeks; Academy of Military Medical Sciences) were used in the current study and all procedures were approved by the Medical Ethics Committee of Peking University People's Hospital (Beijing; permit number, 2012-13). The control group consisted of 5 mice. The remaining 80 mice were randomly divided into 8 groups, with 10 mice per group. The were fed a standard diet and maintained in a specific-pathogen free environment in our hospital, with a 12 h light/dark cycle, and a house temperature of 20–28°C.
Blood flow reduction in the LCA and flow measurements were performed as previously described (
On days 3, 7, 14 and 28 following surgery, the vasculature was fixed by transcardial perfusion at 100 mmHg with 25 ml 10% paraformaldehyde (ZSGB-BIO, Beijing, China) in sodium phosphate buffer (pH 7.0; Sigma-Aldrich), as previously described (
Immunohistochemical analysis of the paraffin-embedded vessel cross sections was performed as previously described (
Vascular tissues were homogenized in ice-cold buffer (50 mmol/l Tris, pH 7.4; 150 mmol/l NaCl; 0.5% Triton X-100; 1 mmol/l edetic acid; 1 mol/l phenylmeth-ylsulfonyl fluoride; and 5 mg/l aprotinin) and centrifuged at 14,000×g at 4°C for 15 min. The supernatants were then collected as total proteins, which were electrophoresed through 8% SDS-PAGE (Sigma-Aldrich) and electrically transferred to a nitrocellulose membrane. The membrane was incubated at 4°C overnight in Tris(hydroxymethyl)aminomethane buffered saline (TBS) containing 5% milk and the primary antibodies (1:1,000 dilution, Cell Signaling Technology, Inc.) against ROCK1, myosin light chain (MLC) and p-MLC. Subsequent to washing with TBS, the membranes were incubated with the secondary antibody (goat anti-rabbit IgG horseradish peroxidase conjugated) at room temperature for 1 h. The protein expression levels of ROCK1 mAb (cat. no. 4035S), MLC mAb (cat. no. 3672S) and p-MLC mAb (cat. no. 3675S) were determined by western blotting where the relative quantity of protein was determined by densitometric analysis (Graigar DM3011; Graigar Technology Co., Ltd., Shenzhen, China). The densitometric intensities of ROCK1, MLC and p-MLC were normalized to GAPDH values.
Data are represented as the mean ± standard error of the mean. Comparisons for two groups were performed using Student's t-test. The differences among three or more groups were determined using one-way analysis of variance with Fisher's exact post-hoc test (Graphpad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference. Differences among groups were analyzed by repeated measures three times.
The mouse model used in the current study included ligation of the internal, external carotid and occipital arteries. Partial blood flow was maintained through the superior thyroid branch of the external carotid artery (
To determine whether the ROCK signaling pathway participated in the flow-induced vascular remodeling, western blotting was used to analyze the protein expression levels of ROCK1, ROCK2 and their downstream effectors, MLC and p-MLC (
In order to assess how blood flow affects arterial remodeling
Various studies have demonstrated that VSMC proliferation is a driving force for vascular remodeling in response to flow reduction (
To characterize the phenotype of VSMCs, staining for SMC α-actin expression was performed. On day 14 subsequent to ligation, α-actin-positive cells were observed to migrate to the intima-media area in the fasudil-treated and untreated vessels. On day 28, subsequent to ligation, a markedly increased intensity of α-actin-positive cells accumulated in the neointima area of the untreated ligated mice, whereas there was a reduced intensity of α-actin staining in the media and neointima of the fasudil-treated mice (
Previous studies have demonstrated that the inflammatory response is important during vascular remodeling (
In the sham group, quantitative evaluation demonstrated that there were almost no CD45+ cells within the mouse intima-media area and few in the adventitia (
Macrophages in the vessel wall were identified by staining for antigen CD68 (a macrophage marker). In the sham group, there were almost no CD68+ cells observed in the mouse intima-media area and adventitia (
The graph in
Flow-induced vascular remodeling is a complex process, involving the expression of multiple genes and the interaction of multiple cell types (
The current study aimed to investigate the ROCK effects on flow-induced vascular remodeling based on partial ligation of the LCA in C57Bl/6J mice. The left external carotid, internal carotid and occipital arteries were ligated leaving the superior thyroid artery intact. Sham procedures consisted of suturing without ligation. This ligation reduces ~80% of the flow rate in the LCA and increases the flow rate in the right common carotid arteries by ~40% with the endothelium intact (
Previous studies have demonstrated that the ROCK/MLC signaling pathway is extensively described in VSMCs (
In the current study, the mice subjected to 28 days of LCA partial ligation with or without fasudil treatment were observed to exhibit IMT and neointima formation in the vessel walls. Evaluated by H&E and EVG staining, 28 days subsequent to ligation, fasudil-treated mice exhibited a significant reduction in media with a lower quantity of neointima formation when compared with untreated ligated mice (
Migration and proliferation of VSMCs are important factors involved in flow-induced vascular remodeling. In the current study, the number of PCNA-positive cells in the medial and intimal areas was markedly reduced in the fasudil-treated mice when compared with their untreated counterparts (
Vascular inflammation participates in flow-induced vascular remodeling, while ROCK1 has been proposed to serve a critical role in vascular inflammatory diseases (
Concerning the limitations of the current study, as fasudil is a non-selective ROCK inhibitor, it may have additional non-selective effects (
In conclusion, the present study identified the involvement of the ROCK signaling pathway in flow-induced vascular remodeling, observing significant ROCK1 expression level increases in ligated LCA. p-MLC, as the activated downstream effector of ROCK, was also significantly increased in ligated mouse LCA. Fasudil, as a ROCK inhibitor, was demonstrated to reduce ROCK1 and p-MLC expression levels in ligated mice and attenuate the process of flow-induced vascular remodeling (for example, IMT, VSMC migration, and proliferation and lymphocyte accumulation). The current study elucidated the role of ROCK signaling in the regulation of vascular remodeling, and highlighted the role of a ROCK inhibitor, fasudil, in the attenuation of the process of flow-induced vascular remodeling. Therefore, further characterization of ROCK signaling and its pharmacological modulation may lead to the development of novel therapeutic strategies for targeting flow-induced vascular remodeling.
This study was supported by the Peking University People's Hospital Research and Development Funds (grant no. 2118000537 to Dr. Wei Li), Specialized Research Fund for the Doctoral Program of Higher Education of China (grant no. 2109000075 to Dr Xiaoming Zhang) and the National Natural Science Foundation of China (grant no. 81470574 to Dr Wei Li).
(A) Image of the ligation performed in the current study. Black arrows demonstrate the ligation points and the intact thyroid artery is highlighted by a black star. (B) The blood flow rate variations (reduction in LCA and increase in RCA) observed following partial ligation. Blood flow on the two sides was comparable in the sham and fasudil-treated groups prior and subsequent to ligation. Values are presented as the mean ± standard error of the mean, n=8 (number of vessels) ***P<0.001. LCA, left common carotid artery; RCA, right common carotid artery; ECA, external carotid artery; ICA, internal carotid artery.
ROCK expression in sham and ligated mice. (A) Expression of ROCK isoforms and downstream markers of ROCK in LCA from fasudil-treated and untreated mice, with and without LCA partial ligation on days 3, 7, 14, and 28. Quantification of ROCK1, ROCK2, MLC and p-MLC in LCA from fasudil-treated and untreated mice on (B) day 7 and (C) day 28 [n=8 (number of vessels); *P<0.05, **P<0.01 and ***P<0.001]. ROCK, Rho-associated kinase; LCA, left common carotid artery; MLC, myosin light chain; p-MLC, phosphorylated-MLC.
Morphology of the control and fasudil-treated carotid arteries following ligation. (A) Representative light microscopy images of hematoxylin and eosin-stained carotid artery cross sections obtained from the control and fasudil-treated mice. Medial thickening was observed in the control and fasudil-treated mice on day 7, and neointima formation was apparent on days 14 and 28 (magnification, ×40; scale bar, 20 µm). (B) Elastic-van Gieson staining of carotid arteries from LCA cross-sections with or without fasudil treatment four weeks after ligation (sham, sham + fasudil, ligation and ligation + fasudil; magnification, ×40; scale bar, 20 µm). (C) Representative light microscopy images of hematoxylin and eosin-stained RCA cross sections obtained from the sham and ligated mice (magnification, ×40; scale bar, 20 µm). (D) Volumes of vessel components, including the lumen, media and media + intima, in addition to the vessel wall thickness of the media and media + intima. Black and white bars indicate fasudil-treated and untreated groups, respectively. Values are presented as the mean ± standard error of the mean; n=8 (number of vessels). *P<0.05, ***P<0.001 (by analysis of variance). LCA, left common carotid artery; RCA, right common carotid artery.
Flow-induced vascular remodeling is reduced in fasudil-treated mice. Proliferation and migration of vascular smooth muscle cells were identified by immunohistochemistry on paraffin-embedded sections of the carotid arteries from wild-type C57Bl/6J mice. (A) PCNA expression in the LCA of (a) untreated or (b) fasudil-treated arteries (16.1±2.4% vs. 5.2±2.1%; ***P<0.001). Values are presented as the mean ± standard error of the mean; n=8 (number of vessels). (B) Smooth muscle cell α-actin staining of LCA cross-sections from fasudil-treated and untreated mice 14 days and 28 days subsequent to ligation (vascular smooth muscle cells are stained brown; magnification, ×40; scale bar, 20 µm). PCNA, proliferating cell nuclear antigen; LCA, left common carotid artery.
Flow-induced vascular remodeling is associated with the accumulation of inflammatory cells. The accumulation of leukocytes and macrophages was assessed by CD45 and CD68 immunohistochemistry (hematoxylin and eosin counterstaining), respectively. In the ligated mice without fasudil treatment, leukocyte accumulation was apparent in all the layers, while fewer leukocytes accumulated within the ligated mice that were treated with fasudil. (A) Positive cells are represented by black arrows (magnification, ×40; scale bar, 20 µm). (B) Ligation of fasudil-treated mice demonstrated reduced macrophage accumulation in the vessel walls (magnification, 40×; scale bar, 20 µm). (C) Positive cells per area. (D) Positive cells per total cells. (E) Positive cells per area. (F) Positive cells per total cells. Values are presented as the mean ± standard error of the mean, n=8 (number of vessels). ***P<0.001. CD, cluster of differentiation.