ROCK1 mediates leukocyte recruitment and neointima formation following vascular injury

Abstract

Although Rho-associated kinase (ROCK) activity has been implicated in cardiovascular diseases, the tissue- and isoform-specific roles of ROCKs in the vascular response to injury are not known. To address the role of ROCKs in this process, we generated haploinsufficient Rock1 (Rock1(+/-)) and Rock2 (Rock2(+/-)) mice and performed carotid artery ligations. Following this intervention, we found reduced neointima formation in Rock1(+/-) mice compared with that of WT or Rock2(+/-) mice. This correlated with decreased vascular smooth muscle cell proliferation and survival, decreased levels proinflammatory adhesion molecule expression, and reduced leukocyte infiltration. In addition, thioglycollate-induced peritoneal leukocyte recruitment and accumulation were substantially reduced in Rock1(+/-) mice compared with those of WT and Rock2(+/-) mice. To determine the role of leukocyte-derived ROCK1 in neointima formation, we performed reciprocal bone marrow transplantation (BMT) in WT and Rock1(+/-) mice. Rock1(+/-) to WT BMT led to reduced neointima formation and leukocyte infiltration following carotid ligation compared with those of WT to WT BMT. In contrast, WT to Rock1(+/-) BMT resulted in increased neointima formation. These findings indicate that ROCK1 in BM-derived cells mediates neointima formation following vascular injury and suggest that ROCK1 may represent a promising therapeutic target in vascular inflammatory diseases.

Figure 1. Decreased neointima formation after carotid artery ligation in Rock1^+/– mice.

(A) Representative cross sections of contralateral unligated and ligated carotid arteries in WT, Rock1^+/–, and Rock2^+/– mice at 28 days after ligation. Scale bar: 100 μm. (B) Representative immunohistochemical analysis of ROCK expression and activity in carotid arteries from WT and Rock1^+/– mice at 14 days after ligation. Vessels were stained with nonspecific antibody (IgG), and antibodies were directed at total MBS (tMBS), phosphorylated MBS (pMBS), ROCK1, and ROCK2. The I and M indicate the intima and media, respectively. Scale bar: 50 μm.

Figure 2. ROCK expression in leukocytes and carotids of Rock1^+/– and Rock2^+/– mice.

(A) Expression of ROCK isoforms in leukocytes from WT, Rock1^+/–, and Rock2^+/– mice with and without carotid ligation. Representative western blot (upper panel). Quantification of ROCK1 expression (middle panel). Mean ± SEM; n = 3. Quantification of ROCK2 expression (lower panel). Mean ± SEM; n = 3. (B) Expression of ROCK isoforms in unligated and ligated carotid arteries from WT, Rock1^+/–, and Rock2^+/– mice. Representative western blot (upper panel). Quantification of ROCK1 expression (middle panel). Mean ± SEM; n = 4. Quantification of ROCK2 expression (lower panel). Mean ± SEM; n = 4. *P < 0.05 versus unligated WT mice; ^†P < 0.01 versus unligated Rock2^+/– mice; ^ΧP < 0.01 versus ligated WT and Rock2^+/– mice; **P < 0.01 versus unligated WT and Rock1^+/– mice; ^‡P < 0.01 versus ligated WT mice; ^#P < 0.05 versus ligated Rock1^+/– mice; ^##P < 0.01 versus unligated WT and Rock2^+/– mice; ***P < 0.01 versus WT and Rock1^+/– mice.

Figure 3. Decreased leukocyte recruitment to ligated vessels of Rock1^+/– mice.

(A) Representative histological sections from carotid arteries in WT and Rock1^+/– mice stained with CD45 Ab for leukocytes. Arrowheads indicate CD45-positive cells. Scale bar: 50 μm (left panel). Quantitative analysis of the number of CD45-positive leukocytes adherent to the unligated (n = 3–4 in each group) and ligated vessel walls (n = 14 in each group) at 3 days after ligation (right panel). (B) Representative histological sections from carotid arteries in WT and Rock1^+/– mice stained with MOMA-2 Ab for macrophage. Arrowheads indicate MOMA-2–positive cells. Scale bar: 50 μm (left panel). Quantitative analysis of the number of MOMA-2–positive macrophages infiltrated into the unligated (n = 3–4 in each group) and ligated vessel (n = 12–13 in each group) at day 3. *P < 0.01 versus unligated WT mice; ^†P < 0.05 versus unligated Rock1^+/– mice; ^ΧP < 0.05 versus ligated WT mice.

Figure 4. Decreased recruitment and adhesion of leukocytes in Rock1^+/– mice.

Neutrophil and macrophage recruitment was induced by i.p. injection of thioglycollate. (A) Neutrophil recruitment 4 hours after i.p. injection of PBS (control) or 3% thioglycollate into WT, Rock1^+/–, and Rock2^+/– mice (n = 10, 5, and 5, respectively). (B) Macrophage recruitment 4 days after i.p. injection of PBS (control) or 3% thioglycollate into WT, Rock1^+/–, and Rock^2+/– mice (n = 9, 8, and 6, respectively). Confluent monolayers of ECs isolated from WT and Rock1^+/– mice were incubated with PBS (control, n = 4–6 in each group) or 10 units/ml of thrombin for 10 minutes (n = 9–10 in each group). They were then placed under laminar flow at 0.5 (C) or 1.0 dynes/cm² (D). U937 monocytes, stably expressed human L selectin (U937-LAM), were perfused over the EC monolayers, and adherent cells were quantified after 3 minutes. *P < 0.01 versus each control group; ^†P < 0.05 versus each control group; ^‡P < 0.01 versus Rock1^+/– mice with thioglycollate or Rock1^+/– ECs with thrombin; ^ΧP < 0.05 versus Rock1^+/– mice with thioglycollate or Rock1^+/– ECs with thrombin.

Figure 5. Decreased expression of endothelial adhesion molecules in Rock1^+/– mice.

Representative histological sections from unligated and ligated carotid arteries at 7 days after ligation in WT and Rock1^+/– mice stained for ICAM-1 (A) and VCAM-1 (B). Scale bars: 50 μm. (C) Representative result of northern blot analysis of Icam1 and Vcam1 mRNA expression in ECs from WT and Rock1^+/– mice. ECs were stimulated with 5 U/ml of thrombin for 6 hours. Gapdh mRNA expression was used as an internal control. (D) Representative result of western blot analysis of ROCK1 and ROCK2 expressions in ECs from WT and Rock1^+/– mice with or without thrombin stimulation for 5 minutes. Actin was used as an internal control. (E) Representative result of western blot analysis of ROCK and ERK activities in ECs from WT and Rock1^+/– mice. ECs were stimulated with 5 U/ml of thrombin for the indicated time periods.

Figure 6. Decreased expression of PDGF-B, degradation of IκB-α, and activation of NF-κB in macrophages from Rock1^+/– mice.

(A) Representative western blot analysis of IκB-α expression in peritoneal macrophages from WT, Rock1^+/–, and Rock2^+/– mice. Actin was used as an internal control. (B) Representative electrophoretic mobility shift assay of NF-κB in WT, Rock1^+/–, and Rock2^+/– macrophages (left panel). Specificity of NF-κB binding activity was analyzed by the addition of unlabeled probe, by the pretreatment with excess unlabeled probe (cold) or mutant probe (mutant cold), and by anti-p65 Ab (p65 Ab) supershift gel assay (right panel). NF-κB binding band (NF-κB), nonspecific binding band (NS), free probe (free), and the raised bands supershifted by Ab (supershift) are indicated on the right. (C) Expression of Pdgfa and -b were analyzed by quantitative real-time PCR. Total RNA was extracted from thioglycollate-induced peritoneal macrophages in WT, Rock1^+/–, and Rock2^+/– mice (n = 6–8). *P < 0.05 versus WT and Rock2^+/– mice.

Figure 7. Decreased cell proliferation in the neointima of Rock1^+/– mice.

(A) Representative histological sections from carotid arteries in WT and Rock1^+/– mice stained for PCNA at 14 days after ligation. Arrowheads indicate PCNA-positive cells. Scale bars: 50 μm (left panels). Quantitative analysis of the ratio of PCNA-positive cells to total cell number in the intima and the media (n = 10–16) (right panel). *P < 0.01 versus WT mice. (B) Cell proliferation in response to serum of VSMCs from WT and Rock1^+/– mice. Experiments were performed 6 times in triplicates. (C) DNA synthesis in response to PDGF of VSMCs from WT and Rock1^+/– mice (n = 12). *P < 0.01 versus without PDGF (control). (D) Cell migration in response to PDGF of VSMCs from WT and Rock1^+/– mice (n = 8­–9). *P < 0.01 versus without PDGF (control); ^†P < 0.01 versus control; ^ΧP < 0.01 versus WT. (E) Representative western blot analysis of ROCK and ERK activities in VSMCs of WT and Rock1^+/– mice. VSMCs were stimulated with 10 ng/ml of PDGF for the indicated time periods.

Figure 8. ROCK1 in BM-derived cells mediates to neointima formation.

(A) Representative cross sections of contralateral unligated and ligated carotid arteries in WT to WT, WT to Rock1^+/–, and Rock1^+/– to WT BMT mice at 28 days after ligation. Scale bars: 100 μm. Representative western blot analysis of ROCK expression in leukocytes (B) and ECs (C) from WT and Rock1^+/– BMT mice. Actin was used as an internal control.

Figure 9. BM-derived cells contribute to leukocyte recruitment and cell proliferation in Rock1^+/– to WT BMT mice.

(A) Representative histological sections from carotid arteries in WT and Rock1^+/– BMT mice stained with CD45 Ab for leukocytes. Arrowheads indicate CD45-positive cells. Scale bar: 50 μm (left panel). Quantitative analysis of the number of CD45-positive leukocytes adherent to the unligated (n = 3–4) and ligated vessel wall at 3 days after ligation (n = 8–12). (B) Representative histological sections from carotid arteries in WT and Rock1^+/– BMT mice stained with MOMA-2 Ab for macrophages. Arrowheads indicate MOMA-2–positive cells. Scale bar: 50 μm (left panel). Quantitative analysis of the number of MOMA-2–positive macrophages infiltrated into the unligated (n = 3–4) and ligated vessel wall at days 3 (n = 7–8). (C) Representative histological sections from carotid arteries in WT and Rock1^+/– BMT mice stained for PCNA at 14 days after ligation. Arrowheads indicate PCNA-positive cells. Scale bar: 50 μm (left panel). Quantitative analysis of the ratio of PCNA-positive cells to total cell number in the intima and the media (n = 8–9). *P < 0.05 versus unligated mice; ^†P < 0.01, ^ΧP < 0.05 versus Rock1^+/– to WT mice.