Yoshiyuki Rikitake

Rho GTPases, Statins, and Nitric Oxide

The lipid-lowering drugs, 3-hydroxy-3-methylgulutaryl-coenzyme A (HMG-CoA) reductase inhibitors or statins, are used in the prevention and treatment of cardiovascular diseases. Recent experimental and clinical studies suggest that statins may exert vascular protective effects beyond cholesterol reduction. For example, statins improve endothelial function by cholesterol-dependent and -independent mechanisms. The cholesterol-independent or pleiotropic effects of statins include the upregulation and activation of endothelial NO synthase (eNOS). Because statins inhibit an early step in the cholesterol biosynthetic pathway, they also inhibit the synthesis of isoprenoids such as farnesylpyrophosphate and geranylgeranylpyrophosphate, which are important posttranslational lipid attachments for intracellular signaling molecules such as the Rho GTPases. Indeed, decrease in Rho GTPase responses as a consequence of statin treatment increases the production and bioavailability of endothelium-derived NO. The mechanism involves, in part, Rho/Rho-kinase (ROCK)-mediated changes in the actin cytoskeleton, which leads to decreases in eNOS mRNA stability. The regulation of eNOS by Rho GTPases, therefore, may be an important mechanism underlying the cardiovascular protective effect of statins.

Critical Role of Endothelial Notch1 Signaling in Postnatal Angiogenesis

Notch receptors are important mediators of cell fate during embryogenesis, but their role in adult physiology, particularly in postnatal angiogenesis, remains unknown. Of the Notch receptors, only Notch1 and Notch4 are expressed in vascular endothelial cells. Here we show that blood flow recovery and postnatal neovascularization in response to hindlimb ischemia in haploinsufficient global or endothelial-specific Notch1+/− mice, but not Notch4−/− mice, were impaired compared with wild-type mice. The expression of vascular endothelial growth factor (VEGF) in response to ischemia was comparable between wild-type and Notch mutant mice, suggesting that Notch1 is downstream of VEGF signaling. Treatment of endothelial cells with VEGF increases presenilin proteolytic processing, &ggr;-secretase activity, Notch1 cleavage, and Hes-1 (hairy enhancer of split homolog-1) expression, all of which were blocked by treating endothelial cells with inhibitors of phosphatidylinositol 3-kinase/protein kinase Akt or infecting endothelial cells with a dominant-negative Akt mutant. Indeed, inhibition of &ggr;-secretase activity leads to decreased angiogenesis and inhibits VEGF-induced endothelial cell proliferation, migration, and survival. Overexpression of the active Notch1 intercellular domain rescued the inhibitory effects of &ggr;-secretase inhibitors on VEGF-induced angiogenesis. These findings indicate that the phosphatidylinositol 3-kinase/Akt pathway mediates &ggr;-secretase and Notch1 activation by VEGF and that Notch1 is critical for VEGF-induced postnatal angiogenesis. These results suggest that Notch1 may be a novel therapeutic target for improving angiogenic response and blood flow recovery in ischemic limbs.

Decreased Perivascular Fibrosis but Not Cardiac Hypertrophy in ROCK1 / Haploinsufficient Mice

Background— Rho GTPase and its downstream target, Rho-associated kinase (ROCK), have been implicated in diverse cardiovascular diseases such as cardiac hypertrophy. However, pharmacological inhibitors of ROCK are not entirely specific, nor can they discriminate between the ROCK isoforms ROCK1 and ROCK2. To determine the specific role of ROCK1 in the development of cardiac hypertrophy, we generated ROCK1+/− haploinsufficient mice and determined whether cardiac hypertrophy and remodeling are decreased in these mice. Methods and Results— Litters of ROCK1−/− mice on C57Bl/6 background were markedly underrepresented, suggesting lethality in utero or postnatally. ROCK1+/− mice, however, are viable and fertile with no obvious phenotypic abnormalities. Basal blood pressure, heart rate, and cardiac dimension and function in ROCK1+/− mice were similar to those in wild-type (WT) littermates. Infusion of angiotensin II (400 ng · kg−1 · min−1 for 28 days) or treatment with NG-nitro-l-arginine methyl ester (1 mg/mL in drinking water for 28 days) caused similar increases in systolic blood pressure, left ventricular wall thickness, left ventricular mass, ratio of heart weight to tibial length, and cardiomyocyte size in ROCK1+/− mice and WT littermates. In contrast, perivascular fibrosis in hearts was increased to a lesser extent in ROCK1+/− mice compared with WT littermates. This was associated with decreased expression of transforming growth factor-β, connective tissue growth factor, and type III collagen. In addition, perivascular fibrosis induced by transaortic constriction or myocardial infarction was decreased in ROCK1+/− mice compared with WT littermates. Conclusions— These findings indicate ROCK1 is critical for the development of cardiac fibrosis, but not hypertrophy, in response to various pathological conditions and suggest that signaling pathways leading to the hypertrophic and profibrotic response of the heart are distinct.

ROCK1 mediates leukocyte recruitment and neointima formation following vascular injury.

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.

Rho Kinase Inhibition Improves Endothelial Function in Human Subjects With Coronary Artery Disease

Investigations from basic biology suggest that activation of the Rho/Rho kinase pathway reduces the bioavailability of nitric oxide (NO) and thereby promotes atherosclerosis and its clinical complications. Yet, little information is available about the relationship of the Rho/Rho kinase pathway to NO bioavailability in humans with atherosclerosis. Accordingly, we determined whether inhibition of Rho kinase augments NO bioavailability and improves endothelial function in human subjects with coronary artery disease (CAD). Thirteen CAD subjects and 16 age- and sex-matched healthy controls were randomly assigned to receive the Rho kinase inhibitor, fasudil, or placebo for 1 month each in a double-blind crossover trial. Flow-mediated, endothelium-dependent and nitroglycerin-induced, endothelium-independent vasodilation were assessed by brachial artery ultrasonography. Rho kinase activity was measured in peripheral leukocytes. Fasudil increased endothelium-dependent vasodilation in CAD subjects from 9.4±1.9% to 13.4±1.9% (P=0.001) but not in healthy controls (from 11.3±1.4% to 7.7±1.1%; P=0.07). Endothelium-independent vasodilation was not affected by fasudil in either CAD or healthy subjects. Fasudil reduced Rho kinase activity by 59±18% in CAD subjects (P=0.001) but not in healthy subjects (by 3±6%; P=0.60). The change in endothelium-dependent vasodilation achieved with fasudil relative to placebo was inversely proportional to Rho kinase inhibition (ie, greater Rho kinase inhibition was associated with larger improvement in endothelium-dependent vasodilation) (r=−0.48; P=0.01). These findings suggest that Rho/Rho kinase activation promotes endothelial dysfunction in humans with atherosclerosis. Inhibition of the Rho/Rho kinase pathway should provide a useful strategy to restore NO bioavailability in humans with atherosclerosis.

Rho-Kinase Mediates Hyperglycemia-Induced Plasminogen Activator Inhibitor-1 Expression in Vascular Endothelial Cells

Background—Elevated levels of plasminogen activator inhibitor-1 (PAI-1) are associated with myocardial infarction and stroke, especially in patients with diabetes. The induction of PAI-1 expression by hyperglycemia involves oxidative stress and protein kinase C (PKC). However, the mechanism by which hyperglycemia increases PAI-1 expression is unknown. Methods and Results—Compared with normoglycemia, exposure of human endothelial cells to hyperglycemia, but not mannitol, increased Rho-kinase activity in a time- and concentration-dependent manner. This increase was inhibited by a PKC inhibitor, GF109203X, and antioxidants N-acetylcysteine (NAC) and reduced form of glutathione (GSH). This correlated with inhibition of hyperglycemia-induced PAI-1 expression by GF109203X, NAC, and GSH. Hyperglycemia-increased PAI-1 mRNA and protein levels were inhibited by Rho-kinase inhibitors hydroxyfasudil and Y27632 and by a dominant-negative mutant of Rho-kinase. The mechanism for this inhibition occurs at the level of gene transcription because Rho-kinase inhibitors repress hyperglycemia-stimulated PAI-1 promoter activity without affecting mRNA stability. Hyperglycemia failed to stimulate Rho-kinase activity and PAI-1 expression in heterozygous ROCK I–knockout murine endothelial cells. Conclusions—Hyperglycemia stimulates Rho-kinase activity via PKC- and oxidative stress–dependent pathways, leading to increased PAI-1 gene transcription. These results suggest that inhibition of ROCK I may be a novel therapeutic target for preventing thromboembolic complications of diabetes and cardiovascular disease.

Decreased vascular lesion formation in mice with inducible endothelial-specific expression of protein kinase Akt

To determine whether endothelial Akt could affect vascular lesion formation, mutant mice with a constitutively active Akt transgene, which could be inducibly targeted to the vascular endothelium using the tet-off system (EC-Akt Tg mice), were generated. After withdrawal of doxycycline, EC-Akt Tg mice demonstrated increased endothelial-specific Akt activity and NO production. After blood flow cessation caused by carotid artery ligation, neointimal formation was attenuated in induced EC-Akt Tg mice compared with noninduced EC-Akt Tg mice and control littermates. To determine the role of eNOS in mediating these effects, mice were treated with N-nitro-L-arginine methyl ester (L-NAME). Neointimal formation was attenuated to a lesser extent in induced EC-Akt Tg mice treated with L-NAME, suggesting that some of the vascular protective effects were NO independent. Indeed, endothelial activation of Akt resulted in less EC apoptosis in ligated arteries. Immunostaining demonstrated decreased inflammatory and proliferative changes in induced EC-Akt Tg mice after vascular injury. These findings indicate that endothelial activation of Akt suppresses lesion formation via increased NO production, preservation of functional endothelial layer, and suppression of inflammatory and proliferative changes in the vascular wall. These results suggest that enhancing endothelial Akt activity alone could have therapeutic benefits after vascular injury.

ROCKs as therapeutic targets in cardiovascular diseases

There is growing evidence that Rho-kinases (ROCKs), the immediate downstream targets of the small guanosine triphosphate-binding protein Rho, may contribute to cardiovascular disease. ROCKs play a central role in diverse cellular functions such as smooth muscle contraction, stress fiber formation and cell migration and proliferation. Overactivity of ROCKs is observed in cerebral ischemia, coronary vasospasm, hypertension, vascular inflammation, arteriosclerosis and atherosclerosis. ROCKs, therefore, may be an important and still relatively unexplored therapeutic target in cardiovascular disease. Recent experimental and clinical studies using ROCK inhibitors such as Y-27632 and fasudil have revealed a critical role of ROCKs in embryonic development, inflammation and oncogenesis. This review will focus on the potential role of ROCKs in cellular functions and discuss the prospects of ROCK inhibitors as emerging therapy for cardiovascular diseases.

Deficiency of ROCK1 in bone marrow-derived cells protects against atherosclerosis in LDLR−/− mice

Rho kinases (ROCKs) are serine‐threonine protein kinases that regulate the actin cytoskeleton. Recent studies suggest that ROCKs also play an important role in cardiovascular disease. However, the isoform‐and tissue‐specific role of ROCKs in mediating this process is unknown. Using homologous recombination, we generated mutant mice harboring alleles with homozygous deletion of ROCK1 (ROCK1−/−). Most ROCK1−/− mice die perinatally. However, a few ROCK1−/− mice survive to adulthood, are phenotypically normal, and have no apparent compensatory changes in ROCK2. Using these ROCK1−/− mice, we show that ROCK1 in bone marrow‐derived macrophages is critical to the development of atherosclerosis, in part, by mediating foam cell formation and macrophage chemotaxis. Lipid accumulation and atherosclerotic lesions were reduced in atherosclerosis‐prone LDLR−/− mice, whose bone marrows have been replaced with bone marrows derived from ROCK1−/− mice. Bone marrow‐derived ROCK1‐deficient macrophages exhibited impaired chemotaxis to monocyte chemotactic protein‐1 and showed reduced ability to take up lipids and to develop into foam cells when exposed to modified low‐density lipoprotein. These findings indicate that ROCK1 in bone marrow‐derived cells is a critical mediator of atherogenesis and suggest that macrophage ROCK1 may be an important therapeutic target for vascular inflammation and atherosclerosis.—Wang, H.‐W., Liu, P.‐Y., Oyama, N., Rikitake, Y., Kitamoto, S., Gitlin, J., Liao, J. K., Boisvert, W. A. Deficiency of ROCK1 in bone marrow‐derived cells protects against atherosclerosis in LDLR–/– mice. FASEB J. 22, 3561–3570 (2008)

Inhibition of Rho-kinase attenuates endothelial-leukocyte interaction during ischemia-reperfusion injury.

Resuscitation from hemorrhagic shock induces endothelial dysfunction and activates inflammatory cascades leading to organ damage. Following restoration of blood flow to ischemic vascular beds, leukocyte–endothelium interactions leading to leukocyte infiltration into the vascular wall occur very early due, in part, to the loss of endothelium-derived nitric oxide (NO). The mechanism by which ischemia–reperfusion injury impairs endothelium-derived NO is not completely understood. We hypothesized that inhibition of Rho-kinase could exert beneficial effects following hemorrhagic shock by preserving endothelial function and attenuating leukocyte trafficking in the microcirculation. Using intravital microscopy, we found that resuscitation from hemorrhage acutely increased the number of rolling and adherent leukocytes in the mouse splanchnic microcirculation. Treatment of mice with the Rho-kinase inhibitor fasudil, markedly attenuated leukocyte–endothelium interaction in response to hemorrhage/reinfusion. The beneficial effect of fasudil was not observed in endothelial nitric oxide synthase (eNOS)−/− mice. In conclusion, inhibition of Rho-kinase prevents inflammatory leukocyte trafficking in the microcirculation via an eNOS-dependent mechanism. Our data support a role for Rho-kinase inhibitors in the treatment of ischemia–reperfusion injury.