Ping-Yen Liu

Evidence for Statin Pleiotropy in Humans: Differential Effects of Statins and Ezetimibe on Rho-Associated Coiled-Coil Containing Protein Kinase Activity, Endothelial Function, and Inflammation

Background— By inhibiting 3-hydroxy-3-methylglutaryl coenzyme A reductase, statins not only reduce cholesterol biosynthesis but also decrease the formation of isoprenoids, which are important for mediating signaling through the Rho-associated coiled-coil containing protein kinase (ROCK) pathway. Increased ROCK activity has been implicated in endothelial dysfunction and vascular inflammation. We hypothesize that ezetimibe, which inhibits intestinal cholesterol absorption, may not exert similar cholesterol-independent or pleiotropic effects of statins and, when used with a lower dose of statin, have less effect on ROCK activity than a higher dose of statin. Methods and Results— In a prospective, randomized, observer-blinded study, we treated 60 dyslipidemic subjects without cardiovascular disease with simvastatin 40 mg/d, simvastatin/ezetimibe 10/10 mg/d, or placebo tablets for 28 days (n=20 in each arm). We evaluated baseline demographics and lipid levels, ROCK activity, C-reactive protein, and flow-mediated dilation before and after treatment. Compared with the placebo group, both treatment regimens decreased low-density lipoprotein cholesterol by 38% and C-reactive protein by 38% to 40% after 28 days (P<0.01 for both compared with placebo). Although the low-density lipoprotein cholesterol and C-reactive protein reductions were comparable with either lipid-lowering regimen, only simvastatin 40 mg reduced ROCK activity and improved flow-mediated dilation (P<0.01 for both compared with baseline). Reduction in ROCK activity with simvastatin 40 mg remained significant even after controlling for changes in low-density lipoprotein cholesterol (P=0.01) and correlated with improvement in flow-mediated dilation (R2=−0.78, P<0.01). No correlation was found between changes in flow-mediated dilation and changes in low-density lipoprotein cholesterol or C-reactive protein. Conclusion— These results indicate that high-dose statin monotherapy exerts greater effects on ROCK activity and endothelial function, but not on C-reactive protein, than low-dose statin plus ezetimibe. These findings provide additional evidence of statin benefits beyond cholesterol lowering.

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.

Statins inhibit Rho kinase activity in patients with atherosclerosis.

BACKGROUND In addition to inhibiting cholesterol synthesis, statins (HMG-CoA reductase inhibitors) decrease the formation of isoprenoid intermediates required for the activation of key signaling pathways, including Rho/Rho kinase (ROCK). In experimental settings, statins inhibit ROCK and reverse vascular dysfunctions in atherosclerosis, independent of cholesterol reduction. It is not known whether statins inhibit ROCK activity in humans with atherosclerosis. METHODS We investigated 35 patients with stable atherosclerosis in a randomized, double-blind study comparing treatment with high-dose (80mg/d) or low-dose (10mg/d) atorvastatin to placebo for 28 days. Blood samples for leukocyte ROCK activity, fasting lipids, and high-sensitivity C-reactive protein (hs-CRP) were obtained on days 0, 7, 14, and 28 after randomization and change over time with the two statin treatments relative to placebo was examined. RESULTS Atorvastatin 80mg/d reduced ROCK activity (p=0.002 vs. placebo). This decline was rapid and significant within 2 weeks of treatment. The inhibition of ROCK by atorvastatin (80mg/d) remained significant even after controlling for changes in low-density lipoprotein cholesterol (LDL-C) and triglycerides (p=0.01). Furthermore, there was no correlation between changes in ROCK activity and changes in LDL-C (r=0.2, p=0.25) or triglycerides (r=0.1, p=0.55). There was a modest correlation between ROCK inhibition and change in hs-CRP among patients randomized to atorvastatin 80mg/d (r=0.6, p=0.07). CONCLUSIONS These first-in-man findings demonstrate that high-dose atorvastatin rapidly inhibits the pro-atherogenic Rho/ROCK pathway, independent of cholesterol reduction. This inhibition may contribute to the clinical benefits of statins. Rho/ROCK may provide a useful therapeutic target in patients with atherosclerosis.

Increased Vascular Senescence and Impaired Endothelial Progenitor Cell Function Mediated by Mutation of Circadian Gene Per2

Background— Alteration of the circadian rhythm and increased vascular senescence are linked to cardiovascular disease. Per2, a circadian gene, is known to regulate endothelium-dependent vasomotion. However, the mechanism by which Per2 affects endothelial function is unknown. We hypothesize that endothelial dysfunction in Per2 mutant (Per2m/m) mice is mediated in part by increased vascular senescence and impaired endothelial progenitor cell (EPC) function. Methods and Results— Endothelial cells from Per2m/m mice exhibit increased protein kinase Akt signaling, greater senescence, and impaired vascular network formation and proliferation. Indeed, Per2m/m mice have impaired blood flow recovery and developed autoamputation of the distal limb when subjected to hind-limb ischemia. Furthermore, matrigel implantation into Per2m/m mice resulted in less neovascularization. Because EPCs contribute to angiogenesis, we studied the role of Per2 in these cells using bone marrow transplantation. Basal EPC levels were similar between wild-type and Per2m/m mice. However, compared with wild-type bone marrow transplantation mice, EPC mobilization was impaired in Per2m/m bone marrow transplantation mice in response to ischemia or VEGF stimulation. Bone marrow transplantation or infusion of wild-type EPC restored blood flow recovery and prevented autoamputation in Per2m/m mice. Conclusion— These findings indicate that mutation of Per2 causes Akt-dependent senescence and impairs ischemia-induced revascularization through the alteration of EPC function.

Smooth muscle notch1 mediates neointimal formation after vascular injury

Background— Notch1 regulates binary cell fate determination and is critical for angiogenesis and cardiovascular development. However, the pathophysiological role of Notch1 in the postnatal period is not known. We hypothesize that Notch1 signaling in vascular smooth muscle cells (SMCs) may contribute to neointimal formation after vascular injury. Methods and Results— We performed carotid artery ligation in wild-type, control (SMC-specific Cre recombinase transgenic [smCre-Tg]), general Notch1 heterozygous deficient (N1+/−), SMC-specific Notch1 heterozygous deficient (smN1+/−), and general Notch3 homozygous deficient (N3−/−) mice. Compared with wild-type or control mice, N1+/− and smN1+/− mice showed a 70% decrease in neointimal formation after carotid artery ligation. However, neointimal formation was similar between wild-type and N3−/− mice. Indeed, SMCs derived from explanted aortas of either N1+/−- or smN1+/− mice showed decreased chemotaxis and proliferation and increased apoptosis compared with control or N3−/− mice. This correlated with decreased staining of proliferating cell nuclear antigen–positive cells and increased staining of cleaved caspase-3 in the intima of N1+/−- or smN1+/− mice. In SMCs derived from CHF1/Hey2−/− mice, activation of Notch signaling did not lead to increased SMC proliferation or migration. Conclusions— These findings indicate that Notch1, rather than Notch3, mediates SMC proliferation and neointimal formation after vascular injury through CHF1/Hey2 and suggest that therapies that target Notch1/CHF1/Hey2 in SMCs may be beneficial in preventing vascular proliferative diseases.

Increased Rho Kinase Activity in a Taiwanese Population With Metabolic Syndrome

OBJECTIVES We sought to determine whether Rho kinase (ROCK) activity is increased in a Taiwanese population with metabolic syndrome (MetS). BACKGROUND Recent studies suggest that ROCK may be involved in the pathogenesis of MetS, but clinical studies linking ROCK with MetS are lacking. METHODS We studied 40 Taiwanese subjects (60% men, mean age 55.5 +/- 5.6 years) who were diagnosed with MetS with National Cholesterol Educational Program Adult Treatment Panel III criteria and 40 age- and gender-matched control subjects. Subject demographics were recorded, and blood samples were obtained. RESULTS Compared with control subjects, ROCK activity, as determined by phosphorylation of myosin binding subunit (MBS) in leukocytes, was greater in MetS subjects (mean phospho-MBS/MBS ratio 0.46 vs. 0.35, p = 0.002). A cutoff value for ROCK activity of 0.39 predicted the presence of MetS with specificity and sensitivity rates of 70%. Plasma high-sensitivity C-reactive protein was greater (5.5 mg/l, 95% confidence interval [CI] 3.1 to 7.2 mg/l vs. 2.8 mg/l, 95% CI 1.1 to 3.9 mg/l, p = 0.01) and adiponectin was lower (4.9 microg/ml, 95% CI 3.2 to 6.1 microg/ml vs. 5.9 microg/ml, 95% CI 4.2 to 7.5 microg/ml, p = 0.01) in MetS subjects compared with control subjects, but plasma levels of interleukin-6 and tumor necrosis factor-alpha were not different (p > 0.05 for both). Body mass index, waist circumference, fasting glucose, high-sensitivity C-reactive protein, and triglyceride levels were associated with increased levels of ROCK activity. The risk of increased ROCK activity increased with the number of MetS components (p for trend <0.001). CONCLUSIONS Rho kinase activity is increased in Taiwanese subjects with MetS and is associated with each component of MetS and markers of inflammation. These findings suggest that ROCK activity may be a novel serological marker of MetS.

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)

A Method for Measuring Rho Kinase Activity in Tissues and Cells

The Rho-associated kinases (ROCKs) can regulate cell shape and function by modulating the actin cytoskeleton. ROCKs are serine-threonine protein kinases that can phosphorylate adducin, ezrin-radixin-moesin proteins, LIM kinase, and myosin light chain phosphatase. In the cardiovascular system, the RhoA/ROCK pathway has been implicated in angiogenesis, atherosclerosis, cerebral and coronary vasospasm, cerebral ischemia, hypertension, myocardial hypertrophy, and neointima formation after vascular injury. ROCKs consist of two isoforms: ROCK1 and ROCK2. They share overall 65% homology in their amino acid sequence and 92% homology in their amino kinase domains. However, these two isoforms have different subcellular localizations and exert biologically different functions. In particular, ROCK1 appears to be more important for immunological functions, whereas ROCK2 is more important for endothelial and vascular smooth muscle function. Thus, the ability to measure ROCK activity in tissues and cells would be important for understanding mechanisms underlying cardiovascular disease. This chapter describes a method for measuring ROCK activity in peripheral blood, tissues, and cells.

Comparison of effects of rosuvastatin (10 mg) versus atorvastatin (40 mg) on rho kinase activity in caucasian men with a previous atherosclerotic event.

In addition to inhibiting cholesterol biosynthesis, statins also inhibit the formation of isoprenoid intermediates, which are required for the activation of the Rho/Rho kinase (ROCK) pathway. Increased ROCK activity has been implicated in causing endothelial dysfunction and atherosclerosis. However, it is not known whether statins, at doses used to lower cholesterol levels, inhibit ROCK activity in humans with atherosclerosis. Furthermore, it is not known whether lipophilic and hydrophilic statins differ in their ability to inhibit ROCK activity. Accordingly, we enrolled 30 men with stable atherosclerosis (low-density lipoprotein [LDL] > or =100 mg/dL) in a randomized, double-blind study comparing equivalent LDL-lowering doses of a hydrophilic statin (rosuvastatin 10 mg once a day) with a lipophilic statin (atorvastatin 40 mg once a day) for 28 days. We assessed the change in lipids, ROCK activity, and flow-mediated dilation (FMD) of the brachial artery before and after statin therapy. Both treatment groups exhibited comparable 30% to 32% and 42% to 45% reductions in total and LDL cholesterol, respectively. Only atorvastatin reduced triglycerides, and neither statin altered high-density lipoprotein cholesterol. Whereas both statins inhibited ROCK activity (p <0.0001), the extent of inhibition was greater with rosuvastatin (18 +/- 2% vs 8 +/- 2%, p = 0.0006). Statins also improved FMD from 7.4 +/- 0.6 to 9.3 +/- 0.4 (p = 0.003) with rosuvastatin being slightly better than atorvastatin. The inhibition of ROCK activity by statins did not correlate with reductions in LDL (p = 0.57) but was associated with improvement in FMD. In conclusion, these findings provide direct clinical evidence that statins, at clinically relevant doses, could differentially inhibit ROCK activity and improve endothelial function by cholesterol-independent mechanism.

Platelet-activating factor-acetylhydrolase A379V (exon 11) gene polymorphism is an independent and functional risk factor for premature myocardial infarction

Summary.  Background: Oxidation of low density lipoproteins is an initial step of atherogenesis that generates pro‐inflammatory phospholipids, including platelet‐activating factor (PAF). PAF is degraded by PAF‐acetylhydrolase (PAF‐AH), which has been postulated to be a risk factor for myocardial infarction (MI). The role of PAF‐AH for the onset of premature MI is unclear. Methods: Polymorphisms located in putatively functional regions were investigated in a cohort of patients having premature MI onset prior to 46 years of age (n = 200) and a sex‐age‐matched control group (n = 200). The activity of PAF‐AH and coronary angiograms were evaluated for the severity of coronary atherosclerosis. Results: The V allele of A379V (exon 11) polymorphism on PAF‐AH gene was more frequent in patients with premature MI (P = 0.001). This V allele polymorphism was also associated with a lower activity of plasma PAF‐AH and a more complex coronary atherosclerosis (p Trends <0.05). Multiple logistic regression analysis showed that this polymorphism was an independent risk factor (Odds Ratio [OR] 1.66, 95% CI 1.14.1 to 5.80, P = 0.008) as well as smoking (OR 3.72, 95% CI 1.77 to 9.28, P = 0.001), diabetes mellitus (OR 2.25, 95% CI 1.40 to 5.32, P = 0.007) and hypertension (OR 1.88, 95% CI 1.25 to 5.36, P = 0.003) for the onset of premature MI. Conclusion: We conclude that a functional and significant association between the A379V polymorphism on exon 11 of PAF‐AH gene and premature MI exists in this Taiwanese population. This polymorphism is significantly associated with the PAF‐AH activity and the severity of coronary atherosclerosis.