Toshiyuki Sasaguri

Laminar Shear Stress Inhibits Vascular Endothelial Cell Proliferation by Inducing Cyclin-Dependent Kinase Inhibitor p21Sdi1/Cip1/Waf1

Alterations in the functions of vascular endothelial cells (ECs) induced by fluid shear stress may play a pivotal role in both the development and prevention of vascular diseases. We found that DNA synthesis of bovine aortic and human umbilical vein ECs, determined by [(3)H]thymidine incorporation, was inhibited by steady laminar shear stress (5 and 30 dyne/cm(2)). This growth inhibition due to shear stress was associated with suppression of cell transition from the G(1) to S phase of the cell cycle. Therefore, we studied G(1)-phase events to find the molecules responsible for this cell cycle arrest. Shear stress inhibited the phosphorylation of a retinoblastoma protein (pRb) and the activity of cyclin-dependent kinase (cdk) 2 and cdk4, which phosphorylate pRb. The level of cdk inhibitor p21(Sdi1/Cip1/Waf1) protein, but not that of p27(Kip1), increased as a result of shear stress, and the amount of p21 protein associated with cdk2 also increased, although the protein level of cdk2 was unchanged. Shear stress markedly elevated the mRNA level of p21, and this elevation in mRNA faded after the release of cells from shear stress, concomitant with a recovery of DNA synthesis. These results suggest that steady laminar shear stress induces cell cycle arrest by upregulating p21. Derangement of the steady laminar flow may release cells from this inhibition and induce cell proliferation, which, in turn, may cause atherosclerosis through the induction of EC stability disruption.

Knock-In Mouse Model of Dilated Cardiomyopathy Caused by Troponin Mutation

We created knock-in mice in which a deletion of 3 base pairs coding for K210 in cardiac troponin (cTn)T found in familial dilated cardiomyopathy patients was introduced into endogenous genes. Membrane-permeabilized cardiac muscle fibers from mutant mice showed significantly lower Ca2+ sensitivity in force generation than those from wild-type mice. Peak amplitude of Ca2+ transient in cardiomyocytes was increased in mutant mice, and maximum isometric force produced by intact cardiac muscle fibers of mutant mice was not significantly different from that of wild-type mice, suggesting that Ca2+ transient was augmented to compensate for decreased myofilament Ca2+ sensitivity. Nevertheless, mutant mice developed marked cardiac enlargement, heart failure, and frequent sudden death recapitulating the phenotypes of dilated cardiomyopathy patients, indicating that global functional defect of the heart attributable to decreased myofilament Ca2+ sensitivity could not be fully compensated by only increasing the intracellular Ca2+ transient. We found that a positive inotropic agent, pimobendan, which directly increases myofilament Ca2+ sensitivity, had profound effects of preventing cardiac enlargement, heart failure, and sudden death. These results verify the hypothesis that Ca2+ desensitization of cardiac myofilament is the absolute cause of the pathogenesis of dilated cardiomyopathy associated with this mutation and strongly suggest that Ca2+ sensitizers are beneficial for the treatment of dilated cardiomyopathy patients affected by sarcomeric regulatory protein mutations.

Transcriptional and Posttranscriptional Regulation of Cyclooxygenase-2 Expression by Fluid Shear Stress in Vascular Endothelial Cells

Objective—Fluid shear stress induces cyclooxygenase (COX)-2 gene expression in vascular endothelial cells. We investigated the underlying mechanism of this induction. Methods and Results—Exposure of human umbilical vein endothelial cells to laminar shear stress in the physiological range (1 to 30 dyne/cm2) upregulated the expression of COX-2 but not COX-1, a constitutive isozyme of COX. The expression of COX-2 mRNA began to increase within 0.5 hour after the loading of shear stress and reached a maximal level at 4 hours. Roles of the promoter region and the 3′-untranslated region in the human COX-2 gene were evaluated by the transient transfection of luciferase reporter vectors into bovine arterial endothelial cells. Shear stress elevated luciferase activity via the region between −327 and 59 bp. Mutation analysis indicated that cAMP-responsive element (−59/−53 bp) was mainly involved in this response. On the other hand, shear stress selectively stabilized COX-2 mRNA. Moreover, shear stress elevated luciferase activity when a 3′-untranslated region of COX-2 gene containing 17 copies of the AUUUA mRNA instability motif was inserted into the vector. Conclusions—Transcriptional activation and posttranscriptional mRNA stabilization contribute to the rapid and sustained expression of COX-2 in response to shear stress.

Fluid Shear Stress Induces Lipocalin-Type Prostaglandin D2 Synthase Expression in Vascular Endothelial Cells

Abstract —Ligands for peroxisome proliferator–activated receptor γ, such as the thiazolidinedione class of antidiabetic drugs and 15-deoxy-Δ 12,14 -prostaglandin J 2 (15d-PGJ 2 ), modulate various processes in atherogenesis. In search of cells that generate prostaglandin D 2 (PGD 2 ), the metabolic precursor of 15d-PGJ 2 , we identified PGD 2 from culture medium of endothelial cells. To study how PGD 2 production is regulated in endothelial cells, we investigated the role of fluid shear stress in the metabolism of PGD 2 . Endothelial cells expressed the mRNA for the lipocalin-type PGD 2 synthase (L-PGDS) both in vitro and in vivo. Loading laminar shear stress using a parallel-plate flow chamber markedly enhanced the gene expression of L-PGDS, with the maximal effect being obtained at 15 to 30 dyne/cm 2 . The expression began to increase within 6 hours after loading shear stress and reached the maximal level at 18 to 24 hours. In contrast, shear stress did not alter the expression levels of PGI 2 synthase and thromboxane A 2 synthase. In parallel with the increase in the expression level of L-PGDS, endothelial cells released PGD 2 and 15d-PGJ 2 into culture medium. These results demonstrate that shear stress promotes PGD 2 production by stimulating L-PGDS expression and suggest the possibility that a peroxisome proliferator–activated receptor γ ligand is produced in vascular wall in response to blood flow.

Nitroglycerine- and isoprenaline-induced vasodilatation: assessment from the actions of cyclic nucleotides.

1 To investigate the vasodilator actions of nitroglycerine and isoprenaline, the effects of these agents, dibutyryl cyclic AMP (db cyclic AMP) and 8‐bromo cyclic GMP (8‐Br cyclic GMP) on intact muscle tissue, and of cyclic AMP and cyclic GMP on skinned muscle of the rabbit mesenteric artery were investigated. 2 In porcine coronary artery, nitroglycerine (>0.1 μm) increased the production of cyclic GMP with no change in the amount of cyclic AMP, while isoprenaline (>0.1 μm) significantly increased the production of cyclic AMP with no change in the amount of cyclic GMP. 3 In the rabbit mesenteric artery, nitroglycerine or isoprenaline inhibited the tonic component of the 39 mm [K]o‐induced contraction to a greater extent than the phasic component. Nitroglycerine and 8‐Br cyclic GMP showed a stronger inhibitory action on the K‐induced contraction than did isoprenaline and db cyclic AMP. 4 The sources of Ca utilized for the generation of contraction by noradrenaline and caffeine were estimated to be the same as those determined from the amplitudes of contractions evoked in Ca‐free solution by various concentrations of noradrenaline or caffeine. 5 In intact muscle tissues, the effects of nitroglycerine or 8‐Br cyclic GMP on the amount of Ca stored in cells were estimated from the caffeine‐induced contraction in Ca‐free solution. Both agents inhibited the contractions due to a reduction in the amount of Ca in the cells. When the effects of isoprenaline or db cyclic AMP were observed, both agents inhibited the caffeine‐induced contraction but the accumulation of Ca into cells was greater than the control. 6 In saponin skinned muscles, the pCa‐tension relationship in the presence of cyclic AMP and cyclic AMP‐dependent protein kinase (cyclic AMP‐PK) shifted to the right and to a lower level in comparison with the control. Applications of cyclic GMP with cyclic GMP‐dependent protein kinase (cyclic GMP PK) also inhibited the contraction induced by low concentrations of Ca. 7 In skinned muscles, cyclic AMP exhibited dual actions on Ca store sites, i.e. in the presence of high concentrations of Ca or prolonged superfusion of Ca, cyclic AMP reduced the amount of Ca due to activation of the Ca‐induced Ca release mechanism by excess accumulation of Ca. On the other hand, cyclic GMP consistently inhibited the amplitude of the caffeine‐induced contraction due to a reduction in the amount of Ca in the store sites. 8 These results indicate that nitroglycerine and isoprenaline increase the amount of cyclic GMP and cyclic AMP, respectively. The main effect of cyclic GMP is activation of Ca extrusion, thus reducing the amount of Ca stored in the cell, while the main effect of cyclic AMP is to increase the amount of Ca stored in the cell. Both cyclic AMP with cyclic AMP‐PK and cyclic GMP with cyclic GMP‐PK inhibit the phosphorylation of myosin. Consequently both cyclic nucleotides reduce the free Ca in the myoplasm and promote relaxation, but by different mechanisms.

Ca2+/Calmodulin-Dependent Kinase IIδ Causes Heart Failure by Accumulation of p53 in Dilated Cardiomyopathy

Background— Dilated cardiomyopathy (DCM), characterized by dilatation and dysfunction of the left ventricle, is an important cause of heart failure. Many mutations in various genes, including cytoskeletal protein genes and contractile protein genes, have been identified in DCM patients, but the mechanisms of how such mutations lead to DCM remain unknown. Methods and Results— We established the mouse model of DCM by expressing a mutated cardiac &agr;-actin gene, which has been reported in patients with DCM, in the heart (mActin-Tg). mActin-Tg mice showed gradual dilatation and dysfunction of the left ventricle, resulting in death by heart failure. The number of apoptotic cardiomyocytes and protein levels of p53 were increased in the hearts of mActin-Tg mice. Overexpression of Bcl-2 or downregulation of p53 decreased the number of apoptotic cardiomyocytes and improved cardiac function. This mouse model showed a decrease in myofilament calcium sensitivity and activation of calcium/calmodulin-dependent kinase II&dgr; (CaMKII&dgr;). The inhibition of CaMKII&dgr; prevented the increase in p53 and apoptotic cardiomyocytes and ameliorated cardiac function. Conclusion— CaMKII&dgr; plays a critical role in the development of heart failure in part by accumulation of p53 and induction of cardiomyocyte apoptosis in the DCM mouse model.

Large scale isolation of non-uniform shear stress-responsive genes from cultured human endothelial cells through the preparation of a subtracted cDNA library.

To investigate the molecular mechanisms responsible for the regional selectivity of early atherogenesis, we have applied a non-uniform shear stress to cultured human umbilical vein endothelial cells (HUVEC). We used a microcarrier culture system and a combination of subtraction and reverse-subtraction methods to isolate a number of genes upregulated by shear stress. The resultant subtracted library includes several known genes (e.g. MCP-1, TM) whose responsiveness to shear stress has been previously reported, indicating that the library is enriched for genes upregulated by shear stress. Also included are atherosclerosis-related genes (e.g. CTGF, IL-8) whose responsiveness to shear stress had not been demonstrated, other known genes whose relationship to atherosclerosis had not been reported, and novel genes. Some responsive to centrifugal force and shear stress (RECS) genes are also upregulated following stimulation by steady laminar shear stress in a parallel plate chamber. Interestingly, the library includes ET-1 and PAI, which are well known atherogenic factors that are downregulated by laminar shear stress. This implies that turbulent shear stress has effects on HUVEC that are different from those elicited by laminar shear stress. Importantly, analysis of specimens taken from human aorta showed that several RECS genes are transcriptionally upregulated in atherosclerotic lesions, suggesting that the subtracted library includes novel therapeutic targets for the treatment of atherosclerosis.

Shear Stress Induces Expression of Vascular Endothelial Growth Factor Receptor Flk-1/KDR Through the CT-Rich Sp1 Binding Site

Fluid shear stress is 1 of the major factors that control gene expression in vascular endothelial cells. We investigated the role of shear stress in the regulation of the expression of fetal liver kinase-1/kinase domain region (Flk-1/KDR), a vascular endothelial growth factor receptor, by using human umbilical vein endothelial cells. Laminar shear stress (15 dyne/cm2) elevated Flk-1/KDR mRNA levels by ≈3-fold for 8 hours, and the expression was upregulated within the range of 5 to 40 dyne/cm2. Deletion analysis of the 5′-flanking region of the Flk-1/KDR gene promoter by use of a luciferase reporter vector revealed that a shear stress–responsive element resided in the sequence between −94 and −31 bp, which contained putative nuclear factor-&kgr;B, activator protein-2, and GC-rich Sp1 and CT-rich Sp1 binding sites. Electrophoretic mobility shift assay demonstrated that nuclear extract was bound to the GC-rich Sp1 sites and the CT-rich Sp1 site with a similar pattern. However, shear stress enhanced the DNA-protein interactions only on the CT-rich Sp1 site but not on the GC-rich Sp1 sites. A 3-bp mutation in the CT-rich Sp1 site eliminated the response to shear stress in electrophoretic mobility shift assay and luciferase reporter assay. These results suggest that shear stress induces Flk-1/KDR expression through the CT-rich Sp1 binding site.

Association between arterial stiffness and cerebral white matter lesions in community-dwelling elderly subjects.

The presence of cerebral white matter lesions (WMLs) on MRI is suggested to be a predictive factor for vascular dementia and stroke. To investigate the relationship between arterial stiffness and WMLs, we performed brain MRI to evaluate the presence of two subtypes of WML—periventricular hyperintensities (PVH) and deep white matter lesions (DWML)—and furthermore, determined the brachial-ankle pulse wave velocity (ba-PWV) as a marker of arterial stiffness in 132 elderly asymptomatic subjects (49 men and 83 women, 70.3±9.0 years). PVH and DWML were observed in 41 (31.0%) and 53 (40.2%) subjects, respectively. The ba-PWV values were significantly greater in subjects with PVH than in those without. DWML also tended to be associated with ba-PWV, but the correlation was not statistically significant. In multiple logistic regression analysis, age and decreased DBP were independently associated with PVH. ba-PWV was also detected as an independent factor for the appearance of PVH (adjusted odds ratio: 2.84, p=0.015) but not DWML. These results indicate that the increase in arterial stiffness contributes to the pathogenesis of PVH rather than DWML. Although further study is needed to clarify the difference between WML subtypes, our study suggests that the measurement of ba-PWV is a simple and useful tool for detecting cerebral arterial dysfunction. (Hypertens Res 2008; 31: 75−81)

Protein tyrosine kinase inhibitors inhibit chemotaxis of vascular smooth muscle cells.

The effects of protein tyrosine kinase inhibitors on platelet-derived growth factor (PDGF)-induced chemotaxis in cultured rat aortic smooth muscle cells (SMCs) were investigated to elucidate the role of tyrosine phosphorylation in the chemotaxis of vascular SMCs. Two tyrosine kinase inhibitors, methyl 2,5-dihydroxycinnamate and genistein, inhibited PDGF-induced chemotaxis, the IC50 being 5 and 150 mumol/L, respectively. Methyl cinnamate and genistein partly inhibited the adhesion of SMC to collagen-coated dishes. A chemotaxis assay using double-well culture dishes revealed that both agents also inhibited cell migration after adhesion. H-7, a C kinase inhibitor, did not inhibit either chemotaxis or SMC adhesion at 100 mumol/L. Western blot analysis using anti-phosphotyrosine revealed that the tyrosine kinase inhibitors inhibited the tyrosine phosphorylation of at least two proteins of molecular weight 85 and 95 kD under our experimental conditions. An immunocytochemical study revealed that these inhibitors eliminated tyrosine phosphorylation along the cell margins; these agents also inhibited the reorganization of microtubules and stress fibers, both of which are involved in directional cell locomotion. These findings suggest that tyrosine kinases may play an important role in SMC chemotaxis.