Toshio Ogihara

Apoptosis Signal-Regulating Kinase 1 Mediates Cellular Senescence Induced by High Glucose in Endothelial Cells

Vascular ageing is accelerated in patients with diabetes. However, the underlying mechanism remains unclear. Here, we show that high glucose induces activation of apoptosis signal-regulating kinase 1 (ASK1), an apoptosis-inducing signal that mediates endothelial cell senescence induced by hyperglycemia. High glucose induced a time-dependent increase in the levels of ASK1 expression and its activity in human umbilical vein endothelial cells (HUVECs). Incubation of endothelial cells with high glucose increased the proportion of cells expressing senescence-associated β-galactosidase (SA-β-gal) activity. However, transfection with an adenoviral construct including a dominant negative form of ASK1 gene significantly inhibited SA-β-gal activity induced by high glucose. In addition, infection with an adenoviral construct expressing the constitutively active ASK1 gene directly induced an increase in the levels of SA-β-gal activity. Activation of the ASK1 signal also enhanced plasminogen activator inhibitor-1 (PAI-1) expression in HUVECs. Induction of senescent endothelial cells in aortas and elevation of plasma PAI-1 levels were observed in streptozotocin (STZ) diabetic mice, whereas these changes induced by STZ were attenuated in ASK1-knockout mice. Our results suggest that hyperglycemia accelerates endothelial cell senescence and upregulation of PAI-1 expression through activation of the ASK1 signal. Thus, ASK1 may be a new therapeutic target to prevent vascular ageing and thrombosis in diabetic patients.

Anti-oxidative effect of Klotho on endothelial cells through cAMP activation.

Klotho, a regulatory factor implicated in countering the aging process, has been reported to ameliorate endothelial dysfunction inxa0vivo. To clarify whether Klotho protein directly affects endothelial cell function, we studied the effects of membrane-form Klotho on manganese superoxide dismutase (Mn-SOD) expression and nitric oxide production in human umbilical vein endothelial cells (HUVEC). We incubated HUVEC with conditioned medium from COS-1 cells transfected with expression vector, pCAGGS-klotho (Klotho-CM) or a recombinant, purified 6His-tagged Klotho protein. Both Klotho-CM and 6His-tagged Klotho protein enhanced Mn-SOD expression by approximately two-fold, partially via activation of the cAMP signaling pathway. Furthermore, Klotho-CM increased nitric oxide production, which also contributed to the up-regulation of Mn-SOD. Using the oxidation-sensitive dye dihydroethidium, we found that Klotho inhibited angiotensin II-induced reactive oxygen species production in HUVEC. These findings provide new insights into the mechanisms of Klotho action and support the therapeutic potential of membrane-form Klotho to regulate endothelial function.

Apop-1, a novel protein inducing cyclophilin D-dependent but Bax/Bak-related channel-independent apoptosis

In the intrinsic pathway of apoptosis, mitochondria play a crucial role by releasing cytochrome c from the intermembrane space into the cytoplasm. Cytochrome c release through Bax/Bak-dependent channels in mitochondria has been well documented. In contrast, cyclophilin D (CypD), an important component of permeability transition pore-dependent protein release, remains largely undefined, and no apoptogenic proteins that act specifically in a CypD-dependent manner have been reported to date. Here, we describe a novel and evolutionarily conserved protein, apoptogenic protein (Apop). Mouse Apop-1 expression induces apoptotic death by releasing cytochrome c from mitochondria into the cytosolic space followed by activation of caspase-9 and -3. Apop-1-induced apoptosis is not blocked by Bcl-2 or Bcl-xL, inhibitors of Bax/Bak-dependent channels, whereas it is completely blocked by cyclosporin A, an inhibitor of permeability transition pore. Cells lacking CypD were resistant to Apop-induced apoptosis. Moreover, inhibition of Apop expression prevented the cell death induced by apoptosis-inducing substances. Our findings, thus, indicate that the expression of Apop-1 induces apoptosis though CypD-dependent pathway and that Apop-1 plays roles in cell death under physiological conditions.

Potential Role of CYLD (Cylindromatosis) as a Deubiquitinating Enzyme in Vascular Cells

Data from several studies suggest that the ubiquitin-proteasome system may play a role in the progression of atherosclerosis. Here, we examined the potential role of the deubiquitinating enzyme CYLD (cylindromatosis), mutation of which has been reported to cause familial cylindromatosis. Northern blot analysis revealed expression of CYLD mRNA in the aorta, as well as in cultured human aortic endothelial cells (ECs) and vascular smooth muscle cells. Treatment with recombinant tumor necrosis factor (TNF)-alpha significantly increased CYLD expression in ECs and vascular smooth muscle cells. Immunostaining showed CYLD expression in atherosclerotic lesions from human carotid arteries and up-regulation of CYLD expression in the neointima of rat carotid arteries after balloon injury. Overexpression of CYLD in ECs resulted in inhibition of TNF-alpha-induced nuclear factor-kappaB activity through deubiquitination of TNFR-associated factor 2 (TRAF2), whereas overexpression of catalytically inactive CYLD had no effect. CYLD overexpression also inhibited expression of cyclin D1 and activation of the E2F pathway through deubiquitination of the upstream molecule Bcl-3 and inhibition of its translocation into the nucleus. Overexpressed CYLD also significantly inhibited cell viability. Furthermore, overexpression of CYLD in rat balloon-injured carotid artery attenuated neointimal formation through inactivation of nuclear factor-kappaB and E2F. In conclusion, these data demonstrate that the deubiquitinating enzyme CYLD may inhibit inflammation and proliferation in vascular cells and may represent a novel target for the treatment or prevention of atherosclerosis.

Aspirin prevents adhesion of T lymphoblasts to vascular smooth muscle cells

In the development of atherosclerosis, inflammatory cells adhere to and migrate into the vascular walls by interacting with vascular smooth muscle cells. To investigate the mechanism of aspirins anti‐atherogenic activity, we examined whether aspirin inhibits the adhesion of lymphocytes to human aortic smooth muscle cells (AoSMC). Aspirin inhibited T‐cell adhesion to AoSMC activated by interleukin 1β (IL‐1β) in a dose‐dependent manner. Antibodies to the adhesion molecules ICAM‐1 or VCAM‐1, but not to E‐selectin, prevented T‐cell adhesion. ICAM‐1 and VCAM‐1 expression stimulated by IL‐1β was reduced by the treatment with aspirin, whereas the expression of E‐selectin was unaffected. Nuclear factor κB (NF‐κB) activity was enhanced by IL‐1β and reduced by aspirin, indicating that decreased ICAM‐1 and VCAM‐1 expression was due to reduced NF‐κB activity.Thus, aspirin inhibits the adhesion of Jurkat T cells to IL‐1β‐activated AoSMC by reducing NF‐κB activity and decreasing expression of ICAM‐1 and VCAM‐1, and may prevent the development of atherosclerosis.

Klotho gene delivery suppresses oxidative stress in vivo

Objective:u2003 Mice deficient in the klotho gene exhibit a syndrome resembling premature human aging. A recent report also suggested that klotho transgenic mice exhibited a long lifespan, which shows that klotho is an antisenescence gene. Previously, klotho has been reported to improve endothelial dysfunction, and also to have a preventive effect against oxidative stress. In the present study, we investigated the effect of klotho gene delivery on blood pressure and oxidative stress in vivo.

Tissue Inhibitor of Metalloproteinase-3 Plays Important Roles in the Kidney Following Unilateral Ureteral Obstruction

Tissue inhibitor of metalloproteinase-3 (Timp-3), an inhibitor of matrix-degrading enzymes, is an important molecule for maintenance of the extracellular matrix. In this study, we generated Timp-3−deficient mice and used them to examine the effect of Timp-3-deficiency on blood pressure and to investigate the role of Timp-3 in the kidney following unilateral ureteral obstruction. The blood pressure and heart rate of Timp-3−deficient mice were not significantly different from those of wild-type mice. On the other hand, the obstructed kidneys of Timp-3−deficient mice developed more severe hydronephrosis than those of wild-type animals. Matrix metalloproteinase activities assessed by in situ zymography and transforming growth factor-β expression were elevated in Timp-3−deficient mice. The renal tissues were thinner and the ratio of renal medulla to cortex was significantly lower in the obstructed Timp-3−deficient kidneys. These findings indicate that Timp-3-deficiency does not substantially affect the blood pressure in mice, and that Timp-3 plays an important role in the maintenance of renal macrostructure after unilateral ureteral obstruction.

Pulmonary venous flow and risk of cardiovascular disease in essential hypertension.

Objective The prognostic significance of the pulmonary venous flow in essential hypertensive patients was investigated. Methods and results Doppler transthoracic echocardiograms were analyzed in 705 essential hypertensive subjects with no prior cardiovascular disease. At baseline, most subjects had ‘normal diastolic function’ or ‘mild diastolic dysfunction’. During follow-up (mean, 32 months), 56 participants developed cardiovascular disease. Sex-specific median values were used to separate the higher group from the lower group of the peak velocity ratio of the pulmonary venous systolic to diastolic wave (S/D) (male < 1.51, female < 1.66), and of the transmitral velocity ratio of early diastolic to atrial filling (E/A) (male < 0.84, female < 0.82). Kaplan–Meier curves with log-rank tests showed significantly poorer event-free survival rates in the groups with high S/D (P < 0.01) and low E/A (P < 0.01), respectively. In multivariate Cox regression analysis, the S/D ratio (HR 1.07 for each 0.1 increase, P = 0.03) or E/A ratio (P < 0.01) was an independent predictor of cardiovascular disease events. When divided into four groups based on the respective sex-specific median levels of S/D in the E/A ≥ median and E/A < median groups, the group with high S/D and low E/A (S/D; male ≥ 1.77, female ≥ 1.81) had a significantly poorer event-free survival rate (χ2 = 28.06, P < 0.01), and the adjusted-hazard ratio by multivariate Cox regression analysis was 2.16 (95% CI; 1.40–3.07, P < 0.01). Conclusion Increased S/D or decreased E/A is associated with an increased cardiovascular disease risk, and the combination of high S/D and low E/A may be a powerful predictor of cardiovascular disease in essential hypertension. Pulmonary venous flow evaluation may provide clinically important prognostic information in patients with essential hypertension.

Akt activation prevents Apop-1-induced death of cells

Apop-1 is a novel protein identified in cultured atherosclerotic smooth muscle cells of ApoE-deficient mice, and the expression of the Apop-1 protein induces the death of cultured cells. Insulin-like growth factor-1 (IGF-1) is a well-characterized survival factor for VSMC; however, the interaction between Apop-1 and survival factor IGF-1 in the mediation of cell death is poorly understood. In this report, we show that the IGF-1 signaling cascade protects VSMC against Apop-1-induced death. Furthermore, our data indicate that the inhibition of Apop-1-induced death by IGF-1 is mediated by the activation of the PI3K/Akt signaling pathway.

Fas Ligand mRNA Levels of Circulating Leukocytes Reflect Endothelial Dysfunction in Hyperlipidemic but Not in Non-Hyperlipidemic Patients

To find a novel marker for identifying patients at high-risk for endothelial dysfunction among patients with atherosclerosis, we examined the correlation between mRNA levels of Fas ligand (FasL), an apoptosis-inducing factor, in circulating leukocytes and clinical parameters in these patients. FasL mRNA levels of circulating leukocytes were measured with the TaqMan-PCR method. A negative correlation was observed between brachial artery flow-mediated dilatation (%FMD) and FasL mRNA levels of leukocytes in hyperlipidemic but not in non-hyperlipidemic patients. %FMD was more impaired in patients with a high level of FasL mRNA than in those with a low level of FasL mRNA. Interestingly, the improvement of %FMD by treatment with a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor (simvastatin) was greater in the group showing a decrease in FasL mRNA than in the group with no such decrease. Additionally, simvastatin suppressed the FasL mRNA expression in leukocytes and decreased plasma oxidized low-density lipoprotein (OxLDL) levels. Furthermore, the supernatant of cultured leukocytes from hyperlipidemic patients induced cell death in Jurkat T cells, which was neutralized by an antibody against FasL. These findings suggest that high FasL mRNA expression in circulating leukocytes may be a marker of high-risk for endothelial dysfunction in hyperlipidemic but not in non-hyperlipidemic patients. This information may provide a novel basis for targeting of statin therapy in patients with vulnerable plaques.