Potential Impact on Lipoprotein Subfractions in Type 2 Diabetes.

INTRODUCTION: Recently, the sodium-glucose cotransporter2 (SGLT2) inhibitor empagliflozin has been shown to lower cardiovascular risk among diabetic patients. It is intriguing that some SGLT2 inhibitors have been found to increase low-density lipoprotein (LDL) cholesterol levels, while the relevance to high-density lipoprotein (HDL) cholesterol is unknown. Although the inhibitory effect of SGLT2 inhibitors on glucose reabsorption may accelerate compensatory lipid metabolism and subsequently reduce body weight and affect the lipid profile, much remains unclear about this mechanism. Therefore, we conducted this study to investigate in detail how canagliflozin affects lipoprotein fractions including LDL and HDL subclasses. MATERIALS AND METHODS: This study is a multicenter prospective study. The participants were patients with 22 type 2 diabetes (60.7 ± 11.6 years, 59.1% of men) who had HbA1c ⩾ 7.0% and consented to participate in the study. They were administered 100 mg canagliflozin orally once per day. Biochemistry test and cholesterol levels of 20 lipoprotein fractions (G1-G20) using high performance liquid chromatography methods were examined before and after 12 weeks of treatment period. RESULTS: Significant decreases were observed in the participants' body weight (69.7 to 67.9 kg, P < .001), systolic blood pressure (129.3 to 119.5 mm Hg, P < .01), and HbA1c (8.5% to 7.4%, P < .001). Cholesterol levels in the 20 lipoprotein fractions increased for very large HDL (G14, G15) and large HDL (G16) (P < .05). CONCLUSIONS: Reduction in body weight, improvement of blood glucose levels, and increases in very large HDL and large HDL subclasses were observed after canagliflozin treatment. These beneficial changes might contribute to subsequent suppression of cardiovascular outcomes.

[Introduction to drug therapy].

Diet and exercise therapy is the main treatment of high triglycerides(TG). However, this disease is often require medication in additon to diet and exercise therapy. In this paper, we described when to start treatment for high TG, and whether to use any medicine. According to the guidelines of the Japan Atherosclerosis Society, the start of the treatment should be determined, and drug could be selected with reference to the WHO classification of dyslipidemia. Fibrates plays a major role in the treatment of TG in various medicines. Moreover, we indicated the usefulness of the formulation EPA/DHA of novel therapeutics agent for high TG and atherosclerosis.

Reduction of indoxyl sulfate by AST-120 attenuates monocyte inflammation related to chronic kidney disease.

Accelerated cardiovascular disease is a frequent complication of CKD. Monocyte-mediated inflammation and adhesion of monocytes to vascular endothelium are key events in atherogenesis. An oral adsorbent, AST-120, retards renal function deterioration by lowering IS, which is known to accumulate in CKD patients. However, the effect of AST-120 on CKD-related monocyte activation is unknown. We aimed to determine whether AST-120 improves monocyte-mediated inflammation through IS reduction. Flow cytometric analysis showed that Mac-1 expression and ROS production were significantly higher in peripheral blood monocytes of subtotal Nx CKD mice than in sham-operated mice. AST-120 treatment significantly decreased Mac-1 expression and ROS production in CKD model mice. Furthermore, administration of IS induced monocyte-mediated inflammation and ROS generation. In vitro studies indicated that IS dose-dependently increased THP-1 monocytic cell adhesion to IL-1ß-activated HUVECs under physiological flow conditions. IS also induced monocyte-mediated inflammation and ROS production in THP-1 cells. Phosphorylation of p38 MAPK and membrane translocation of NAD(P)H oxidase subunit p47phox in THP-1 cells were induced by IS. Both SB203580 (p38 MAPK inhibitor) and apocynin [NAD(P)H oxidase inhibitor] reduced THP-1 cell adhesion to HUVECs. Apocynin also inhibited IS-induced ROS production in THP-1 cells. IS induced monocyte-driven inflammation through NAD(P)H oxidase- and p38 MAPK-dependent pathways in monocytes. The main finding of this study was that AST-120 inhibited monocyte activation by reducing IS in vivo. This provides new insights on how AST-120 attenuates the progression of atherosclerosis in CKD.

Yin Yang-1 inhibits tumor cell growth and inhibits p21WAF1/Cip1 complex formation with cdk4 and cyclin D1.

The GLI-Krüppel zinc finger factor yin yang-1 (YY1) is a complex protein that regulates a variety of processes including transcription, proliferation, development and differentiation. YY1 inhibits cell growth in a cell type-specific manner. The role played by YY1 in its control of tumor cell growth is unclear and controversial. We show here that YY1 can suppress the growth of different tumor cell types in vitro, including human breast carcinoma cells and glioblastoma cells. YY1 also blocked the growth of 13762 MAT mammary adenocarcinoma isografts in rats. YY1 inhibited 13762 MAT tumor growth by approximately 80% compared with the GFP alone group 21 days after injection. YY1 inhibited proliferating cell nuclear antigen (PCNA) expression and pRbSer249/Thr252 phosphorylation without influencing tumor microvascular density. Moreover, YY1 inhibited p21WAF1/Cip1 complex formation with cdk4 and cyclin D1. These findings demonstrate that YY1 can negatively regulate the growth of multiple malignant cell types.

Small dense LDL enhances THP-1 macrophage foam cell formation.

AIM: Increased levels of small dense low-density lipoproteins (sd-LDL) have been reported more atherogenic compared to total low-density lipoprotein (LDL); however, no definitive experiments using macrophages have examined this concept in vitro. METHOD AND RESULT: In this study, we isolated fractions of total LDL (density 1.019-1.063 g/ml) and sd-LDL (density 1.044-1.063 g/ml) from the plasma of subjects with modest hypertriglycidemia. Oxidizabilty as assessed by copper-induced generation (1.6 µmol/L CuSO(4),12 h) of thiobarbituric acid reactive substances (TBARS) was significantly greater (7-fold higher, p < 0.01) for sd-LDL (4.3 ± 1.1 nmol/mg) than for total LDL (0.6 ± 0.2 nmol/mg) at the same cholesterol concentrations. Moreover, oxidized sd-LDL induced more lipid staining in macrophages than oxidized total LDL. When non-oxidized sd-LDL were incubated with THP1 macrophages, there was much greater lipid accumulation as assessed by oil red O staining, and more than a 2-fold increase (p < 0.05) in intracellular triglyceride content as compared to non-oxidized total LDL. Furthermore, non-oxidized sd-LDL in contrast to non-oxidized total LDL enhanced macrophage lectin-like oxidized LDL receptor-1 (LOX-1) protein expression and significantly LOX-1 mRNA levels (+158%, p < 0.05), with no effect on scavenger receptor A or CD36 gene expression. These effects of non-oxidized sd-LDL on LOX-1 gene expression were suppressed when Toll-like receptor 4 was inactivated either by RNAi or antibody. CONCLUSION: Our data indicate for the first time that sd-LDL is much more effective in promoting macrophage triglyceride accumulation and LOX-1 gene expression than total LDL.

[Platelets, coagulation, and fibrinolysis in atherosclerosis formation].

Atherosclerosis is thought to be a chronic inflammatory disease, and many studies show platelets play important role in atherosclerosis formation. Activated platelets adhere to endothelial cells and make leukocyte arrest on endothelial cells. Platelets can interact with both leukocytes and with endothelial cells. Leykocytes or endothelial cells adhere to platelets secrete various inflammatory factors and promote inflammation. On the other hand, inflammation inhibits anti-thrombotic effect on endothelial cells. Annexin A2 is a co-receptor of tissue plasminogen activator and plasminogen and facilitates plasmin generation. We showed annexin A2 can inhibit formation of thrombus in injured blood vessel. Thus, platelets, coagulation, and fibrinolysis may be key factors in inflamed blood wall.

Indoxyl sulfate induces leukocyte-endothelial interactions through up-regulation of E-selectin.

Despite a positive correlation between chronic kidney disease and atherosclerosis, the causative role of uremic toxins in leukocyte-endothelial interactions has not been reported. We thus examined the effects of indoxyl sulfate, a uremic toxin, on leukocyte adhesion to activated endothelial cells and the underlying mechanisms. Pretreatment of human umbilical vein endothelial cells (HUVEC) with indoxyl sulfate significantly enhanced the adhesion of human monocytic cells (THP-1 cell line) to TNF-α-activated HUVEC under physiological flow conditions. Treatment with indoxyl sulfate enhanced the expression level of E-selectin, but not that of ICAM-1 or VCAM-1, in HUVEC. Indoxyl sulfate treatment enhanced the activation of JNK, p38 MAPK, and NF-κB in TNF-α-activated HUVEC. Inhibitors of JNK and NF-κB attenuated indoxyl sulfate-induced E-selectin expression in HUVEC and subsequent THP-1 adhesion. Furthermore, treatment with the NAD(P)H oxidase inhibitor apocynin and the glutathione donor N-acetylcysteine inhibited indoxyl sulfate-induced enhancement of THP-1 adhesion to HUVEC. Next, we examined the in vivo effect of indoxyl sulfate in nephrectomized chronic kidney disease model mice. Indoxyl sulfate-induced leukocyte adhesion to the femoral artery was significantly reduced by anti-E-selectin antibody treatment. These findings suggest that indoxyl sulfate enhances leukocyte-endothelial interactions through up-regulation of E-selectin, presumably via the JNK- and NF-κB-dependent pathway.

[Inflammatory cytokines].

Diabetic macroangiopathy is severe complication for diabetes mellitus, and inflammatory cytokines induces various reactions for the formation of atherosclerosis in diabetes. In addition, recent reports indicate the accumulation of macrophages in the adipose tissue occurs in diabetes and these macrophages secret an inflammatory cytokines. It is also known macrophages produce most of circulating TNF-alpha. Accordingly, inflammatory cytokines, produced in adipose tissue, influence systemic inflammation possibly. Leukocyte-endothelial interactions are critical in the progression of inflammation and atherosclerosis. Recruitment of leukocytes to the sites of inflammation or atherosclerosis prone vasculature involves multistep complex cascades of adhesion events, which inflammatory cytokines induce. Inflammatory cytokines are not the only factor in diabetic macroangiopathy, however, it is certain that inflammation plays important role in diabetes and diabetic vascular complication.

MMP-9 inhibition by ACE inhibitor reduces oxidized LDL-mediated foam-cell formation.

AIM: Angiotensin-converting enzyme inhibitors (ACEIs) have been shown to block matrix metalloproteinase (MMP)-9 activity, which plays a role in atherogenesis. MMP-9 activity of macrophages is increased during foam cell formation. To investigate the contribution of ACEIs to foam cell formation, we studied the effects of an ACEI, imidaprilat, on THP-1 macrophages and the underlying molecular mechanisms in vitro. METHODS AND RESULTS: Pre-treatment of THP-1 macrophages with imidaprilat (100 nmol/L, 4 hours) significantly decreased foam cell formation induced by oxidized LDL (OxLDL). Imidaprilat reduced the protein level of MMP-9 in THP-1 macrophages and attenuated OxLDL-induced MMP-9 activity in the culture supernatants. Indeed, pretreatment of THP-1 macrophages with an MMP-2/9 inhibitor (20 micromol/L, 4 hours) attenuated OxLDL-induced foam-cell formation. Imidaprilat or the MMP-2/9 inhibitor blocked OxLDL-induced expressions of LOX-1 and scavenger receptor-A (SR-A), but not that of CD36, in THP-1 macrophages. In addition, OxLDL-induced activation of p38 mitogen-activated protein kinase (MAPK) and ERK, but not JNK, was blunted by imidaprilat or the MMP-2/9 inhibitor. Finally, siRNA against MMP-9 inhibited foam cell formation as well as lipid accumulation in THP-1 macrophages. CONCLUSION: These findings suggest that imidaprilat reduces OxLDL-triggered foam-cell formation in THP-1 macrophages via modulation of MMP-9 activity and may indicate a novel antiinflamma-tory mechanism of imidaprilat in atherogenesis.

Casein-derived tripeptide, Val-Pro-Pro (VPP), modulates monocyte adhesion to vascular endothelium.

AIM: A food-derived bioactive tripeptide, Val-Pro-Pro (VPP), has been shown to possess angiotensin I-converting enzyme (ACE) inhibitory activity and foods containing such peptides exhibit an anti-hypertensive effect in clinical settings. METHODS: The present study focused on the effect of VPP on monocyte adhesion to endothelium under flow conditions using phorbol 12-myristate 13-acetate (PMA)-stimulated monocytic THP-1 cells. RESULTS: Pre-incubation of THP-1 cells with VPP (1 mM, 24 hours) significantly decreased the PMA-induced adhesion of THP-1 cells (p<0.05) to human umbilical vein endothelial cells (HUVECs). PMA-induced up-regulation of beta1 and beta2 integrin activation in THP-1 cells was downregulated by VPP, which significantly suppressed only the PMA-induced phosphorylation of JNK (p<0.05) in THP-1 cells. In addition, preincubation of THP-1 with SP600125, a specific inhibitor of JNK, resulted in significant reduction of the PMA-induced adhesion of THP-1. Interestingly, another tripeptide with comparable ACE inhibitory activity, Leu-Gly-Pro (LGP), failed to reduce the PMA-induced adhesion of THP-1, suggesting a distinct anti-inflammatory effect of VPP on THP-1 adhesion. CONCLUSION: These observations suggest that VPP moderates monocyte adhesion to inflamed endothelia via attenuation of the JNK pathway in monocytes, which might contribute to the primary prevention of atherosclerosis.

Inhibition of hepatic Niemann-Pick C1-like 1 improves hepatic insulin resistance.

The present study attempted to define the role of hepatic Niemann-Pick C1-like 1 (NPC1L1), a cholesterol transporter, in hepatic insulin resistance as well as hepatic steatosis. The inhibition of NPC1L1 and its molecular consequences were examined in Zucker obese fatty (ZOF) rats and cultured steatotic hepatocytes using ezetimibe, a pharmacoloigcal inhibitor of NPC1L1, and short hairpin RNA (shRNA) of NPC1L1. Ezetimibe improved hepatic insulin signaling as well as hepatic steatosis in ZOF rats. It also restored insulin sensitivity in steatotic hepatocytes in vitro through a reduction in hepatic reactive oxygen species (ROS) generation, JNK activation, and ER stress. In addition, ezetimibe recovered insulin-induced Akt activation and reduced gluconeogenic genes in the liver of ZOF rats and cultured steatotic hepatocytes. Transfection of NPC1L1 shRNA into hepatocytes also reduced ROS generation and ER stress. These results indicate that NPC1L1 contributes to hepatic insulin resistance through cholesterol accumulation, and its inhibition could be a potential therapeutic target of hepatic insulin resistance.

Enhanced adhesion of early endothelial progenitor cells to radiation-induced senescence-like vascular endothelial cells in vitro.

The effects of ionizing radiation (IR) on tumor neovascularization are still unclear. We previously reported that vascular endothelial cells (ECs) expressing the IR-induced senescence-like (IRSL) phenotype exhibit a significant decrease in angiogenic activity in vitro. In this study, we examined the effects of the IRSL phenotype on adhesion to early endothelial progenitor cells (early EPCs). Adhesion of human peripheral blood-derived early EPCs to human umbilical vein endothelial cells (HUVECs) expressing the IRSL phenotype was evaluated by an adhesion assay under static conditions. It was revealed that the IRSL HUVECs supported significantly more adhesion of early EPCs than normal HUVECs. Expressions of ICAM-1, VCAM-1 and E-selectin were up-regulated in IRSL HUVECs. Pre-treatment of IRSL HUVECs with adhesion-blocking monoclonal antibodies against E-selectin and VCAM-1 significantly reduced early EPC adhesion to IRSL HUVECs, suggesting a potential role for the E-selectin and VCAM-1 in the adhesion between IRSL ECs and early EPCs. Therefore, the IRSL phenotype expressed in ECs may enhance neovascularization via increased homing of early EPCs. Our findings are first to implicate the complex effects of this phenotype on tumor neovascularization following irradiation.

Angiotensin II type-I receptor blocker, candesartan, improves brachial-ankle pulse wave velocity independent of its blood pressure lowering effects in type 2 diabetes patients.

BACKGROUND: Hypertension in diabetic patients has been shown to accelerate the progression of atherosclerosis and angiotensin II type-I (AT1) receptor blocker (ARB) has been shown to have anti-atherosclerosis action independent of its action on blood pressure. METHODS AND RESULTS: Type 2 diabetic patients with hypertension received either ARB (candesartan), or a calcium channel blocker (CCB; amlodipine or nifedipine) for 12 weeks. Though clinical parameters such as blood glucose, HbA1c, and systolic and diastolic pressure were not significantly changed between the two groups, brachial-ankle pulse wave velocity (baPWV) was significantly reduced in the candesartan group when compared to CCB groups. Moreover, candesartan treatment exhibited a tendency of reduction in inflammation markers such as high sensitive C-reactive protein (hsCRP) and interleukin-6 (IL-6). CONCLUSION: We conclude that candesartan may improve vascular inflammation independent of its effect on hypertension in diabetes, thus suggesting its efficacy in diabetic patients.

Ezetimibe improves liver steatosis and insulin resistance in obese rat model of metabolic syndrome.

Non-alcoholic fatty liver disease (NAFLD) is associated with the metabolic syndrome characterized by dislipidemia and insulin resistance. We hypothesized that ezetimibe, an inhibitor of NPC1L1, improves these metabolic disorders in Zucker obese fatty rats (ZOF). Ezetimibe significantly lowered total cholesterol and triglycerides in ZOF with prominent reduction in the remnant lipoprotein fraction and small dense low density lipoprotein fraction. Moreover, lipid deposition and fibrosis of liver were decreased by ezetimibe. Interestingly, ezetimibe improved insulin and plasma glucose response after intraperitoneal glucose injection. Further, ezetimibe enhanced insulin signaling in cultured hepatocytes. Our results indicate the potential of ezetimibe in treating the metabolic syndrome and NAFLD.

Cilostazol inhibits monocytic cell adhesion to vascular endothelium via upregulation of cAMP.

AIM: Cilostazol is clinically used as an inhibitor of platelet aggregation. Although several reports have demonstrated its anti-inflammatory effect, its effect on monocytes and their adhesive interaction to vascular endothelium remains unclear. We thus examined the potential role of cilostazol towards monocyte endothelial interaction under physiological flow conditions. METHODS: THP-1 cells, a monocytic cell line, were pretreated with cilostazol (5 microM) for 48 hours. The cells were then perfused over TNF-alpha (5 microg/mL for 4 hours)-stimulated monolayers of human umbilical vein endothelial cells (HUVECs) at shear stress of 1.0 dyen/cm(2). RESULTS: TNF-alpha-activated HUVECs supported significantly more monocyte adhesion to HUVECs (7.32+/-1.25/HPF) compared to inactivated HUVECs (0.74+/-0.15/HPF), and the amount of adhesion to TNF-alpha-activated HUVECs was markedly reduced (3.63+/-0.55/HPF) when THP-1 cells were incubated in the presence of cilostazol at 5 microM. Interestingly, surface expressions of integrins were not dramatically changed after cilostazol treatment. Intracellular concentration of cAMP was significantly increased after cilostazol treatment, and treatment with Forskolin and Dibutyryl-cAMP, potent inducers of cAMP, dramatically increased THP-1 adhesion to HUVECs. CONCLUSION: These data suggest that cilostazol has a potential anti-inflammatory effect on monocyte-endothelial interactions via the upregulation of intracellular cAMP.

Efficacy of recombinant annexin 2 for fibrinolytic therapy in a rat embolic stroke model: a magnetic resonance imaging study.

Efficacy of recombinant annexin 2 (rAN II) in a rat model of embolic stroke was examined using a magnetic resonance imaging (MRI) and histology. The right middle cerebral artery of male Wistar rats was occluded by autologous clots under anesthesia. Four doses of rAN II (0.125, 0.25, 0.5 and 1.0 mg/kg, n=10 for each group) or saline (1 ml/kg, n=10) were administrated intravenously within 5 min before clot infusion. Serial changes in apparent diffusion coefficient (ADC) and relative blood flow (CBF) were measured with the use of MRI in half of the animals in each group. The remaining half of the animals in each group was evaluated for hemorrhage and final infarct size by histology at 48 h after embolization. At 3 h after embolization, lesion volumes with ADC were abnormality and CBF in the peripheral lesion was improved in groups treated with 0.25, 0.5 and 1.0 mg/kg, but not 0.125 mg/kg, of rAN II in comparison with the saline-treated group (P<0.05). Histological analyses were consistent with MRI findings. More importantly, no hemorrhagic transformation was documented in rats treated with 0.125 and 0.25 mg/kg of rAN II, whereas it was observed at higher doses. We concluded that rAN II at 0.25 mg/kg significantly reduced infarct size and improved CBF without hemorrhagic complications. rAN II is a novel compound that has the potential to be a promising fibrinolytic agent to treat embolic stroke.

Yin Yang-1 inhibits vascular smooth muscle cell growth and intimal thickening by repressing p21WAF1/Cip1 transcription and p21WAF1/Cip1-Cdk4-cyclin D1 assembly.

Vascular injury initiates a cascade of phenotype-altering molecular events. Transcription factor function in this process, particularly that of negative regulators, is poorly understood. We demonstrate here that the forced expression of the injury-inducible GLI-Krüppel zinc finger protein Yin Yang-1 (YY1) inhibits neointima formation in human, rabbit and rat blood vessels. YY1 inhibits p21(WAF1/Cip1) transcription, prevents assembly of a p21(WAF1/Cip1)-cdk4-cyclin D1 complex, and blocks downstream pRb(Ser249/Thr252) phosphorylation and expression of PCNA and TK-1. Conversely, suppression of endogenous YY1 elevates levels of p21(WAF1/Cip1), PCNA, pRb(Ser249/Thr252) and TK-1, and increases intimal thickening. YY1 binds Sp1 and prevents its occupancy of a distinct element in the p21(WAF1/Cip1) promoter without YY1 itself binding the promoter. Additionally, YY1 induces ubiquitination and proteasome-dependent degradation of p53, decreasing p53 immunoreactivity in the artery wall. These findings define a new role for YY1 as both an inducer of p53 instability in smooth muscle cells, and an indirect repressor of p21(WAF1/Cip1) transcription, p21(WAF1/Cip1)-cdk4-cyclin D1 assembly and intimal thickening.

Recombinant annexin-2 inhibits the progress of diabetic nephropathy in a diabetic mouse model via recovery of hypercoagulability.

Diabetic nephropathy, a major complication of diabetes mellitus that leads to mortality, has been shown to involve a dysregulation of the coagulation system. Annexin-2, a co-receptor for plasminogen and tissue plasminogen activator on endothelial cells, is one of the molecules required to maintain the antithrombogenic properties of endothelial cells. Previously, we showed that recombinant annexin-2 protein (rAN II) modulated impaired fibrinolytic activity in the carotid arteries of rats. In the present study, to investigate its protective effects against diabetic nephropathy, rAN II was administered to KK-Ay mice, a murine model of type 2 diabetes, for eight weeks, and albuminuria, kidney size, and histological glomerular lesions were investigated. The mean weight of kidneys from KK-Ay mice treated with rAN II was significantly less than that of those treated with PBS (control) (p < 0.02). Furthermore, the level of albuminuria observed in rAN II-treated KK-Ay mice was significantly less than that of the control group (rAN II, 0.90+/-0.12 microg/day; PBS, 1.55+/-0.31 microg/day; p < 0.01); also, the area of diffuse glomerular lesions was significantly smaller (rAN II, 41.51+/-4.54%; PBS, 81.81+/-8.10%; p < 0.01). Bleeding time, prothrombin time (PT), and active partial thromboplastin time (APTT) did not significantly differ between the two groups. Our results suggest that rAN II may inhibit the progression of diabetic nephropathy in KK-Ay mice without influencing the coagulation system, indicating that annexin-2 may be considered as a possible new therapeutic tool for patients with diabetic nephropathy.