NLRP3 Inflammasome Activation Through Heart-Brain Interaction Initiates Cardiac Inflammation and Hypertrophy During Pressure Overload.

BACKGROUND: Mechanical stress on the heart, such as high blood pressure, initiates inflammation and causes hypertrophic heart disease. However, the regulatory mechanism of inflammation and its role in the stressed heart remain unclear. IL-1ß (interleukin-1ß) is a proinflammatory cytokine that causes cardiac hypertrophy and heart failure. Here, we show that neural signals activate the NLRP3 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing 3) inflammasome for IL-1ß production to induce adaptive hypertrophy in the stressed heart. METHODS: C57BL/6 mice, knockout mouse strains for NLRP3 and P2RX7 (P2X purinoceptor 7), and adrenergic neuron-specific knockout mice for SLC17A9, a secretory vesicle protein responsible for the storage and release of ATP, were used for analysis. Pressure overload was induced by transverse aortic constriction. Various animal models were used, including pharmacological treatment with apyrase, lipopolysaccharide, 2'(3')-O-(4-benzoylbenzoyl)-ATP, MCC950, anti-IL-1ß antibodies, clonidine, pseudoephedrine, isoproterenol, and bisoprolol, left stellate ganglionectomy, and ablation of cardiac afferent nerves with capsaicin. Cardiac function and morphology, gene expression, myocardial IL-1ß and caspase-1 activity, and extracellular ATP level were assessed. In vitro experiments were performed using primary cardiomyocytes and fibroblasts from rat neonates and human microvascular endothelial cell line. Cell surface area and proliferation were assessed. RESULTS: Genetic disruption of NLRP3 resulted in significant loss of IL-1ß production, cardiac hypertrophy, and contractile function during pressure overload. A bone marrow transplantation experiment revealed an essential role of NLRP3 in cardiac nonimmune cells in myocardial IL-1ß production and cardiac phenotype. Pharmacological depletion of extracellular ATP or genetic disruption of the P2X7 receptor suppressed myocardial NLRP3 inflammasome activity during pressure overload, indicating an important role of ATP/P2X7 axis in cardiac inflammation and hypertrophy. Extracellular ATP induced hypertrophic changes of cardiac cells in an NLRP3- and IL-1ß-dependent manner in vitro. Manipulation of the sympathetic nervous system suggested sympathetic efferent nerves as the main source of extracellular ATP. Depletion of ATP release from sympathetic efferent nerves, ablation of cardiac afferent nerves, or a lipophilic ß-blocker reduced cardiac extracellular ATP level, and inhibited NLRP3 inflammasome activation, IL-1ß production, and adaptive cardiac hypertrophy during pressure overload. CONCLUSIONS: Cardiac inflammation and hypertrophy are regulated by heart-brain interaction. Controlling neural signals might be important for the treatment of hypertensive heart disease.

Toll-Like Receptor 9 Plays a Pivotal Role in Angiotensin II-Induced Atherosclerosis.

Background Toll-like receptor ( TLR ) 9 recognizes bacterial DNA , activating innate immunity, whereas it also provokes inflammation in response to fragmented DNA released from mammalian cells. We investigated whether TLR 9 contributes to the development of vascular inflammation and atherogenesis using apolipoprotein E-deficient ( Apoe -/-) mice. Methods and Results Tlr9-deficient Apoe -/- ( Tlr9 -/- Apoe -/-) mice and Apoe -/- mice on a Western-type diet received subcutaneous angiotensin II infusion (1000 ng/kg per minute) for 28 days. Angiotensin II increased the plasma level of double-stranded DNA, an endogenous ligand of TLR 9, in these mice. Genetic deletion or pharmacologic blockade of TLR 9 in angiotensin II-infused Apoe -/- mice attenuated atherogenesis in the aortic arch ( P<0.05), reduced the accumulation of lipid and macrophages in atherosclerotic plaques, and decreased RNA expression of inflammatory molecules in the aorta with no alteration of metabolic parameters. On the other hand, restoration of TLR 9 in bone marrow in Tlr9 -/- Apoe -/- mice promoted atherogenesis in the aortic arch ( P<0.05). A TLR 9 agonist markedly promoted proinflammatory activation of Apoe -/- macrophages, partially through p38 mitogen-activated protein kinase signaling. In addition, genomic DNA extracted from macrophages promoted inflammatory molecule expression more effectively in Apoe -/- macrophages than in Tlr9 -/- Apoe -/- macrophages. Furthermore, in humans, circulating double-stranded DNA in the coronary artery positively correlated with inflammatory features of coronary plaques determined by optical coherence tomography in patients with acute myocardial infarction ( P<0.05). Conclusions TLR 9 plays a pivotal role in the development of vascular inflammation and atherogenesis through proinflammatory activation of macrophages. TLR 9 may serve as a potential therapeutic target for atherosclerosis.

Activation of Toll-Like Receptor 9 Impairs Blood Flow Recovery After Hind-Limb Ischemia.

Background: Peripheral artery disease causes significant functional disability and results in impaired quality of life. Ischemic tissue injury releases various endogenous ligands for Toll-like receptors (TLRs), suggesting the involvement of TLRs in blood flow recovery. However, the role of TLR9, which was originally known as a sensor for bacterial DNA, remains unknown. This study investigated the role of TLR9 in blood flow recovery in the ischemic limb using a mouse hind-limb ischemia model. Methods and Results: Unilateral femoral artery ligation was performed in TLR9-deficient (Tlr9 -/-) mice and wild-type mice. In wild-type mice, femoral artery ligation significantly increased mRNA expression of TLR9 in the ischemic limb (P < 0.001) and plasma levels of cell-free DNA (cfDNA) as determined by single-stranded DNA (ssDNA) (P < 0.05) and double-stranded DNA (dsDNA) (P < 0.01), which are endogenous ligands for TLR9, compared with the sham-operated group. Laser Doppler perfusion imaging demonstrated significantly improved ratio of blood flow in the ischemic to non-ischemic limb in Tlr9 -/- mice compared with wild-type mice at 2 weeks after ligation (P < 0.05). Tlr9 -/- mice showed increased capillary density and reduced macrophage infiltration in ischemic limb. Genetic deletion of TLR9 reduced the expression of TNF-α, and attenuated NF-κB activation in ischemic muscle compared with wild-type mice (P < 0.05, respectively) at 3 days after the surgery. ODN1826, a synthetic agonistic oligonucleotide for TLR9, or plasma obtained from mice with ischemic muscle promoted the expression of TNF-α in wild-type macrophages (P < 0.05), but not in Tlr9 -/- macrophages. ODN1826 also activated NF-κB signaling as determined by the degradation of IκBα in wild-type macrophages (P < 0.05), but not in Tlr9 -/- macrophages. In vitro experiments using human umbilical vein endothelial cells demonstrated that TNF-α, or conditioned medium obtained from wild-type macrophages treated with ODN1826 accelerated cell death as determined by MTS assay (P < 0.05 and P < 0.01, respectively). Conclusion: Our results suggest that ischemic muscle releases cfDNA, which activates TLR9 and enhances inflammation, leading to impairment of blood flow recovery in the ischemic limb. cfDNA-TLR9 signaling may serve as a potential therapeutic target in ischemic limb disease.

Ticagrelor, a P2Y12 antagonist, attenuates vascular dysfunction and inhibits atherogenesis in apolipoprotein-E-deficient mice.

BACKGROUND AND AIMS: Ticagrelor reduces cardiovascular events in patients with acute coronary syndrome (ACS). Recent studies demonstrated the expression of P2Y12 on vascular cells including endothelial cells, as well as platelets, and suggested its contribution to atherogenesis. We investigated whether ticagrelor attenuates vascular dysfunction and inhibits atherogenesis in apolipoprotein E-deficient (apoe-/-) mice. METHODS: Eight-week-old male apoe-/- mice were fed a western-type diet (WTD) supplemented with 0.1% ticagrelor (approximately 120 mg/kg/day). Non-treated animals on WTD served as control. Atherosclerotic lesions were examined by en-face Sudan IV staining, histological analyses, quantitative RT-PCR analysis, and western blotting. Endothelial function was analyzed by acetylcholine-dependent vasodilation using aortic rings. Human umbilical vein endothelial cells (HUVEC) were used for in vitro experiments. RESULTS: Ticagrelor treatment for 20 weeks attenuated atherosclerotic lesion progression in the aortic arch compared with control (p < 0.05). Ticagrelor administration for 8 weeks attenuated endothelial dysfunction (p < 0.01). Ticagrelor reduced the expression of inflammatory molecules such as vascular cell adhesion molecule-1, macrophage accumulation, and lipid deposition. Ticagrelor decreased the phosphorylation of JNK in the aorta compared with control (p < 0.05). Ticagrelor and a JNK inhibitor ameliorated impairment of endothelium-dependent vasodilation by adenosine diphosphate (ADP) in wild-type mouse aortic segments. Furthermore, ticagrelor inhibited the expression of inflammatory molecules which were promoted by ADP in HUVEC (p < 0.001). Ticagrelor also inhibited ADP-induced JNK activation in HUVEC (p < 0.05). CONCLUSIONS: Ticagrelor attenuated vascular dysfunction and atherogenesis through the inhibition of inflammatory activation of endothelial cells. These effects might be a potential mechanism by which ticagrelor decreases cardiovascular events in patients with ACS.

Inhibition of activated factor X by rivaroxaban attenuates neointima formation after wire-mediated vascular injury.

Accumulating evidence suggests that activated factor X (FXa), a key coagulation factor, plays an important role in the development of vascular inflammation through activation of many cell types. Here, we investigated whether pharmacological blockade of FXa attenuates neointima formation after wire-mediated vascular injury. Transluminal femoral artery injury was induced in C57BL/6 mice by inserting a straight wire. Rivaroxaban (5mg/kg/day), a direct FXa inhibitor, was administered from one week before surgery until killed. At four weeks after surgery, rivaroxaban significantly attenuated neointima formation in the injured arteries compared with control (P<0.01). Plasma lipid levels and blood pressure were similar between the rivaroxaban-treated group and non-treated group. Quantitative RT-PCR analyses demonstrated that rivaroxaban reduced the expression of inflammatory molecules (e.g., IL-1ß and TNF-α) in injured arteries at seven days after surgery (P<0.05, respectively). In vitro experiments using mouse peritoneal macrophages demonstrated that FXa increased the expression of inflammatory molecules (e.g., IL-1ß and TNF-α), which was blocked in the presence of rivaroxaban (P<0.05). Also, in vitro experiments using rat vascular smooth muscle cells (VSMC) demonstrated that FXa promoted both proliferation and migration of this cell type (P<0.05), which were blocked in the presence of rivaroxaban. Inhibition of FXa by rivaroxaban attenuates neointima formation after wire-mediated vascular injury through inhibition of inflammatory activation of macrophages and VSMC.

A variant of death-receptor 3 associated with rheumatoid arthritis interferes with apoptosis-induction of T cell.

Rheumatoid arthritis (RA) is a chronic polyarthritis of unknown etiology. To unravel the molecular mechanisms in RA, we performed targeted DNA sequencing analysis of patients with RA. This analysis identified a variant of the death receptor 3 (DR3) gene, a member of the family of apoptosis-inducing Fas genes, which contains four single-nucleotide polymorphisms (SNPs) and a 14-nucleotide deletion within exon 5 and intron 5. We found that the deletion causes the binding of splicing regulatory proteins to DR3 pre-mRNA intron 5, resulting in a portion of intron 5 becoming part of the coding sequence, thereby generating a premature stop codon. We also found that this truncated DR3 protein product lacks the death domain and forms a heterotrimer complex with wildtype DR3 that dominant-negatively inhibits ligand-induced apoptosis in lymphocytes. Myelocytes from transgenic mice expressing the human DR3 variant produced soluble truncated DR3, forming a complex with TNF-like ligand 1A (TL1A), which inhibited apoptosis induction. In summary, our results reveal that a DR3 splice variant that interferes with ligand-induced T cell responses and apoptosis may contribute to RA pathogenesis.

Teneligliptin, a dipeptidyl peptidase-4 inhibitor, attenuated pro-inflammatory phenotype of perivascular adipose tissue and inhibited atherogenesis in normoglycemic apolipoprotein-E-deficient mice.

BACKGROUND: Dipeptidyl peptidase-4 (DPP-4) inhibitors have various cellular effects that are associated with vascular protection. Here, we examined whether teneligliptin alters the pro-inflammatory phenotype of perivascular adipose tissue (PVAT) and inhibits atherogenesis. METHODS AND RESULTS: Teneligliptin (60mg/kg/day) was administered orally to apolipoprotein-E-deficient (ApoE-/-) mice for 20weeks. Teneligliptin significantly inhibited the development of atherosclerosis in the aortic arch compared with vehicle (P<0.05), without alteration of blood glucose level or blood pressure. Histological analyses demonstrated that teneligliptin decreased lipid deposition and MCP-1 expression (P<0.05, respectively), and tended to decrease macrophage accumulation in atherosclerotic plaques. The results of quantitative RT-PCR analysis demonstrated that teneligliptin reduced the expression of inflammatory molecules such as TNF-α and MCP-1 in the abdominal aorta. Furthermore, teneligliptin reduced the expression of a macrophage marker and Nox-4, a major NADPH oxidase subunit in adipocytes, in PVAT around the aortic arch. Administration of teneligliptin for 8weeks ameliorated endothelium-dependent vasodilation and reduced oxidative stress as determined by urinary 8-OHdG excretion (P<0.05) compared with vehicle. In vitro experiments demonstrated that exendin-4 (Ex-4), a GLP-1 analog, decreased the expression of inflammatory molecules in RAW264.7 cells. Also, Ex-4 decreased Nox4 expression in 3T3-L1 adipocytes. CONCLUSION: Teneligliptin inhibited atherogenesis with attenuation of the inflammatory phenotype in PVAT. A GLP-1 analog suppressed pro-inflammatory activation of macrophages and adipocytes. Suppression of the pro-inflammatory phenotype of PVAT might contribute, at least partially, to the cardioprotective effects of teneligliptin.

Combination of n-3 polyunsaturated fatty acids reduces atherogenesis in apolipoprotein E-deficient mice by inhibiting macrophage activation.

BACKGROUND AND AIMS: Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are major components of n-3 polyunsaturated fatty acids (n-3 PUFAs) which inhibit atherogenesis, although few studies have examined the effects of the combination of EPA and DHA on atherogenesis. The aim of this study was to investigate whether DHA has additional anti-atherosclerotic effects when combined with EPA. METHODS: Male 8-week-old apolipoprotein E-deficient (Apoe-/-) mice were fed a western-type diet supplemented with different amounts of EPA and DHA; EPA (2.5%, w/w), low-dose EPA + DHA (2.5%, w/w), or high-dose EPA + DHA (5%, w/w) for 20 weeks. The control group was fed a western-type diet containing no n-3 PUFA. Histological and gene expression analysis were performed in atherosclerotic lesions in the aorta. To address the mechanisms, RAW264.7 cells were used. RESULTS: All n-3 PUFA treatments significantly attenuated the development and destabilization of atherosclerotic plaques compared with the control. The anti-atherosclerotic effects were enhanced in the high-dose EPA + DHA group (p < 0.001), whereas the pure EPA group and low-dose EPA + DHA group showed similar results. EPA and DHA additively attenuated the expression of inflammatory molecules in RAW264.7 cells stimulated with LPS. DHA or EPA + DHA suppressed LPS-induced toll-like receptor 4 (TLR4) expression in lipid rafts on RAW264.7 cells (p < 0.05). Lipid raft disruption by methyl-ß-cyclodextrin suppressed mRNA expression of inflammatory molecules in LPS-stimulated macrophages. CONCLUSION: n-3 PUFAs suppressed atherogenesis. DHA combined with EPA had additional anti-inflammatory effects and inhibited atherogenesis in Apoe-/- mice. The reduction of TLR4 expression in lipid rafts in macrophages by DHA might be involved in this mechanism, at least partially.

Obesity-induced DNA released from adipocytes stimulates chronic adipose tissue inflammation and insulin resistance.

Obesity stimulates chronic inflammation in adipose tissue, which is associated with insulin resistance, although the underlying mechanism remains largely unknown. Here we showed that obesity-related adipocyte degeneration causes release of cell-free DNA (cfDNA), which promotes macrophage accumulation in adipose tissue via Toll-like receptor 9 (TLR9), originally known as a sensor of exogenous DNA fragments. Fat-fed obese wild-type mice showed increased release of cfDNA, as determined by the concentrations of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) in plasma. cfDNA released from degenerated adipocytes promoted monocyte chemoattractant protein-1 (MCP-1) expression in wild-type macrophages, but not in TLR9-deficient (Tlr9 (-/-) ) macrophages. Fat-fed Tlr9 (-/-) mice demonstrated reduced macrophage accumulation and inflammation in adipose tissue and better insulin sensitivity compared with wild-type mice, whereas bone marrow reconstitution with wild-type bone marrow restored the attenuation of insulin resistance observed in fat-fed Tlr9 (-/-) mice. Administration of a TLR9 inhibitory oligonucleotide to fat-fed wild-type mice reduced the accumulation of macrophages in adipose tissue and improved insulin resistance. Furthermore, in humans, plasma ssDNA level was significantly higher in patients with computed tomography-determined visceral obesity and was associated with homeostasis model assessment of insulin resistance (HOMA-IR), which is the index of insulin resistance. Our study may provide a novel mechanism for the development of sterile inflammation in adipose tissue and a potential therapeutic target for insulin resistance.

Rivaroxaban, a novel oral anticoagulant, attenuates atherosclerotic plaque progression and destabilization in ApoE-deficient mice.

OBJECTIVE: Activated factor X (FXa) plays a key role in the coagulation cascade, whereas accumulating evidence suggests that it also contributes to the pathophysiology of chronic inflammation on the vasculature. In this study, we assessed the hypothesis that rivaroxaban (Riv), a direct FXa inhibitor, inhibits atherogenesis by reducing macrophage activation. METHODS AND RESULTS: Expression levels of PAR-1 and PAR-2, receptors for FXa, increased in the aorta of apolipoprotein E-deficient (ApoE(-/-)) mice compared with wild-type mice (P < 0.01, P < 0.05, respectively). Administration of Riv (5 mg/kg/day) for 20 weeks to 8-week-old ApoE(-/-) mice reduced atherosclerotic lesion progression in the aortic arch as determined by en-face Sudan IV staining compared with the non-treated group (P < 0.05) without alteration of plasma lipid levels and blood pressure. Histological analyses demonstrated that Riv significantly decreased lipid deposition, collagen loss, macrophage accumulation and matrix metallopeptidase-9 (MMP-9) expression in atherosclerotic plaques in the aortic root. Quantitative RT-PCR analyses using abdominal aorta revealed that Riv significantly reduced mRNA expression of inflammatory molecules, such as MMP-9, tumor necrosis factor-α (TNF-α). In vitro experiments using mouse peritoneal macrophages or murine macrophage cell line RAW264.7 demonstrated that FXa increased mRNA expression of inflammatory molecules (e.g., interleukin (IL)-1ß and TNF-α), which was blocked in the presence of Riv. CONCLUSIONS: Riv attenuates atherosclerotic plaque progression and destabilization in ApoE(-/-) mice, at least in part by inhibiting pro-inflammatory activation of macrophages. These results indicate that Riv may be particularly beneficial for the management of atherosclerotic diseases, in addition to its antithrombotic activity.

HMGB1 plays a critical role in vascular inflammation and lesion formation via toll-like receptor 9.

OBJECTIVE: Endogenous ligands such as high-mobility group box 1 (HMGB1) and nucleic acids are released by dying cells and bind to Toll-like receptors (TLRs). As TLR9 is involved in both microbial and sterile inflammation by detecting both bacterial and endogenous DNA, we investigated its role in inflammation and lesion formation in a mouse model of vascular injury. METHODS AND RESULTS: C57BL/6 (WT) and TLR9 KO mice were subjected to wire-mediated vascular injury. Anti-HMGB1 antibody and purified HMGB1 protein were chronically delivered around the injured arteries by gelatin hydrogel, and neointima formation at 4 weeks after injury was evaluated. In addition, the same vascular injury was performed in bone-marrow chimeric mice (WT bone marrow into TLR KO mice; TLR9 KO bone marrow into WT mice). We also evaluated the production of inflammatory cytokines by mouse macrophages in response to HMGB1 and CpG-ODN. In wild-type mice after vascular injury, anti-HMGB1 antibody significantly reduced neointima formation and HMGB1 protein accelerated neointima hyperplasia. HMGB1 failed to accelerate lesion formation in TLR9 KO mice. The bone marrow transplantation study revealed that TLR9 in bone marrow-derived cells played a fundamental role in neointima formation. In vitro, HMGB1 and CpG-ODN synergistically induced the production of inflammatory cytokines by macrophages. CONCLUSIONS: HMGB1 serves as an endogenous mediator of inflammation and lesion formation via the TLR9 pathway in response to vascular injury. Blockade of HMGB1 and/or TLR9 may represent a novel approach to treating atherosclerosis.

Toll-like receptor-2 mediates adaptive cardiac hypertrophy in response to pressure overload through interleukin-1ß upregulation via nuclear factor κB activation.

BACKGROUND: Inflammation is induced in the heart during the development of cardiac hypertrophy. The initiating mechanisms and the role of inflammation in cardiac hypertrophy, however, remain unclear. Toll-like receptor-2 (TLR2) recognizes endogenous molecules that induce noninfectious inflammation. Here, we examined the role of TLR2-mediated inflammation in cardiac hypertrophy. METHODS AND RESULTS: At 2 weeks after transverse aortic constriction, Tlr2(-/-) mice showed reduced cardiac hypertrophy and fibrosis with greater left ventricular dilatation and impaired systolic function compared with wild-type mice, which indicated impaired cardiac adaptation in Tlr2(-/-) mice. Bone marrow transplantation experiment revealed that TLR2 expressed in the heart, but not in bone marrow-derived cells, is important for cardiac adaptive response to pressure overload. In vitro experiments demonstrated that TLR2 signaling can induce cardiomyocyte hypertrophy and fibroblast and vascular endothelial cell proliferation through nuclear factor-κB activation and interleukin-1ß upregulation. Systemic administration of a nuclear factor-κB inhibitor or anti-interleukin-1ß antibodies to wild-type mice resulted in impaired adaptive cardiac hypertrophy after transverse aortic constriction. We also found that heat shock protein 70, which was increased in murine plasma after transverse aortic constriction, can activate TLR2 signaling in vitro and in vivo. Systemic administration of anti-heat shock protein 70 antibodies to wild-type mice impaired adaptive cardiac hypertrophy after transverse aortic constriction. CONCLUSIONS: Our results demonstrate that TLR2-mediated inflammation induced by extracellularly released heat shock protein 70 is essential for adaptive cardiac hypertrophy in response to pressure overload. Thus, modulation of TLR2 signaling in the heart may provide a novel strategy for treating heart failure due to inadequate adaptation to hemodynamic stress.

Exendin-4, a glucagon-like peptide-1 receptor agonist, attenuates neointimal hyperplasia after vascular injury.

Exendin-4 is a glucagon-like peptide-1 receptor agonist that has been used as a drug for treatment of type 2 diabetes. To investigate the effect of exendin-4 on the cardiovascular system, we investigated the impact of exendin-4 on neointimal hyperplasia of the femoral artery after vascular injury. We performed wire-mediated endovascular injury in C57BL/6 mice, followed by administration of exendin-4 24 nmol/kg/day via infusion pump. Four weeks after the injury, exendin-4 treatment significantly attenuated neointimal hyperplasia of the injured artery, although it did not affect glucose metabolism and lipid profile in wild-type mice. Immunofluorescence study revealed abundant expression of GLP-1 receptor on α-smooth muscle actin-positive cells in the injured vessel. Cell proliferation assay using rat aortic smooth muscle cells showed that exendin-4 reduced PDGF-BB induced smooth muscle cell proliferation through the cAMP/PKA pathway. Exendin-4 also inhibited TNFα production by peritoneal macrophages in response to inflammatory stimulus. Our findings indicate that a GLP-1 receptor agonist attenuated neointimal formation after vascular injury. GLP-1 receptor agonists or drugs that raise endogenous GLP-1 level might be effective in the treatment of vascular diseases.

Src kinase-mediates androgen receptor-dependent non-genomic activation of signaling cascade leading to endothelial nitric oxide synthase.

Our previous study has demonstrated that testosterone rapidly activates endothelial nitric oxide synthase (eNOS), enhancing nitric oxide (NO) release from endothelial cells (ECs) via the phosphatidylinositol 3-kinase/Akt (PI3-kinase/Akt) pathway. The upstream regulators of this pathway are unknown. In this study, we further investigated the non-genomic action of testosterone in human aortic ECs. Acute (30 min) activation of eNOS caused by testosterone was unaffected by pretreatment with a transcriptional inhibitor, actinomycin D. Non-permeable testosterone-BSA rapidly induced Akt and eNOS phosphorylation. In contrast, luciferase reporter assay showed that the transcriptional activity of the androgen-responsive element (ARE) was increased by testosterone, but not by testosterone-BSA at 2h after stimulation. Immunostaining displayed co-localization of androgen receptor (AR) with caveolin-1. Fractional analysis showed that AR was expressed in caveolae-enriched membrane fractions. Immunoprecipitation assays revealed the association of AR with caveolin-1 and c-Src, suggesting complex formation among them. Testosterone rapidly increased the phosphorylation of c-Src on Tyr416, which was inhibited by an AR antagonist and by siRNA for AR. PP2, a specific-inhibitor of Src kinase, abolished the testosterone-induced phosphorylation of Akt and eNOS. Our data indicate that testosterone induces rapid assembly of a membrane signaling complex among AR, caveolin-1 and c-Src, which then facilitates activation of the c-Src/ PI3-kinase/Akt cascade, resulting in activation of eNOS.

The ATP-binding cassette transporter ABCG2 protects against pressure overload-induced cardiac hypertrophy and heart failure by promoting angiogenesis and antioxidant response.

OBJECTIVE: ATP-binding cassette transporter subfamily G member 2 (ABCG2), expressed in microvascular endothelial cells in the heart, has been suggested to regulate several tissue defense mechanisms. This study was performed to elucidate its role in pressure overload-induced cardiac hypertrophy. METHODS AND RESULTS: Pressure overload was induced in 8- to 12-week-old wild-type and Abcg2-/- mice by transverse aortic constriction (TAC). Abcg2-/- mice showed exaggerated cardiac hypertrophy and ventricular remodeling after TAC compared with wild-type mice. In the early phase after TAC, functional impairment in angiogenesis and antioxidant response in myocardium was found in Abcg2-/- mice. In vitro experiments demonstrated that ABCG2 regulates transport of glutathione, an important endogenous antioxidant, from microvascular endothelial cells. Besides, glutathione transported from microvascular endothelial cells in ABCG2-dependent manner ameliorated oxidative stress-induced cardiomyocyte hypertrophy. In vivo, glutathione levels in plasma and the heart were increased in wild-type mice but not in Abcg2-/- mice after TAC. Treatment with the superoxide dismutase mimetic ameliorated cardiac hypertrophy in Abcg2-/- mice after TAC to the same extent as that in wild-type mice, although cardiac dysfunction with impaired angiogenesis was observed in Abcg2-/- mice. CONCLUSION: ABCG2 protects against pressure overload-induced cardiac hypertrophy and heart failure by promoting angiogenesis and antioxidant response.

Augmented angiogenesis in adventitia promotes growth of atherosclerotic plaque in apolipoprotein E-deficient mice.

OBJECTIVE: Accumulating evidence suggests that exaggerated formation of vasa vasorum (VV) plays an important role in the pathogenesis of atherosclerosis. However, it remains unclear whether augmented angiogenesis in the adventitia could promote hyperlipidemia-induced atherosclerotic lesion formation. METHODS AND RESULTS: First, we analyzed the time course of VV development in apolipoprotein E-deficient (ApoE-/-) mice. VV proliferation was observed only after atherosclerotic lesion formation. Next, we investigated whether forced perivascular angiogenesis could promote plaque progression. Basic fibroblast growth factor (bFGF) (100 µg/body) incorporated in acid gelatin hydrogel microspheres (AGHM) (bFGF+AGHM group, n=10), AGHM alone (AGHM group, n=7), or PBS (control group, n=8) was administered into the periaortic area of the retroperitoneal space in 10- to 11-week-old male ApoE-/- mice. At 13 weeks after the operation, lesions were significantly larger in the bFGF+AGHM group than in others (bFGF+AGHM: 3.4 ± 0.7 × 10(4)µm(2); AGHM: 0.1 ± 0.1 × 10(4)µm(2); control: 0 µm(2); p<0.0001), which was associated with increased neovascularization in the adventitia. The number of adventitial capillaries correlated with plaque size (r=0.69, p<0.0001). In the bFGF+AGHM group, an increase in the number of VV and accumulation of Mac3-positive macrophages were observed prior to atherosclerotic lesion formation. CONCLUSIONS: Our findings demonstrated that local administration of bFGF in the adventitia induced development of VV and accelerated plaque progression in ApoE-/- mice, supporting the notion that VV formation plays a crucial role in the pathogenesis of atherosclerosis.

The ATP-binding cassette transporter BCRP1/ABCG2 plays a pivotal role in cardiac repair after myocardial infarction via modulation of microvascular endothelial cell survival and function.

OBJECTIVE: To clarify the impact of breast cancer resistance protein 1 (BCRP1)/ATP-binding cassette transporter subfamily G member 2 (ABCG2) expression on cardiac repair after myocardial infarction (MI). METHODS AND RESULTS: The ATP-binding cassette transporter BCRP1/ABCG2 is expressed in various organs, including the heart, and may regulate several tissue defense mechanisms. BCRP1/ABCG2 was mainly expressed in endothelial cells of microvessels in the heart. MI was induced in 8- to 12-week-old wild-type (WT) and Bcrp1/Abcg2 knockout (KO) mice by ligating the left anterior descending artery. At 28 days after MI, the survival rate was significantly lower in KO mice than in WT mice because of cardiac rupture. Echocardiographic, hemodynamic, and histological assessments showed that ventricular remodeling was more deteriorated in KO than in WT mice. Capillary, myofibroblast, and macrophage densities in the peri-infarction area at 5 days after MI were significantly reduced in KO compared with WT mice. In vitro experiments demonstrated that inhibition of BCRP1/ABCG2 resulted in accumulation of intracellular protoporphyrin IX and impaired survival of microvascular endothelial cells under oxidative stress. Moreover, BCRP1/ABCG2 inhibition impaired migration and tube formation of endothelial cells. CONCLUSIONS: BCRP1/ABCG2 plays a pivotal role in cardiac repair after MI via modulation of microvascular endothelial cell survival and function.

Role of vascular progenitor cells in cardiovascular disease.

It is a widely accepted view that vascular repair results from migration and proliferation of adjacent vascular cells. On the other hand, accumulating evidence suggests that bone marrow cells can give rise to endothelial-like cells and smooth muscle-like cells that potentially contribute to vascular healing, remodeling and lesion formation under physiological and pathological conditions. However, some recent reports indicated controversial results and cast a doubt on the specificity of the method to detect differentiation of ectopic cells. Here, we overview recent findings on the role of vascular progenitor cells in cardiovascular diseases and provide possible explanation why different conclusions have been drawn from different animal experiments.

A new constitutively active mutant of AMP-activated protein kinase inhibits anoxia-induced apoptosis of vascular endothelial cell.

The inhibition of apoptotic changes in vascular endothelial cells is important for preventing vascular damage from hypoxia. AMP-activated protein kinase (AMPK) has recently been identified as playing a role in vascular protection. Although the chemical reagent 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) has been used to stimulate AMPK activity, AICAR has been associated with several nonspecific reactions. We therefore constructed a new constitutively active mutant of AMPK alpha 1 (NcaAMPK), which lacks the autoinhibitory domain in AMPK alpha 1 and in which threonine 172 has been replaced with aspartate. We investigated whether NcaAMPK has an anti-apoptotic effect in vascular endothelial cells under anoxic conditions. NcaAMPK, or green fluorescent protein (GFP) as a control, was overexpressed in human umbilical vein endothelial cells (HUVECs). After HUVECs were incubated for 40 h under normoxic or anoxic conditions, we examined cell viability, caspase 3/7 activity, and expression and phosphorylation levels of apoptosis-related proteins. Cell viabilities under anoxic conditions were improved in NcaAMPK-overexpressing cells. Anoxia increased caspase 3/7 activity, but NcaAMPK reduced this increase significantly. NcaAMPK overexpression increased protein kinase B/Akt Ser473 and endothelial nitric oxide synthase Ser1177 phosphorylation, but pretreatment with the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) did not decrease the viability of NcaAMPK-overexpressing HUVECs. Furthermore, co-expression of a dominant-negative Akt reduced the improvement in cell viability and the suppression of poly (ADP-ribose) polymerase cleavage by NcaAMPK under anoxic conditions. In conclusion, NcaAMPK inhibited anoxia-induced apoptosis in vascular endothelial cells through Akt activation, suggesting that activation of AMPK might protect against ischemic vascular injury.