Heart failure with preserved vs reduced ejection fraction following cardiac rehabilitation: impact of endothelial function.

There is no proven therapy for heart failure with preserved ejection fraction (HFpEF). Research has shown beneficial responses to cardiac rehabilitation (CR) among HF patients. To date, there are no reports comparing those responses between patients with HFpEF and those with reduced ejection fraction (HFrEF). The purpose of this study was to compare responses to CR in patients with HFpEF versus those with HFrEF. We included 78 consecutive patients (mean age 69 ± 15 years; 80% male) with HF in our CR unit who underwent cardiopulmonary exercise testing and brachial artery flow-mediated dilation (FMD) testing pre- and 5 months post-CR. Patients were judged as HFpEF (n = 40) or HFrEF (n = 38) using a left ventricular ejection fraction (LVEF) cut-off of 50%, and endothelial dysfunction was defined as FMD ≤ 5.0%. Following 155 ± 11 days and 44 ± 8 sessions, peak oxygen uptake ([Formula: see text]) and plasma B-type natriuretic peptide concentrations improved significantly in both groups. The percentage change in peak [Formula: see text] of HFrEF patients was significantly greater than compared with the HFpEF patients (P < 0.01). To further investigate whether a combination of LVEF and FMD values predicts the effect of CR, we divided patients into four groups according to LVEF of 50% and FMD of 50%. Post hoc analysis showed a significant difference between HFrEF patients without endothelial dysfunction and HFpEF patients with endothelial dysfunction (P = 0.01). In conclusion, although CR improves prognosis in HF patients, a larger effect can be expected in HFrEF patients than in HFpEF patients, and endothelial function may enhance the effect.

Acute Deterioration of Pulmonary Arterial Hypertension (PAH) in a Patient with Neurofibromatosis Type 1 (NF1).

A 56-year-old woman was diagnosed as having chronic obstructive pulmonary disease with heavy smoking. Mild pulmonary hypertension (mean pulmonary arterial pressure: 31 mmHg) was detected at the first visit. She was diagnosed with pulmonary hypertension due to pulmonary disease and medicated only with bronchodilators. Simultaneous, multiple freckling in the trunk of her body and café au lait macules in her back with some cutaneous neurofibromas were also detected. A plastic surgeon removed one of the neurofibromas and pathologically diagnosed it as neurofibromatosis type 1 (NF1). We finally rediagnosed her with pulmonary hypertension with unclear and/or multifactorial factors when she deteriorated 1 year after being treated only with bronchodilators. We then administrated upfront combination therapy with macitentan and tadalafil. Mean pulmonary arterial pressure rapidly improved. Learning Objective. Pulmonary arterial hypertension (PAH) in neurofibromatosis type 1 (NF1) can occur due to lung disease or due to certain involvement of pulmonary arteries, or a combination of both. Increased awareness of PAH in NF1 is very important for patients survival. The current therapeutic strategy is almost identical to that of idiopathic PAH; however, there is no clinical evidence. Insights gained from clinical experiences should help identify promising novel therapeutic approaches in NF1-PAH.

Statin treatment upregulates vascular neuronal nitric oxide synthase through Akt/NF-kappaB pathway.

OBJECTIVE: Three-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) are known to enhance vascular expression of endothelial (eNOS) and inducible nitric oxide synthase (iNOS). In this study, we examined whether statins also upregulate vascular expression of neuronal NOS (nNOS). METHODS AND RESULTS: In cultured rat aortic smooth muscle cells, treatment with atorvastatin significantly increased nNOS expression, associated with activation of Akt and NF-kappaB. Inhibition of Akt by dominant-negative Akt suppressed atorvastatin-induced nNOS expression as well as Akt and NF-kappaB activation. Inhibition of NF-kappaB by dominant-negative IkappaB also attenuated atorvastatin-induced nNOS expression and NF-kappaB activation, but not Akt activation. We further examined whether atorvastatin also enhances nNOS expression in isolated mouse aorta, and if so, how much nNOS-derived NO accounts for atorvastatin-induced NOx production. In isolated aortas of wild-type mice, atorvastatin significantly increased all three NOS isoform expression and NOx production. In isolated aortas of doubly i/eNOS(-/-), n/eNOS(-/-), and n/iNOS(-/-) mice, which express only nNOS, iNOS, and eNOS, respectively, atorvastatin-induced NOx production was approximately 25%, 25%, and 50% to that of wild-type mice, respectively, suggesting that nNOS accounts for 25% of the atorvastatin-mediated NOx production. CONCLUSIONS: These results indicate that atorvastatin upregulates vascular nNOS through Akt/NF-kappaB pathway, demonstrating a novel nNOS-mediated vascular effect of the statin.

Extracellular superoxide dismutase overexpression reduces cuff-induced arterial neointimal formation.

The mechanisms of neointimal formation in cuff-injury models are still uncertain. To examine whether extracellular superoxide dismutase (EC-SOD) can reduce neointimal formation in a cuff-injury model, adenoviruses expressing EC-SOD (AxCAEC-SOD) or Escherichia coli beta-galactosidase (AxCALacZ) was injected between the cuff and the adventitia of rat femoral arteries. As a result, EC-SOD protein was effectively produced in the adventitia, as assessed by immunohistochemical staining. In comparison with cuff-treated control arteries and AxCALacZ-transfected arteries, neointimal formation was significantly reduced in AxCAEC-SOD-transfected arteries. Furthermore, proliferating smooth muscle cells in neointima and media were reduced by EC-SOD treatment. Similarly, augmented iNOS expression, apoptosis and collagen content in the vascular wall were also reduced by EC-SOD treatment. Reactive oxygen species (ROS) generation in tissue was reduced by EC-SOD expression, as assessed by dihydroethidium staining and coelenterazine chemiluminescence. These results suggest that ROS, especially superoxide anions at an adventitia, are responsible for neointimal formation in a cuff-injury model.

Valvular and supravalvular aortic stenosis in heterozygous familial hypercholesterolemia, a case report.

Familial hypercholesterolemia (FH) is an autosomal dominant disorder characterized by a high level of LDL-cholesterol and frequent coronary atherosclerosis. We studied a 64 year old woman with heterozygous (hetero) FH, who showed symptoms of chest pain and dyspnea with no other coronary risk factors than post-menopause and hypercholesterolemia. Although her coronary symptoms didn't reveal significant stenosis on coronary angiography, she had severe aortic valvular and supravalvular stenosis at the ascending aorta, which qualified her for aortic valve replacement. Moreover, a coronary flow study revealed functional ischemia with a reduction of the coronary flow reserve. We report a case of valvular and supravalvular aortic stenosis corrected by aortic valve replacement, a rare complication of hetero FH.

Evaluation of improved coronary flow velocity reserve using transthoracic Doppler echocardiography after single LDL apheresis.

UNLABELLED: The purpose of this study was to clarify whether coronary flow velocity reserve (CFVR), evaluated by adenosine 5'-triphosphate-induced hyperemia, is improved by single low-density lipoprotein (LDL) apheresis. Lipid lowering therapy is known to improve endothelium-dependent vasodilatation in forearm or coronary resistant vessels. However, few reports have studied the effect of acute LDL reduction on CFVR. METHODS: Seven patients with familial hypercholesterolemia and significant coronary stenosis except in the left anterior descending artery (LAD) were enrolled in this study. Coronary flow velocity reserve was estimated before and after LDL apheresis using transthoracic Doppler echocardiography (TTDE), which detects the flow velocity at the distal site of the LAD. Although the averaged diastolic peak velocity (ADPV) during ATP-induced hyperemia was similar before and after LDL apheresis, the ADPV at baseline decreased from 30.69 to 25.56 cm/s, resulting in an increased CFVR from 1.78 to 2.10 (P < 0.001). Plasma bradykinin and 6 keto PGF1alpha increased while fibrinogen and plasma viscosity decreased after apheresis. Single LDL apheresis improves CFVR according to TTDE analysis because of the decreasing ADPV at baseline, which is thought to be induced by epicardial coronary artery dilatation and improved microvessel function. This is the result of various factors, such as changes in plasma LDL cholesterol, bradykinin and PGI2 levels with LDL apheresis.

Role of copper transport protein antioxidant 1 in angiotensin II-induced hypertension: a key regulator of extracellular superoxide dismutase.

Extracellular superoxide dismutase (SOD3) is a secretory copper enzyme involved in protecting angiotensin II (Ang II)-induced hypertension. We found previously that Ang II upregulates SOD3 expression and activity as a counterregulatory mechanism; however, underlying mechanisms are unclear. Antioxidant 1 (Atox1) is shown to act as a copper-dependent transcription factor, as well as a copper chaperone, for SOD3 in vitro, but its role in Ang II-induced hypertension in vivo is unknown. Here we show that Ang II infusion increases Atox1 expression, as well as SOD3 expression and activity, in aortas of wild-type mice, which are inhibited in mice lacking Atox1. Accordingly, Ang II increases vascular superoxide production, reduces endothelium-dependent vasodilation, and increases vasoconstriction in mesenteric arteries to a greater extent in Atox1(-/-) than in wild-type mice. This contributes to augmented hypertensive response to Ang II in Atox1(-/-) mice. In cultured vascular smooth muscle cells, Ang II promotes translocation of Atox1 to the nucleus, thereby increasing SOD3 transcription by binding to Atox1-responsive element in the SOD3 promoter. Furthermore, Ang II increases Atox1 binding to the copper exporter ATP7A, which obtains copper from Atox1, as well as translocation of ATP7A to plasma membranes, where it colocalizes with SOD3. As its consequence, Ang II decreases vascular copper levels, which is inhibited in Atox1(-/-) mice. In summary, Atox1 functions to prevent Ang II-induced endothelial dysfunction and hypercontraction in resistant vessels, as well as hypertension, in vivo by reducing extracellular superoxide levels via increasing vascular SOD3 expression and activity.

Loss of fibulin-5 binding to beta1 integrins inhibits tumor growth by increasing the level of ROS.

Tumor survival depends in part on the ability of tumor cells to transform the surrounding extracellular matrix (ECM) into an environment conducive to tumor progression. Matricellular proteins are secreted into the ECM and impact signaling pathways that are required for pro-tumorigenic activities such as angiogenesis. Fibulin-5 (Fbln5) is a matricellular protein that was recently shown to regulate angiogenesis; however, its effect on tumor angiogenesis and thus tumor growth is currently unknown. We report that the growth of pancreatic tumors and tumor angiogenesis is suppressed in Fbln5-null (Fbln5(-/-)) mice compared with wild-type (WT) littermates. Furthermore, we observed an increase in the level of reactive oxygen species (ROS) in tumors grown in Fbln5(-/-) animals. Increased ROS resulted in elevated DNA damage, increased apoptosis of endothelial cells within the tumor, and represented the underlying cause for the reduction in angiogenesis and tumor growth. In vitro, we identified a novel pathway by which Fbln5 controls ROS production through a mechanism that is dependent on beta1 integrins. These results were validated in Fbln5(RGE/RGE) mice, which harbor a point mutation in the integrin-binding RGD motif of Fbln5, preventing its interaction with integrins. Tumor growth and angiogenesis was reduced in Fbln5(RGE/RGE) mice, however treatment with an antioxidant rescued angiogenesis and elevated tumor growth to WT levels. These findings introduce a novel function for Fbln5 in the regulation of integrin-induced ROS production and establish a rationale for future studies to examine whether blocking Fbln5 function could be an effective anti-tumor strategy, alone or in combination with other therapies.

Novel mechanism for regulation of extracellular SOD transcription and activity by copper: role of antioxidant-1.

Extracellular superoxide dismutase (SOD3), a secretory copper-containing antioxidant enzyme, plays an important role in various oxidative stress-dependent cardiovascular diseases. Although cofactor copper is required for SOD3 activity, it remains unknown whether it can regulate SOD3 transcription. We previously demonstrated that SOD3 activity requires the copper chaperone antioxidant-1 (Atox1), involved in copper delivery to SOD3 at the trans-Golgi network (TGN). Here we show that copper treatment in mouse fibroblasts significantly increases mRNA and protein levels of SOD3, but not SOD1, which is abolished in Atox1-deficient cells. Copper promotes Atox1 translocation to the nucleus. Promoter deletion analysis identifies copper- and Atox1-response elements (REs) at the SOD3 promoter. Gel-shift and ChIP assays reveal that Atox1 directly binds to the Atox1 RE in a copper-dependent manner in vitro and in vivo. Adenovirus-mediated reexpression in Atox1(-/-) cells of nucleus-targeted Atox1 (Atox1-NLS), but not TGN-targeted Atox1 (Atox1-TGN), increases SOD3 transcription without affecting SOD3 activity. Importantly, reexpression of both Atox1-NLS and Atox1-TGN together, but not either alone, in Atox1(-/-) cells increases SOD3 activity. SOD3 transcription is positively regulated by copper through the transcription factor function of Atox1, whereas the full activity of SOD3 requires both the copper chaperone and the transcription factor functions of Atox1. Thus, Atox1 is a potential therapeutic target for oxidant stress-dependent cardiovascular disease.

Role of Menkes ATPase in angiotensin II-induced hypertension: a key modulator for extracellular superoxide dismutase function.

The extracellular superoxide dismutase (SOD3), a secretory copper-containing enzyme, regulates angiotensin II (Ang II)-induced hypertension by modulating levels of extracellular superoxide anion. The present study was designed to determine the role of the copper transporter Menkes ATPase (MNK) in Ang II-induced SOD3 activity and hypertension in vivo. Here we show that chronic Ang II infusion enhanced systolic blood pressure and vascular superoxide anion production in MNK mutant (MNK(mut)) mice as compared with those in wild-type mice, which are associated with impaired acetylcholine-induced endothelium-dependent vasorelaxation in MNK(mut) mice. These effects in MNK(mut) mice are rescued by infusion of the SOD mimetic Tempol. By contrast, norepinephrine-induced hypertension, which is not associated with an increase in vascular superoxide anion production, is not affected in MNK(mut) mice. Mechanistically, basal and Ang II infusion-induced increase in vascular SOD3-specific activity is significantly inhibited in MNK(mut) mice. Coimmunoprecipitation analysis reveals that Ang II stimulation promotes association of MNK with SOD3 in cultured vascular smooth muscle cell and in mouse aortas, which may contribute to SOD3-specific activity by increasing copper delivery to SOD3 through MNK. In summary, MNK plays an important role in modulating Ang II-induced hypertension and endothelial function by regulating SOD3 activity and vascular superoxide anion production and becomes a potential therapeutic target for oxidant stress-dependent cardiovascular diseases.

Novel role of antioxidant-1 (Atox1) as a copper-dependent transcription factor involved in cell proliferation.

Copper plays a fundamental role in regulating cell growth. Many types of human cancer tissues have higher copper levels than normal tissues. Copper can also induce gene expression. However, transcription factors that mediate copper-induced cell proliferation have not been identified in mammals. Here we show that antioxidant-1 (Atox1), previously appreciated as a copper chaperone, represents a novel copper-dependent transcription factor that mediates copper-induced cell proliferation. Stimulation of mouse embryonic fibroblasts (MEFs) with copper markedly increased cell proliferation, cyclin D1 expression, and entry into S phase, which were completely abolished in Atox1(-/-) MEFs. Promoter analysis and EMSA revealed that copper stimulates the Atox1 binding to a previously undescribed cis element in the cyclin D1 promoter. The ChIP assay confirms that copper stimulates Atox1 binding to the DNA in vivo. Transfection of Atox1 fused to the DNA-binding domain of Gal4 demonstrated a copper-dependent transactivation in various cell types, including endothelial and cancer cells. Furthermore, Atox1 translocated to the nucleus in response to copper through its highly conserved C-terminal KKTGK motif and N-terminal copper-binding sites. Finally, the functional role of nuclear Atox1 is demonstrated by the observation that re-expression of nuclear-targeted Atox1 in Atox1(-/-) MEFs rescued the defective copper-induced cell proliferation. Thus, Atox1 functions as a novel transcription factor that, when activated by copper, undergoes nuclear translocation, DNA binding, and transactivation, thereby contributing to cell proliferation.

PTEN reduces cuff-induced neointima formation and proinflammatory cytokines.

An inflammatory response followed by vascular injury plays an important role in neointima formation and development of atherosclerotic lesions, which are in part mediated by proinflammatory cytokines. Using a cuff injury model, we examined the effects of adenovirus-mediated overexpression of phosphatase and tensin homology deleted on chromosome 10 (PTEN) on neointima formation and the proinflammatory response. A cuff was placed around the femoral artery, and adenovirus expressing human PTEN type 1 (AdPTEN) or Escherichia coli beta-galactosidase (AdLacZ) was injected between the cuff and the adventitia. After 14 days, the arteries were examined histopathologically and by Western blotting. The significant reduction of neointima formation by AdPTEN compared with AdLacZ was accompanied by reduced cell proliferation and increased adventitial cell apoptosis. AdPTEN also reduced expression of phosphorylated I kappa B-alpha, but not nonphosphorylated I kappa B-alpha. Western blotting revealed that AdPTEN reduced the cuff injury-induced expression levels of monocyte chemoattractant protein-1, TNF-alpha, and IL-1 beta and their expression in all layers of the arterial wall. In contrast, cuff-induced macrophage invasion, which was also inhibited by AdPTEN, was detected only at the intimal surface and in the adventitia. In cultured vascular smooth muscle cells, PTEN directly inhibited ANG II-induced monocyte chemoattractant protein-1 expression as quantified by real-time PCR and Western blotting. Our results suggest that overexpression of PTEN reduces neointima formation, possibly in part through inhibition of the inflammatory response by macrophage invasion and proinflammatory cytokine expression.

Oxidized low-density lipoprotein-induced apoptosis is attenuated by insulin-activated phosphatidylinositol 3-kinase/Akt through p38 mitogen-activated protein kinase.

1. The aim of the present study was to investigate whether p38 mitogen-activated protein kinase (p38 MAPK) is involved in oxidized low-density lipoprotein (oxLDL)-induced apoptosis of human umbilical vein endothelial cells (HUVECs). We also sought to determine whether this apoptosis is regulated by the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. 2. Low-density lipoprotein was oxidized with CuSO4 and used as oxLDL. Using HUVEC, we determined whether LDL/oxLDL induces apoptosis by DNA fragmentation and the cell cycle distribution (SubG1 method). The mechanism and activation of p38 MAPK and Akt were determined by western blot analysis. 3. The results showed that oxLDL induced DNA fragmentation, whereas cell cycle distribution showed that it also significantly increased the rate of cell death compared with the LDL group. SB203580 significantly inhibited cell death induced by oxLDL, as did the administration of insulin. Western blot analysis showed the activation of p38 MAPK by oxLDL, but not with LDL. It was found that Akt was activated in the presence of insulin. In the presence of either SB203580 or insulin, activation of p38 MAPK was significantly inhibited compared with stimulation by oxLDL alone. However, application of both insulin and wortmannin resulted in no significant difference compared with HUVEC stimulated by oxLDL only. 4. The results showed that apoptosis in HUVEC can be induced by oxLDL and involves p38 MAPK. It was also demonstrated that insulin inhibited oxLDL-induced apoptosis and may inhibit the activation of p38 MAPK through the PI3K/Akt pathway.

Association between plasma lipoprotein(a) and endothelial dysfunction in normocholesterolemic and non-diabetic patients with angiographically normal coronary arteries.

The present study was designed to examine whether elevated levels of lipoprotein(a) (Lp(a)) are related to the impairment of the endothelium-dependent vasoresponse to acetylcholine (ACh) in normocholesterolemic and non-diabetic human normal coronary arteries. ACh (30 microg) was injected into the left main coronary artery of 31 patients (serum low-density cholesterol <160mg/dl and fasting plasma glucose <126mg/dl) with angiographically normal coronary arteries, and the relation between diameter change and lipid levels was analyzed. The mean diameter change of all coronary segments examined (segments 6, 8, 11 and 13) was reduced by 14.6+/-26.5% in response to ACh, but increased by 23.3+/-6.0% in response to nitroglycerin, suggesting endothelial dysfunction in those arteries. The mean diameter change of the left anterior descending artery or left circumflex artery in each patient was negatively correlated only with the level of Lp(a). Stepwise multiple regression analysis also revealed that only Lp(a) among the lipids showed significant correlation with impaired vasodilation (p=0.033). These findings suggest that elevated levels of plasma Lp(a) might be a strong predictor of endothelial dysfunction in normocholesterolemic and non-diabetic subjects.

Hyperhomocysteinemia is associated with human coronary atherosclerosis through the reduction of the ratio of endothelium-bound to basal extracellular superoxide dismutase.

BACKGROUND: Homocysteine is involved in coronary atherosclerosis through oxidative stress, so the present study investigated the association between plasma concentrations of homocysteine and extracellular superoxide dismutase (EC-SOD) in coronary artery disease (CAD). METHODS AND RESULTS: The study group comprised 154 consecutive male patients with suspected CAD who had undergone angiography. Plasma concentrations of homocysteine and EC-SOD, which was determined before (basal) and after heparin therapy, were measured and the difference was designated as endothelium-bound EC-SOD. The EC-SOD ratio (endothelium-bound/basal EC-SOD) was also evaluated as an index of binding capacity. The plasma homocysteine concentration in the stenosis (+) group (n=97, 12.0+/-4.6 micromol/L) was significantly higher than that of the stenosis (-) group (n=57, 10.2+/-3.0 micromol/L, p=0.004). Plasma homocysteine correlated positively with the basal EC-SOD (r=0.377, p<0.001) and negatively with the EC-SOD ratio (r=-0.199, p=0.014). When the group was subdivided according to either homocysteine or the EC-SOD ratio, there were 2 groups with high homocysteine concentration and of these atherosclerosis was reduced in the group with a high EC-SOD ratio. CONCLUSIONS: In CAD patients, homocysteine is involved in the significant release of EC-SOD from the endothelium. Furthermore, the higher EC-SOD binding capacity, even at high concentrations of homocysteine, suggested that homocysteine-induced atherosclerosis was suppressed.