PCSK9 inhibitors suppress oxidative stress and inflammation in atherosclerotic development by promoting macrophage autophagy.

OBJECTIVES: Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors, a novel class of cholesterol-lowering drugs, can reduce atherosclerosis independent of systemic lipid changes. However, the mechanism by which PCSK9 inhibition protects against arteriosclerosis has not been fully elucidated. Recent evidence has demonstrated a correlation between PCSK9 inhibitors and oxidative stress, which accelerates atherosclerotic development. Moreover, an increasing number of studies have shown that autophagy protects the vasculature against atherosclerosis. Therefore, the aims of this study were to investigate the effect of PCSK9 inhibition on oxidative stress and autophagy in atherosclerosis and determine whether autophagy regulates PCSK9 inhibition-mediated oxidative stress and inflammation in macrophages. METHODS: Male apolipoprotein E (ApoE)-/- mice were fed a high-fat diet (HFD) for 8 weeks and then received the PCSK9 inhibitor (evolocumab), vehicle, or evolocumab plus chloroquine (CQ) for another 8 weeks. ApoE-/- mice in the control group were fed a regular (i.e., non-high-fat) diet for 16 weeks. Additional in vitro experiments were performed in oxidized low-density lipoprotein (ox-LDL)-treated human acute monocytic leukemia cell line THP-1-derived macrophages to mimic the pathophysiologic process of atherosclerosis. RESULTS: PCSK9 inhibitor treatment reduced oxidative stress, lipid deposition, and plaque lesion area and induced autophagy in HFD-fed ApoE-/- mice. Most importantly, the administration of chloroquine (CQ), an autophagy inhibitor, significantly reduced the beneficial effects of PCSK9-inhibitor treatment on oxidative stress, lipid accumulation, inflammation, and atherosclerotic lesions in HFD-fed ApoE-/- mice. The in vitro experiments further showed that the PCSK9 inhibitor enhanced autophagic flux in ox-LDL-treated THP-1-derived macrophages, as indicated by increases in the numbers of autophagosomes and autolysosomes. Moreover, the autophagy inhibitor CQ also reduced PCSK9 inhibition-mediated protection against oxidative stress, generation of reactive oxygen species (ROS) and inflammation in ox-LDL-treated THP-1-derived macrophages. CONCLUSIONS: This study reveals a novel protective mechanism by which PCSK9 inhibition enhances autophagy and thereby reduces oxidative stress and inflammation in atherosclerosis.

Improving CBCT image quality to the CT level using RegGAN in esophageal cancer adaptive radiotherapy.

OBJECTIVE: This study aimed to improve the image quality and CT Hounsfield unit accuracy of daily cone-beam computed tomography (CBCT) using registration generative adversarial networks (RegGAN) and apply synthetic CT (sCT) images to dose calculations in radiotherapy. METHODS: The CBCT/planning CT images of 150 esophageal cancer patients undergoing radiotherapy were used for training (120 patients) and testing (30 patients). An unsupervised deep-learning method, the 2.5D RegGAN model with an adaptively trained registration network, was proposed, through which sCT images were generated. The quality of deep-learning-generated sCT images was quantitatively compared to the reference deformed CT (dCT) image using mean absolute error (MAE), root mean square error (RMSE) of Hounsfield units (HU), and peak signal-to-noise ratio (PSNR). The dose calculation accuracy was further evaluated for esophageal cancer radiotherapy plans, and the same plans were calculated on dCT, CBCT, and sCT images. RESULTS: The quality of sCT images produced by RegGAN was significantly improved compared to the original CBCT images. ReGAN achieved image quality in the testing patients with MAE sCT vs. CBCT: 43.7 ± 4.8 vs. 80.1 ± 9.1; RMSE sCT vs. CBCT: 67.2 ± 12.4 vs. 124.2 ± 21.8; and PSNR sCT vs. CBCT: 27.9 ± 5.6 vs. 21.3 ± 4.2. The sCT images generated by the RegGAN model showed superior accuracy on dose calculation, with higher gamma passing rates (93.3 ± 4.4, 90.4 ± 5.2, and 84.3 ± 6.6) compared to original CBCT images (89.6 ± 5.7, 85.7 ± 6.9, and 72.5 ± 12.5) under the criteria of 3 mm/3%, 2 mm/2%, and 1 mm/1%, respectively. CONCLUSION: The proposed deep-learning RegGAN model seems promising for generation of high-quality sCT images from stand-alone thoracic CBCT images in an efficient way and thus has the potential to support CBCT-based esophageal cancer adaptive radiotherapy.

[Convenient Approach to Improve Correlation between Geometry and Dosimetric Parameters for Automatic Segmentation in Radiotherapy].

OBJECTIVE: To design a series of geometric indexes, which can improve the correlation between geometric parameters and dosimetric parameters. METHODS: 48 cases of upper abdomen were selected. Manual and automatic segmentation were performed for two organs at risk, which were stomach and duodenum. Three overlapping structures, which were the overlaps with target expanded by 5 mm, 10 mm and 20 mm, were generated for each organ at risk. The geometric parameters of overlapping structures were calculated. The relationship between these geometric parameters and the dosimetric parameters of organs was investigated. RESULTS: When the geometric parameters of overlapping structures related to the target expand 5 mm, 10 mm and 20 mm were larger than 0.4, 0.6 and 0.8 respectively, the maximum dose differences of manual and automatic segmentation were less than 3 Gy. For the case with no overlaps between the organs and the target expansions, the overlap structure corresponding to target expanding 20 mm were recommended for safety considerations. CONCLUSIONS: For organs at risk in the upper abdomen, the overlapping geometric parameters were closely related to the maximum dose of organs. Overlapping geometric parameters could predict whether the difference of maximum dose caused by automaticsegmentation was clinically acceptable or not.

Effects of the ketogenic diet in mice with hind limb ischemia.

BACKGROUND: The ketogenic diet (KD) has anti-tumor and anti-diabetic effects in addition to its anti-epileptic role. It could also improve cardiac function and attenuate neurological insult. However, the effect of KD on blood perfusion or tissue recovery after ischemia remains largely unknown. Thus, we observed blood flow and ischemic tissue recovery following hind limb ischemia (HLI) in mice. METHODS: C57 mice were fed with either a KD or normal diet (ND) for 2 weeks, before inducing hind limb ischemia, blood perfusion of ischemic limb tissue was observed at 0, 7, and 21 days post operation. RESULTS: KD not only decreased blood perfusion of ischemic limb tissue but also delayed muscle recovery after ischemia, induced muscle atrophy of non-ischemic tissue compared to mice fed with ND. Furthermore, KD delayed wound healing at the surgical site and aggravated inflammation of the ischemic tissue. At the cellular level, KD altered the metabolic status of limb tissue by decreasing glucose and ketone body utilization while increasing fatty acid oxidation. Following ischemia, glycolysis, ketolysis, and fatty acid utilization in limb tissue were all further reduced by KD, while ketogenesis was mildly increased post KD in this mice model. CONCLUSION: The KD may cause impaired tissue recovery after ischemia and possible muscle atrophy under a prolonged diet. Our results hint that patients with limb ischemia should avoid ketogenic diet.

Voxel-Level BED Corrected Dosimetric and Radiobiological Assessment of 2 Kinds of Hybrid Radiotherapy Planning Methods for Stage III NSCLC.

Background/purpose: To access the comparative dosimetric and radiobiological advantages of two methods of intensity-modulated radiation therapy (IMRT)-based hybrid radiotherapy planning for stage III nonsmall cell lung cancer (NSCLC). Methods: Two hybrid planning methods were respectively characterized by conventional fraction radiotherapy (CFRT) and stereotactic body radiotherapy (SBRT) and CFRT and simultaneous integrated boost (SIB) planning. All plans were retrospectively completed using the 2 methods for 20 patients with stage III NSCLC. CFRT and SBRT dose regimes 2 Gy × 30 f and 12.5 Gy × 4 f were, respectively, used for planning target volume of lymph node (PTVLN) and planning target volume of the primary tumor (PTVPT), while dose regimes 2 Gy × 26 f for PTVLN and sequential 2 Gy × 4 f for PTVLN combined with 12.5 Gy × 4 f for PTVPT were adopted for CFRT and SIB plans. SBRT and SIB EQD2 dose were calculated voxel by voxel, and then, respectively, superimposed with 30-fraction and 26-fraction CFRT plan dose to achieve biological equivalent dose (BED) dosimetric parameters of CFRT and SBRT and CFRT and SIB plans. Tumor control probability (TCP)/normal tissue complication probability (NTCP) was, respectively, calculated by equivalent uniform dose/Lyman-Kutcher-Burman models. BED plan parameters and TCP/NTCP were analyzed between 2 methods of hybrid planning. Primary tumor/lymph node (LN)/total TCP values were, respectively, evaluated as a function of the radiation dose needed to control 50% of tumor (TCD50) for 20 patients. Dosimetric errors were analyzed by nontransit electronic portal imaging device dosimetry measurement during hybrid plan delivery. Results: Statistically lower BED plan parameters of PTVLN D2 and homogeneity index resulted in slightly lower averaged LN/total TCP curves by CFRT and SIB planning. The gaps between Max and Min LN/total TCP curves were significantly closer for CFRT and SIB planning, which indicated better robustness of LN/total TCPs. A lower esophagus dose resulted in a lower esophagus NTCP by CFRT and SIB planning, which may be compromised by 1 week shorter overall treatment time by CFRT and SIB irradiation. Spinal cord Dmax was significantly reduced by CFRT and SIB plans. The dose verification results of the subplans involved in hybrid plans were acceptable, which showed that the 2 methods of hybrid planning could be delivered accurately in our center. Conclusion: CFRT and SIB plannings have more advantages on BED plan parameters and TCP/NTCP than CFRT and SBRT planning, and both methods of IMRT-based hybrid planning could be executed accurately for stage III NSCLC. The effectiveness of the results needs to be validated in the hybrid trial.

A Tryptophan Metabolite of the Microbiota Improves Neovascularization in Diabetic Limb Ischemia.

Diabetes mellitus (DM) is accompanied by a series of macrovascular and microvascular injuries. Critical limb ischemia is the most severe manifestation of peripheral artery disease (PAD) caused by DM and is almost incurable. Therapeutic modulation of angiogenesis holds promise for the prevention of limb ischemia in diabetic patients with PAD. However, no small-molecule drugs are capable of promoting diabetic angiogenesis. An endogenous tryptophan metabolite, indole-3-aldehyde (3-IAld), has been found to have proangiogenic activity in endothelial cells. Nevertheless, the role of 3-IAld in diabetic angiogenesis remains unknown. Here, we found that 3-IAld ameliorated high glucose-induced mitochondrial dysfunction, decreasing oxidative stress and apoptosis and thus improving neovascularization.

Legumain Is an Endogenous Modulator of Integrin αvß3 Triggering Vascular Degeneration, Dissection, and Rupture.

BACKGROUND: The development of thoracic aortic dissection (TAD) is closely related to extracellular matrix degradation and vascular smooth muscle cell (VSMC) transformation from contractile to synthetic type. LGMN (legumain) degrades extracellular matrix components directly or by activating downstream signals. The role of LGMN in VSMC differentiation and the occurrence of TAD remains elusive. METHODS: Microarray datasets concerning vascular dissection or aneurysm were downloaded from the Gene Expression Omnibus database to screen differentially expressed genes. Four-week-old male Lgmn knockout mice (Lgmn-/-), macrophage-specific Lgmn knockout mice (LgmnF/F;LysMCre), and RR-11a-treated C57BL/6 mice were given BAPN (ß-aminopropionitrile monofumarate; 1 g/kg/d) in drinking water for 4 weeks for TAD modeling. RNA sequencing analysis was performed to recapitulate transcriptome profile changes. Cell interaction was examined in macrophage and VSMC coculture system. The reciprocity of macrophage-derived LGMN with integrin αvß3 in VSMCs was tested by coimmunoprecipitation assay and colocalization analyses. RESULTS: Microarray datasets from the Gene Expression Omnibus database indicated upregulated LGMN in aorta from patients with TAD and mice with angiotensin II-induced AAA. Elevated LGMN was evidenced in aorta and sera from patients with TAD and mice with BAPN-induced TAD. BAPN-induced TAD progression was significantly ameliorated in Lgmn-deficient or inhibited mice. Macrophage-specific deletion of Lgmn alleviated BAPN-induced extracellular matrix degradation. Unbiased profiler polymerase chain reaction array and Gene Ontology analysis displayed that LGMN regulated VSMC phenotype transformation. Macrophage-specific deletion of Lgmn ameliorated VSMC phenotypic switch in BAPN-treated mice. Macrophage-derived LGMN inhibited VSMC differentiation in vitro as assessed by macrophages and the VSMC coculture system. Macrophage-derived LGMN bound to integrin αvß3 in VSMCs and blocked integrin αvß3, thereby attenuating Rho GTPase activation, downregulating VSMC differentiation markers and eventually exacerbating TAD development. ROCK (Rho kinase) inhibitor Y-27632 reversed the protective role of LGMN depletion in vascular dissection. CONCLUSIONS: LGMN signaling may be a novel target for the prevention and treatment of TAD.

ß-Hydroxybutyrate Exacerbates Hypoxic Injury by Inhibiting HIF-1α-Dependent Glycolysis in Cardiomyocytes-Adding Fuel to the Fire?

PURPOSE: Ketone body oxidation yields more ATP per mole of consumed oxygen than glucose. However, whether an increased ketone body supply in hypoxic cardiomyocytes and ischemic hearts is protective or not remains elusive. The goal of this study is to determine the effect of ß-hydroxybutyrate (ß-OHB), the main constituent of ketone bodies, on cardiomyocytes under hypoxic conditions and the effects of ketogenic diet (KD) on cardiac function in a myocardial infarction (MI) mouse model. METHODS: Human peripheral blood collected from patients with acute myocardial infarction and healthy volunteers was used to detect the level of ß-OHB. N-terminal proB-type natriuretic peptide (NT-proBNP) levels and left ventricular ejection fractions (LVEFs) were measured to study the relationship between plasma ß-OHB and cardiac function. Adult mouse cardiomyocytes and MI mouse models fed a KD were used to research the effect of ß-OHB on cardiac damage. qPCR, western blot analysis, and immunofluorescence were used to detect the interaction between ß-OHB and glycolysis. Live/dead cell staining and imaging, lactate dehydrogenase, Cell Counting Kit-8 assays, echocardiography, and 2,3,5-triphenyltetrazolium chloride staining were performed to evaluate the cardiomyocyte death, cardiac function, and infarct sizes. RESULTS: ß-OHB level was significantly higher in acute MI patients and MI mice. Treatment with ß-OHB exacerbated cardiomyocyte death and decreased glucose absorption and glycolysis under hypoxic conditions. These effects were partially ameliorated by inhibiting hypoxia-inducible factor 1α (HIF-1α) degradation via roxadustat administration in hypoxia-stimulated cardiomyocytes. Furthermore, ß-OHB metabolisms were obscured in cardiomyocytes under hypoxic conditions. Additionally, MI mice fed a KD exhibited exacerbated cardiac dysfunction compared with control chow diet (CD)-fed MI mice. CONCLUSION: Elevated ß-OHB levels may be maladaptive to the heart under hypoxic/ischemic conditions. Administration of roxadustat can partially reverse these harmful effects by stabilizing HIF-1α and inducing a metabolic shift toward glycolysis for energy production.

Dietary Cholesterol Exacerbates Statin-Induced Hepatic Toxicity in Syrian Golden Hamsters and in Patients in an Observational Cohort Study.

PURPOSE: Statins are inhibitors of 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) reductase, which is involved in cholesterol synthesis. The major side effects of statins include muscle- and liver-related toxicity. Muscle toxicity is highly associated with polymorphisms in cytochrome P450 proteins (CYPs), as predicted by pharmacogenomics. However, the mechanisms of hepatotoxicity are not well understood. Due to differences in cholesterol metabolism, statins are well tolerated in mice. In contrast, hamsters exhibit metabolic traits similar to humans and are suitable for studying the hepatotoxicity of statins. METHODS: We investigated the effect of rosuvastatin (RSV) on liver damage in wild-type (WT) hamsters fed a high-cholesterol diet (HCD) and LDLR knockout (LDLR-/-) hamsters that developed spontaneous hypercholesterolemia. Two cohorts of clinical subjects (clinical registry number: 2017001) taking atorvastatin (ATV) were recruited for direct (assessment of cholesterol intake individually, n = 44) and indirect (celebratory meals/holiday season, n = 1993) examination of dietary cholesterol intake and liver damage, as indicated by elevation of alanine aminotransferase (ALT). RESULTS: RSV at a dose of 10 mg/kg caused fatal liver damage only in HCD-fed WT hamsters, while LDLR-/- hamsters with the same cholesterol levels were resistant to this toxic effect. In the human studies, we observed that the incidence of hepatic toxicity in patients receiving long-term ATV treatment was higher in patients with greater dietary cholesterol intake and in patients who consumed more food during Chinese holidays. CONCLUSION: Our results propose, for the first time, that dietary cholesterol significantly contributes to statin-related hepatotoxicity, providing valuable insight into the clinical use of statins.

[Cardiac protective effect of the autoantibody against ß3-adrenoceptor in rats with experimental heart failure].

OBJECTIVE: To explore the effect of the autoantibody against the ß3-adrenoceptor on rats with experimental heart failure. METHOD: The peptide corresponding to the sequence of ß3 adrenoceptor was synthesized to actively immunize the rats, ELISA was used to detect the serum level of autoantibody against the ß3-adrenoceptor (ß3AA). Total IgGs were extracted from the serum containing ß3AA in immunized rats. Aortic banding surgery was used to establish the heart failure model in male Wistar rats and rats were divided into the sham group (n = 8), heart failure group(n = 8),ß3AA-immunized heart failure group (HF+ß3AA, n = 8) and corresponding negative IgG-immunized heart failure group (HF+ IgG, n = 8).In 6 weeks and 8 weeks after aortic banding surgery, the serum levels of NT-pro brain natriuretic peptide (NT-proBNP) were assayed with ELISA assay and cardiac function was assessed by echocardiography. RESULTS: ß3AA was used to immunize rat with heart failure, the serum level of ß3AA was stable at 50 days post immunization. At 8 weeks after aortic banding surgery, heart failure group showed significantly increased LVEDD [(6.92 ± 0.22) mm vs.(5.62 ± 0.19) mm, P < 0.001], LVESD [(4.63 ± 0.23) mm vs.(3.50 ± 0.20) mm, P < 0.01] and IVS [(2.44 ± 0.06) mm vs.(2.28 ± 0.05) mm, P < 0.05], and decreased LVEF[(62.07 ± 3.99)% vs.(79.63 ± 3.02)%, P < 0.01] and LVFS [(31.46 ± 3.22)% vs.(43.65 ± 2.68) %, P < 0.05] compared with the sham group.HF+ß3AA IgG group showed decreased LVEDD [(6.07 ± 0.30) mm vs.(6.92 ± 0.24) mm, P < 0.05] and LVESD [(3.92 ± 0.22) mm vs.(4.68 ± 0.23) mm, P < 0.05], and higher LVEF [(70.29 ± 1.78)% vs.(61.95 ± 3.03)%, P < 0.05] and LVFS [(38.08 ± 2.32)% vs.(30.50 ± 1.82)%, P < 0.05] compared to the HF+ IgG group.In addition, compared with the HF+ IgG group, HF+ß3AA IgG group showed decreased serum levels of NT-proBNP [(196.43 ± 6.56) pg/ml vs.(242.13 ± 7.86) pg/ml, P < 0.01]. CONCLUSION: Our results demonstrate that ß3AA can improve cardiac function and reduce the serum levels of NT-proBNP in rat with heart failure.

Autoantibodies against the ß3-adrenoceptor protect from cardiac dysfunction in a rat model of pressure overload.

ß3-Adrenoceptors (ß3-ARs) mediate a negative inotropic effect in human ventricular cardiomyocytes, which is opposite to that of ß1- and ß2-ARs. It has been previously demonstrated that autoantibodies against the ß1/ß2-AR exist in the sera of some patients with heart failure (HF) and these autoantibodies display agonist-like effects. Our aim in this study was to observe whether autoantibodies against the ß3-AR (ß3-AR Abs) exist in the sera of patients with HF and to assess the effects of ß3-AR Abs on rat model of pressure overload cardiomyopthy. In the present study, the level of ß3-AR Abs in the sera of HF patients was screened by ELISA. ß3-AR Abs from HF patients were administrated to male adult rats with abdominal aortic banding (AAB), and the cardiac function was measured by echocardiographic examination and hemodynamic studies. The biological effects of this autoantibody on cardiomyocytes were evaluated using a motion-edge detection system, intracellular calcium transient assay, and patch clamp techniques. Compared to healthy subjects, the frequency of occurrence and titer of ß3-AR Abs in the sera of HF patients were greatly increased, and ß3-AR Abs could prevent LV dilation and improve the cardiac function of rats with AAB. ß3-AR Abs exhibited negative chronotropic and inotropic effects and were accompanied by a decreased intracellular Ca(2+) transient and membrane L-type Ca(2+) current in cardiomyocytes. Our results demonstrated the existence of ß3-AR Abs in the sera of patients with HF and found that this autoantibody could alleviate the cardiac dysfunction induced by pressure-overload in AAB rats.

Vascular dysfunction in the offspring of AT1 receptor antibody-positive pregnant rats during high-salt diet.

Antibody against the angiotensin AT1 receptor (AT1-Ab) could disturb placental development. The placenta is the key organ between mother and fetus. Placental damage will seriously impair fetal growth and development in utero, leading to intrauterine growth restriction (IUGR). Based on the fetal origins of adult disease (FOAD) hypothesis, IUGR could increase a propensity to develop adult onset cardiovascular disease (CVD). The present study was designed to determine whether vascular function has changed in the adult offspring of AT1-Ab positive pregnant rats. Twenty four female rats (8-week-old, AT1-Ab negative) were randomly divided into two groups, immunized and vehicle groups. Immunized group received active immunization to establish AT1-Ab-positive model, while vehicle group was subjected to Freund's adjuvant without antigen. After 8 weeks of immunization, the antibody titers in sera from the female rats were detected by enzyme-linked immunosorbent assay (ELISA). Then all the female rats were mated with normal Wistar male rats and became pregnant. Immunized/vehicle group offspring rats (I offspring/V offspring) were raised to 40-week-old under standard chow feeding. Then the two groups' offspring rats were given a high-salt diet for 12 weeks (4% NaCl in chow feeding). Systolic blood pressure (SBP) was measured dynamically by noninvasive blood pressure system. The vascular ring experiment was performed to detect vascular function and reactivity. As detected by ELISA, the titers of antibody peaked at the 8th week (OD values: 2.75 ± 0.08 vs 0.33 ± 0.01, P < 0.01 vs vehicle group at the same time point). There was no significant difference of SBP between the two groups' offspring rats during the high-salt diet (P > 0.05). Isolated thoracic aortic rings of I offspring had significantly decreased constriction under norepinephrine treatment (P < 0.01 vs V offspring) and significantly decreased dilation under acetylcholine treatment (P < 0.05 vs V offspring). These results suggest that the offspring of AT1-Ab-positive pregnant rats are more susceptible to vascular functional abnormality while being fed high-salt diet.

[The proarrhythmic effects of autoantibody against beta1 adrenergic receptor].

OBJECTIVE: To investigate the distribution characteristics of autoantibody against beta1 adrenergic receptor (beta1 AR) in the sera of arrhythmia patients and whether the autoantibody could induce arrhythmia. METHODS: Healthy subjects and patients with arrhythmia or coronary artery disease were chosen. The autoantibody against beta1 AR in the sera was screened by enzyme-linked immunosorbent assay (ELISA). IgG in the positive autoantibody sera from arrhythmia patients were purified and administrated to normal rats; then the ECGs were dynamic monitored. RESULTS: The positive rate of autoantibody against beta1 AR in arrhythmia patients was 52.8%, which was significantly higher than that in coronary heart disease group (24%, P < 0.01) and healthy people group (5%, P < 0.01), respectively. Moreover, the autoantibody against beta1 AR could lead to the occurring of arrhythmia in normal rats, most of which were ventricular arrhythmia. CONCLUSION: In the sera of arrhythmia patients, the autoantibody against beta1 AR has a high titer and it could lead to the arrhythmia of rats in vivo.

Inhibition of iNOS protects endothelial-dependent vasodilation in aged rats.

AIM: To examine whether iNOS contributes to endothelial dysfunction in aged rats. METHODS: Male Sprague Dawley rats were divided into three groups: young rats, aged rats treated with vehicle and aged rats treated with N-[3-(Aminomethyl) benzyl] acetamidine (1400W, 1 mg/kg, ip). Vasorelaxation was measured in isolated thoracic aorta. iNOS expression of thoracic aortic arteries was detected using immunohistochemistry and Western blot. Nitrotyrosine (a marker for peroxynitrite formation) content and expression in thoracic aortic tissue were determined using enzyme linked immunosorbent assay and immunohistochemistry. RESULTS: Maximal relaxation induced by acetylcholine (10⁻9 to 10⁻5 mol/L) in the aged rats treated with vehicle was significantly decreased (70%±15%, P<0.01), as compared with the young rats (95%±8%). However, the maximal relaxation induced by acidified NaNO2 (an endothelium-independent vasodilator) had no significant difference between the two groups. Moreover, iNOS and nitrotyrosine expression increased in the vessels of the aged rats. In the aged rats treated with 1400W (a highly selective iNOS inhibitor) nitrotyrosine expression was reduced and acetylcholine-induced vasorelaxation was markedly improved (maximal relaxation was increased to 87%±8%, P<0.05), but the acidified NaNO2-induced vasorelaxation had no significant change. CONCLUSION: Our study demonstrated that inhibition of iNOS by 1400W increased endothelium-dependent vasodilation in aged rats. The mechanism was related with attenuation of peroxynitrite formation.

Hyperlipidemia does not prevent the cardioprotection by postconditioning against myocardial ischemia/reperfusion injury and the involvement of hypoxia inducible factor-1alpha upregulation.

Hyperlipidemia is regarded as an independent risk factor in the development of ischemic heart disease, and it can increase the myocardial susceptibility to ischemia/reperfusion (I/R) injury. Ischemic postconditioning (Postcon) has been demonstrated to attenuate the myocardial injury induced by I/R in normal conditions. But the effect of ischemic Postcon on hyperlipidemic animals is unknown. Hypoxia inducible factor-1 (HIF-1) has been demonstrated to play a central role in the cardioprotection by preconditioning, which is one of the protective strategies except for Postcon. The aim of this study was to determine whether Postcon could reduce myocardial injury in hyperlipidemic animals and to assess whether HIF-1 was involved in Postcon mechanisms. Male Wistar rats underwent the left anterior descending coronary occlusion for 30 min followed by 180 min of reperfusion with or without Postcon after fed with high fat diet or normal diet for 8 weeks. The detrimental indices induced by the I/R insult included infarct size, plasma creatine kinase activity and caspase-3 activity. Results showed that hyperlipidemia remarkably enhanced the myocardial injury induced by I/R, while Postcon significantly decreased the myocardial injury in both normolipidemic and hyperlipidemic rats. Moreover, both hyperlipidemia and I/R promoted the HIF-1alpha expression. Most importantly, we have for the first time demonstrated that Postcon further induced a significant increase in HIF-1alpha protein level not only in normolipidemic but also in hyperlipidemic conditions. Thus, Postcon reduces the myocardial injury induced by I/R in normal and hyperlipidemic animals, and HIF-1alpha upregulation may involve in the Postcon-mediated cardioprotective mechanisms.

[Establishment and application of double-antibody sandwich ELISA for determination of 3-nitrotyrosine].

AIM: To prepare the working standards of 3-nitrotyrosine (3-NT) and establish a two-antibody-sandwich ELISA for determining the concentration of peroxynitrite in the tissue. METHODS: Nitrated bovine serum albumin was prepared by additions of an alkaline stock solution of peroxynitrite which was synthesized by a quenched-flow reactor. The monoclone anti-3-NT antibody from mouse was used as coating antibody and the polyclone anti-3-NT antibody from as labeling antibody to prepare the standard work curve by orthogonal design. The concentrations of 3-NT in cardiac tissue from rats subjected to myocardial ischemia and reperfusion (MI/R) were analyzed. RESULTS: A two-antibody-sandwich ELISA method for measuring 3-NT content in biological fluids and homogenates was successfully established. The detecting limit was 0.1 ng x ml(-1) and the linear range of standard work curve was 0.15 - 7.50 ng x ml(-1) (r2 = 0.995). The 3-NT concentration in cardiac tissue from rats subjected to MI/R (1022.42 +/- 97.35 ng x mg pro(-1)) was significantly higher than that in the sham group (246.58 +/- 56.52 ng x mg pro(-1), P < 0.01). CONCLUSION: A two-antibody-sandwich ELISA was established for determining the 3-NT concentration in the tissue and conveniently, quickly, accurately quantitative analysis of the content of 3-NT. The assay provides a new method for quantitative analysis of the peroxyinitrite in the future.