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.

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.

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.

[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.

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.

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.

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.

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.

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.