Melatonin Ameliorates Atherosclerotic Plaque Vulnerability by Regulating PPARδ-Associated Smooth Muscle Cell Phenotypic Switching.

Vulnerable atherosclerotic plaque rupture, the leading cause of fatal atherothrombotic events, is associated with an increased risk of mortality worldwide. Peroxisome proliferator-activated receptor delta (PPARδ) has been shown to modulate vascular smooth muscle cell (SMC) phenotypic switching, and, hence, atherosclerotic plaque stability. Melatonin reportedly plays a beneficial role in cardiovascular diseases; however, the mechanisms underlying improvements in atherosclerotic plaque vulnerability remain unknown. In this study, we assessed the role of melatonin in regulating SMC phenotypic switching and its consequential contribution to the amelioration of atherosclerotic plaque vulnerability and explored the mechanisms underlying this process. We analyzed features of atherosclerotic plaque vulnerability and markers of SMC phenotypic transition in high-cholesterol diet (HCD)-fed apolipoprotein E knockout (ApoE-/-) mice and human aortic SMCs (HASMCs). Melatonin reduced atherosclerotic plaque size and necrotic core area while enhancing collagen content, fibrous cap thickness, and smooth muscle alpha-actin positive cell coverage on the plaque cap, which are all known phenotypic characteristics of vulnerable plaques. In atherosclerotic lesions, melatonin significantly decreased the synthetic SMC phenotype and KLF4 expression and increased the expression of PPARδ, but not PPARα and PPARγ, in HCD-fed ApoE-/- mice. These results were subsequently confirmed in the melatonin-treated HASMCs. Further analysis using PPARδ silencing and immunoprecipitation assays revealed that PPARδ plays a role in the melatonin-induced SMC phenotype switching from synthetic to contractile. Collectively, we provided the first evidence that melatonin mediates its protective effect against plaque destabilization by enhancing PPARδ-mediated SMC phenotypic switching, thereby indicating the potential of melatonin in treating atherosclerosis.

Activation of heme oxygenase-1 by laminar shear stress ameliorates high glucose-induced endothelial cell and smooth muscle cell dysfunction.

High glucose (HG)-induced endothelial cell (EC) and smooth muscle cell (SMC) dysfunction is critical in diabetes-associated atherosclerosis. However, the roles of heme oxygenase-1 (HO-1), a stress-response protein, in hemodynamic force-generated shear stress and HG-induced metabolic stress remain unclear. This investigation examined the cellular effects and mechanisms of HO-1 under physiologically high shear stress (HSS) in HG-treated ECs and adjacent SMCs. We found that exposure of human aortic ECs to HSS significantly increased HO-1 expression; however, this upregulation appeared to be independent of adenosine monophosphate-activated protein kinase, a regulator of HO-1. Furthermore, HSS inhibited the expression of HG-induced intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and reactive oxygen species (ROS) production in ECs. In an EC/SMC co-culture, compared with static conditions, subjecting ECs close to SMCs to HSS and HG significantly suppressed SMC proliferation while increasing the expression of physiological contractile phenotype markers, such as α-smooth muscle actin and serum response factor. Moreover, HSS and HG decreased the expression of vimentin, an atherogenic synthetic phenotypic marker, in SMCs. Transfecting ECs with HO-1-specific small interfering (si)RNA reversed HSS inhibition on HG-induced inflammation and ROS production in ECs. Similarly, reversed HSS inhibition on HG-induced proliferation and synthetic phenotype formation were observed in co-cultured SMCs. Our findings provide insights into the mechanisms underlying EC-SMC interplay during HG-induced metabolic stress. Strategies to promote HSS in the vessel wall, such as continuous exercise, or the development of HO-1 analogs and mimics of the HSS effect, could provide an effective approach for preventing and treating diabetes-related atherosclerotic vascular complications.

Adipose Tissue-Derived CCL5 Enhances Local Pro-Inflammatory Monocytic MDSCs Accumulation and Inflammation via CCR5 Receptor in High-Fat Diet-Fed Mice.

The C-C chemokine motif ligand 5 (CCL5) and its receptors have recently been thought to be substantially involved in the development of obesity-associated adipose tissue inflammation and insulin resistance. However, the respective contributions of tissue-derived and myeloid-derived CCL5 to the etiology of obesity-induced adipose tissue inflammation and insulin resistance, and the involvement of monocytic myeloid-derived suppressor cells (MDSCs), remain unclear. This study used CCL5-knockout mice combined with bone marrow transplantation (BMT) and mice with local injections of shCCL5/shCCR5 or CCL5/CCR5 lentivirus into bilateral epididymal white adipose tissue (eWAT). CCL5 gene deletion significantly ameliorated HFD-induced inflammatory reactions in eWAT and protected against the development of obesity and insulin resistance. In addition, tissue (non-hematopoietic) deletion of CCL5 using the BMT method not only ameliorated adipose tissue inflammation by suppressing pro-inflammatory M-MDSC (CD11b+Ly6G-Ly6Chi) accumulation and skewing local M1 macrophage polarization, but also recruited reparative M-MDSCs (CD11b+Ly6G-Ly6Clow) and M2 macrophages to the eWAT of HFD-induced obese mice, as shown by flow cytometry. Furthermore, modulation of tissue-derived CCL5/CCR5 expression by local injection of shCCL5/shCCR5 or CCL5/CCR5 lentivirus substantially impacted the distribution of pro-inflammatory and reparative M-MDSCs as well as macrophage polarization in bilateral eWAT. These findings suggest that an obesity-induced increase in adipose tissue CCL5-mediated signaling is crucial in the recruitment of tissue M-MDSCs and their trans-differentiation to tissue pro-inflammatory macrophages, resulting in adipose tissue inflammation and insulin resistance.

Post-recurrence survival analysis in patients with oligo-recurrence after curative esophagectomy.

BACKGROUND: Recurrent esophageal cancer is associated with dismal prognosis. There is no consensus about the role of surgical treatments in patients with limited recurrences. This study aimed to evaluate the role of surgical resection in patients with resectable recurrences after curative esophagectomy and to identify their prognostic factors. METHODS: We retrospectively reviewed patients with recurrent esophageal cancer after curative esophagectomy between 2004 and 2017 and included those with oligo-recurrence that was amenable for surgical intent. The prognostic factors of overall survival (OS) and post-recurrence survival (PRS), as well as the survival impact of surgical resection, were analyzed. RESULTS: Among 654 patients after curative esophagectomies reviewed, 284 (43.4%) had disease recurrences. The recurrences were found resectable in 63 (9.6%) patients, and 30 (4.6%) patients received surgery. The significant prognostic factors of PRS with poor outcome included mediastinum lymph node (LN) recurrence and pathologic T3 stage. In patients with and without surgical resection for recurrence cancer, the 3-year OS rates were 65.6 and 47.6% (p = 0.108), while the 3-year PRS rates were 42.9 and 23.5% (p = 0.100). In the subgroup analysis, surgery for resectable recurrence, compared with non-surgery, could achieve better PRS for patients without any comorbidities (hazard ratio 0.36, 95% CI: 0.14 to 0.94, p = 0.038). CONCLUSIONS: Mediastinum LN recurrence or pathologic T3 was associated with worse OS and PRS in patients with oligo-recurrences after curative esophagectomies. No definite survival benefit was noted in patients undergoing surgery for resectable recurrence, except in those without comorbidities.

PPARα, δ and FOXO1 Gene Silencing Overturns Palmitate-Induced Inhibition of Pyruvate Oxidation Differentially in C2C12 Myotubes.

The molecular mechanisms by which free fatty acids (FFA) inhibit muscle glucose oxidation is still elusive. We recently showed that C2C12 myotubes treated with palmitate (PAL) presented with greater protein expression levels of PDK4 and transcription factors PPARα and PPARδ and lower p-FOXO/t-FOXO protein ratios when compared to control. This was complemented with the hallmarks of metabolic inflexibility (MI), i.e., reduced rates of glucose uptake, PDC activity and maximal pyruvate-derived ATP production rates (MAPR). However, the relative contribution of these transcription factors to the increase in PDK4 and reduced glucose oxidation could not be established. Therefore, by using a similar myotube model, a series of individual siRNA gene silencing experiments, validated at transcriptional and translation levels, were performed in conjunction with measurements of glucose uptake, PDC activity, MAPR and concentrations of metabolites reflecting PDC flux (lactate and acetylcarnitine). Gene silencing of PPARα, δ and FOXO1 individually reduced PAL-mediated inhibition of PDC activity and increased glucose uptake, albeit by different mechanisms as only PPARδ and FOXO1 silencing markedly reduced PDK4 protein content. Additionally, PPARα and FOXO1 silencing, but not PPARδ, increased MAPR with PAL. PPARδ silencing also decreased FOXO1 protein. Since FOXO1 silencing did not alter PPARδ protein, this suggests that FOXO1 might be a PPARδ downstream target. In summary, this study suggests that the molecular mechanisms by which PAL reduces PDC-mediated glucose-derived pyruvate oxidation in muscle occur primarily through increased PPARδ and FOXO1 mediated increases in PDK4 protein expression and secondarily through PPARα mediated allosteric inhibition of PDC flux. Furthermore, since PPARδ seems to control FOXO1 expression, this may reflect an important role for PPARδ in preventing glucose oxidation under conditions of increased lipid availability.

Preoperative consolidation-to-tumor ratio is effective in the prediction of lymph node metastasis in patients with pulmonary ground-glass component nodules.

BACKGROUND: Preoperative positron emission tomography/computed tomography (PET/CT) is recommended as a guideline for staging of lung cancer. However, for patients with pulmonary ground-glass opacity (GGO) nodules who are supposed to have a relatively low risk of incidence of lymphatic metastasis, it remains uncertain whether PET/CT is more effective than consolidation-to-tumor ratio (CTR) in the prediction of regional lymphatic metastasis. METHODS: The data on patients who underwent surgery for lung cancer from 2011 to 2016 were collected retrospectively, which included CTR, results of PET/CT, and pathological characteristics. The patients who had undergone preoperative PET/CT were identified to find the risk factors for lymphatic metastasis. A receiver operating characteristic (ROC) curve and multiple logistic regression was utilized to clarify the predictive value of CTR and main tumor maximal standardized uptake value (SUVmax). RESULTS: Among 217 patients who had PET/CT before lobectomy, chest computed tomography revealed that 75 patients had CTR greater than 62%. The patients with lymphatic metastasis were shown to have higher CTR and higher main tumor SUVmax. Multiple logistic regression showed that younger age (<60 years), higher main tumor SUVmax on PET/CT, and greater CTR were independent predictive factors for lymphatic metastasis. The area under the ROC curve was comparable, 0.817 for CTR, and 0.816 for main tumor SUVmax. CONCLUSIONS: The present study revealed that CTR was not inferior to main tumor SUVmax considering the predictive power for lymphatic metastasis preoperatively in lung cancer patients with a GGO component. PET/CT might not be necessary preoperatively in selected patients.

Significance of preoperative biopsy in radiological solid-dominant clinical stage I non-small-cell lung cancer.

OBJECTIVES: The present study aimed to clarify the association between preoperative biopsy and surgical outcomes in clinical stage I non-small-cell lung cancer (NSCLC) with different proportions of ground-glass opacity (GGO). METHODS: Data on patients who underwent pulmonary resection for NSCLC from 2006 to 2016 were drawn from a prospective registered database and analysed retrospectively. Patient characteristics collected included tumour size, location and staging, surgical approach, consolidation-tumour ratio, histopathology and the presence or absence of preoperative biopsy to identify the independent prognostic factors of disease-free survival (DFS) and cancer-specific survival. A 1:1 propensity score matching was conducted between the preoperative biopsy and reference groups based on their baseline characteristics measured before the decision for preoperative biopsy. RESULTS: A total of 1427 patients were collected to achieve an overall 5-year DFS as 84.5% (median follow-up: 67.3 months), stratified to be 99.5% in the GGO-dominant group (n = 430) and 78.2% in the solid-dominant group (n = 997). Only 2 patients (0.5%) in the GGO-dominant group experienced tumour recurrence. For solid-dominant tumours matched with propensity scores (279 in preoperative biopsy vs 279 in reference group), the independent predictors of DFS included preoperative biopsy, sublobar resection, pathological staging and angiolymphatic invasion. Preoperative biopsy was a predictor of cancer-specific survival in univariable analysis but was not in multivariable analysis. Significant differences were also found between matched groups in those with late-delay surgery, but not in patients receiving preoperative biopsy with early-delay surgery (≤21 days). CONCLUSION: Preoperative biopsy may worsen surgical outcomes in patients with clinical stage I, solid-dominant NSCLC.

PPARδ and FOXO1 Mediate Palmitate-Induced Inhibition of Muscle Pyruvate Dehydrogenase Complex and CHO Oxidation, Events Reversed by Electrical Pulse Stimulation.

The mechanisms behind the reduction in muscle pyruvate dehydrogenase complex (PDC)-controlled carbohydrate (CHO) oxidation during chronic high-fat dietary intake are poorly understood, as is the basis of CHO oxidation restoration during muscle contraction. C2C12 myotubes were treated with (300 µM) palmitate or without (control) for 16 h in the presence and absence of electrical pulse stimulation (EPS, 11.5 V, 1 Hz, 2 ms). Compared to control, palmitate reduced cell glucose uptake (p < 0.05), PDC activity (p < 0.01), acetylcarnitine accumulation (p < 0.05) and glucose-derived mitochondrial ATP production (p < 0.01) and increased pyruvate dehydrogenase kinase isoform 4 (PDK4) (p < 0.01), peroxisome proliferator-activated receptor alpha (PPARα) (p < 0.01) and peroxisome proliferator-activated receptor delta (PPARδ) (p < 0.01) proteins, and reduced the whole-cell p-FOXO1/t-FOXO1 (Forkhead Box O1) ratio (p < 0.01). EPS rescued palmitate-induced inhibition of CHO oxidation, reflected by increased glucose uptake (p < 0.01), PDC activity (p < 0.01) and glucose-derived mitochondrial ATP production (p < 0.01) compared to palmitate alone. EPS was also associated with less PDK4 (p < 0.01) and PPARδ (p < 0.01) proteins, and lower nuclear p-FOXO1/t-FOXO1 ratio normalised to the cytoplasmic ratio, but with no changes in PPARα protein. Collectively, these data suggest PPARδ, and FOXO1 transcription factors increased PDK4 protein in the presence of palmitate, which limited PDC activity and flux, and blunted CHO oxidation and glucose uptake. Conversely, EPS rescued these metabolic events by modulating the same transcription factors.

Importance of PLC-Dependent PI3K/AKT and AMPK Signaling in RANTES/CCR5 Mediated Macrophage Chemotaxis.

Regulated upon activation, normal T cell expressed, and secreted (RANTES), also known as chemokine ligand 5 (CCL5), has been reported to facilitate macrophage migration, which plays a crucial role in tissue inflammation. The aim of this study is to investigate the characteristics and underlying mechanism of RANTES on macrophage chemotaxis under physiological and pathological conditions. The study was conducted on macrophage RAW264.7 cell and bone marrow-derived macrophages (BMDM) isolated from CCL receptor 5 (CCR5) knockout mice. The macrophage migration and glucose uptake was assessed in time and dose dependent manners. Moreover, reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis were used to characterize mRNA and protein level related to the underlying mechanism. The present result showed that the maraviroc, a selective CCR5 inhibitor, dose-dependently suppressed RANTES-induced rapid increases in glucose uptake and cell migration in RAW264.7 cells. Similar effects were observed in the BMDM isolated from CCR5 knockout mice compared with wild type control. RANTES treatment promptly enhanced membrane glucose transporter 1 (GLUT1) expression, glucose uptake as well as phosphorylation of AKT on Thr308, Ser473 within min and has prolonged effect on phosphorylation of AMP-activated protein kinase (AMPK) on Thr172, which were abrogated by maraviroc, CCR5 siRNA or phospholipase C (PLC) inhibitor in RAW264.7 cells. Inhibition of PI3K and AMPK by LY294002 and Compound C significantly suppress RANTES-stimulated macrophage glucose uptake and migration, respectively. RANTES has biphasic effect on activating PLC signaling including prompt action on PI3K/AKT phosphorylation and prolong action on AMPK phosphorylation via CCR5 which leads to increased GLUT1-mediated glucose uptake and macrophage migration under physiopathological states.

Tubeless single-port thoracoscopic sublobar resection: indication and safety.

BACKGROUND: The tubeless technique, defined as non-intubated general anesthesia with omission of chest drainage after video-assisted thoracoscopic surgery (VATS), is a new concept to further minimize surgical trauma. However, there has been little investigation into the associated feasibility and safety. Minimization of postoperative pneumothorax is challenging. We set up a "tubeless protocol" to select patients for tubeless single-port VATS with monitoring of a digital drainage system (DDS). METHODS: From November 2016 to September 2017, 50 consecutive non-intubated single-port VATS for pulmonary resection were performed. In our study, patients with small and peripheral pulmonary lesions indicated for sublobar resections, as diagnostic or curative intent, were included. After excluding patients having tumors >2 cm, or intrapleural adhesions noted during operation, or forced expiratory volume in the 1 second <1.5 L, 36 patients were selected for tubeless protocol. The clinical characteristics and perioperative outcomes of these patients are presented. RESULTS: Among 36 cases, 5 patients had minor air leaks detected using the DDS and required intercostal drainage after wound closure. Among the remaining 31 patients in whom the DDS showed no air leak, the chest drainage was removed immediately after wound closure. A postoperative chest roentgenogram on the surgery day showed full expansion in all patients without pneumothorax. Only 7 (19.4%) patients developed minor subclinical pneumothorax on the first postoperative day without the need for chest drainage. All patients were discharged uneventfully without the need for intervention. CONCLUSIONS: Our tubeless protocol utilizes DDS to select patients who can have intercostal drainage omitted after non-intubated single-port VATS for pulmonary resection. Using objective DDS parameters, we believe that this is an effective way to reduce the rate of pneumothorax after tubeless single-port VATS in selected patients.

Critical airway compression caused by a large mediastinal tumour with spontaneous haemorrhage.

We report the case of a 77-year-old woman presenting with out-of-hospital cardiac arrest, which was then interpreted as an acute, life-threatening critical airway compression by a huge mediastinal tumour without appropriate diagnosis. Emergency extracorporeal membrane oxygenation was cannulated for sufficient respiratory support after spontaneous circulation was regained. After the multidisciplinary team, involving thoracic surgeons, discussed the resectability of the mediastinal tumour, the patient underwent successful resection of the mediastinal tumour through a median sternotomy. The pathological report demonstrated an intrathoracic goitre with spontaneous haemorrhage and haematoma formation, and the patient was discharged with favourable respiratory and neurological outcomes.

The prognostic value of metastatic lymph node number and ratio in oesophageal squamous cell carcinoma patients with or without neoadjuvant chemoradiation.

OBJECTIVES: We aim to evaluate the prognostic value of metastatic lymph node number (MLN) and ratio (MLR) in oesophageal squamous cell carcinoma (OSCC) patients with or without neoadjuvant chemoradiation. METHODS: Two thousand one hundred and fifty-one OSCC patients receiving oesophagectomy with (n = 850) or without (n = 1301) neoadjuvant chemoradiation were included. The MLN was categorized into 0 (N0), 1-2 (N1), 3-6 (N2) and more than 7 (N3); the MLR was categorized into 0, 0-0.2 and >0.2. The prognostic value was evaluated with survival analysis using the Cox proportional hazards regression model and the Kaplan-Meier method. RESULTS: In patients without neoadjuvant chemoradiation, the 3-year overall survival rates were 54.8, 34.4, 21.8 and 6.5% with MLN = 0, 1-2, 3-6 and more than 7, respectively (P < 0.001). The 3-year overall survival rates were 54.7, 31.2 and 14.2% with MLR = 0, 0-0.2 and more than 0.2, respectively (P < 0.001). In patients with neoadjuvant chemoradiation, the 3-year overall survival rates were 49.0, 28.4, 12.5 and 0.0% with MLN = 0, 1-2, 3-6 and more than 7, respectively (P < 0.001). However, the survival curves of MLN = 3-6 and MLN ≥7 overlapped on the Kaplan-Meier plots. In contrast, MLR demonstrated good ability to show the survival differences on the Kaplan-Meier plots. The 3-year overall survival rates were 48.9, 27.3 and 0.0% with MLR = 0, 0-0.2 and more than 0.2, respectively (P < 0.001). CONCLUSIONS: Both MLN and MLR were significant prognostic factors in OSCC patients regardless of neoadjuvant chemoradiation. But in patients with neoadjuvant chemoradiation, the survival rates were similar between ypN2 and ypN3 patients, suggesting that there was no necessity of separating patients into ypN2 and ypN3 stages.