ACSL4 upregulates IFI44 and IFI44L expression and promotes the proliferation and invasiveness of head and neck squamous cell carcinoma cells.

Reprogramming of cellular energy metabolism, including deregulated lipid metabolism, is a hallmark of head and neck squamous cell carcinoma (HNSCC). However, the underlying molecular mechanisms remain unclear. Long-chain acyl-CoA synthetase 4 (ACSL4), which catalyzes fatty acids to form fatty acyl-CoAs, is critical for synthesizing phospholipids or triglycerides. Despite the differing roles of ACSL4 in cancers, our data showed that ACSL4 was highly expressed in HNSCC tissues, positively correlating with poor survival rates in patients. Knockdown of ACSL4 in HNSCC cells led to reduced cell proliferation and invasiveness. RNA sequencing analyses identified interferon-induced protein 44 (IFI44) and interferon-induced protein 44-like (IFI44L), encoded by two interferon-stimulated genes, as potential effectors of ACSL4. Silencing IFI44 or IFI44L expression in HNSCC cells decreased cell proliferation and invasiveness. Manipulating ACSL4 expression or activity modulated the expression levels of JAK1, tyrosine kinase 2 (TYK2), signal transducer and activator of transcription 1 (STAT1), interferon α (IFNα), IFNß, and interferon regulatory factor 1 (IRF1), which regulate IFI44 and IFI44L expression. Knockdown of IRF1 reduced the expression of JAK1, TYK2, IFNα, IFNß, IFI44, or IFI44L and diminished cell proliferation and invasiveness. Our results suggest that ACSL4 upregulates interferon signaling, enhancing IFI44 and IFI44L expression and promoting HNSCC cell proliferation and invasiveness. Thus, ACSL4 could serve as a novel therapeutic target for HNSCC.

Impact of contrast-enhanced mammography in surgical management of breast cancers for women with dense breasts: a dual-center, multi-disciplinary study in Asia.

OBJECTIVE: To evaluate the impact of pre-operative contrast-enhanced mammography (CEM) in breast cancer patients with dense breasts. METHODS: We conducted a retrospective review of 232 histologically proven breast cancers in 200 women (mean age: 53.4 years ± 10.2) who underwent pre-surgical CEM imaging across two Asian institutions (Singapore and Taiwan). Majority (95.5%) of patients had dense breast tissue (BI-RADS category C or D). Surgical decision was recorded in a simulated blinded multi-disciplinary team setting on two separate scenarios: (i) pre-CEM setting with standard imaging, and clinical and histopathological results; and (ii) post-CEM setting with new imaging and corresponding histological findings from CEM. Alterations in surgical plan (if any) because of CEM imaging were recorded. Predictors CEM of patients who benefitted from surgical plan alterations were evaluated using logistic regression. RESULTS: CEM resulted in altered surgical plans in 36 (18%) of 200 patients in this study. CEM discovered clinically significant larger tumor size or extent in 24 (12%) patients and additional tumors in 12 (6%) patients. CEM also detected additional benign/false-positive lesions in 13 (6.5%) of the 200 patients. Significant predictors of patients who benefitted from surgical alterations found on multivariate analysis were pre-CEM surgical decision for upfront breast conservation (OR, 7.7; 95% CI, 1.9-32.1; p = 0.005), architectural distortion on mammograms (OR, 7.6; 95% CI, 1.3-42.9; p = .022), and tumor size of ≥ 1.5 cm (OR, 1.5; 95% CI, 1.0-2.2; p = .034). CONCLUSION: CEM is an effective imaging technique for pre-surgical planning for Asian breast cancer patients with dense breasts. KEY POINTS: • CEM significantly altered surgical plans in 18% (nearly 1 in 5) of this Asian study cohort with dense breasts. • Significant patient and imaging predictors for surgical plan alteration include (i) patients considered for upfront breast-conserving surgery; (ii) architectural distortion lesions; and (iii) tumor size of ≥ 1.5 cm. • Additional false-positive/benign lesions detected through CEM were uncommon, affecting only 6.5% of the study cohort.

Evaluation of an AI-Based TB AFB Smear Screening System for Laboratory Diagnosis on Routine Practice.

The most robust and economical method for laboratory diagnosis of tuberculosis (TB) is to identify mycobacteria acid-fast bacilli (AFB) under acid-fast staining, despite its disadvantages of low sensitivity and labor intensity. In recent years, artificial intelligence (AI) has been used in TB-smear microscopy to assist medical technologists with routine AFB smear microscopy. In this study, we evaluated the performance of a TB automated system consisting of a microscopic scanner and recognition program powered by artificial intelligence and machine learning. This AI-based system can detect AFB and classify the level from 0 to 4+. A total of 5930 smears were evaluated on the performance of this automatic system in identifying AFB in daily lab practice. At the first stage, 120 images were analyzed per smear, and the accuracy, sensitivity, and specificity were 91.3%, 60.0%, and 95.7%, respectively. In the second stage, 200 images were analyzed per smear, and the accuracy, sensitivity, and specificity were increased to 93.7%, 77.4%, and 96.6%. After removing disqualifying smears caused by poor staining quality and smear preparation, the accuracy, sensitivity, and specificity were improved to 95.2%, 85.7%, and 96.9%, respectively. Furthermore, the automated system recovered 85 positive smears initially identified as negative by manual screening. Our results suggested that the automated TB system could achieve higher sensitivity and laboratory efficiency than manual microscopy under the quality control of smear preparation. Automated TB smear screening systems can serve as a screening tool at the first screen before manual microcopy.

Distinct JNK/VEGFR signaling on angiogenesis of breast cancer-associated pleural fluid based on hormone receptor status.

Malignant pleural effusion is a common complication in metastatic breast cancer (MBC); however, changes in the pleural microenvironment are poorly characterized, especially with respect to estrogen receptor status. Histologically, MBC presents with increased microvessels beneath the parietal and visceral pleura, indicating generalized angiogenic activity. Breast cancer-associated pleural fluid (BAPF) was collected and cultured with HUVECs to recapitulate the molecular changes in subpleural endothelial cells. The clinical progression of triple-negative breast cancer (TNBC) is much more aggressive than that of hormone receptor-positive breast cancer (HPBC). However, BAPF from HPBC (BAPF-HP) and TNBC (BAPF-TN) homogeneously induced endothelial proliferation, migration, and angiogenesis. In addition, BAPF elicited negligible changes in the protein marker of endothelial-mesenchymal transition. Both BAPF-HP and BAPF-TN exclusively upregulated JNK signaling among all MAPKs in HUVECs. By contrast, the response to the JNK inhibitor was insignificant in Transwell and tube formation assays of the HUVECs cultured with BAPF-TN. The distinct contribution of p-JNK to endothelial angiogenesis was consequently thought to be induced by BAPF-HP and BAPF-TN. Due to increased angiogenic factors in HUVECs cultured with BAPF, vascular endothelial growth factor receptor 2 (VEGFR2) inhibitor was applied accordingly. Responses to VEGFR2 blockade were observed in both BAPF-HP and BAPF-TN concerning endothelial migration and angiogenesis. In conclusion, the above results revealed microvessel formation in the pleura of MBC and the underlying activation of p-JNK/VEGFR2 signaling. Distinct responses to blocking p-JNK and VEGFR2 in HUVECs cultured with BAPF-HP or BAPF-TN could lay the groundwork for future investigations in treating MBC based on hormone receptor status.

Astaxanthin attenuates joint inflammation induced by monosodium urate crystals.

Gouty arthritis is the one of the most painful arthritis and is caused by an inflammatory reaction. This study investigated whether astaxanthin (AXT), which has documented anti-inflammatory and antioxidant properties, exhibits protective effects against monosodium urate (MSU) crystal-induced inflammation. Cell viability of J774A.1 murine macrophages was assessed by AXT dose-dependent incubation by MTT assays, and expression levels of iNOS and COX-2 proteins as well as secretion of IL-1ß were also analyzed under MSU crystals stimulation with or without AXT treatment. The production of inflammatory mediators was found to significantly decrease with AXT treatment, and the formation of the inflammasome complex was also attenuated when cells were co-stimulated with MSU crystals and AXT. Furthermore, we found that expression of the MAPK pathway was downregulated in J774A.1 cells. AXT also inhibited the induction of COX-2 and IL-6 in human chondrocytes and synovial fibroblasts by western blots. Finally, an MSU crystal intra-articular injection rat model for gouty arthritis was utilized in which treatment groups received 5-daily intraperitoneal injections of AXT prior to MSU crystal stimulation, or once intra-articular injections of AXT following MSU crystal stimulation for 6 hours. Results of synovitis score analysis revealed that inflammation was significantly attenuated in the group which received intraperitoneal AXT injection prior to MSU crystal stimulation compared to the group which received MSU only. These results indicate that AXT attenuates the effects of MSU crystal-induced inflammation by suppressing the production of pro-inflammatory cytokines and inflammatory mediators. Our findings that the anti-inflammatory activities of AXT may be beneficial in the treatment of MSU crystal-induced arthritis.

Architecture distortion score (ADS) in malignancy risk stratification of architecture distortion on contrast-enhanced digital mammography.

OBJECTIVE: To develop a risk predictor model in evaluation of tomosynthesis-detected architectural distortion (AD) based on characteristics of contrast-enhanced digital mammography (CEDM). METHODS: Ninety-four AD lesions on CEDM in combination with tomosynthesis were retrospectively reviewed from 92 consecutive women (mean age, 52.4 years ± 7.9) with abnormal diagnostic or screening mammography. CEDM results were correlated with histology of ADs using cross-tabulation for statistical analysis. Predictors for risk of malignancy from CEDM characteristics (background parenchyma enhancement, degree of AD enhancement, enhancing morphology, size of enhancement, and enhancing spiculations) and patient's age were evaluated using logistic regression. We propose a sum score, termed AD score (ADS), for risk stratification and corresponding suggested BI-RADS category. RESULTS: Thirty-three of ninety-four (35.1%) of detected AD lesions were malignant. The sensitivity, specificity, PPV, and NPV of CEDM in evaluation of malignant AD are 100%, 42.6%, 48.5%, and 100%, respectively. Absence of AD enhancement on CEDM is highly indicative of no underlying malignancy. On multivariate analysis, the predictors on CEDM with statistical significance are (1) marked intensity of AD enhancement (OR, 22.6; 95%CI 3.1, 166.6; p = .002); and (2) presence of enhancing spiculations (OR, 9.1; 95%CI 2.2, 36.5; p = .002). A prediction model whose scores (ADS) given by ranking of OR of all predictors with AUC of 0.934 and Brier score of 0.0956 was developed. CONCLUSION: ADS-based lesion characterization on CEDM enables risk assessment of tomosynthesis-detected AD lesions. KEY POINTS: • Architecture distortions presenting with marked enhancement intensity and presence of enhancing spiculations are highly associated with risk of malignancy. • Absence of architecture distortion enhancement in minimal or mild background parenchyma enhancement on CEDM indicates low risk of breast malignancy (NPV = 100%).

Ameliorative Effects of Cardamonin on Monosodium Urate-Induced Gouty Arthritis through Inhibiting NLRP3 Inflammasome Mediation.

Background and Objectives: Gouty arthritis is an acute inflammatory response caused by the precipitation of monosodium urate (MSU) crystals in joints. The triggering of MSU leads to increased production of inflammatory cytokines, such as interleukin-1ß, which in turn lead to the formation of macromolecular complexes, referred to as inflammasomes. Thorough characterization of the NLRP3 inflammasome can be used as an indicator of an immune response against harmful stimuli. Cardamonin is a chalcone, mainly found in the seeds of Alpinia katsumadai, and exhibits anti-inflammatory activity by inhibiting the release of pro-inflammatory cytokines in vitro. However, the mechanism by which cardamonin treatment alleviates gouty arthritis has yet to be fully elucidated. Materials and Methods: In vitro or in vivo models were used to study whether cardamonimn inhibited NLRP3 inflammasome activation or suppressed gouty inflammation. Results: In the current study, we determined that most NLRP3 was released passively after MSU stimulation, and this release of NLRP3 promoted caspase-1 activation and IL-1ß secretion. Cardamonin was shown to decrease both the activity of caspase-1 and secretion of IL-1ß in J774A.1 macrophage cells subjected to MSU stimulation. Cardamonin was also shown to attenuate the production of COX-2 in MSU-stimulated J774A.1 macrophage cells. Finally, cardamonin reduced the thickness of the synovial lining and the infiltration of gouty arthritis in a rat model. Conclusions: Overall, cardamonin significantly attenuated IL-1ß secretion, caspase-1 activity, and COX-2 production stimulated by MSU. These findings provide new insights into the molecular mechanisms underlying the effects of cardamonin treatment for gouty arthritis.

Effect of malignant-associated pleural effusion on endothelial viability, motility and angiogenesis in lung cancer.

Malignant pleural effusion (MPE) and paramalignant pleural effusion (PPE) remain debilitating complications in lung cancer patients with poor prognosis and limited treatment options. The role of vascular endothelial cells has not been explored in the pleural environment of lung cancer. By integrating MPE and PPE as malignant-associated pleural fluid (MAPF), the current study aimed to evaluate the effect of MAPF on cell proliferation, migration and angiogenesis of HUVEC. First, increased capillaries were identified in the subpleural layer of lung adenocarcinoma. Compatible with pathological observations, the ubiquitous elevation of HUVEC survival was identified in MAPF culture regardless of the underlying cancer type, the driver gene mutation, prior treatments and evidence of malignant cells in pleural fluid. Moreover, MAPF enhanced HUVEC motility with the formation of lamellipodia and filopodia and focal adhesion complex. Tube formation assay revealed angiogenic behavior with the observation of sheet-like structures. HUVEC cultured with MAPF resulted in a significant increase in MAPK phosphorylation. Accompanied with VEGFR2 upregulation in MAPF culture, there was increased expressions of p-STAT3, HIF-1α and Nf-kB. VEGF/VEGFR2 blockade regressed endothelial migration and angiogenesis but not cell proliferation. Our data indicate the angiogenic activities of MAPF on vascular endothelial cells that revealed increased pleural capillaries in lung cancer. Targeting the VEGF/VEGFR2 pathway might modulate the angiogenic propensity of MAPF in future clinical investigations.

Psychiatric disorders risk in patients with iron deficiency anemia and association with iron supplementation medications: a nationwide database analysis.

BACKGROUND: It has been shown that iron deficiency anemia (IDA) is associated with psychosocial consequences and psychiatric morbidity. However, the association between adults with IDA and psychiatric disorders has not been clarified. The purpose of this study was to investigate the psychiatric disorder morbidity of an IDA group in comparison with a non-IDA group and to examine the risk of psychiatric disorders in IDA patients treated with iron supplementation. METHODS: All study subjects were 20 years of age or over with newly diagnosed IDA enrolled in the Taiwan National Health Insurance Database from 2000 to 2012. We matched IDA and non-IDA subjects according to age and gender in a 1:2 ratio. Our primary outcome was diagnosis of psychiatric disorders and the patients were monitored until the end of 2013. A multivariate Cox proportional hazards regression model was used to explore the risk of psychiatric disorders in patients with IDA after adjustment for confounders, including demographic characteristics and comorbidities. RESULTS: The adjusted hazard ratios (aHRs) of psychiatric disorders was 1.52 (95% CI = 1.45-1.59) in the IDA group compared with the non-IDA group. Among the different types of psychiatric disorders, the IDA group was associated with significantly higher incidence and risks of anxiety disorders, depression, sleep disorders, and psychotic disorders (p <  0.05). Furthermore, iron supplementation in IDA subjects was associated with a significantly lower risk of psychiatric disorders compared to non-iron supplementation in IDA patients. CONCLUSIONS: Our study indicates that IDA subjects had an increased risk of psychiatric disorders, regardless of other confounders. In IDA patients, iron supplementation was associated with a decreased risk of psychiatric disorders. Moreover, IDA patients receiving iron supplementation also had a lower risk of sleep disorders.

Glucose reduces the osmopressor response in connection with the tyrosine phosphorylation of focal adhesion kinase in red blood cells.

Glucose ingestion attenuates the water ingestion-induced increase in the total peripheral vascular resistance and orthostatic tolerance. We investigated the gastrointestinal physiology of glucose by examining the effect of glucose ingestion on the functional expression of focal adhesion kinase (FAK) in red blood cell (RBC) membrane. This study was performed in 24 young, healthy subjects. Blood samples were collected at 5 min before and 25 min and 50 min after an ingestion of 10% glucose water 500 mL, water 500 mL, or normal saline 500 mL. We determined glucose and osmolality in plasma, and phosphorylation of aquaporin 1 (AQP1), glucose transporter 1 (Glut1), and FAK in RBC membrane. Our results showed that glucose ingestion reduced the rise of peripheral vascular resistance after water ingestion and upregulated the serine phosphorylation of Glut1. It also lowered both the serine phosphorylation of FAK and tyrosine phosphorylation of AQP1, compared with the ingestion of either water or saline. In an ex vivo experiment, glucose activated the Glut1 receptor and subsequently reduced the expression of FAK compared with 0.8% saline alone. We concluded that glucose activates Glut1 and subsequently lowers the functional expression of FAK, a cytoskeleton protein of RBCs. The functional change in the RBC membrane proteins in connection with the attenuation of osmopressor response may elucidate the pathophysiology of glucose in postprandial hypotension.

Histopathology of fluoroscopy-induced radiation ulcer: a case series study in comparison with morphea.

BACKGROUND AND OBJECTIVES: Histopathologic diagnosis of fluoroscopy-induced radiation ulcer (FIRU) can be challenging if the past history of radiation exposure is unknown. Morphea is the most important differential diagnosis. This study was intended to identify clinical and pathologic features that can be used to distinguish FIRU from morphea. PATIENTS AND METHODS: We performed a retrospective study on 25 specimens from 15 patients with FIRU and 21 specimens from 21 patients with morphea. Clinical findings and pathological features were analyzed. RESULTS: Thirteen of 15 patients (86.7 %) with FIRU underwent angioplasty for coronary artery disease, and eleven patients had lesions in the right subscapular area. Compared with morphea, FIRU patients were more likely to display non-inflammatory infiltrates (28 %), bizarre fibroblasts (100 %), sclerosis (48 %), telangiectasia (96 %), vascular damage (64 %), and loss of skin appendages (100 %). In morphea, bizarre fibroblasts were rare (14 %), while telangiectasia (62 %) and loss of skin appendages (62 %) were variable. Loss of CD34+ cells and compression of elastic fibers could not be used to distinguish between FIRU and morphea. CONCLUSIONS: Skin lesion in the right subscapular area with presence of bizarre fibroblasts, sclerosis, telangiectasia, and loss of cutaneous appendages as seen with histology are highly characteristic of the radiation damage associated with fluoroscopic angiography.

Increased risk of fractures in patients with polycystic ovary syndrome: a nationwide population-based retrospective cohort study.

Polycystic ovary syndrome (PCOS) is a complex disorder; various features of this disorder may influence bone metabolism and skeletal mass. The contribution of PCOS to lower bone mineral density has been recognized. However, the impact of PCOS on the long-term risks for fractures remains inconclusive. The aim of this study was to determine the risk of overall fracture and fractures at different anatomic sites in patients with PCOS. Using a nationwide health insurance claims database, we included 11,106 subjects, aged 15-80 years, with newly diagnosed PCOS (ICD-9-CM: 254.4X) during 2000-2012. Patients with PCOS and respective age-matched (1:4) controls without PCOS were enrolled. The occurrence of fracture was monitored until the end of 2013. Cox regression and computed hazard ratios (HR) with 95% confidence intervals (95% CI) were used to determine the risk of PCOS among women with fractures. The PCOS and non-PCOS groups were comprised of 11,106 patients with PCOS and 44,424 participants without PCOS, respectively. Patients with PCOS had a higher incidence of any fractures compared with non-PCOS group (10.16 versus 8.07 per 1000 person-years) and a greater risk of any fractures [adjusted hazard ratio (aHR) = 1.23, 95% CI = 1.13-1.33], osteoporotic fractures (aHR = 1.33, 95% CI = 1.15-1.54), spine fractures (aHR = 1.36, 95% CI = 1.11-1.66) and forearm fractures (aHR = 1.39, 95% CI = 1.07-1.80), but the risk for femur or hip fracture, humerus, wrist and non-osteoporotic fractures were not increased. In conclusion, the PCOS group had a higher occurrence rate of fractures than the non-PCOS group. These results provide evidence for the adverse effects of PCOS on the risk of fractures.

Dexamethasone Down-regulates Osteocalcin in Bone Cells through Leptin Pathway.

Glucocorticoid therapy, especially at higher doses, is associated with significant adverse side effects including osteoporosis. Leptin, secreted from adipose tissue, has diverse effects on bone tissue regulation. As glucocorticoids stimulate leptin synthesis and secretion directly in adipose tissue we hypothesised that dexamethasone (DEX) induced osteoporosis may, in part, be mediated by an osteoblast dependent leptin-leptin receptor pathway. Human bone cells expressed leptin and leptin receptors (Ob-Ra and Ob-Rb). DEX increased leptin, Ob-Ra and Ob-Rb expression in a dose-dependent manner while decreasing expression of osteocalcin. In the presence of leptin, Cbfa1 and osteonectin expression showed no significant change, whereas osteocalcin expression was decreased. Recombinant human quadruple antagonist leptin suppressed DEX-induced osteocalcin downregulation. The signaling pathway involved up-regulation of JAK2. In conclusion, upregulation of leptin and Ob-Rb in human bone cells by DEX is associated with down-regulation of osteocalcin expression. The down regulation of osteocalcin by DEX was partially through a leptin autocrine/paracrine loop. Adverse effects of DEX on the skeleton may be modified by targeting leptin signaling pathways.

Infusion of Porcine-Derived Amniotic Fluid Stem Cells for Treatment of Experimental Colitis in Mice.

Recently, stem cells have offered an alternative treatment for inflammatory bowel disease (IBD) or colitis to overcome the poor outcomes associated with current therapies. Amniotic fluid-derived stem cells (AFSCs) have the potential for the regeneration of impaired organs and the recovery of normal physiologic functions of damaged tissues without ethical concerns or risk of tumor formation. In this work, we aimed to examine the therapeutic effects of infusion of porcine AFSCs (pAFSCs) in dextran sulfate sodium (DSS)-induced colitis in mice. Treatment with pAFSCs was shown to inhibit the shortening of the colon after induction of colitis and dramatically ameliorated the body weightloss induced by the DSS treatment. In addition, pAFSCs could also reduce the extent of the inflamed area represented by epithelial mesenchymal transformation in the colitis mice. The levels of the inflammatory cytokines interleukin 6 (IL-6) and interferon gamma (IFN-γ) were also reduced in colitis mice transplanted with pAFSCs. In conclusion, pAFSCs can ameliorate experimental colitis in mice, suggesting that they may be a potential treatment for IBD or colitis.

CB1 cannabinoid receptor antagonist attenuates left ventricular hypertrophy and Akt-mediated cardiac fibrosis in experimental uremia.

Cannabinoid receptor type 1 (CB1R) plays an important role in the development of myocardial hypertrophy and fibrosis-2 pathological features of uremic cardiomyopathy. However, it remains unknown whether CB1R is involved in the pathogenesis of uremic cardiomyopathy. Here, we aimed to elucidate the role of CB1R in the development of uremic cardiomyopathy via modulation of Akt signalling. The heart size and myocardial fibrosis were evaluated by echocardiography and immunohistochemical staining, respectively, in 5/6 nephrectomy chronic kidney disease (CKD) mice treated with a CB1R antagonist. CB1R and fibrosis marker expression levels were determined by immunoblotting in H9c2 cells exposed to the uremic toxin indoxyl sulfate (IS), with an organic anion transporter 1 inhibitor or a CB1R antagonist or agonist. Akt phosphorylation was also assessed to examine the signaling pathways downstream of CB1R activation induced by IS in H9c2 cells. CKD mice exhibited marked left ventricular hypertrophy and myocardial fibrosis, which were reversed by treatment with the CB1R antagonist. CB1R, collagen I, transforming growth factor (TGF)-ß, and α-smooth muscle actin (SMA) expression showed time- and dose-dependent upregulation in H9c2 cells treated with IS. The inhibition of CB1R by either CB1R antagonist or small interfering RNA-mediated knockdown attenuated the expression of collagen I, TGF-ß, and α-SMA in IS-treated H9c2 cells, while Akt phosphorylation was enhanced by CB1R agonist and abrogated by CB1R antagonist in these cells. In summary, we conclude that CB1R blockade attenuates LVH and Akt-mediated cardiac fibrosis in a CKD mouse model. Uremic toxin IS stimulates the expression of CB1R and fibrotic markers and CB1R inhibition exerts anti-fibrotic effects via modulation of Akt signaling in H9c2 myofibroblasts. Therefore, the development of drugs targeting CB1R may have therapeutic potential in the treatment of uremic cardiomyopathy.

Hyperphosphatemia induces protective autophagy in endothelial cells through the inhibition of Akt/mTOR signaling.

OBJECTIVE: Hyperphosphatemia-induced endothelial dysfunction has been shown to play a pathogenic role in the development of atherosclerosis in chronic kidney disease (CKD) through unclear mechanisms. Emerging evidence indicates that autophagy is involved in the maintenance of normal cardiovascular function. However, it is unclear whether autophagy participates in the molecular mechanism underlying high phosphate (Pi)-induced endothelial dysfunction. METHODS: The autophagy activity was determined by the immunofluorescence staining of the expression of endothelial microtubule-associated protein 1 light chain 3 (LC3) in the 5/6 nephrectomy rat model of CKD and sham-operated control rats. The LC3-II/LC3-I ratio and the activation of the Akt/mammalian target of rapamycin (mTOR) signaling pathway were determined in cultured human microvascular endothelial cell (HMEC-1) endothelial cells that were exposed to a high concentration of Pi with or without the Pi influx blocker phosphonoformic acid, the autophagy inhibitor 3-methyladenine, and the autophagy inducer rapamycin. The impacts of autophagy on Pi-induced apoptotic damage were assessed by flow cytometry. RESULTS: The in vivo rat model of CKD revealed that hyperphosphatemia is associated with increased endothelial LC3 staining. The exposure of HMEC-1 cells to high Pi induced both dose-dependent and time-dependent increases in the LC3-II/LC3-I expression ratio accompanied by the inhibition of the Akt/mTOR signaling pathway. In HMEC-1 cells, high Pi-induced autophagy and the inhibition of Akt/mTOR signaling were reversed by phosphonoformic acid through the blockage of Pi influx. Apoptosis, characterized by the levels of cleaved caspase 3 and poly(ADP-ribose) polymerase, along with autophagy was induced by high Pi, and the inhibition of autophagy by 3-methyladenine significantly aggravated high Pi-induced apoptosis. The flow cytometry results confirmed that the blockage of autophagy promoted the apoptosis of endothelial cells. CONCLUSIONS: Hyperphosphatemia induces endothelial autophagy, possibly through the inhibition of the Akt/mTOR signaling pathway, which may play a protective role against high Pi-induced apoptosis.

Squamous Cell Carcinoma Arising From Massive Localized Lymphedema of Scrotum Mimicking Scrotal Smooth Muscle Hamartoma of Dartos: A Case Report.

Massive localized lymphedema (MLL) is an uncommon benign skin lesion typically presenting with prominent edema and vascular proliferation in the adipose tissue of lower limbs. When rarely occurring in scrotum, it instead is characterized by a striking proliferation of dermal smooth muscle bundles mimicking acquired smooth muscle hamartoma of dartos. The authors report a rare case of scrotal MLL. A 57-year-old obese man with a history of previous surgery for rectal adenocarcinoma, 20 years earlier, presented with progressive nodular enlargement of the scrotum for 2 years, causing discomfort, difficulty in ambulation, and cosmetic problems. The preoperative radiographic investigation revealed thickening of the scrotal wall with multiple soft-tissue nodules. The patient underwent a wide excision of the scrotal wall, perineum, and penile skin. The pathological examination showed a scrotal MLL associated with well-differentiated squamous cell carcinoma. The authors speculate that prior radiotherapy and surgery together with morbid obesity led to long-standing lymphedema that triggered the proliferation of smooth muscle cells, chronic epidermal change, and finally squamous cell carcinoma.

Nerve growth factor increases MMP9 activity in annulus fibrosus cells by upregulating lipocalin 2 expression.

PURPOSE: Nerve growth factor (NGF) expression and activity is important in chronic lower back pain but may also act as a pro-catabolic factor in the pathogenesis of intervertebral disc (IVD) degeneration. Lipocalin 2 (Lcn2) expression in IVD was upregulated by NGF stimulation in our previous study. The current study was undertaken to identify potential mechanisms of the latter effect including potential interactions between Lcn2 and matrix metalloproteinase 9 (MMP9). METHODS: Rat annulus fibrosus (AF) cells were stimulated by NGF and subjected to microarray analysis, subsequent real-time PCR, western immunoblotting, and immunofluorescence. Cells were treated with NGF in the absence or presence of the NGF inhibitor Ro 08-2750. Zymography and functional MMP9 assays were used to determine MMP9 activity, whilst the dimethyl-methylene blue assay was used to quantify the release of glycosaminoglycans (GAGs) reflecting catabolic effects following NGF treatment. Immunoprecipitation with immunoblotting was used to identify interactions between MMP9 and Lcn2. RESULTS: Increased expression of Lcn2 gene and protein following NGF stimulation was confirmed by microarray analysis, real-time PCR, western blot and immunofluorescence. Zymography showed that NGF enhanced 125-kDa gelatinase activity, identified as a Lcn2/MMP9 complex by immunoprecipitation and immunoblotting. Functional assays showed increased MMP9 activity and GAG release in the presence of NGF. The effects of NGF were neutralized by the presence of Ro 08-2750. CONCLUSIONS: NGF upregulates Lcn2 expression and increases MMP9 activity in AF cells; processes which are likely to potentiate degeneration of AF tissue in vivo. Anti-NGF treatment may have benefit for management of pain relief and slowing down progression of AF tissue degeneration.

The hMLH1 -93G>A promoter polymorphism is associates with outcomes in oral squamous cell carcinoma patients.

BACKGROUND: The aim of this study was to investigate the impact of hMLH1 polymorphisms on treatment outcomes in patients with oral squamous cell carcinoma (OSCC). METHODS: Genotypings were performed by direct DNA sequencing in peripheral blood leukocytes from 185 male OSCC patients. Patients received primary surgery with or without adjuvant radiotherapy. Two hMLH1 tag single nucleotide polymorphisms (SNPs)-rs1800734 (-93G>A in the promoter) and rs1540354 (in the third intron)-were chosen from the HapMap project. Overall survival (OS) and disease-free survival (DFS) were compared between different genotypes. RESULTS: The hMLH1 rs1800734 and rs1540354 polymorphisms were in weak linkage disequilibrium (r (2) = 0.456). OSCC patients with the rs1800734 AA genotype had a significantly poor prognosis in both OS and DFS. This SNP can also predict the outcomes of OSCC patients with postoperative adjuvant radiotherapy, especially in advanced stage; however, no significant differences in patient outcomes were found for the hMLH1 rs1540354 genotypes. CONCLUSIONS: Our results demonstrate that the hMLH1 -93G>A SNP is found to be associated with patient outcomes in OSCC. This SNP can also predict their treatment outcome of radiotherapy.