He-Wei Granule enhances anti-tumor activity of cyclophosphamide by changing tumor microenvironment.

Objective: He-Wei Granule (HWKL) is a modern product derived from the modified formulation of traditional Chinese medicine Banxia Xiexin Decoction (BXD), which remarkedly enhanced the anti-proliferation activity of cyclophosphamide (CTX) on HepG2 and SGC-7901 cell lines in vitro in our previous research. The aim of the study was to investigate the synergistic effects of HWKL and CTX using a transplanted H22 hepatocellular carcinoma mouse model. Methods: The CTX-toxic-reducing efficacy of HWKL was evaluated by hematology indexes, organ indexes and marrow DNA detection. To investigate the underlying mechanisms, histopathology test, immunohistochemistry test and TUNEL staining were conducted. The efficacy of HWKL on the micro-vessel density (MVD) in tumor tissue was also evaluated by measuring CD34 level. Results: High dose HWKL (6.75 g/kg) markedly attenuated CTX-induced hepatotoxicity and myelosuppression while significantly enhanced CTX anticancer efficacy in vivo. Further mechanism investigation suggested that high dose HWKL significantly increased cleaved Caspase 3 level and promoted apoptosis in tumor tissue by up-regulating Bax expression and down-regulating Bcl-2 and FasL expressions. Compared with CTX alone group, the decrease in LC-3B and Beclin 1 levels suggested that the autophagy in H22 carcinoma was significantly inhibited with addition of high dose HWKL. ELISA assay results indicated that the autophagy inhibition was achieved by decreasing p53 expression, blocking PI3K/AKT/mTOR pathway and recovering Th1/Th2 cytokine balance. In addition, CD34 and EGFR immunohistochemistry assay suggest that high dose HWKL could significantly decrease micro-vessel density (MVD) and inhibit angiogenesis in H22 carcinoma. Conclusion: It can be concluded that high-dose HWKL enhanced CTX efficacy by promoting apoptosis, inhibiting autophagy and angiogenesis in tumor tissue while significantly alleviated CTX-induced toxicity, and could be applied along with CTX in clinical treatment as a supplement agent.

Effects of Anti-Cancer Drug Sensitivity-Related Genetic Differences on Therapeutic Approaches in Refractory Papillary Thyroid Cancer.

Thyroid cancer (TC) includes tumors of follicular cells; it ranges from well differentiated TC (WDTC) with generally favorable prognosis to clinically aggressive poorly differentiated TC (PDTC) and undifferentiated TC (UTC). Papillary thyroid cancer (PTC) is a WDTC and the most common type of thyroid cancer that comprises almost 70-80% of all TC. PTC can present as a solid, cystic, or uneven mass that originates from normal thyroid tissue. Prognosis of PTC is excellent, with an overall 10-year survival rate >90%. However, more than 30% of patients with PTC advance to recurrence or metastasis despite anti-cancer therapy; consequently, systemic therapy is limited, which necessitates expansion of improved clinical approaches. We strived to elucidate genetic distinctions due to patient-derived anti-cancer drug-sensitive or -resistant PTC, which can support in progress novel therapies. Patients with histologically proven PTC were evaluated. PTC cells were gained from drug-sensitive and -resistant patients and were compared using mRNA-Seq. We aimed to assess the in vitro and in vivo synergistic anti-cancer effects of a novel combination therapy in patient-derived refractory PTC. This combination therapy acts synergistically to promote tumor suppression compared with either agent alone. Therefore, genetically altered combination therapy might be a novel therapeutic approach for refractory PTC.

Synergistic Anticancer Activity of N-Hydroxy-7-(2-Naphthylthio) Heptanomide, Sorafenib, and Radiation Therapy in Patient-Derived Anaplastic Thyroid Cancer Models.

Anaplastic thyroid cancer (ATC) is an undifferentiated and advanced form of thyroid cancer, accompanied with a high ratio of epigenetic adjustment, which occurs more than genetic mutations. In this study, we aimed to evaluate the synergistic anticancer effect (in vitro and in vivo) of the new combination of N-hydroxy-7-(2-naphthylthio) heptanomide (HNHA) and sorafenib with radiation therapy in pre-clinical models of ATC. The ATC cell lines, YUMC-A1 and YUMC-A2, were isolated from the current patients who were treated with HNHA and sorafenib, either as monotherapy or combination therapy. Synergistic anticancer effect of the combination therapy on the intracellular signaling pathways and cell cycle was assessed via flow cytometry and immunoblot analysis. To examine tumor shrinkage activity in vivo, an ATC cell line-derived mouse xenograft model was used. Results showed that the combination therapy of HNHA and sorafenib with radiation promoted tumor suppression via caspase cleavage and cell cycle arrest in patient-derived ATC. In addition, the combination therapy of HNHA and sorafenib with radiation was more effective against ATC than therapy with HNHA or sorafenib with radiation. Thus, the combination of HNHA and sorafenib with radiation may be used as a novel curative approach for the treatment of ATC.

Synergistic anticancer activity of sorafenib, paclitaxel, and radiation therapy on anaplastic thyroid cancer in vitro and in vivo.

BACKGROUND: This study aimed to investigate the antitumor activity of paclitaxel with radiation and sorafenib in anaplastic thyroid cancer (ATC) cells in vitro and in vivo. METHODS: The 8505C ATC cell line was exposed to radiation, sorafenib, and paclitaxel each or in combination. The effects of combined treatment on the cell cycle and intracellular signaling pathways were assessed using flow cytometry and western blot analysis. An ATC cell line xenograft model was used to examine antitumor activity in vivo. RESULTS: Radiation, paclitaxel plus sorafenib synergistically decreased cell viability in ATC cells and significantly increased apoptotic cell death. The combination of paclitaxel, sorafenib with radiation reduced the antiapoptotic factor in ATC. This combination therapy significantly reduced the tumor volume and increased survival in the ATC xenograft model. CONCLUSIONS: These results suggest that the combination of radiation and paclitaxel plus sorafenib has significant anticancer activity in preclinical models.

Thyroid Hormone Supplementation Therapy for Differentiated Thyroid Cancer After Lobectomy: 5 Years of Follow-Up.

Background: Lobectomy with preservation of the contralateral lobe has already become the most preferred surgical method for patients with low-risk thyroid cancer. The incidence of and risk factors for the development of hypothyroidism after lobectomy for thyroid cancer remains unclear. The previous practice of levothyroxine supplementation post-thyroidectomy, to bring about thyroid stimulating hormone (TSH) suppression, had some serious side effects. This study aimed to evaluate the incidence of hypothyroidism and to identify the factors associated with hypothyroidism requiring thyroid hormone replacement. Methods: We retrospectively reviewed the charts of 256 consecutive patients with differentiated thyroid cancer treated with lobectomy at the Gangnam Severance Hospital between April and December 2014 who were followed-up for more than 5 years. Patients were evaluated using a thyroid function test at the time of outpatient visit every 6 months for the 1st year, with an annual follow-up thereafter. Results: After 5 years, 66.0% (169) of the patients needed levothyroxine supplementation to maintain euthyroid status. The incidence of hypothyroidism requiring levothyroxine supplementation increased until 3 years but showed no significant change in the 4 and 5th year. Recurrence showed no difference between the group with and without levothyroxine supplementation. The presence of thyroiditis and preoperative TSH levels were correlated with postoperative levothyroxine supplementation to maintain euthyroid status, in univariate and multivariate analyses. Conclusion: High preoperative TSH levels and/or thyroiditis indicate a significantly increased likelihood of developing hypothyroidism requiring thyroid hormone supplementation after a thyroid lobectomy. Patients with an increased risk of postoperative hypothyroidism must be aware of their risk factors and should undergo more intensive follow-ups.

Chemical composition and pharmacological mechanism of Qingfei Paidu Decoction and Ma Xing Shi Gan Decoction against Coronavirus Disease 2019 (COVID-19): In silico and experimental study.

The Coronavirus Disease 2019 (COVID-19) pandemic has become a huge threaten to global health, which raise urgent demand of developing efficient therapeutic strategy. The aim of the present study is to dissect the chemical composition and the pharmacological mechanism of Qingfei Paidu Decoction (QFPD), a clinically used Chinese medicine for treating COVID-19 patients in China. Through comprehensive analysis by liquid chromatography coupled with high resolution mass spectrometry (MS), a total of 129 compounds of QFPD were putatively identified. We also constructed molecular networking of mass spectrometry data to classify these compounds into 14 main clusters, in which exhibited specific patterns of flavonoids (45 %), glycosides (15 %), carboxylic acids (10 %), and saponins (5 %). The target network model of QFPD, established by predicting and collecting the targets of identified compounds, indicated a pivotal role of Ma Xing Shi Gan Decoction (MXSG) in the therapeutic efficacy of QFPD. Supportively, through transcriptomic analysis of gene expression after MXSG administration in rat model of LPS-induced pneumonia, the thrombin and Toll-like receptor (TLR) signaling pathway were suggested to be essential pathways for MXSG mediated anti-inflammatory effects. Besides, changes in content of major compounds in MXSG during decoction were found by the chemical analysis. We also validate that one major compound in MXSG, i.e. glycyrrhizic acid, inhibited TLR agonists induced IL-6 production in macrophage. In conclusion, the integration of in silico and experimental results indicated that the therapeutic effects of QFPD against COVID-19 may be attributed to the anti-inflammatory effects of MXSG, which supports the rationality of the compatibility of TCM.

Genetic and metabolic links between the murine microbiome and memory.

BACKGROUND: Recent evidence has linked the gut microbiome to host behavior via the gut-brain axis [1-3]; however, the underlying mechanisms remain unexplored. Here, we determined the links between host genetics, the gut microbiome and memory using the genetically defined Collaborative Cross (CC) mouse cohort, complemented with microbiome and metabolomic analyses in conventional and germ-free (GF) mice. RESULTS: A genome-wide association analysis (GWAS) identified 715 of 76,080 single-nucleotide polymorphisms (SNPs) that were significantly associated with short-term memory using the passive avoidance model. The identified SNPs were enriched in genes known to be involved in learning and memory functions. By 16S rRNA gene sequencing of the gut microbial community in the same CC cohort, we identified specific microorganisms that were significantly correlated with longer latencies in our retention test, including a positive correlation with Lactobacillus. Inoculation of GF mice with individual species of Lactobacillus (L. reuteri F275, L. plantarum BDGP2 or L. brevis BDGP6) resulted in significantly improved memory compared to uninoculated or E. coli DH10B inoculated controls. Untargeted metabolomics analysis revealed significantly higher levels of several metabolites, including lactate, in the stools of Lactobacillus-colonized mice, when compared to GF control mice. Moreover, we demonstrate that dietary lactate treatment alone boosted memory in conventional mice. Mechanistically, we show that both inoculation with Lactobacillus or lactate treatment significantly increased the levels of the neurotransmitter, gamma-aminobutyric acid (GABA), in the hippocampus of the mice. CONCLUSION: Together, this study provides new evidence for a link between Lactobacillus and memory and our results open possible new avenues for treating memory impairment disorders using specific gut microbial inoculants and/or metabolites. Video Abstract.

Guaiane type of sesquiterpene with NO inhibitory activity from the root of Wikstroemia indica.

Roots of Wikstroemia indica is widely used in China as a folk medicine in treatment of many diseases. However, active compounds of guaiane type of sesquiterpene remain largely unknown. In the present work, five new guaiane of type sesquiterpene compounds wikstronone A-E, along with one known guaiane type of sesquiterpene compound were isolated from the petroleum and CH2Cl2 fraction of roots of Wikstroemia indica. Structures of these compounds including absolute configuration were determined by extensive NMR and CD spectroscopic analysis. The inhibitory activity of all isolated compounds were assayed against LPS-induced NO production in RAW 264.7 macrophages by Griess reagent. Among all compounds, wikstronone A (1) showed remarkable NO inhibitory activity comparable to that of positive control Indometacin. Wikstronone D and E (4 and 5) showed weak NO inhibitory activity. The isolated guaiane type of sesquiterpene may be potent NO synthetase inhibitors.

SoLAT (Sorafenib Lenvatinib alternating treatment): a new treatment protocol with alternating Sorafenib and Lenvatinib for refractory thyroid Cancer.

BACKGROUND: In the last decade, several tyrosine kinase inhibitors (TKIs), which disrupt pathways involved in the proliferation and tumorigenesis of thyroid cancer, have been extensively studied. Two different TKIs, lenvatinib and sorafenib, were recently approved by both the US FDA and European Medicine Agency. Until date, the duration of the TKI response is not sufficient and resistance eventually occurs. The goal of this study was to investigate a new treatment protocol, SoLAT, using sorafenib and lenvatinib alternatively on refractory thyroid cancer. METHODS: Patient-derived aggressive papillary thyroid cancer (PTC) cell lines from patients with biochemical and histologically proven aggressive RAI-refractory papillary thyroid cancer were exposed to sorafenib and lenvatinib alternatively. Human thyroid cancer cell xenografts were obtained by injecting patient-derived aggressive PTC cell lines into the flank of female BALB/c nude mice. Tumor-bearing mice were treated with sorafenib and lenvatinib alternatively. Cell viability assay, immunofluorescence analysis, confocal imaging, immunoblot analysis, flow cytometry analysis of cell cycle and a tube formation assay were performed. RESULTS: SoLAT was more effective for advanced PTC cell lines than individual treatment. Immunoblot analysis showed that SoLAT markedly increased levels of cell cycle inhibitors (p53 and p21), and pro-apoptotic factors (Apaf-1 and cleaved caspase 3) and decreased levels of positive cell cycle regulators (cyclin D1, CDK4, CDK6) and anti-apoptotic factors (p-NFκB, Bcl-2). Increased sub-G0/G1 population was observed in the SoLAT group, leading to apoptosis, cell cycle arrest, and strong inhibition of advanced PTC cell viability. SoLAT reduced the level of EMT markers such as vimentin, E-cadherin, Snail and Zeb1 by FGFR inhibition. In the xenograft model, individual treatment with sorafenib or lenvatinib did not markedly suppress patient-derived aggressive PTC cell xenograft tumors, whereas SoLAT significantly suppressed the proliferation of these tumors. CONCLUSIONS: SoLAT was more effective than individual treatment with sorafenib or lenvatinib in inhibiting PTC progression by inducing cell cycle arrest. Studies using both in vitro cell culture and an in vivo xenograft model provided evidence of tumor shrinkage with SoLAT. We suggest that these effects may be due to reduced EMT-mediated drug resistance in the aggressive PTC model.

Lignans from the root of Wikstroemia indica and their cytotoxic activity against PANC-1 human pancreatic cancer cells.

Six new compounds, wikstronin A (1), wikstronin B (2), wikstresinol (3), acetylwikstresinol (4), bis-5',5'-(+)-matairesinol (5), bis-5,5'-(+)-matairesinol (6), together with 20 known compounds (7-26) were isolated from the CH2Cl2 extract of roots of Wikstroemia indica. Structures of compounds 1-6 were determined by extensive NMR and CD spectroscopic analysis. In vitro preferential cytotoxicity of all the isolates was evaluated against a PANC-1 human pancreatic cell line. Compounds 8 and 12 displayed mild preferential cytotoxicity in the nutrient-deprived medium (NDM) and without causing toxicity in normal nutrient-rich conditions.

Screening of the optimized prescription from Suqingwan in terms of its therapeutic effect on DSS-induced ulcerative colitis by its regulation of inflammatory and oxidative mediators.

ETHNOPHARMACOLOGICAL RELEVANCE: Suqingwan (SQW), a traditional Chinese medicine used for treating ulcerative colitis (UC), is composed of 13 kinds of Traditional Chinese medicines (TCMs). According to TCM theory, we investigated whether a simplified prescription composed of the herbs with some functions, would have similar effects to SQW and examined its potential treatment mechanism of action. MATERIALS AND METHODS: We categorized the herbs in SQW into four groups according to their traditional functions and used an orthogonal experimental design to obtain nine separated prescriptions (SPs) of SQW. A dextran sulfate sodium (DSS)-induced UC mouse model was used to evaluate the anti-ulcer colitis effects of the nine SPs and the calculated prescription (CP) was obtained based on the orthogonal t values of the disease activity index (DAI) of the nine SPs. The effect of the CP and SP8 were verified in the DSS-induced UC model, and the DAI and histopathology of the UC mice were examined. Myeloperoxidase (MPO), malondialdehyde (MDA), tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, IL-4 and IL-10 of the mice in SP8 were investigated to explore the mechanism of action of the optimized prescription with regard to anti-inflammatory and anti-oxidation effects. RESULTS: Among the 9 SPs, separate prescription 6, 7 and 8 (SP6, SP7 and SP8) and the SQW formulation all significantly reduced the DAI of the UC mice and, in particular, SP8 had an effect similar to SQW, which consists of Sanguisorba officinalis L., Rehmannia glutinosa Libosch. and four other herbal medicines. In a further investigation, SP8 was found to improve the ulcerative colitis in mice in terms of both clinical symptoms and histopathology. The mortality of mice in the SP8 group was 33.3%, better than CP based on the orthogonal t values (83.3%). SP8 could also reduce the levels of TNF-α, IL-1ß, IL-6, MPO and MDA and increase the levels of IL-4 and IL-10 in colon tissue of UC mice in comparison with those of the model group (p<0.05). CONCLUSIONS: An optimized prescription (SP8) from SQW was obtained based on an orthogonal experimental design, which involved 6 herbal medicines, with significantly fewer herbs than in the original prescription. SP8 displayed a similar anti-ulcerative colitis activity to SQW, and its in vivo mechanism of action is related to up-regulation of anti-inflammatory cytokines and down-regulation of pro-inflammatory and oxidative factors.

Parathyroid score can predict the duration of required calcium supplementation after total thyroidectomy.

BACKGROUND: Postoperative hypoparathyroidism is the most common complication after total thyroidectomy, owing to unintentional injury or decreased blood flow to the parathyroid glands. Prediction of postoperative hypoparathyroidism would be helpful for surgeons to manage postoperative hypocalcemia. In this study, we scored the discoloration of the parathyroid glands using a new parathyroid scoring system and evaluated the correlation between the parathyroid score and duration of required calcium supplementation after total thyroidectomy. METHODS: A total of 316 patients undergoing total thyroidectomy between November 2009 and April 2010 were enrolled in this retrospective study. Parathyroid scoring was performed by one experienced surgeon. The status of each of the 4 parathyroid glands was classified as normal color (3 points), slightly discolored (2 points), dark discoloration (1 point), or loss of the gland (0 points), resulting in possible total scores of 0-12. Serum parathyroid hormone (PTH), serum calcium, and ionized calcium concentrations were measured at 2 hours, 2 weeks, 3 months, 6 months, and 1 year after surgery. Patients were also divided into three groups based on the duration of required calcium supplementation: no required supplementation (n = 260, 82.3%), required supplementation for <6 months (n = 38, 12%), and required supplementation for ≥6 months (n = 18, 5.75%). RESULTS: Parathyroid scores were positively correlated with ionized PTH concentrations at 2 hours (r = 0.053, p < 0.001), 2 weeks (r = 0.056, p < 0.001), 3 months (r = 0.032, p<0.001), 6 months (r = 0.072, p < 0.001), and 1 year (r = 0.071, p < 0.001) after thyroidectomy. Parathyroid scores were significantly and inversely associated with the duration of required calcium supplementation (p = 0.001). CONCLUSIONS: Parathyroid scores at the end of surgery might be helpful for predicting the degree of postoperative hypocalcemia after total thyroidetomy.

Changing trends in the management of well-differentiated thyroid carcinoma in Korea.

A questionnaire administered in 2009 found that members of the Korean Association of Thyroid-Endocrine Surgeons (KATES) favored more aggressive treatment of well-differentiated thyroid carcinoma (WDTC) than physicians from other countries. This study assessed the changes in practical management of WDTC in Korea from the previous survey. Questionnaires were sent by e-mail to KATES members. A total of 101 members completed the questionnaire. Their responses were compared with response for the 2009 survey. Of the respondents, 53.5% and 80.2% indicated that they would perform fine-needle aspiration cytology on nodules that were <0.5 cm and 0.5-1.0 cm in diameter, respectively. If the cytology was positive, a large number of respondents favored surgical treatment, regardless of tumor size. Compared with the 2009 survey, a slightly higher percentage favored observation for patients with tumors that were <0.5 cm in diameter, and a larger percentage recommended less-than-total thyroidectomy for patients with T1 cancers. Respondents in 2014 favored aggressive lymph node dissection less, irrespective of tumor size, preferring short-term treatment with thyroid stimulating hormone suppressors. The percentage preferring postoperative high-dose radioactive iodine therapy slightly increased, whereas the percentage favoring external irradiation decreased, in 2014 compared with 2009. The management of Korean patients with WDTC changed from 2009 to 2014. In 2009, Korean respondents favored more aggressive treatment of WDTC compared with respondents from other countries. In 2014, however, Korean respondents favored a more conservative approach, especially in patients with microcarcinomas.

Recovery from Permanent Hypoparathyroidism After Total Thyroidectomy.

BACKGROUND: Permanent hypoparathyroidism after total thyroidectomy is a rare but potentially serious iatrogenic complication. The aim of this study was to investigate the rate of recovery from postoperative, permanent hypoparathyroidism in patients undergoing thyroidectomy without parathyroid autotransplantation. METHODS: This study was a prospective case series with a postoperative follow-up of up to 3 years. We enrolled patients with thyroid cancer who underwent total thyroidectomy with central compartment dissection, with or without lateral neck dissection, and who had postoperative permanent hypoparathyroidism, defined as serum levels of intact parathyroid hormone (PTH) <15 pg/mL for at least 1 year. In the postoperative follow-up period, the serum levels of PTH and calcium were measured regularly. Recovery from permanent hypoparathyroidism was defined as return to normal serum levels of PTH (15-65 pg/mL) and calcium (8.5-10.1 mg/dL) without calcium and/or vitamin D supplementation. RESULTS: In the 1467 patients who underwent total thyroidectomy, 22 presented with permanent postoperative hypoparathyroidism. In 5 of these 22 patients, the PTH levels increased steadily and returned to normal in 27.6±2.9 months, after which supplementation of calcium and vitamin D could be discontinued. CONCLUSIONS: Although recovery from permanent hypoparathyroidism is rare, patients should be monitored for serum PTH levels so that unnecessary treatments such as calcium and vitamin D supplementation can be avoided.

Low iodine diet for one week is sufficient for adequate preparation of high dose radioactive iodine ablation therapy of differentiated thyroid cancer patients in iodine-rich areas.

BACKGROUND: Most current guidelines suggest one or two weeks of low iodine diet (LID) before radioactive iodine ablation therapy (RAIT) to increase its efficacy in differentiated thyroid cancer (DTC) patients after total thyroidectomy. LID duration is particularly important for patients living in iodine excess areas. However, there is no standardized LID protocol and there are limited reports regarding the relationship between LID and ablation outcome. Therefore, we aimed to evaluate the optimal LID duration and define clinical features that affect ablation outcome. METHODS: A total of 202 papillary thyroid cancer patients with total thyroidectomy preparing for RAIT were enrolled. All patients had undergone two weeks of LID before (131)I administration. Morning spot urine specimens were obtained twice (one week or two weeks after LID, respectively) from each patient. Urine iodine excretion (UIE) values were used to evaluate LID efficacy. Successful ablation was defined using two definitions: (i) no visible uptake on a follow-up diagnostic (131)I scans, and (ii) no visible uptake on a follow-up diagnostic (131)I scans and stimulated serum thyroglobulin (Tg) levels <1 ng/mL. RESULTS: The UIE median values after LID for one and two weeks were lower than 50 µg/L, and the median UIE values were not significantly different according to the LID duration. Based on the first criterion for successful ablation, 175 of the 195 patients were successfully ablated. There were no significant differences in mean and median UIE levels between the ablated and non-ablated groups after LID for two weeks. The rate of ablation did not differ between the mild and moderate iodine deficient groups. Based on the second criterion for successful ablation, 149 of 188 patients were successfully ablated. The ablation success rate did not differ between UIE levels. When we analyzed clinical factors that affect ablation outcome, serum Tg level at the time of ablation was the only significant variable in multivariate logistic analysis. CONCLUSION: Strict LID for one week was sufficient to achieve target UIE values for RAIT preparation, even in iodine-rich areas.

Hyperbaric oxygen activates discoidin domain receptor 2 via tumour necrosis factor-alpha and the p38 MAPK pathway to increase vascular smooth muscle cell migration through matrix metalloproteinase 2.

DDR2 (discoidin domain receptor 2) regulates collagen turnover mediated by SMCs (smooth muscle cells) in atherosclerosis. HBO (hyperbaric oxygen) has been used in medical practice; however, the molecular mechanism of the beneficial effects of HBO is poorly understood. Furthermore, the effect of HBO on DDR2 has not been reported previously. In the present study, we investigated the cellular and molecular mechanisms of DDR2 regulation by HBO in VSMCs (vascular SMCs). Cells were exposed to 2.5 ATA (atmosphere absolute) of oxygen in a hyperbaric chamber. DDR2 protein (3.63-fold) and mRNA (2.34-fold) expression were significantly increased after exposure to 2.5 ATA HBO for 1 h. Addition of SB203580 and p38 MAPK (mitogen-activated protein kinase) siRNA (small interfering RNA) 30 min before HBO inhibited the induction of DDR2 protein. HBO also significantly increased DNA-protein binding activity of Myc/Max. Addition of SB203580 and an anti-TNF-alpha (tumour necrosis factor-alpha) monoclonal antibody 30 min before HBO abolished the DNA-protein binding activity induced by HBO. HBO significantly increased the secretion of TNF-alpha from cultured VSMCs. Exogenous addition of TNF-alpha significantly increased DDR2 protein expression, whereas anti-TNF-alpha and anti-(TNF-alpha receptor) antibodies blocked the induction of DDR2 protein expression. HBO significantly increased VSMC migration and proliferation, whereas DDR2 siRNA inhibited the migration induced by HBO. HBO increased activated MMP2 (matrix metalloproteinase 2) protein expression, and DDR2 siRNA abolished the induction of activated MMP2 expression induced by HBO. In conclusion, HBO activates DDR2 expression in cultured rat VSMCs. HBO-induced DDR2 is mediated by TNF-alpha and at least in part through the p38 MAPK and Myc pathways.

Wogonin inhibits IL-6-induced angiogenesis via down-regulation of VEGF and VEGFR-1, not VEGFR-2.

So far, no antiangiogenic activity of wogonin, a flavonoid, on human umbilical vein endothelial cells (HUVECs) has been demonstrated. The aim of this study was to investigate the effects of wogonin on IL-6-induced angiogenesis in HUVEC cultures and chorioallantoic membrane (CAM) neovascularization. The in vivo CAM model was applied to evaluate the percentage of new vessel formations, followed by measurement of endothelial migration and tube formation in HUVEC cultures. The results revealed that wogonin (10(-8) approximately 10(-5)M) concentration-dependently inhibited IL-6-induced angiogenesis. The signaling pathway was through down-regulation of the autocrine loop of VEGF and VEGFR-1, but not of VEGFR-2. Furthermore, the regulating loop of the IL-6 receptor complex was also attenuated via expression of sIL-6Ralpha and gp130, but not of IL-6/IL-6R binding density. We conclude that wogonin is a suppressive agent of the autoregulated loop of VEGF, VEGFR-1 and the IL-6 receptor complex.

Chrysin inhibits lipopolysaccharide-induced angiogenesis via down-regulation of VEGF/VEGFR-2(KDR) and IL-6/IL-6R pathways.

The relationship between chrysin and inflammation-induced angiogenesis remains unclear. The aim of this study was to evaluate the suppressive effects of chrysin on lipopolysaccharide (LPS)-induced angiogenesis in chicken chorioallantoic membrane (CAM) as well as in human umbilical endothelial cells (HUVEC). The IN VIVO CAM model was applied to evaluate the percentage of new vessels formation, followed by measuring endothelial migration and tube formation in HUVEC cultures. The mechanisms of the suppressive effect of chrysin on LPS-induced angiogenesis, in terms of VEGF, VEGF receptors (VEGFR), interleukin 6 (IL-6) and IL-6 receptor gene expressions, were analyzed by Western blot, ELISA cytokine assay, and quantitative real time PCR. The results showed that chrysin (10(-8) - 10(-5) M) inhibited LPS-induced CAM neovascular density. There was a significant down-regulation of VEGF and VEGFR-2 (KDR) but not VEGFR-1 (Flt-1) gene expression by chrysin in LPS-treated HUVEC cultures. Besides, chrysin concentration-dependently inhibited the auto-regulation loop of IL-6/IL-6R in LPS-treated HUVEC cells. We conclude that chrysin suppresses both IN VITRO and IN VIVO LPS-induced angiogenesis.

Hyperbaric oxygen selectively induces angiopoietin-2 in human umbilical vein endothelial cells.

One of the biological effects of hyperbaric oxygen (HBO) therapy in enhancing ischemia-related wound healing is the induction of angiogenesis. To elucidate the mechanism(s) underlying the HBO-induced angiogenesis, we studied the expression of several angiogenesis-related genes in human umbilical vein endothelial cells exposed to HBO. Western blot analyses showed that HBO enhanced the expression of angiopoietin-2 (Ang2) with no effect on the expression of Tie2, angiopoietin-1, and VEGF. The induction of Ang2 was further confirmed by immunohistochemistry, quantitative PCR, and Northern blot analyses. Inhibition of endothelial nitric oxide synthase blocked the HBO-induced Ang2 expression, but failed to block hypoxia-induced Ang2 expression. These data indicated that HBO-induced Ang2 expression may be through transcriptional stimulation, and requires the nitric oxide signaling pathway, which may play an important role in HBO-induced angiogenesis.