Enhancement of fibronectin synthesis and fibrillogenesis by BMP-4 in cultured rat osteoblast.

The skeletal extracellular matrix produced by osteoblasts contains the glycoprotein fibronectin (Fn), which regulates the adhesion, differentiation, and function of osteoblasts. Fn fibrillogenesis is involved in the process of bone mineralization. Bone morphogenetic proteins (BMPs) can be isolated from organic bone matrix and are able to initiate de novo cartilage and bone formation. In this study, the effect of BMP-4 on Fn fibrillogenesis in cultured rat osteoblasts was examined. BMP-4 enhanced Fn synthesis and extracellular Fn assembly in primary cultured osteoblasts. In addition, the extracellular assembly of Fn from exogenously applied soluble human Fn was also increased by BMP-4. It has been reported that alpha5beta1 integrin is related to Fn fibrillogenesis. The synthesis of both alpha5 and beta1 integrins was upregulated by BMP-4. Immunocytochemistry showed that the clustering of alpha5 and beta1 integrins was also increased by BMP-4. BMP-4 increased fibril formation of Fn and the adhesion of osteoblasts onto Fn matrix, which was inhibited by disintegrin triflavin and Gly-Arg-Gly-Asp-Ser (GRGDS) peptide. Phosphorylation of extracellular signal-regulated kinase (ERK) and focal adhesion kinase (FAK) was increased by BMP-4. Enhancement of extracellular Fn fibrillogenesis and the mRNA expression of beta1 integrin by BMP-4 were inhibited by ERK kinase (MEK) inhibitor PD98059. These results suggest that the enhancement of extracellular Fn fibrillogenesis by BMP-4 in cultured osteoblasts is related to the increase of the synthesis of Fn and clustering of alpha5 and beta1 integrins. ERK is involved in the signaling pathway of BMP-4 in regulating Fn fibrillogenesis in osteoblasts.     

Rhodostomin inhibits thrombin-enhanced adhesion of ROS 17/2.8 cells through the blockade of alphavbeta3 integrin.

Osteosarcoma is a very malignant bone tumor which has a high metastatic potential and usually lead to poor prognosis. The adhesion of tumor cells to the endothelium or extracellular matrix (ECM) is an essential step in the metastatic cascade. We investigated the effect of thrombin on the adhesion activity of the osteosarcoma cell line, ROS 17/2.8. Incubation with the low concentrations of thrombin (0.01-5 U/ml, 5 min to 24 h) elevated the adhesion activity of ROS 17/2.8 to both human umbilical vein endothelial cells (HUVEC) and extracellular matrix, with the peak effect at the concentration of 0.5 U/ml for 30 min at 37 degrees C. The ROS 17/2.8 cells responded to thrombin by a peak effect of increased adhesion to HUVEC (5.5 folds vs. control) and fibronectin (4.8 folds) after thrombin pretreatment (0.5 U/ml, 30 min, 37 degrees C). Pretreatment with monoclonal antibodies against beta3 integrins, including anti-alphavbeta3, 10E5 and 7E3, effectively antagonized the thrombin-enhanced cell adhesion activity, whereas anti-alpha3beta1 and anti-alpha5beta1 did not antagonize the enhanced cell adhesion. Rhodostomin, an Arg-Gly-Asp (RGD)-containing snake venom peptide, and synthetic peptide RGDS also blocked the thrombin-enhanced ROS 17/2.8 cell adhesion. This study demonstrated that thrombin enhanced the cell adhesion of ROS 17/2.8 cells to HUVEC or ECM through an upregulation of beta3 integrins, and rhodostomin was a strong inhibitor on thrombin-enhanced cell adhesion, either to HUVEC or fibronectin substratum.     

Bradykinin promotes vascular endothelial growth factor expression and increases angiogenesis in human prostate cancer cells

Prostate cancer is the most commonly diagnosed malignancy in men and shows a tendency for metastasis to distant organs. Angiogenesis is required for metastasis. Bradykinin (BK) is an inflammatory mediator involved in tumor growth and metastasis, but its role in vascular endothelial growth factor (VEGF) expression and angiogenesis in human prostate cancer remains unknown. The aim of this study was to examine whether BK promotes prostate cancer angiogenesis via VEGF expression. We found that exogenous BK increased VEGF expression in prostate cancer cells and further promoted tube formation in endothelial progenitor cells and human umbilical vein endothelial cells. Pretreatment of prostate cancer with B2 receptor antagonist or small interfering RNA (siRNA) reduced BK-mediated VEGF production. The Akt and mammalian target of rapamycin (mTOR) pathways were activated after BK treatment, and BK-induced VEGF expression was abolished by the specific inhibitor and siRNA of the Akt and mTOR cascades. BK also promoted nuclear factor-κB (NF-κB) and activator protein 1 (AP-1) activity. Importantly, BK knockdown reduced VEGF expression and abolished prostate cancer cell conditional medium-mediated angiogenesis. Taken together, these results indicate that BK operates through the B2 receptor, Akt, and mTOR, which in turn activate NF-κB and AP-1, activating VEGF expression and contributing to angiogenesis in human prostate cancer cells.     

Insight into Mechanisms of Cellular Uptake of Lipid Nanoparticles and Intracellular Release of Small RNAs

Purpose

Understanding mechanisms of cellular uptake and intracellular release would enable better design of nanocarriers for delivery of nucleic acids such as siRNA and microRNA (miRNA).

Method

In this study, we investigated cellular pharmacokinetics of siRNA by co-encapsulating fluorescently labeled siRNA and molecular beacon (MB) in four different formulations of cationic lipid nanoparticles (LNPs). A miRNA mimic was also used as a probe for investigating cellular pharmacokinetics, which correlated well with RNAi activities.

Results

We tried to find the best LNP formulation based on the combination of DOTMA and DODMA. When the DOTMA/DODMA ratio was at 5/40, the LNP containing a luciferase siRNA produced the highest gene silencing activity. The superior potency of DOTMA/DODMA could be attributed to higher uptake and improved ability to facilitate siRNA release from endosomes subsequent to uptake.

Conclusions

Our findings may provide new insights into RNAi transfection pathways and have implications on cationic LNP design.     

Analysis of Different Types of Interferon-Associated Retinopathy in Patients with Chronic Hepatitis C Virus Infection Treated with Pegylated Interferon Plus Ribavirin

This retrospective cohort study aims to investigate interferon (IFN)-associated retinopathy incidence in patients with chronic hepatitis C virus (HCV) infection treated with pegylated interferon (PegIFN) plus ribavirin (RBV). We selected 1688 patients undergoing PegIFN/RBV therapy for HCV (HCV-treated cohort), 3376 patients not receiving HCV treatment (HCV-untreated cohort) and 16,880 controls without HCV (non-HCV cohort) from the Taiwan Longitudinal Health Insurance Database. The patients were frequency-matched by age, sex, and index date at a 1:2:10 ratio, and followed up until the end of 2013. Cox proportional hazard regression models were used to compare the incidences of any retinal vascular events, including subtypes, among the three cohorts. Compared with the non-HCV cohort, the HCV-treated cohort had a significantly increased risk of retinopathy (hazard ratio (HR) = 4.98, 95% confidence interval (CI): 2.02–12.3). The risk was particularly prominent for retinal hemorrhage (HR = 12.7, 95% CI: 3.78–42.9). When the HCV-untreated cohort was used as the reference, the aforementioned HRs increased to 9.02 (95% CI: 3.04–26.8) and 32.3 (95% CI: 3.94–265), respectively. This study suggested that PegIFN/RBV therapy significantly increased the risk of retinal hemorrhage but not retinal vascular occlusions in the HCV-treated cohort.     

Risk of Pneumonia Among Patients With Chronic Kidney Disease in Outpatient and Inpatient Settings: A Nationwide Population-Based Study

Patients with chronic kidney disease (CKD) are more at risk for pneumonia than the general population. Patients with pneumonia are usually treated as outpatients. However, previous studies were conducted on the basis of inpatient pneumonia. This method may underestimate the risk of pneumonia in patients with CKD. Therefore, we investigated the risk of pneumonia among CKD patients in both outpatient and inpatient settings.A total of 15,562 patients with CKD and 62,109 individuals without CKD (matched for age and gender) were taken as subjects in the Longitudinal Health Insurance Database of Taiwan National Insurance from 1996 to 2010. The incidence density rates of inpatient and outpatient pneumonia were calculated. The risk factors associated with pneumonia were analyzed using Cox proportional hazard models with adjustments for confounders.The incidence density rate of pneumonia was 65.6 per 1000 person-years in patients with CKD and 28.4 per 1000 person-years in individuals without CKD. The incidence density rate of inpatient pneumonia was 43.3 per 1000 person-years in patients with CKD and 16.6 per 1000 person-years in individuals without CKD. CKD was associated with increased risk of pneumonia (adjusted hazard ratio [aHR], 1.97; 95% confidence interval [CI], 1.89–2.05; P < 0.001), outpatient pneumonia (aHR, 1.40; 95% CI, 1.31–1.49), and inpatient pneumonia (aHR, 2.17; 95% CI, 2.07–2.29, P < 0.001). Patients’ comorbidities, including diabetes, cardiovascular disease (CVD), asthma, and chronic obstructive pulmonary disease (COPD), were independently associated with increased risk of pneumonia.CKD is associated with the increased risk of both outpatient and inpatient pneumonia. This association is independent of comorbid diabetes, CVD, asthma, and COPD.     

Basic fibroblast growth factor promotes doxorubicin resistance in chondrosarcoma cells by affecting XRCC5 expression

Chondrosarcoma is the second most common form of bone cancer and is characterized by its ability to produce an extracellular matrix of the cartilage. High-grade chondrosarcoma is highly aggressive and can metastasize to other parts of the body. Chondrosarcoma is resistant to both conventional chemotherapy and radiotherapy; hence, the current main treatment is still surgical resection. Doxorubicin (Dox) has been shown to significantly improve patient survival compared with untreated chondrosarcoma. However, for patients with metastasis, surgical resection alone can hardly treat them. In addition, drug resistance is one of the leading causes of death in patients with chondrosarcoma. Secreted proteins can mediate cell-cell interactions in the cancer microenvironment, which may be associated with the development of drug resistance. In the present study, chondrosarcoma cells were treated with Dox, the conditioned medium was then collected and changes in secreted proteins were analyzed using the antibody array. Results showed that the Dox-treated group had the highest secretion of basic fibroblast growth factor (bFGF), indicating the effect of bFGF on Dox sensitivity in chondrosarcoma. Furthermore, lentiviral-mediated knockdown and treatment of exogenous recombinant protein were employed to further investigate the effect of bFGF on Dox resistance. Results demonstrated that bFGF can promote the expression of X-ray repair cross-complementing protein 5 (XRCC5), leading to Dox resistance. Secreted bFGF is likely to be detected in serum, in addition to being a biomarker for predicting Dox resistance, the combination of Dox and bFGF/XRCC5 blockers may be a new therapeutic strategy to improve the efficacy of Dox in future.     

Changes of coronary risk factors after eradication of Helicobacter pylori infection

Several epidemiological studies have shown a positive correlation between chronic gastric infection with Helicobacter pylori (HP) and coronary artery disease. A number of reports also claimed that there are strong relationships between HP infection and coronary risk factors. However, clinical studies concerning the changes of coronary risk factors after eradication of HP infection are few and contradictory. We conducted a prospective study aiming to compare sugar, lipid and fibrinolytic profiles before HP eradication with those after HP eradication. HP infection was confirmed by endoscope-based examinations and eradicated by a standard OAC (omeperazole-amoxicillin-clarithromycin) regimen. We measured and compared pre- and post-eradication blood sugar, lipid profiles (total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and triglyceride) and fibrinolytic profiles (tissue-plasminogen activator, plasminogen activator inhibitor-1, fibrinogen, and D-dimer levels). Forty-eight patients (male:female, 25:23; mean age, 50.8 +/- 11.3 years) with gastric HP infection were enrolled in this study. Although HP infection was confirmed to have been successfully eradicated, no significant changes of blood fasting sugar, lipids or fibrinolytic profiles were found in patients after treatment. Coronary risk factors including fasting sugar, lipid and fibrinolytic profiles were not changed after successful HP eradication treatment. The relationship between HP infection and coronary artery disease needs to be clarified.