We have previously shown that ritonavir (RTV), a highly active anti-retroviral therapy (HAART) drug, can cause endothelial dysfunction through oxidative stress. Several antioxidants including ginsenoside Rb1, a compound with antioxidant effect, can effectively block this side effect of RTV in endothelial cells. In the current study, we explored a mechanism by which ginsenoside Rb1 could protect these cells via binding of estrogen receptors (ERs). We found that several human endothelial cell lines differentially expressed ER-β and had very low levels of ER-α. RTV treatment significantly increased the production of reactive oxygen species (ROS) and decreased the expression of endothelial nitric oxidase synthase (eNOS) and superoxide dismutase (SOD) in HUVECs, while Rb1 effectively blocked these effects of RTV. These effects of Rb1 were effectively inhibited by silencing ER-β, indicating that ginsenoside Rb1 requires ER-β for its antioxidant activity in inhibiting the deleterious effect of RTV in human endothelial cells. Furthermore, Rb1 specifically activated ER-β transactivation activity by ER-β luciferase reporter assay. Rb1 competitively bound to ER-β, which was determined by the high sensitive fluorescent polarization assay.

The latent HIV-1 reservoir of memory CD4+ T cells that persists during combination antiviral therapy prevents a cure of infection. Insight into mechanisms of latency and viral reactivation are essential for the rational design of strategies to reduce the latent reservoir. In this study, we quantified the levels of >2,600 proteins in the CCL19 primary CD4+ T cell model of HIV-1 latency. We profiled proteins under conditions that promote latent infection and after cells were treated with phorbol 12-myristate 13-acetate (PMA) + ionomycin, which is known to efficiently induce reactivation of latent HIV-1. In an analysis of cells from two healthy blood donors, we identified 61 proteins that were upregulated ≥2-fold, and 36 proteins that were downregulated ≥2-fold under conditions in which latent viruses were reactivated. These differentially expressed proteins are, therefore, candidates for cellular factors that regulate latency or viral reactivation. Two unexpected findings were obtained from the proteomic data: (1) the interactions among the majority of upregulated proteins are largely undetermined in published protein-protein interaction networks and (2) downregulated proteins are strongly associated with Gene Ontology terms related to mitochondrial protein synthesis. This proteomic data set provides a useful resource for future mechanistic studies of HIV-1 latency.

Cessation of highly active antiretroviral therapy (HAART) in HIV-infected individual leads to a rebound of viral replication due to reactivation of a viral reservoir composed largely of latently infected memory CD4+ T cells. Efforts to deplete this reservoir have focused on reactivation of transcriptionally silent latent proviruses. HIV provirus transcription depends critically on the positive transcription elongation factor b (P-TEFb), whose core components are cyclin-dependent kinase 9 (CDK9) and cyclin T1. In resting CD4+ cells, the functional levels of P-TEFb are extremely low. Cellular activation upregulates cyclin T1 protein levels and CDK9 T-loop (T186) phosphorylation. The broad-spectrum histone deacetylase inhibitors (HDACis) vorinostat and panobinostat have been shown to reactivate latent virus in vivo in HAART-treated individuals. In this study, we have found that vorinostat and panobinostat activate P-TEFb in resting primary CD4+ T cells through induction of CDK9 T-loop phosphorylation. In contrast, tacedinaline and romidepsin, HDAC 1 and 2 inhibitors, were unable to activate CDK9 T-loop phosphorylation. We used a CCL19 primary CD4+ T-cell model HIV latency to assess the correlation between induction of CDK9 T-loop phosphorylation and reactivation of latent HIV virus by HDACis. Vorinostat and panobinostat treatment of cells harboring latent HIV increased CDK9 T-loop phosphorylation and reactivation of latent virus, whereas tacedinaline and romidepsin failed to induce T-loop phosphorylation or reactivate latent virus. We conclude that the ability of vorinostat and panobinostat to induce latent HIV is, in part, likely due to the ability of the broad-spectrum HDACis to upregulate P-TEFb through increased CDK9 T-loop phosphorylation.

Cardiovascular disease remains the number one killer in the United States and many other countries. Each year, there are enormous research efforts on its pathogenesis, prevention and treatment led by scientists worldwide. One of the most significant research areas is the impact and mechanisms of existing or new cardiovascular risk factors on the vascular system. The current review provides the most updated research advances in the area of the regulation of the endothelial nitric oxide synthase-nitric oxide (eNOS-NO) system by several cardiovascular risk factors. There are many exciting discoveries made from the studies of several major cardiovascular risk factors such as hypertension, cigarette smoking, dyslipidemia and diabetes mellitus as well as emerging risk factors such as HIV infection, antiretroviral therapy, genomic variability, and cytokines. In general, cardiovascular risk factors could impair the eNOS-NO system with a variety of molecular mechanisms including decrease in NO bioavailability by excess reactive oxygen species, inhibition of eNOS expression and activity, and deficiency of eNOS cofactors. Special attention is paid to the impact of several new or emerging risk factors on cardiovascular disease and the eNOS-NO system. These mechanistic studies are clinically significant because they may lead towards new and effective strategies for the prevention and treatment of cardiovascular disease.

Resistin has been linked to obesity, insulin resistance, atherosclerosis, and the development of cardiovascular disease. Nevertheless, the effects and the molecular mechanisms of resistin on endothelial permeability, a key event in the development of atherosclerosis, inflammation, and vascular disease, are largely unknown. In order to determine the effect of resistin on endothelial permeability, human coronary artery endothelial cells (HCAECs) were treated with clinically relevant concentrations of resistin and the endothelial permeability was measured using the Transwell system with a Texas-Red-labeled dextran tracer. The permeability of HCAEC monolayers treated with resistin (80 ng/mL) was 51% higher than the permeability of control monolayers (P<0.05). The mRNA levels of tight junction proteins zonula occludens-1 (ZO-1) and occludin in resistin-treated cells were 37% and 42% lower, respectively, than the corresponding levels in untreated cells. The protein levels of these molecules in resistin-treated cells were significantly reduced by 35% and 37%, respectively (P<0.05), as shown by flow cytometry and Western blot analysis. Superoxide dismutase (SOD) mimetic MnTBAP effectively blocked the resistin-mediated reduction of ZO-1 and occludin levels in HCAECs. In addition, superoxide anion production was increased from 21% (untreated cells) to 55% (cells treated with 40 ng/mL resistin), and 64% (resistin, 80 mg/mL) (P<0.05). The natural antioxidant Ginkgolide A effectively inhibited resistin-induced increase in permeability and the increase in superoxide anion production in HCAECs. Furthermore, resistin treatment significantly activated p38 MAPK, but not ERK1/2. Pretreatment of HCAECs with a p38 inhibitor effectively blocked resistin-induced permeability. These results provide new evidence that resistin may contribute to the vascular lesion formation via increasing endothelial permeability through the mechanism of oxidative stress and the activation of p38 MAPK.

Pancreatic cancer (PC) is an aggressive malignancy with poor survival. The discovery of microRNAs (miRNAs) has provided a new opportunity to study the disease. Thus far, altered expression of miRNAs has been reported in a wide range of malignancies, including PC, and some miRNAs are associated with PC cell proliferation, invasion, chemoresistance, and patient survival. This review summarizes recent advances with respect to the roles and mechanisms of miRNAs in PC and discusses potential clinical applications.

miRNAs are small, endogenously expressed noncoding RNAs that regulate gene expression, mainly at the post-transcriptional level, via degradation or translational inhibition of their target mRNAs. Functionally, an individual miRNA can regulate the expression of multiple target genes. The study of miRNAs is rapidly growing and recent studies have revealed a significant role of miRNAs in vascular biology and disease. Many miRNAs are highly expressed in the vasculature, and their expression is dysregulated in diseased vessels. Several miRNAs have been found to be critical modulators of vascular pathologies, such as atherosclerosis, lipoprotein metabolism, inflammation, arterial remodeling, angiogenesis, smooth muscle cell regeneration, hypertension, apoptosis, neointimal hyperplasia and signal transduction pathways. Thus, miRNAs may serve as novel biomarkers and/or therapeutic targets for vascular disease. This article summarizes the current studies related to the disease correlations and functional roles of miRNAs in the vascular system and discusses the potential applications of miRNAs in vascular disease.

Highly active antiretroviral therapy (HAART) is often associated with endothelial dysfunction and cardiovascular complications. In this study, we determined whether HIV non-nucleoside reverse transcriptase inhibitor efavirenz (EFV) could increase endothelial permeability. Human coronary artery endothelial cells (HCAECs) were treated with EFV (1, 5 and 10μg/ml) and endothelial permeability was determined by a transwell system with a fluorescence-labeled dextran tracer. HCAECs treated with EFV showed a significant increase of endothelial permeability in a concentration-dependent manner. With real time PCR analysis, EFV significantly reduced the mRNA levels of tight junction proteins claudin-1, occludin, zonula occluden-1 and junctional adhesion molecule-1 compared with controls (P<0.05). Protein levels of these tight junction molecules were also reduced substantially in the EFV-treated cells by western blot and flow cytometry analyses. In addition, EFV also increased superoxide anion production with dihydroethidium and cellular glutathione assays, while it decreased mitochondrial membrane potential with JC-staining. Antioxidants (ginkgolide B and MnTBAP) effectively blocked EFV-induced endothelial permeability and mitochondrial dysfunction. Furthermore, EFV increased the phosphorylation of MAPK JNK and IκBα, thereby increasing NFκB translocation to the nucleus. Chemical JNK inhibitor and dominant negative mutant JNK and IκBα adenoviruses effectively blocked the effects of EFV on HCAECs. Thus, EFV increases endothelial permeability which may be due to the decrease of tight junction proteins and the increase of superoxide anion. JNK and NFκB activation may be directly involved in the signal transduction pathway of EFV action in HCAECs.

Objective— The objective of this study was to determine the effects and molecular mechanisms of eotaxin, a newly discovered chemokine (CCL11), on endothelial permeability in the human coronary artery endothelial cells (HCAECs). Methods and Results— Cells were treated with eotaxin, and the monolayer permeability was studied by using a costar transwell system with a Texas Red–labeled dextran tracer. Eotaxin significantly increased monolayer permeability in a concentration-dependent manner. In addition, eotaxin treatment significantly decreased the mRNA and protein levels of endothelial junction molecules including zonula occludens-1 (ZO-1), occludin, and claudin-1 in a concentration-dependent manner as determined by real-time RT-PCR and Western blot analysis, respectively. Increased oxidative stress was observed in eotaxin-treated HCAECs by analysis of cellular glutathione levels. Furthermore, eotaxin treatment substantially activated the phosphorylation of MAPK p38. HCAECs expressed CCR3. Consequently, antioxidants (ginkgolide B and MnTBAP), specific p38 inhibitor SB203580, and anti-CCR3 antibody effectively blocked the eotaxin-induced permeability increase in HCAECs. Eotaxin also increased the phosphorylation of Stat3 and nuclear translocation of NF-κB in HCAECs. Conclusions— Eotaxin increases vascular permeability through CCR3, the downregulation of tight junction proteins, increase of oxidative stress, and activation of MAPK p38, Stat3, and NF-kB pathways in HCAECs.

Objective-Our previous studies revealed upregulation of stanniocalcin-1 (STC1) in cardiac vessels in dilated cardiomyopathy. However, the functional significance of STC1 is unknown. The objective of this study was to determine the effects of STC1 on TNF-alpha-induced monolayer permeability of human coronary artery endothelial cells (HCAECs).Methods and Results-Cells were pretreated with STC1 for 30 minutes followed by treatment with TNF-alpha (2 ng/mL) for 24 hours. Monolayer permeability was studied using a transwell system. STC1 pretreatment significantly blocked TNF-alpha-induced monolayer permeability in a concentration-and time-dependent manner. STC1 effectively blocked TNF-alpha-induced downregulation of endothelial tight junction proteins zonula occluden-1 and claudin-1 at both mRNA and protein levels. STC1 also significantly decreased TNF-alpha-induced superoxide anion production. The inhibitory effect of STC1 was specific to TNF-alpha, as it failed to inhibit VEGF-induced endothelial permeability. Furthermore, STC1 partially blocked NF-kappa B and JNK activation in TNF-alpha-treated endothelial cells. JNK inhibitor and antioxidant also effectively blocked TNF-alpha-induced NF-kappa B activation and monolayer permeability in HCAECs.Conclusions-STC1 maintains endothelial permeability in TNF-alpha-treated HCAECs through preservation of tight junction protein expression, suppression of superoxide anion production, and inhibition of the activation of NF kappa B and JNK, suggesting an important role for STC1 in regulating endothelial functions during cardiovascular inflammation.

The purpose of this study was to determine the effects and mechanisms of sCD40L on endothelial dysfunction in both human coronary artery endothelial cells (HCAECs) and porcine coronary artery rings. HCAECs treated with sCD40L showed significant reductions of endothelial nitric oxide synthase (eNOS) mRNA and protein levels, eNOS mRNA stability, eNOS enzyme activity, and cellular NO levels, whereas superoxide anion (O(2)(-)) production was significantly increased. sCD40L enhanced eNOS mRNA 3'UTR binding to cytoplasmic molecules and induced a unique expression pattern of 95 microRNAs. sCD40L significantly decreased mitochondrial membrane potential, and catalase and SOD activities, whereas it increased NADPH oxidase (NOX) activity. sCD40L increased phosphorylation of MAPKs p38 and ERK1/2 as well as IkappaBalpha and enhanced NF-kappaB nuclear translocation. In porcine coronary arteries, sCD40L significantly decreased endothelium-dependent vasorelaxation and eNOS mRNA levels, whereas it increased O(2)(-) levels. Antioxidant seleno-l-methionine; chemical inhibitors of p38, ERK1/2, and mitochondrial complex II; as well as dominant negative mutant forms of IkappaBalpha and NOX4 effectively blocked sCD40L-induced eNOS down-regulation in HCAECs. Thus, sCD40L reduces eNOS levels, whereas it increases oxidative stress through the unique molecular mechanisms involving eNOS mRNA stability, 3'UTR-binding molecules, microRNAs, mitochondrial function, ROS-related enzymes, p38, ERK1/2, and NF-kappaB signal pathways in endothelial cells.

In this study, the effects of acetylsalicylic acid (aspirin) on the expression of uPAR and the mechanism by which it regulates expression of uPAR was examined in two different colon cancer cell lines HCT116 and GEO, respectively. The study shows that under physiological concentration, aspirin upregulates steady-state level expression of uPAR mRNA as well as expression of uPAR protein. Using a transient transfection assay, a region corresponding to -1 to -398 region of uPAR promoter has been identified which shows maximum responsiveness to aspirin treatment and found that this region is sufficient for the aspirin-induced up-regulation of uPAR. A stable integration of a single copy of this region coupled to luciferase reporter gene into the HCT116 genome also behaved similarly. Using gel mobility shift assays, it is found that the distal AP1 region between -171 and -186 is responsible for the aspirin-induced up-regulation of uPAR. Mutation of this region reduced up-regulation. Supershift assays identify that the bound proteins at this region are c-Jun and Fra-1. Real-time PCR analysis showed more than 4-fold increase in the binding of c-Jun and a 1.6-fold increase in the binding of Fra-1 in this region and this up-regulation corresponds to an increased binding of acetylated histone H4 in this region. Since an increase in the expression of uPAR corresponds to an increase in the migration of the cell, a migration assay was performed and result showed a 3-fold increased migration of HCT116 cells through the vitronectin-coated layer. Thus, an AP1 mediated pathway for aspirin induced up-regulation of uPAR has been identified.

The phosphatidylinositol 3-kinase (PI3K)/Akt pathway plays important roles for prostate cancer cell survival, and the androgen receptor (AR) plays essential roles for prostate cancer cell proliferation. How these two signals cooperate to control cell growth and death, however, remains unclear and debated. Here we provide the first linkage by the identification of Forkhead transcription factor FOXO3a, the PI3K/Akt downstream substrate, as a positive regulator for the induction of AR gene expression. Both Western blot and real time PCR assays demonstrate that FOXO3a can induce AR expression at the protein and mRNA levels, and gel shift and chromatin immunoprecipitation assays further demonstrate that FOXO3a can induce 5' AR promoter activity via binding to the consensus DNA-binding sequence in the AR 5' promoter - 1290 to - 1297 (5'-TTGTTTCA-3'). Under normal growth conditions, blocking PI3K/Akt signals by LY294002 causes LNCaP cell arrest in G(1) phase rather than apoptosis. However, further blocking of AR functions by AR small interfering RNA leads to dramatic LNCaP cell death, suggesting that AR may play important protective roles when the PI3K/Akt signal pathway is blocked by LY294002. Together, our data provide the first model to explain how PI3K/Akt and AR can cooperate to control LNCaP cell growth and death under normal conditions.

Glycogen synthase kinase (GSK)-3beta is a constitutively active, proline-directed serine/threonine kinase that controls growth modulation and tumorigenesis through multiple intracellular signaling pathways. How GSK-3beta regulates signaling pathways induced by cytokines such as tumor necrosis factor (TNF) is poorly understood. In this study, we used fibroblasts derived from GSK-3beta gene-deleted mice to understand the role of this kinase in TNF signaling. TNF induced NF-kappaB activation as measured by DNA binding in wild-type mouse embryonic fibroblasts, but deletion of GSK-3beta abolished this activation. This inhibition was due to suppression of IkappaBalpha kinase activation and IkappaBalpha phosphorylation, ubiquitination, and degradation. TNF-induced NF-kappaB reporter gene transcription was also suppressed in GSK-3beta gene-deleted cells. NF-kappaB activation induced by lipopolysaccharide, interleukin-1beta, or cigarette smoke condensate was completely suppressed in GSK-3beta(-/-) cells. Deletion of GSK-3beta also abolished TNF-induced c-Jun N-terminal kinase and p44/p42 mitogen-activated kinase activation. Most surprisingly, TNF-induced Akt activation also required the presence of GSK-3beta. TNF induced expression of the NF-kappaB-regulated gene products cyclin D1, COX-2, MMP-9, survivin, IAP 1, IAP 2, Bcl-x(L), Bfl-1/A1, TRAF1, and FLIP in wild-type mouse embryonic fibroblasts but not in GSK-3beta(-/-) cells, and this correlated with potentiation of TNF-induced apoptosis as indicated by cell viability, annexin V staining, and caspase activation. Overall, our results indicate that GSK-3beta plays a critical role in TNF signaling and in the signaling of other inflammatory stimuli and that its suppression can be exploited as a potential target to inhibit angiogenesis, proliferation, and survival of tumor cells.