Vascular Endothelial Growth Factor Vegf Research Articles

The NK1 receptor antagonist NKP608 inhibits proliferation of human colorectal cancer cells via Wnt signaling pathway

BackgroundNeurokinin1 (NK1) receptor has played a vital role in the development of tumor. However, NKP608 as a NK1 receptor antagonist whether has the effect of the resistance of colorectal cancer is still unclear. Thereby, in this study, we investigated the role of NKP608 on human colorectal cancer and explored the underlying mechanism.MethodsThe cell proliferation of colorectal cancer cells was detected by cell counting kit-8 (CCK8) assay, cell migration and invasion were assessed by transwell assay, the apoptotic ratio of cells was assessed by Annexin V-fluorescein isothiocyanate/propidium iodide stained and flow cytometry. The involvement of molecular mechanisms was examined by western blot.ResultsIn this study, we found that NKP608 inhibited the proliferation, migration/invasion of HCT116 cells. In addition, NKP608 reduced expressions of Wnt-3a, β-catenin, Cyclin D1, and (vascular endothelial growth factor) VEGF while induced expression of E-Cadherin. Furthermore, flow cytometry analyzed that NKP608 induced apoptosis of HCT116 cells, consistently, western blotting detecting of apoptosis-related proteins revealed that NKP608 downregulated Bcl-2 while upregulated Bax and Active-Caspase-3.ConclusionsTaken together, our results demonstrated that NKP608 inhibited colorectal cancer cell proliferation, migration and invasion via suppressing the Wnt/β-catenin signaling pathway. Therefore, NKP608 might represent a promising therapeutic agent in the treatment of colorectal cancer.

Obstructive Lymphangitis Precedes Colitis in Murine Norovirus–Infected Stat1-Deficient Mice

Murine norovirus (MNV) is an RNA virus that can prove lethal in mice with impaired innate immunity. We found that MNV-4 infection of Stat1-/- mice was not lethal, but produced a 100% penetrant, previously undescribed lymphatic phenotype characterized by chronic-active lymphangitis with hepatitis, splenitis, and chronic cecal and colonic inflammation. Lesion pathogenesis progressed from early ileal enteritis and regional dilated lymphatics to lymphangitis, granulomatous changes in the liver and spleen, and, ultimately, typhlocolitis. Lesion development was neither affected by antibiotics nor reproduced by infection with another enteric RNA virus, rotavirus. MNV-4 infection in Stat1-/- mice decreased expression of vascular endothelial growth factor (Vegf) receptor 3, Vegf-c, and Vegf-d and increased interferon (Ifn)-γ, tumor necrosis factor-α, and inducible nitric oxide synthase. However, anti-IFN-γ and anti-tumor necrosis factor-α antibody treatment did not attenuate the histologic lesions. Studies in Ifnαβγr-/- mice suggested that canonical signaling via interferon receptors did not cause MNV-4-induced disease. Infected Stat1-/- mice had increased STAT3 phosphorylation and expressed many STAT3-regulated genes, consistent with our findings of increased myeloid cell subsets and serum granulocyte colony-stimulating factor, which are also associated with increased STAT3 activity. In conclusion, in Stat1-/- mice, MNV-4 induces lymphatic lesions similar to those seen in Crohn disease as well as hepatitis, splenitis, and typhlocolitis. MNV-4-infected Stat1-/- mice may be a useful model to study mechanistic associations between viral infections, lymphatic dysfunction, and intestinal inflammation in a genetically susceptible host.

Vascular-Derived Vegfa Promotes Cortical Interneuron Migration and Proximity to the Vasculature in the Developing Forebrain.

Vascular endothelial growth factor (Vegfa) is essential for promoting the vascularization of the embryonic murine forebrain. In addition, it directly influences neural development, although its role in the forming forebrain is less well elucidated. It was recently suggested that Vegfa may influence the development of GABAergic interneurons, inhibitory cells with crucial signaling roles in cortical neuronal circuits. However, the mechanism by which it affects interneuron development remains unknown. Here we investigated the developmental processes by which Vegfa may influence cortical interneuron development by analyzing transgenic mice that ubiquitously express the Vegfa120 isoform to perturb its signaling gradient. We found that interneurons reach the dorsal cortex at mid phases of corticogenesis despite an aberrant vascular network. Instead, endothelial ablation of Vegfa alters cortical interneuron numbers, their intracortical distribution and spatial proximity to blood vessels. We show for the first time that vascular-secreted guidance factors promote early-migrating interneurons in the intact forebrain in vivo and identify a novel role for vascular-Vegfa in this process.

The protective roles of L-borneolum, D-borneolum and synthetic borneol in cerebral ischaemia via modulation of the neurovascular unit

ObjectiveBorneol has been used to treat stroke in China since ancient times. In our previous research, we demonstrated the effect of borneol on cerebral ischaemia injury via meta-analysis. The neurovascular unit (NVU) is the structural basis of the preservation of the brain microenvironment and is believed to be a promising target in treating stroke. In this research, we explored the roles of three kinds of borneol, namely, L-borneolum (B1), D-borneolum (B2) and synthetic borneol (B3), in the NVU with permanent middle cerebral artery occluded (pMCAO) rats. MethodsThe Longa scoring method was used to evaluate nerve function deficits in the pMCAO rats. Awakening time, brain water content, brain index and brain edema rate were also measured. TTC staining was used to calculate the cerebral infarction rate. The morphology of the ischaemia penumbra brain tissue was observed via HE staining, and the neuronal denatured cell index (DCI) was calculated. An enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of vascular endothelial growth factor VEGF and TNF-α in the serum. Moreover, the ultrastructures of the neurons and of the blood-brain barrier (BBB) were observed using transmission electron microscopy. The expression levels of Claudin-5, Bcl-2 and Bax in the ischaemia penumbra of pMCAO rats were detected using real-time PCR and immunohistochemistry. ResultsPretreatment with B1, B2 and B3 delayed the recovery time (P < 0.01). B1 remarkably ameliorated neurological deficits 24 h after cerebral ischaemia (P < 0.05). Moreover, B1 and B3 were both able to ameliorate brain edema and the area of cerebral infarction. In addition, B1, B2 and B3 all increased serum VEGF levels and decreased serum TNF-α levels (P < 0.01). For the ultrastructure determination, the BBB and the nerve centre were significantly improved by B1, B2 and B3. The mechanistic exploration revealed that B2 and B3 protected the brain by reducing the Bax/Bcl-2 ratio (P < 0.05, P < 0.01, respectively). Immunohistochemistry suggested that B1, B2 and B3 could also enhance the expression of Claudin-5 (P < 0.01). ConclusionThe three kinds of borneol demonstrated different protective effects on cerebral ischaemia injury. L-Borneolum displayed the most prominent anti-cerebral ischaemia effect among them. The mechanism was most likely executed via anti-apoptosis and anti-inflammation effects and maintenance of the stability of the BBB and TJs to comprehensively improve NVU function.

IMMUNOSUPPRESSIVE POTENTIAL OF PERIPHERAL REGULATORY T-LYMPHOCYTES IN THE PROCESS OF TUMOR PROGRESSION IN PATIENTS WITH METASTATIC SOFT TISSUE SARCOMAS

In tumor microenvironment the self-maintenance condition of T-regulatory lymphocytes (Treg) are created by tumor cells due to the production of vascular endothelial growth factor VEGF and chemokine CCL2. In the present work there was evaluated the quantitative content of these proteins in cultured cell supernatants of metastatic soft tissue sarcomas (STS) as well as characterized the immunophenotype of peripheral blood Treg by flow cytometry. The study included 35 patients who received treatment at the N.N. Petrov National Medical Research Center of Oncology. For the study- samples of metastatic tumor were taken to obtain sarcoma cell culture and samples of peripheral blood of patients in the absence of tumor growth (stable disease-SD) or disease progression (PD). The statistically significant differences were found in the quantitative content of CCR10+Treg (9.1 % and 4.5 %, respectively, p=0.001), CCR4+Treg (10 % and 3.3 %, respectively, p=0.001), neuropilin-1 (Nrp1+) Treg (6 % and 4.5 %, respectively, p=0.021) in patients with PD and SD. A direct correlation of high strength was found between production of VEGF, CCL2 by metastatic STS cells and expression of Nrp1 (r=0.93, p=0.001), VEGFR-2(r=0.88, p=0.007), CCR4(r=0.81, p=0.024) by Treg cells. Statistically significant differences in the CCR10+Treg (9.5 % and 2.93 %, respectively, p=0.012) and CCR4+Treg (68, 3 % and 3.95 %, respectively, p=0.007) were detected in the group of patients with liposarcoma and synovial sarcoma. Thus in patients with metastatic STS - there was directional chemotaxis of Treg into tumor microenvironment providing the creation of tumor-induced tolerance, which could be associated with DP. The revealed regularities could be used to plan adjuvant and palliative treatment of STS patients.

The effect of vascular endothelial growth factor VEGF-A gene polymorphisms and intravitreal anti-VEGF treatment outcomes in patients with exudative age-related macular degeneration

The effect of vascular endothelial growth factor VEGF-A gene polymorphisms and intravitreal anti-VEGF treatment outcomes in patients with exudative age-related macular degeneration

P 42 Down-regulation of The Nr2-regulated gene heme oxygenase-1 (HO-1) in the placenta from patients with early-onset preeclampsia

Introduction and objective Increased oxidative stress and diminished oxidative capacity are distinctive features in the pregnancy-specific syndrome preeclampsia (PE). Activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) initiates and enhances the transcription of many antioxidative enzymes such as heme oxygenase-1 (HO-1), which participates in the exposure to reactive oxygen species and protects the trophoblast-like cells against oxidative injury. HO-1 is an important mediator of the pro-angiogenic activity of the vascular endothelial growth factor VEGF. In this study we analysed the placental protein expression of Nrf2 and HO-1 in the placentas from normal pregnancies and those complicated with early-onset preeclampsia. Patients and methods 12 placentas from patients complicated with early-onset preeclampsia were analysed and compared to normal placentae from 11 healthy normotensive women (29–33 weeks gestation). The protein extracts were subjected to western blotting using anti-Nrf2 and anti-HO-1 antibodies. The serum levels of VEGF and sFlt-1 were estimated using ELISA. Results As expected the sFlt-1/VEGF ratio was increased in patients with early-onset preeclampsia R > 440 compared to normotensive ones (p Conclusion These data reveal a novel mechanism by which the decreased expression of HO-1 during PE may be involved in the pathogenesis of this syndrome. Interestingly, this does not correlate with the expression of Nrf2 in preeclamptic placentas. Nrf2 is the major transcriptional regulator of HO-1 and possible alterations in post-translational Nrf2 regulation of antioxidative enzymes in early-onset PE will be a subject of our further research.

HIF1A overexpression using cell-penetrating DNA-binding protein induces angiogenesis in vitro and in vivo

Hypoxia-inducible factor-1 alpha (HIF1A) is an important transcription factor for angiogenesis. Recent studies have used the protein transduction domain (PTD) to deliver genes, but the PTD has not been used to induce the expression of HIF1A. This study aimed at using a novel PTD (Hph-1-GAL4; ARVRRRGPRR) to overexpress the HIF1A and identify the effects on angiogenesis in vitro and in vivo. Overexpression of HIF1A was induced using Hph-1-GAL4 in human umbilical vein/vascular endothelium cells (HUVEC). The expression levels of genes were analyzed by the quantitative real-time polymerase chain reaction (qPCR) after 2 and 4days, respectively. An in vitro tube formation was performed using Diff-Quik staining. HIF1A and Hph-1-GAL4 were injected subcutaneously into the ventral area of each 5-week-old mouse. All of the plugs were retrieved after 1week, and the gene expression levels were evaluated by qPCR. Each Matrigel plug was evaluated using the hemoglobin assay and hematoxylin and eosin (HE) staining. The expression levels of HIF1A and HIF1A target genes were significantly higher in HIF1A-transfected HUVEC than in control HUVEC in vitro. In the in vivo Matrigel plug assay, the amount of hemoglobin was significantly higher in the HIF1A-treatment group than in the PBS-treatment group. Blood vessels were identified in the HIF1A-treatment group. The expression levels of HIF1A, vascular endothelial growth factor (Vegf), and Cd31 were significantly higher in the HIF1A-treatment group than in the PBS-treatment group. These findings suggest that using Hph-1-G4D to overexpress HIF1A might be useful for transferring genes and regenerating tissues.

Melittin suppresses tumor progression by regulating tumor-associated macrophages in a Lewis lung carcinoma mouse model.

Tumor-associated macrophages (TAM) are a major component of tumor stroma. It has been reported that TAMs have M2-like phenotype and facilitate tumor progression by promoting angiogenesis and immunosuppression. Melittin, a major polypeptide of bee venom, has been widely studied as an anti-cancer drug due to its cytotoxicity to malignant cells. However, very little is known regarding the effect of melittin on immune cells in the tumor microenvironment. This study focuses on the effect of melittin on TAMs in a Lewis lung carcinoma mouse model. Melittin inhibited the rapid tumor growth compared to the control in vivo. Melittin increased the M1/M2 ratio of TAMs by selectively reducing the number of CD206+ M2-like TAMs while not altering the population of CD86+ M1-like TAMs. Melittin also preferentially binds to M2 macrophages, and this binding was not associated with phagocytosis. Gene and protein expression of vascular endothelial growth factor (Vegf) and mannose receptor C type 1 (Mrc1/CD206) was reduced in M2-like bone marrow-derived macrophages by melittin treatment, but there was no significant change in the gene level of Vegf and FMS-like tyrosine kinase 1 (Flt1/VEGFR1) in tumor cells in vitro. Additionally, the levels of VEGF and CD31, markers of angiogenesis, were significantly decreased by melittin treatment in tumor tissues. This study revealed a novel role for melittin in tumor treatment and suggested that melittin could be a promising therapeutic agent for targeting M2-like TAMs.

Podocytes regulate the glomerular basement membrane protein nephronectin by means of miR-378a-3p in glomerular diseases

The pathophysiology of many proteinuric kidney diseases is poorly understood, and microRNAs (miRs) regulation of these diseases has been largely unexplored. Here, we tested whether miR-378a-3p is a novel regulator of glomerular diseases. MiR-378a-3p has two predicted targets relevant to glomerular function, the glomerular basement membrane matrix component, nephronectin (NPNT), and vascular endothelial growth factor VEGF-A. In zebrafish (Danio rerio), miR-378a-3p mimic injection or npnt knockdown by a morpholino oligomer caused an identical phenotype consisting of edema, proteinuria, podocyte effacement, and widening of the glomerular basement membrane in the lamina rara interna. Zebrafish vegf-A protein could not rescue this phenotype. However, mouse Npnt constructs containing a mutated 3'UTR region prevented the phenotype caused by miR-378a-3p mimic injection. Overexpression of miR-378a-3p in mice confirmed glomerular dysfunction in a mammalian model. Biopsies from patients with focal segmental glomerulosclerosis and membranous nephropathy had increased miR-378a-3p expression and reduced glomerular levels of NPNT. Thus, miR-378a-3p-mediated suppression of the glomerular matrix protein NPNT is a novel mechanism for proteinuria development in active glomerular diseases.

Melittin suppresses tumor progression by regulating tumor-associated macrophages in a Lewis lung carcinoma mouse model

Abstract Tumor-associated macrophages (TAM) are a major component of tumor stroma. It has been reported that TAMs have M2-like phenotype and facilitate tumor progression by promoting angiogenesis and immunosuppression. Melittin, a major polypeptide of bee venom, has been widely studied as an anti-cancer drug due to its cytotoxicity to malignant cells. However, very little is known regarding the effect of melittin on immune cells in the tumor microenvironment. We demonstrated here that melittin inhibited the rapid tumor growth compared to the control in vivo. Melittin increased the M1/M2 ratio of TAMs by selectively reducing the number of CD206+ M2-like TAMs, while not affecting the number of CD86+ M1-like TAMs. Melittin also preferentially bound to CD11b+ cells and this binding was not associated with phagocytosis. Gene expression of vascular endothelial growth factor (Vegf) and mannose receptor C type 1 (Mrc1/CD206) was reduced in M2-like bone marrow-derived macrophages by melittin treatment, but there was no significant change in the gene level of Vegf and FMS-like tyrosine kinase 1 (Flt1/VEGFR1) in tumor cells. Additionally, the levels of VEGF and CD31, markers of angiogenesis, were significantly decreased by melittin treatment in tumor tissues. This study revealed a novel role for melittin in tumor treatment and suggested that melittin could be a promising therapeutic agent for targeting M2-like TAMs.

Vegf signaling promotes vascular endothelial differentiation by modulating etv2 expression

Vasculogenesis involves the differentiation of vascular endothelial progenitors de novo from undifferentiated mesoderm, their migration and coalescence to form the major embryonic vessels and the acquisition of arterial or venous identity. Vascular Endothelial Growth Factor (Vegf) signaling plays multiple roles during vascular development. However, its function during embryonic vasculogenesis has been controversial. Previous studies have implicated Vegf signaling in either regulating arteriovenous specification or overall vascular endothelial differentiation. To clarify the role of Vegf in embryonic vasculogenesis and identify its downstream targets, we used chemical inhibitors of Vegf receptor (Vegfr) signaling in zebrafish embryos as well as zebrafish genetic mutants. A high level of chemical inhibition of Vegfr signaling resulted in the reduction of overall vascular endothelial marker gene expression, including downregulation of both arterial and venous markers, ultimately leading to the apoptosis of vascular endothelial cells. In contrast, a low level of Vegfr inhibition specifically blocked arterial specification while the expression of venous markers appeared largely unaffected or increased. Inhibition of Vegfr signaling prior to the initiation of vasculogenesis reduced overall vascular endothelial differentiation, while inhibition of Vegfr signaling starting at mid-somitogenesis stages largely inhibited arterial specification. Conversely, Vegf overexpression resulted in the expansion of both arterial and pan-endothelial markers, while the expression of several venous-specific markers was downregulated. We further show that Vegf signaling affects overall endothelial differentiation by modulating the expression of the ETS transcription factor etv2/ etsrp. etv2 expression was downregulated in Vegfr- inhibited embryos, and expanded in Vegfaa-overexpressing embryos. Furthermore, vascular-specific overexpression of etv2 in Vegfr-inhibited embryos rescued defects in vascular endothelial differentiation. Similarly, vegfaa genetic mutants displayed a combination of the two phenotypes observed with chemical Vegfr inhibition: the expression of arterial and pan-endothelial markers including etv2 was downregulated while the expression of most venous markers was either expanded or unchanged. Based on these results we propose a revised model which explains the different phenotypes observed upon inhibition of Vegf signaling: low levels of Vegf signaling promote overall vascular endothelial differentiation and cell survival by upregulating etv2 expression, while high levels of Vegf signaling promote arterial and inhibit venous specification.

Tumor Blood Vessels and Vasculogenic Mimicry – Current Knowledge and Searching for New Cellular/Molecular Targets of Anti-Angiogenic Therapy

Summary Blood vessel formation in tumor is defined as tumor angiogenesis. So far, the most known its mechanism is sprouting, which means formation of blood vessels from existing ones, as a result of the proliferation and migration of endothelial cells. The main mitogenic factor of these cells is vascular endothelial growth factor VEGF, acting by VEGFR-2 receptors. Recent studies have provided knowledge about the ability of tumors to form vessel-like structures. The phenomenon was called vascular mimicry. Tumor cells show a high plasticity and they can undergo differentiation to the ones with phenotype similar to endothelial cells. Each of the known tumor angiogenesis mechanisms is a result of many different factors and cell cooperation in tumor microenvironment. Tumor ability to the heterogeneous vascularization forces developing of complex, anti-angiogenic therapy directed to different molecular and cellular targets. Therapies, used so far, often lead to drug-induced hypoxia, which increases tumor cell aggressiveness and metastasis.

Expression, purification and functionality of bioactive recombinant human vascular endothelial growth factor VEGF165 in E. coli

Vascular endothelial growth factor (VEGF) is associated with tumour growth and metastasis. Because VEGF is the major player in both angiogenesis and vascular permeability and the most explored factor in angio-inhibitory therapies, many expression procedures have been developed to produce functional VEGF165 in convenient yield. In this study, recombinant human VEGF165 was cloned and expressed in Escherichia coli (BL21)-DE3 cells and large scale production was performed by fermentation. A high yield of active soluble protein was obtained after protein extraction employing both lysozyme and sonication treatment. Inclusion bodies were also isolated from the cell lysate and subjected to a simple protocol of solubilisation and refolding. Single-step purification was performed using nickel affinity chromatography and the purified proteins were able to recognize monoclonal Anti-poly-His antibody. The biological activity of the VEGF165 was successfully tested using the Chicken chorioallantoic membrane assay, wound-healing migration and proliferation assay on human umbilical vein endothelial cells (HUVEC).

Endogenous lysophosphatidic acid participates in vascularisation and decidualisation at the maternal–fetal interface in the rat

Lysophosphatidic acid (LPA) affects several female reproductive functions through G-protein-coupled receptors. LPA contributes to embryo implantation via the lysophospholipid LPA3 receptor. In the present study we investigated the participation of endogenous LPA signalling through the LPA3 receptor in vascularisation and decidualisation, two crucial events at the maternal-fetal interface. Pregnant rats were treated with diacylglycerol pyrophosphate (DGPP), a highly selective antagonist of LPA3 receptors, on Day 5 of gestation. Pregnant rats received intrauterine (i.u.) injections of single doses of DGPP (0.1mgkg-1) in a total volume of 2μL in the left horn (treated horn) in the morning of GD5. DGPP treatment produced aberrant embryo spacing and increased embryo resorption. The LPA3 receptor antagonist decreased the cross-sectional length of the uterine and arcuate arteries and induced histological anomalies in the decidua and placentas. Marked haemorrhagic processes, infiltration of immune cells and tissue disorganisation were observed in decidual and placental tissues from sites of resorption. The mRNA expression of three vascularisation markers, namely interleukin 10 (Il10), vascular endothelial growth factor (Vegfa) and vascular endothelial growth factor receptor 1 (Vegfr1), was reduced at sites of resorption from Day 8. The results show that the disruption of endogenous LPA signalling by blocking the LPA3 receptor modified the development of uterine vessels with consequences in the formation of the decidua and placenta and in the growth of embryos.

Plasma levels and diagnostic utility of VEGF, MMP-2 and TIMP-2 in the diagnostics of breast cancer patients

Objective: We investigated plasma levels and diagnostic utility of vascular endothelial growth factor VEGF, matrix metalloproteinase-2 (MMP-2) and tissue inhibitors of metalloproteinase-2 (TIMP-2) in comparison to cancer antigen 15-3 (CA 15-3).Methods: Plasma levels of tested parameters were determined using enzyme-linked immunosorbent assay (ELISA) while CA 15-3 with chemiluminescent microparticle immunoassay (CMIA).Results: The plasma levels of VEGF, TIMP-2 showed significantly higher than CA 15-3 values of the diagnostic sensitivity, the predictive values of positive and negative test results (PPV, NPV) and the area under the receiver-operating characteristics (ROC) curve (AUC) in early stages of breast cancer (BC). The combined use of the tested parameters with CA 15-3 resulted in the increase in sensitivity, NPV and AUC, especially in the combination with VEGF (83%; 72%; 0.888) and TIMP-2 (83%; 72%; 0.894). The highest values were obtained for combination of all three parameters (93%; 85%; 0.923).Conclusions: These findings suggest the usefulness of the tested parameters in the diagnosis of BC, especially VEGF and TIMP-2 with CA 15-3 in early stages of BC, which could be a new diagnostic panel.

Efficient delivery of therapeutic siRNA into glioblastoma cells using multifunctional dendrimer-entrapped gold nanoparticles.

To synthesize the arginine-glycine-aspartic (RGD) functionalized dendrimer-entrapped gold nanoparticles (Au DENPs) for siRNA delivery to induce gene silencing of cancer cells in vitro and in vivo. Au DENPs modified with RGD peptide via a polyethylene glycol spacer were used as a vector of two distinct small interfering RNAs (siRNAs) (VEGFvascular endothelial growth factor siRNA and B-cell lymphoma/leukemia-2 siRNA), and the physicochemical properties, cytocompatibility and transfection efficiency of Au DENP/siRNA polyplexes were characterized. The Au DENP/siRNA polyplexes with good cytocompatibility and highly efficient transfection capacity can be used for the transfection of siRNAs. The developed functional RGD-modified Au DENPs may be used for efficient gene therapy of different types of cancer.

Abstract P137: Mir-122 is Associated With Stress-response Protein, Hemeoxygenase-1, for Regulation of Extracellular Matrix Remodeling in Renal Hypertension

Oxidative stress is a major contributing factor in hypertension-induced kidney injury. Hemeoxygenase-1 (Ho-1) is stress response protein constitutively expressed by the proximal tubular epithelial cells in response to oxidative stress. MicroRNAs are single stranded RNA involved in the regulation of gene expression. MicroRNA-122 has been shown to regulate Ho-1 expression in hepatitis; however whether miR-122 regulates Ho-1 in hypertensive kidney is not known. The purpose of the study was to investigate the miRNA-122 Ho-1 regulation and determine its role in extracellular matrix remodeling in renal hypertension. In vitro experiments were done using mesangial cells, treated with/without 200 μM of Angiotensin-II (Ang-II). Ho-1 was induced by ~3.5 folds with Ang-II treatment. miR-122, Ho-1 regulator, was downregulated by &gt;15 times in Ang-II treated cells. In vivo experiments were performed on WT (C57BL6/J) mice aged 12-14 wk and 75-78 wk. The animals were treated with Ang-II (1000ng/kg/min) for 4 weeks. Ho-1 is ~6 folds less in kidney of aged mice as compared to that in the young mice. Hypertension increases miR-122 expression to a greater extent (~5 folds) in aged animals. In Ho-1 knocked down mesangial cells, the extracellular matrix component, Collagen 1A1 (Col1a1), was increased by ~2 folds. In contrast, vascular endothelial growth factor ( Vegf ) and hypoxia-inducible factor ( Hif1 α ) were downregulated in Ho-1 depleted cells. In conclusion, micro RNA, miR-122, transcriptionally regulates Ho-1 as a repressor in kidney and thus affects Ho-1 mediated regulation of the extracellular remodeling in hypertension-induced renal damage.

Cell-permeable iron inhibits vascular endothelial growth factor receptor-2 signaling and tumor angiogenesis.

Angiogenesis is important for tumor growth and metastasis. Hypoxia in tumors drives this angiogenic response by stabilizing Hypoxia Inducible Factors (HIF) and target genes like Vascular Endothelial Growth Factor (VEGF). HIF stability is regulated by Prolylhydroxylases (PHD)-mediated modification. Iron is an important cofactor in regulating the enzymatic activity of PHDs. Reducing intracellular iron, for instance, mimics hypoxia and induces a pro-angiogenic response. It is hypothesized that increasing the intracellular iron levels will have an opposite, anti-angiogenic effect. We tested this hypothesis by perturbing iron homeostasis in endothelial cells using a unique form of iron, Ferric Ammonium Citrate (FAC). FAC is a cell-permeable form of iron, which can passively enter into cells bypassing the transferrin receptor mediated uptake of transferrin-bound iron. Our studies show that FAC does not decrease the levels of HIF-1α and HIF-2α in endothelial cells but inhibits the autocrine stimulation of VEGF-Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) system by blocking receptor tyrosine kinase phosphorylation. FAC inhibits VEGF-induced endothelial cell proliferation, migration, tube formation and sprouting. Finally, systemic administration of FAC inhibits VEGF and tumor cell-induced angiogenesis in vivo. In conclusion, our studies show that cell-permeable iron attenuates VEGFR-2 mediated signaling and inhibits tumor angiogenesis.

Triazine herbicides inhibit relaxin signaling and disrupt nitric oxide homeostasis

Triazines are herbicides that are widely used worldwide, and we previously observed that the maternal exposure of mice to simazine (50 or 500μg/kg) resulted in smaller ovaries and uteri of their female offspring. Here, we investigated the underlying mechanism that may account for the reproductive dysfunction induced by simazine. We found that following maternal exposure, simazine is transmitted to the offspring, as evidenced by its presence in the offspring ovaries. Analyses of the simazine-exposed offspring revealed that the expression of the relaxin hormone receptor, relaxin-family peptide receptor 1 (RXFP1), prominently decreased in their ovaries and uteri. In addition, downstream target genes of the relaxin pathway including nitric oxide (NO) synthase 2 (Nos2), Nos3, matrix metallopeptidase 9 (Mmp9), and vascular endothelial growth factor (Vegf) were downregulated in their ovaries. Moreover, AKT and extracellular signal-regulated kinases (ERK) levels and their phosphorylated active forms decreased in simazine-exposed ovaries. In vitro exposure of the human ovarian granulosa cells (KGN) and uterine endometrium cells (Hec-1A) to very low concentrations (0.001 to 1nM) of triazines including atrazine, terbuthylazine, and propazine repressed NO production with a concurrent reduction in RXFP1, NOS2, and NOS3. The inhibitory action of triazines on NO release was dependent on RXFP1, phosphoinositol 3-kinase (PI3K)/AKT, and ERK. Radioligand-binding assay also confirmed that triazines competitively inhibited the binding of relaxin to its receptor. Therefore, the present study suggests that triazine herbicides act as endocrine disrupters by interfering with relaxin hormone signaling. Thus, further evaluation of their impact on human health is imperative.