The autophagy protein Ambra1 regulates gene expression by supporting novel transcriptional complexes.

Ambra1 is considered an autophagy and trafficking protein with roles in neurogenesis and cancer cell invasion. Here, we report that Ambra1 also localizes to the nucleus of cancer cells, where it has a novel nuclear scaffolding function that controls gene expression. Using biochemical fractionation and proteomics, we found that Ambra1 binds to multiple classes of proteins in the nucleus, including nuclear pore proteins, adaptor proteins such as FAK and Akap8, chromatin-modifying proteins, and transcriptional regulators like Brg1 and Atf2. We identified biologically important genes, such as Angpt1, Tgfb2, Tgfb3, Itga8, and Itgb7, whose transcription is regulated by Ambra1-scaffolded complexes, likely by altering histone modifications and Atf2 activity. Therefore, in addition to its recognized roles in autophagy and trafficking, Ambra1 scaffolds protein complexes at chromatin, regulating transcriptional signaling in the nucleus. This novel function for Ambra1, and the specific genes impacted, may help to explain the wider role of Ambra1 in cancer cell biology.

New soluble angiopoietin analog of Hepta-ANG1 prevents pathological vascular leakage.

Vascular leak is a key driver of organ injury in diseases, and strategies that reduce enhanced permeability and vascular inflammation are promising therapeutic targets. Activation of the angiopoietin-1 (ANG1)-Tie2 tyrosine kinase signaling pathway is an important regulator of vascular quiescence. Here we describe the design and construction of a new soluble ANG1 mimetic that is a potent activator of endothelial Tie2 in vitro and in vivo. Using a chimeric fusion strategy, we replaced the extracellular matrix (ECM) binding and oligomerization domain of ANG1 with a heptameric scaffold derived from the C-terminus of serum complement protein C4-binding protein α. We refer to this new fusion protein biologic as Hepta-ANG1, which forms a stable heptamer and induces Tie2 phosphorylation in cultured cells, and in the lung following intravenous injection of mice. Injection of Hepta-ANG1 ameliorates vascular endothelial growth factor- and lipopolysaccharide-induced vascular leakage, in keeping with the known functions of Angpt1-Tie2 in maintaining quiescent vascular stability. The new Hepta-ANG1 fusion is easy to produce and displays remarkable stability with high multimericity that can potently activate Tie2. It could be a new candidate ANG1 mimetic therapy for treatments of inflammatory vascular leak, such as acute respiratory distress syndrome and sepsis.

Abnormal morphological and functional nature of bone marrow stromal cells provides preferential support for survival of acute myeloid leukemia cells.

Hematopoietic progenitors, residing in the bone marrow (BM) niche, are supported by mesenchymal stromal cells (MSCs). Cytogenetic and molecular aberrations in these progenitors lead to acute myeloid leukemia (AML). The BM-MSC role in leukemogenesis is not fully elucidated. In the current study, an ex-vivo system of patient's own stroma (POS), best mimicking the in-vivo BM niche, has been developed aiming to unravel interactions and crosstalk between MSCs and AML cells. POS derived from AML patients at diagnosis (Dx), relapse (Rx) and remission (Rm) was compared to healthy donor MSCs in terms of their morphology, growth pattern, support of leukemia cell viability and cytokine profile. Compared to control MSCs, POS (Dx/Rx, Rm) demonstrated a reduced proliferation rate (35%), significantly slower expansion, enlarged cell area (3-4-fold) and provided preferential support to leukemic cells of the same individual. Cytokine profiling showed significantly higher secreted phosphoprotein-1 (SPP1) expression in Dx/Rx and Rm POS compared to healthy MSCs. Additionally, the angiopoietin-1 expression was elevated in Dx/Rx POS with a further increase in the AML cell presence. In conclusion, the fact that POS derived in active disease and remission exhibited similar morphological and functional characteristics, might imply the involvement of the BM niche in leukemogenesis.

Angiogenic and Restorative Abilities of Human Mesenchymal Stem Cells Were Reduced Following Treatment With Serum From Diabetes Mellitus Type 2 Patients.

This experiment investigated the impact of serum from patients with type 2 diabetes mellitus on the angiogenic behavior of human mesenchymal stem cells in vitro. Changes in the level of Ang-1, Ang-2, cell migration, and trans-differentiation into pericytes and endothelial lineage were monitored after 7 days. The interaction of mesenchymal stem cells with endothelial cells were evaluated using surface plasmon resonance technique. Paracrine restorative effect of diabetic stem cells was tested on pancreatic ß cells. Compared to data from FBS and normal serum, diabetic serum reduced the stem cell survival and chemotaxis toward VEGF and SDF-1α (P < 0.05). Diabetic condition were found to decline cell migration rate and the activity of MMP-2 and -9 (P < 0.05). The down-regulation of VEGFR-2 and CXCR-4 was observed with an increase in the level of miR-1-3p and miR-15b-5p at the same time. The paracrine angiogenic potential of diabetic stem cells was disturbed via the changes in the dynamic of Ang-1, Ang-2, and VEGF. Surface plasmon resonance analysis showed that diabetes could induce an aberrant increase in the interaction of stem cells with endothelial cells. After treatment with diabetic serum, the expression of VE-cadherin and NG2 and ability for uptake of Dil-Ac-LDL were reduced (P < 0.01). Conditioned media prepared from diabetic stem cells were unable to decrease fatty acid accumulation in ß-cells (P < 0.05). The level of insulin secreted by ß-cells was not affected after exposure to supernatant from diabetic or non-diabetic mesenchymal stem cells. Data suggest diabetes could decrease angiogenic and restorative effect of stem cells in vitro. J. Cell. Biochem. 119: 524-535, 2018. © 2017 Wiley Periodicals, Inc.

The balance between proliferation and transcription of angiogenic factors of mesenchymal stem cells in hypoxia.

Bridging large bone defects with mesenchymal stromal cells-seeded scaffolds remains a big challenge in orthopedic surgery, due to the lack of vascularization. Within such a cell-scaffold construct, cells are exposed to ischemic conditions. When human mesenchymal stem cells (hMSCs) encounter hypoxic conditions, they show higher cell proliferation than at ambient oxygen levels. However, when hMSCs are exposed to prolonged ischemia, cell proliferation ceases completely. Exposure of hMSCs to hypoxic conditions is known to result in the transcription of angiogenic factors (AGF), which can promote the development of new blood vessels. In this study, we investigated at which oxygen level hMSC proliferation and the transcription of AGF were optimal. Human bone marrow-derived hMSCs were cultured at 0.1, 1, 2, 3, 4, 5, and 21% oxygen. Cell proliferation over 14 days was assayed using a DNA quantification method. hMSC metabolic activity over 14 days was measured using a MTT test. Quantitative RT-PCR was used to assess mRNA levels of angiogenic factors at the tested oxygen percentages. hMSCs showed the highest cell proliferation rate at 1% oxygen. The highest corrected cell metabolic rate was found at 21% oxygen, followed by 2% oxygen. HIF1α transcription did not increase under hypoxic conditions compared to 21% oxygen conditions. However, transcription of VEGF and ANG-1 was significantly higher at 2% oxygen than at 21% O2. The optimum oxygen range at which hMSCs proliferated rapidly and angiogenic factors ANG-1 and VEGF simultaneously came to expression was from 1 to 2% oxygen.

Vasculotide, an angiopoietin-1 mimetic, reduces pulmonary vascular leakage and preserves microcirculatory perfusion during cardiopulmonary bypass in rats.

BACKGROUND: Cardiopulmonary bypass (CPB) during cardiac surgery impairs microcirculatory perfusion and is paralleled by vascular leakage. The endothelial angiopoietin/Tie2 system controls microvascular leakage. This study investigated whether targeting Tie2 with the angiopoietin-1 mimetic vasculotide reduces vascular leakage and preserves microcirculatory perfusion in a rat CPB model. METHODS: Rats were subjected to 75 min of CPB after treatment with vasculotide or phosphate buffered solution as control or underwent a sham procedure. Microcirculatory perfusion and leakage were assessed with intravital microscopy (n=10 per group) and Evans blue dye extravasation (n=13 per group), respectively. Angiopoietin-1, -2, and Tie2 protein and gene expression were determined in plasma, kidney, and lung. RESULTS: CPB immediately impaired microcirculatory perfusion [5 (4-8) vs 10 (7-12) vessels per recording, P=0.002] in untreated CPB rats compared with sham, which persisted after weaning from CPB. CPB increased circulating angiopoeietin-1, -2, and soluble Tie2 concentrations and reduced Tie2 messenger ribonucleic acid (mRNA) expression in kidney and lung. Moreover, CPB increased Evans blue dye leakage in kidney [12 (8-25) vs 7 (1-12) µg g-1, P=0.04] and lung [and 23 (13-60) vs 6 (4-16) µg g-1, P=0.001] compared with sham. Vasculotide treatment preserved microcirculatory perfusion during and after CPB. Moreover, vasculotide treatment reduced Evans blue dye extravasation in lung compared with CPB control [18 (6-28) µg g-1vs 23 (13-60) µg g-1, P=0.04], but not in kidney [10 (3-23) vs 12 (8-25) µg g-1, P=0.38]. Vasculotide did not affect circulating or mRNA expression of angiopoietin-1, -2, and Tie2 concentrations compared with untreated CPB controls. CONCLUSIONS: Treatment with the angiopoietin-1 mimetic vasculotide reduced pulmonary vascular leakage and preserved microcirculatory perfusion during CPB in a rat model.

Overexpression of Angiopoietin-1 Potentiates Endothelial Progenitor Cells for the Treatment of Aneurysm.

BACKGROUND: To investigate whether angiopoietin-1 (Ang-1) could regulate the endothelial progenitor cells (EPCs) survival and the effect of accelerating intra-aneurysmal organization and occlusion of the aneurysm neck. METHODS: EPCs were isolated from Wistar rats. EPCs were cultured and transfected with lentivirus-Ang-1-endothelial progenitor cells (Ang-1-EPCs) and lentivirus-NC-endothelial progenitor cells (NC-EPCs). The effects of Ang-1 on viability and functioning of EPCs were explored via tube formation, migration, and MTT (3-[4,5-dimethylthiazolyl-2]-2,5-diphenyltetrazolium bromide) assays. Eighteen Wistar rats were randomly allocated into 3 groups. Eighteen bare coils were inserted into the ligated external carotid artery (ECA) sacs of rats. The ECA sacs were removed 2 weeks after the coils were implanted and examined by histology assay. RESULTS: Ang-1 significantly promoted EPCs tube formation, migration, and proliferation ability in vitro. Histology analyses revealed that the organized areas in the ECA sacs in the Ang-1-EPCs group are higher than NC-EPCs group and control group at 2 weeks. Immunofluorescence revealed that organized tissues were characterized by an accumulation of cells positive for α-smooth muscle actin-positive cells in aneurysm sacs. CONCLUSIONS: Overexpression of Ang-1 enhanced the tube formation, migration, and proliferation ability of EPCs. Ang-1 gene-modified EPCs accelerated organization within the aneurysms and occlusion of aneurysm neck. Transplantation of Ang-1-transfected EPCs may be a new method for the treatment of aneurysm.

Adenovirus-delivered angiopoietin-1 ameliorates phosgene-induced acute lung injury via inhibition of NLRP3 inflammasome activation.

OBJECTIVE: Angiopoietin-1 (Ang1) is reported to have the ability to attenuate endothelial permeability and inflammation during the stress condition and is considered to play a critical role in vascular stabilization. The aim of this study was to investigate the mechanisms involved in the protective effects of adenovirus-delivered Ang1 in phosgene-induced acute lung injury (ALI). METHODS: ALI was induced in rats by phosgene exposure at 8.33 g/m3 for 5 min, followed by an intravenous injection of adenovirus-Ang1 (Ad/Ang1). The histologic changes of the lung were evaluated with H&E staining. The levels of cytokines in the serum and bronchoalveolar lavage fluid (BALF) were determined by ELISA. NLRP3 inflammasome activation was assessed with immunohistochemistry, RT-PCR, Western blotting and TUNEL staining. RESULTS: Histologic analyses suggested that reduced severity in phosgene-induced ALI with Ad/Ang1 treatment. Reduced levels of IL-1ß, IL-18 and IL-33 were found in both serum and BALF samples from Ad/Ang1-treated ALI rats induced by phosgene. Moreover, immunohistochemistry analysis revealed that Ad/Ang1 treatment inhibited the NLRP3 inflammasome activation. Decreased mRNA and protein levels of NLRP3 and caspase-1 were found in phosgene-exposed rats treated with Ad/Ang1. In addition, TUNEL staining indicated a decrease in pyroptosis in phosgene-exposed rats treated with Ad/Ang1. CONCLUSIONS: Ang1 exerts beneficial effects on phosgene-induced lung injury via inhibition of NLRP3 inflammasome activation. Disruption of NLRP3 inflammasome activation might be served as therapeutic modality for the treatment of phosgene-induced ALI.

Cervical cancer cell-derived angiopoietins promote tumor progression.

Metastatic or recurrent cervical cancer has limited treatment options and a high rate of mortality. Although anti-vascular endothelial growth factor drugs have shown great promise as a therapeutic target for treatment of advanced cervical cancer, drug resistance and class-specific side effects negate long-term benefits. The identification of alternative anti-angiogenic factors will be critical for future drug development for advanced or recurrent cervical cancer. In this study, we found that angiopoietins and Tie receptors were highly expressed in cervical cancer cells. Tie-2 expression in tumor cells predicted poorer prognosis. Wound closure assay and Transwell assay showed that upregulated or downregulated Ang-1 and Ang-2 expression promoted or reduced cervical cancer cell lines migration and invasion, respectively. In subcutaneous xenograft models of cervical cancer, downregulation of Ang-1 and Ang-2 attenuated tumor growth. The expression of vimentin and endomucin and microvessel density were all significantly decreased in the siAng-1 group and siAng-2 group relative to the infection control group. Our data support that dual inhibition of Ang-1 and Ang-2 may be an alternative target for anti-angiogenic adjuvant therapy in advanced or recurrent cervical squamous cell cancer.

VEGF, ANGPT1, ANGPT2, and MMP-9 expression in the autologous hematopoietic stem cell transplantation and its impact on the time to engraftment.

As a site of complicated interactions among cytokines, bone marrow niche has been the subject of many scientific studies, mainly in the context of the proteins influencing damage or recovery of endothelium after allogeneic hematopoietic stem cell transplantation (HSCT). In this study, we aimed at exploring mutual correlations of bone marrow niche cytokines involved in the homing and mobilization of hematopoietic stem cells, as well as in angiogenesis. The aim of our study was to evaluate levels of cytokines: VEGF, angiopoietin-1 (ANGPT1), angiopoietin-2 (ANGPT2), and matrix metalloproteinase 9 (MMP-9) during autologous HSCT and to examine their influence on hematological recovery. Forty-three patients with hematological malignancies (33 multiple myeloma, 10 lymphoma) were enrolled in the study. Plasma samples were taken at five time points: before conditioning treatment (BC), on transplantation day (0) and 7 (+7), 14 (+14), and 21 (+21) days after HSCT. The cytokine levels were evaluated by ELISA method. Our study revealed decreased levels of VEGF, ANGPT1, and MMP-9 in the early post-transplant period as compared to the baseline (BC). ANGPT2 was decreased after conditioning treatment, but tended to increase from day +7. On day +7, positive correlations between ANGPT1 level as well as MMP-9 and the time to engraftment were observed. As opposite to ANGPT1, negative correlation between ANGPT2 level on day +7 after HSCT and the time to hematological recovery was noticed. Our study suggests that investigated cytokines are an important part of bone marrow environment and significantly influence the time to engraftment after HSCT.

Pulmonary vascular inflammation: effect of TLR signalling on angiopoietin/TIE regulation.

Increased pulmonary vascular resistance is a critical complication in sepsis. Toll-like receptor (TLR) as well as angiopoietin (ANG) signalling both contribute to the emergence of pulmonary arterial hypertension. We hypothesized that TLR stimulation by bacterial ligands directly affects expression and secretion of ligands and receptors of the angiopoietin/TIE axis. Microvascular endothelial (HPMEC) and smooth muscle cells (SMC) of pulmonary origin were incubated with thrombin and with ligands for TLR2, -4, -5, and -9. Expression and secretion of ANG1, -2, TIE2 and IL-8 were determined using quantitative real-time PCR and ELISA. TLR stimulation had no impact either on the expression of ANG2 and TIE2 in HPMEC or on that of ANG1 in SMC. However, overall levels of both released ANG1 and -2 were halved upon stimulation with the TLR9 ligand CpG, and ANG2 release was significantly enhanced by TLR4 activation when initially provoked by sequentially performed stimulation. Furthermore, enhanced ANG2 activity increased endothelial permeability, as demonstrated in an in vitro transwell assay. We conclude that sole TLR stimulation by bacterial ligands plays no significant role for altered expression and secretion of ANG1, -2 and TIE2 in human pulmonary vascular cells. The interplay between various stimuli is required to induce imbalances between ANG1 and -2.

Role of vascular endothelial growth factor in inducing production of angiopoetin-1 - in vivo study in Fisher rats.

The aim of the study was to investigate how an intramuscular injection of plasmids with genes coding various pro-angiogenic factors: angiopoetin-1 (ANGPT1), vascular endothelial growth factor (VEGF165) and hepatic growth factor (HGF), influences the production of ANGPT1. 40 Healthy Fisher rats received i.m. injections containing plasmids encoding pro-angiogenic genes in thigh muscles. They were divided into four equal groups. The first group received the plANGPT1 plasmid and the second group- the pIRES/ANGPT1/VEGF165 bicistronic plasmid. The pIRES/VEGF165/HGF bicistronic plasmid was administered to the third group and an empty plasmid (control group) to the fourth group. The animals were euthanized after 12 weeks. In each group, the number of vessels stained with the anti-ANGPT1 antibody was assessed under an optical microscope. The anti-ANGPT1 antibodies stained the vessels in all the groups. There were on average 14.1 ±2.3 vessels in the the plANGPT1 group, 32.5 ±10.5 in the pl/RESANGPT1/VEGF group and 30.8 ±13.3 in the plRES/HGV/VEGF group. There were on average 7.3 ±2.3 stained vessels (p < 0.0001) in the control group . The VEGF plays a role in the induction of the production of ANGPT1. The administration of plasmids only encoding ANGPT1 does not induce its production.

Mechanism of angiopoietin-1 upregulation in Kaposi's sarcoma-associated herpesvirus-infected PEL cell lines.

UNLABELLED: Angiopoietin-1 (ANGPT-1) is a secreted glycoprotein that was first characterized as a ligand of the Tie2 receptor. In a previous study using microarray analysis, we found that the expression of ANGPT-1 was upregulated in Kaposi's sarcoma-associated herpesvirus (KSHV)-infected primary effusion lymphoma (PEL) cell lines compared with that in uninfected Burkitt and other leukemia cell lines. Other authors have also reported focal expression of ANGPT-1 mRNA in biopsy specimens of Kaposi's sarcoma (KS) tissue from patients with AIDS. Here, to confirm these findings, we examined the expression and secretion levels of ANGPT-1 in KSHV-infected PEL cell lines and address the mechanisms of ANGPT-1 transcriptional regulation. We also showed that ANGPT-1 was expressed and localized in the cytoplasm and secreted into the supernatant of KSHV-infected PEL cells. Deletion studies of the regulatory region revealed that the region encompassing nucleotides -143 to -125 of the ANGPT-1-regulating sequence was responsible for this upregulation. Moreover, an electrophoretic mobility shift assay and chromatin immunoprecipitation, followed by quantitative PCR, suggested that some KSHV-infected PEL cell line-specific DNA-binding factors, such as OCT-1, should be involved in the upregulation of ANGPT-1 in a sequence-dependent manner. IMPORTANCE: We confirmed that ANGPT-1 was expressed in and secreted from KSHV-infected PEL cells and that the transcriptional activity of ANGPT-1 was upregulated. A 19-bp fragment was identified as the region responsible for ANGPT-1 upregulation through binding with OCT-1 as a core factor in PEL cells. This study suggests that ANGPT-1 is overproduced in KSHV-infected PEL cells, which could affect the pathophysiology of AIDS patients with PEL.

Phase I study of multi-gene cell therapy in patients with peripheral artery disease.

UNLABELLED: Alternative treatment strategies for claudication are needed and cell-based therapies designed to induce angiogenesis are promising. The purpose of this report was to conduct a Phase I safety, dose-escalating, non-randomized, open-label study of autologous, fully differentiated venous endothelial and smooth muscle cells called MultiGeneAngio (MGA) for claudication due to peripheral artery disease. Twelve subjects, at two centers, received a single intra-arterial infusion of a suspension of equal amounts of transduced autologous venous smooth muscle cells expressing vascular endothelial growth factor (VEGF165) and endothelial cells expressing angiopoietin-1 (Ang-1) (Cohort 1: 1 × 10(7), Cohort 2: 2 × 10(7), Cohort 3: 5 × 10(7), Cohort 4: 7 × 10(7)). The treatment was given unblinded and in the more symptomatic lower extremity. Transduced cells were tested for in vitro doubling time, telomerase activity, and gene expression. The main outcomes were clinical safety and tolerability. Other safety measures included ankle-brachial index (ABI) and walking time on a treadmill. All subjects were male (mean age 60 ± 5 years) including 25% with diabetes mellitus. At 1-year follow-up, there was one serious adverse event possibly related to MGA. Safety endpoints including VEGF and Ang-1 plasma protein levels were within normal ranges in all subjects. The mean maximal walking time increased from baseline to 1 year and the index limb ABI was unchanged, indicating no safety concerns. MGA, an autologous, transduced, cell-based therapy was well tolerated and safe in this Phase I study. Further evaluation is warranted in randomized human studies. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT00390767.

Angiopoietin-1 prevents severe bleeding complications induced by heparin-like drugs and fibroblast growth factor-2 in mice.

Critically ill children can develop bleeding complications when treated with heparin-like drugs. These events are usually attributed to the anticoagulant activity of these drugs. However, previous studies showed that fibroblast growth factor-2 (FGF-2), a heparin-binding growth factor released in the circulation of these patients, could precipitate intestinal hemorrhages in mice treated with the heparin-like drug pentosan polysulfate (PPS). Yet very little is known about how FGF-2 induces bleeding complications in combination with heparin-like drugs. Here, we examined the mechanisms by which circulating FGF-2 induces intestinal hemorrhages in mice treated with PPS. We used a well-characterized mouse model of intestinal hemorrhages induced by FGF-2 plus PPS. Adult FVB/N mice were infected with adenovirus carrying Lac-Z or a secreted form of recombinant human FGF-2, and injected with PPS, at doses that do not induce bleeding complications per se. Mice treated with FGF-2 in combination with PPS developed an intestinal inflammatory reaction that increased the permeability and disrupted the integrity of submucosal intestinal vessels. These changes, together with the anticoagulant activity of PPS, induced lethal hemorrhages. Moreover, a genetically modified form of the endothelial ligand angiopoietin-1 (Ang-1*), which has powerful antipermeability and anti-inflammatory activity, prevented the lethal bleeding complications without correcting the anticoagulant status of these mice. These findings define new mechanisms through which FGF-2 and Ang-1* modulate the outcome of intestinal bleeding complications induced by PPS in mice and may have wider clinical implications for critically ill children treated with heparin-like drugs.

Effect of aging and cardiovascular risk factors on receptor Tie1 expression in human erectile tissue.

INTRODUCTION: Erectile dysfunction is highly prevalent in patients with advanced age or cardiovascular disease risk factors (CVDRFs). These conditions interfere on expression of vascular growth factors and respective receptors causing disturbance in endothelial function. AIM: This study aims to assess the effect of aging and CVDRF on the expression of tyrosine kinase with immunoglobulin-like and EGF-like domains (Tie) 1 in human corpus cavernosum (CC). METHODS: CC fragments obtained from programmed surgeries or organ donors were divided into three groups: young, healthy aged, and aged with CVDRF. Angiopoietin (Ang) 1, Ang2, Tie1, and Tie2 mRNA and protein levels were assessed by real-time polymerase chain reaction and Western blotting, respectively. Dual-immunolabeling of Tie1 with specific markers of endothelium and smooth muscle and Ang1 and Ang2 was performed. MAIN OUTCOME MEASURES: To characterize the expression of Tie1 in human CC and elucidate its potential inhibitory effect in Ang-Tie2 system. RESULTS: Analysis of mRNAs demonstrated a decrease in Tie1 expression in CVDRF individuals compared with aged or young healthy individuals. No variation for Tie2, Ang1, or Ang2 expression was observed among the studied groups. In all analyzed CC fragments, a 125 kDa band, Tie1, was detected. This protein presented a significant age-related decrease, specially in individuals with CVDRF. Immunofluorescence study revealed Tie1 expression in the endothelium of samples of all experimental groups. CONCLUSIONS: Employing different methodological approaches, we show for the first time that Tie1 is expressed in human CC endothelium, and its level of expression diminishes in aged individuals, particularly those with CVDRF. This finding reinforces the view that delivery of Ang1 to the CC of erectile dysfunction affected CVDRF patients is able to activate a beneficial Tie2 response.

Dopamine stabilizes tumor blood vessels by up-regulating angiopoietin 1 expression in pericytes and Kruppel-like factor-2 expression in tumor endothelial cells.

Impaired blood flow in the tumor vascular bed caused by structurally and functionally abnormal blood vessels not only hinders the delivery of chemotherapeutic agents but also aggravates tumor hypoxia, making the tumor cells further resistant to antineoplastic drugs. Therefore, normalization of tumor blood vessels may be an important approach to increase therapeutic efficacy in the treatment of cancer patients. As blood vessels are supplied by sympathetic nerves containing dopamine (DA), and DA regulates functions of normal blood vessels through its receptors present in these vessels, we investigated the effect of DA on tumor vasculature. Here we report loss of sympathetic innervation and endogenous DA in abnormal and immature tumor blood vessels in malignant colon and prostate tumor tissues. In contrast, exogenous administration of DA normalizes the morphology and improves the functions of these vessels by acting on pericytes and endothelial cells, the two major cellular components of blood vessels. DA acts through its D(2) receptors present in these cells to up-regulate directly the expression of angiopoietin 1 (Ang1) in pericytes and the expression of the zinc finger transcriptional factor, Krüppel-like factor-2 (KLF2) in tumor endothelial cells. Importantly, this vessel stabilization by DA also significantly increases the concentration of anticancer drug in tumor tissues. These results show a relationship between vascular stabilization and a neurotransmitter and indicate that DA or its D(2) receptor-specific agonists can be an option for the treatment of cancer and disorders in which normalization of blood vessels may have therapeutic benefits.

Renal ischemia-reperfusion induces a dysbalance of angiopoietins, accompanied by proliferation of pericytes and fibrosis.

Endothelial cells (ECs) are highly susceptible to hypoxia and easily affected upon ischemia-reperfusion (I/R) during renal transplantation. Pericytes and angiopoeitins play important role in modulating EC function. In the present study, we investigate the effect of renal I/R on the dynamics of angiopoietin expression and its association with pericytes and fibrosis development. Male Lewis rats were subjected to unilateral renal ischemia for 45 min followed by removal of the contralateral kidney. Rats were killed at different time points after reperfusion. Endothelial integrity (RECA-1), pericytes [platelet-derived growth factor receptor-ß (PDGFR-ß)], angiopoietin-2 (Ang-2)/angiopoietin-1 (Ang-1) expression, and interstitial collagen deposition (Sirius red and α-smooth muscle actin) were assessed using immunohistochemistry and RT-PCR. Our study shows an increase in protein expression of Ang-2 starting at 5 h and remaining elevated up to 72 h, with a consequently higher Ang-2/Ang-1 ratio after renal I/R (P < 0.05 at 48 h). This was accompanied by an increase in protein expression of the pericytic marker PDGFR-ß and a loss of ECs (both at 72 h after I/R, P < 0.05). Nine weeks after I/R, when renal function was restored, we observed normalization of the Ang-2/Ang-1 ratio and PDGFR-ß expression and increase in cortical ECs, which was accompanied by fibrosis. Renal I/R induces a dysbalance of Ang-2/Ang-1 accompanied by proliferation of pericytes, EC loss, and development of fibrosis. The Ang-2/Ang-1 balance was reversed to baseline at 9 wk after renal I/R, which coincided with restoration of cortical ECs and pericytes. Our findings suggest that angiopoietins and pericytes play an important role in renal microvascular remodeling and development of fibrosis.

Reduced expression of bone morphogenetic protein receptor IA in pancreatic cancer is associated with a poor prognosis.

BACKGROUND: The expression of SMAD4, the central component of the transforming growth factor-ß (TGF-ß) and bone morphogenetic protein (BMP) signalling pathways, is lost in 50% of pancreatic cancers and is associated with a poor survival. Although the TGF-ß pathway has been extensively studied and characterised in pancreatic cancer, there is very limited data on BMP signalling, a well-known tumour-suppressor pathway. BMP signalling can be lost not only at the level of SMAD4 but also at the level of BMP receptors (BMPRs), as has been described in colorectal cancer. METHODS: We performed immunohistochemical analysis of the expression levels of BMP signalling components in pancreatic cancer and correlated these with survival. We also manipulated the activity of BMP signalling in vitro. RESULTS: Reduced expression of BMPRIA is associated with a significantly worse survival, primarily in a subset of SMAD4-positive cancers. In vitro inactivation of SMAD4-dependent BMP signalling increases proliferation and invasion of pancreatic cancer cells, whereas inactivation of BMP signalling in SMAD4-negative cells does not change the proliferation and invasion or leads to an opposite effect. CONCLUSION: Our data suggest that BMPRIA expression is a good prognostic marker and that the BMP pathway is a potential target for future therapeutic interventions in pancreatic cancer.

Vascular endothelial growth factor and angiopoietin production by primate follicles during culture is a function of growth rate, gonadotrophin exposure and oxygen milieu.

STUDY QUESTION: What is the time course of production of vascular endothelial growth factor-A (VEGF-A), angiopoietin (ANGPT)-1 and ANGPT-2 by primate follicles during encapsulated three-dimensional culture, and what conditions affect their production? SUMMARY ANSWER: Primate follicles produce VEGF-A and ANGPT-2 in vitro, particularly after developing to the antral stage, with VEGF production influenced by FSH concentration and O(2) tension. WHAT IS KNOWN ALREADY: Folliculogenesis, i.e. the development of primordial follicles into mature, antral follicles, requires the creation of a vascular network in the follicle wall via a process called angiogenesis. Angiogenic factors including VEGFs and ANGPTs have documented roles in angiogenesis. However, direct studies on the production and regulation of angiogenic factors by individual, growing follicles are limited. STUDY DESIGN, SIZE, DURATION: Ovaries (n = 9 pairs) were obtained from rhesus macaques during the early follicular phase of the menstrual cycle (cycle days 1-4). Secondary (125-225 µm) follicles were isolated mechanically, encapsulated into alginate (0.25% w/v) and cultured for 40 days. MATERIALS, SETTING, METHODS: Individual follicles were cultured in a 5 or 20% O(2) environment in alpha minimum essential medium supplemented with recombinant human (h) FSH. Half of the follicles had recombinant hLH added to the media from Days 30 to 40. Follicle diameters were measured weekly. Follicles were categorized at Week 5 as no-grow (NG; <250 µm in diameter), slow-grow (SG; 251-499 µm) and fast-grow (FG; >500 µm). VEGF-A, ANGPT-1 and -2 concentrations in media were measured by ELISA. MAIN RESULTS AND THE ROLE OF CHANCE: VEGF concentrations were low throughout the culture for NG follicles. SG and FG follicles had detectable VEGF concentrations at Week 2, which continued to rise throughout culture. VEGF concentrations were distinct (P < 0.05) among all three follicle categories during Weeks 4 and 5. VEGF concentrations were higher (P < 0.05) in SG follicles in the presence of high/mid-dose FSH at 5% O(2). In contrast, there were no dose-dependent differences in VEGF production for FG follicles based on FSH concentrations or O(2) tension. At Week 5, follicles that produced metaphase II oocytes, following exposure to an ovulatory hCG dose, secreted higher concentrations of VEGF than those containing germinal vesicle-intact oocytes. Media concentrations of ANGPT-1 were low throughout culture for all three follicle categories. ANGPT-2 concentrations were low throughout culture for NG follicles. In contrast, ANGPT-2 concentrations of SG and FG follicles continued to rise from Weeks 1 to 4. During Weeks 2-4, ANGPT-2 concentrations in FG follicles were significantly higher than those of SG and NG follicles (P < 0.05). LIMITATION, REASONS FOR CAUTION: This study reports VEGF-A, ANGPT-1 and -2 production by in vitro-developed individual primate (macaque) follicles, that is limited to the interval from the secondary to small antral stage. After VEGF and ANGPT-1 assays, the limited remaining samples did not allow assessment of the independent effects of gonadotrophin and O(2) on the ANGPT-2 production by cultured follicles. Findings await translation to human follicles. WIDER IMPLICATION OF THE FINDINGS: The above findings provide novel information on the process of primate follicle maturation. We hypothesize that a symbiotic relationship between elevated concentrations of ANGPT-2 and VEGF allows FG antral follicles to excel in follicle maturation, e.g. by promoting its vascularization. Elevated ANGPT-2 may also offer possible insight into future oocyte quality as early as Week 2, compared with Week 4 for VEGF and follicle size. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by the following grants: NIH U54 RR024347/HD058294/PL1-EB008542 (Oncofertility Consortium), NIH U54-HD018185 (SCCPIR), NIH ORWH/NICHD 2K12HD043488 (BIRCWH), NIH FIC TW/HD-00668, ONPRC 8P51OD011092. There are no conflicts of interest to declare.