Angiogenic Network Research Articles

Von Willebrand Factor (VWF) Regulates Angiogenesis: Studies in Blood-Derived Endothelial Progenitor Cells From Von Willebrand's Disease Patients and VWF-Deficient Mice

Abstract 5311Dysregulation of angiogenesis is implicated in many diseases. Von Willebrand factor (VWF), a large plasma glycoprotein essential for normal haemostasis is synthesized by endothelial cells (EC) and megakaryocytes. Raised VWF plasma levels are a risk factor for arterial thrombosis, whilst deficiency of VWF causes Von Willebrand disease (VWD), the most common congenital bleeding disorder in man. VWD can be associated with angiodysplasia, vascular malformations linked to defective angiogenesis which are responsible for intractable bleeding. We recently showed that VWF is involved in angiogenesis. Inhibition of VWF expression in human umbilical vein EC (HUVEC) with specific siRNA resulted in increased in vitro angiogenesis on Matrigel, proliferation and migration. Mechanism studies implicated the endothelial VWF receptor, integrin αvβ3 and the angiogenesis regulator angiopoietin-2. The findings were confirmed in EC from VWD patients and in VWF-deficient mice. Blood outgrowth endothelial cells (BOEC) isolated from peripheral blood of patients with VWD showed decreased VWF release, compared to control BOEC, and increased in vitro angiogenesis, migration and proliferation, similar to what observed with VWF siRNA-treated HUVEC. In vivo studies using the matrigel model and imaging of blood vessels in the ear showed increased angiogenesis and vascular network in VWF-deficient mice compared to controls. Recent studies in patients BOEC and in VWF deficient mice provide new insight into the complexity of this phenotype. We have used the mouse model of post-natal angiogenesis in the retina to carry out detailed analysis of angiogenic networks in the VWF-deficient mouse, and found increased vascular density and defective vascular network. Moreover, using BOEC from patients with type I and type II VWD, we have studied VWF intracellular distribution by immunofluorescence confocal analysis and found patterns of expression that point to a variety of defects in synthesis, storage and secretion. These studies define a new function for VWF, which may have clinical implications for VWD and for patients at risk of CV disease. Moreover, studies with BOEC from VWF patients provide a novel understanding of the physiopathology of this disease. Disclosures:No relevant conflicts of interest to declare.

Tumor Angiogenesis: Pericytes and Maturation Are Not to Be Ignored

Angiogenesis, an essential component of tumor growth and survival, is regulated by complex interactions between several cell types and soluble mediators. Heterogeneous tumor vasculature originates from the collective effect of the nature of carcinoma and the complexity of the angiogenic network. Although the application of angiogenesis inhibitors in some types of cancers has shown clinical benefits, predictive markers to assess treatment effects have yet to be established. In this review, we focus on tumor vessel maturity as a potential marker for evaluating treatment response.

Abstract A21: DACH1 blocks prostate cancer cell growth and interleukin-6 signaling

Abstract In Drosophila, eye development is coordinately regulated by the retinal determination gene network (RDGN), mainly consisting of dachshund (dac), eyes absent (eya), and sin oculis (so). The mammalian homologues are Dach, Eya, and Six, respectively. RDGN, although best known for its role in eye specification, is essential for the development of many organs including retinal, kidney, brain, and limb in both flies and mammals. Recently, coordinate abnormal expression of the DACH1/Eya/Six complex has been observed in human tumors supporting a tumor promoting role of Six and Eya family members, and a tumor-suppressor function of DACH. Oncogenes sustain host-cell interactions, promoting invasiveness, recruiting inflammatory mediators and developing angiogenic networks. Ras and Myc promote tumor angiogenesis through up-regulation of VEGF and the inflammatory mediator, interleukin-8(IL-8) and interleukin-6 (IL-6). Our previous studies demonstrate DACH1 inhibits migration of MCF10A cells transformed with distinct oncogenes (Ha-Ras, c-Raf, c-Myc, and ErbB2) as well as the highly metastatic MDA-MB-231 human breast cancer cell line. An unbiased proteomic approach to identify candidate proteins regulated by DACH1 expression, using antibody array analysis, demonstrated DACH1 expression correlated with reduced secretion of IL-8 and the related chemokines IL-1, IL-6, GRO, and MIP1 (α, β, γ) from Ras or c-Myc transformed MCF-10A cells and repressed IL-8 promoter activity. Altered expressions of the IL-6 and IL-8 genes are seen in many cancers including prostate cancer. Hormone-resistant prostate carcinoma cell lines produce large amounts of IL-6 and IL-8 compared to hormone responsive prostate cancer cell lines. The expression of IL-8 and IL-6 are linked to poor prognosis and metastatic phenotype in prostate cancer. Recent finding demonstrated that IL-6 regulates the dynamic equilibrium between cancer stem cells and non-stem cancer cells in human breast and prostate cancer. IL-6 alone can transform non-tumorigenic benign prostate epithelial cells and further progress to invasive phenotype. To explore the role of DACH1 in androgen resistant prostate cancer, PC3 cells stably expressing DACH1 were established. DACH1 inhibited proliferation evaluated by MTT assay and growth curve. Microarray analyses demonstrated DACH1 inhibited mRNA expression of GRO, IL-6, IL-8 by 75%, 80% and 90%, respectively. However, there was no change of mRNA expression for IL-6 and IL-8 receptor. It has been reported that stable expression IL-6 in SV40T immortalized non-tumorigenic prostate epithelial cell line p69 induced EMT and acquired malignant phenotypes through autocrine loop IL6 and IGF-1R signaling. To further test the functional interaction of DACH1 with IL-6, a table cells expressing IL-6 alone or IL-6 with DACH1 were deployed. Ectopic expression of DACH1 attenuated activation of IGF-1 R by IL-6 as evaluated by phosphorylation of IGF-1R. Subcutaneous implantation study showed a 60% inhibition of in vivo tumor growth by DACH1. Together, our studies suggest that loss expression of DACH1 is an important cause of prostate tumorigenesis and restoration of DACH1 expression provides a potential therapeutic application. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A21.

Exploring and challenging the network of angiogenesis

Angiogenesis is one of the hallmarks of cancer and, as such, one of the alternative general targets for anticancer therapy. Since angiogenesis is a complex process involving a high number of interconnected components, a network approach would be a convenient systemic way to analyse responses to directed drug attacks. Herein we show that, although the angiogenic network is easily broken by short combinations of directed attacks, it still remains essentially functional by keeping the global patterns and local efficiency essentially unaltered after these attacks. This is a clear sign of its high robustness and resilience and stresses the need of directed, combined attacks for an effective blockade of the process. The results of this theoretical study could be relevant for the design of new antiangiogenic therapies and the selection of their targets.

Pleiotropic and Age-dependent Effects of Decreased Protein Modification by O-Linked N-Acetylglucosamine on Pancreatic β-Cell Function and Vascularization

The hexosamine biosynthesis pathway (HBP) regulates the post-translational modification of nuclear and cytoplasmic protein by O-linked N-acetylglucosamine (O-GlcNAc). Numerous studies have demonstrated increased flux through this pathway contributes to the development of β-cell dysfunction. The effect of decreased O-GlcNAc on the maintenance of normal β-cell function, however, is not well understood. We studied transgenic mice that over express β-N-acetylglucosaminidase (O-GlcNAcase), an enzyme that catalyzes the removal of O-GlcNAc from proteins, in the pancreatic β-cell under control of the rat insulin promoter. 3-4-Month-old O-GlcNAcase transgenic mice have higher glucose excursions with a concomitant decrease in circulating insulin levels, insulin mRNA levels, and total islet insulin content. In older (8-9-month-old) O-GlcNAcase transgenic mice glucose tolerance is no longer impaired. This is associated with increased serum insulin, islet insulin content, and insulin mRNA in the O-GlcNAcase transgenic mice. These improvements in β-cell function with aging are associated with increased angiogenesis and increased VEGF expression, with parallel increases in activation of Akt and expression of PGC1α. The biphasic effects as a function of age are consistent with published observations of mice with increased O-GlcNAc in islets and demonstrate that O-GlcNAc signaling exerts multiple effects on both insulin secretion and islet survival.

ChIP-on-chip analysis reveals angiopoietin 2 (Ang2, ANGPT2) as a novel target of steroidogenic factor-1 (SF-1, NR5A1) in the human adrenal gland.

The nuclear receptor steroidogenic factor-1 (SF-1, NR5A1) is a key regulator of adrenal and gonadal biology. Disruption of SF-1 can lead to disorders of adrenal development, while increased SF-1 dosage has been associated with adrenocortical tumorigenesis. We aimed to identify a novel subset of SF-1 target genes in the adrenal by using chromatin immunoprecipitation (ChIP) microarrays (ChIP-on-chip) combined with systems analysis. SF-1 ChIP-on-chip was performed in NCI-H295R human adrenocortical cells using promoter tiling arrays, leading to the identification of 445 gene loci where SF-1-binding regions were located from 10 kb upstream to 3 kb downstream of a transcriptional start. Network analysis of genes identified as putative SF-1 targets revealed enrichment for angiogenic process networks. A 1.1-kb SF-1-binding region was identified in the angiopoietin 2 (Ang2, ANGPT2) promoter in a highly repetitive region, and SF-1-dependent activation was confirmed in luciferase assays. Angiogenesis is paramount in adrenal development and tumorigenesis, but until now a direct link between SF-1 and vascular remodeling has not been established. We have identified Ang2 as a potentially important novel target of SF-1 in the adrenal gland, indicating that regulation of angiogenesis might be an important additional mechanism by which SF-1 exerts its actions in the adrenal gland.—Ferraz-de-Souza, B., Lin, L., Shah, S., Jina, N., Hubank, M., Dattani, M. T., Achermann, J. C. ChIP-on-chip analysis reveals angiopoietin 2 (Ang2, ANGPT2) as a novel target of steroidogenic factor-1 (SF-1, NR5A1) in the human adrenal gland.

Active immunotherapy induces antibody responses that target tumor angiogenesis.

The inhibition of VEGF signaling with antibodies or small molecules achieves clinical benefits in diverse solid malignancies. Nonetheless, therapeutic effects are usually not sustained, and most patients eventually succumb to progressive disease, indicating that antiangiogenic strategies require additional optimization. Vaccination with lethally irradiated, autologous tumor cells engineered to secrete granulocyte-macrophage colony stimulating factor (GM-CSF) and antibody blockade of cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) trigger a tumor vasculopathy in some long-term responding subjects. These reactions are characterized by disrupted tumor blood vessels in association with lymphocyte and granulocyte infiltrates and zonal areas of ischemic tumor necrosis. However, the mechanisms underlying this immune-mediated destruction of the tumor vasculature remain to be clarified. Here, we show that GM-CSF-secreting tumor cell vaccines and CTLA-4 blockade elicit a functionally important humoral reaction against multiple angiogenic cytokines. Antibodies to angiopoietin-1 and angiopoietin-2 block Tie-2 binding, downstream signaling, endothelial cell tube formation, and macrophage chemotaxis. Antibodies to macrophage inhibitory factor (MIF) attenuate macrophage Tie-2 expression and matrix metalloproteinase-9 (MMP-9) production. Together, these results delineate an immunotherapy-induced host response that broadly targets the angiogenic network in the tumor microenvironment.

Integrin Cytoplasmic Domain–Associated Protein-1 Attenuates Sprouting Angiogenesis

The ICAP1 (integrin cytoplasmic domain-associated protein-1) is a specific intracellular binding protein of beta1-integrins and the cerebral cavernous malformation (CCM) protein CCM1. ICAP1 recruits CCM1 to the cell membrane and activates CCM1 by changing its conformation. Because CCM1 plays a critical role for cardiovascular development, we hypothesized that its activator ICAP1 is involved in vascular differentiation. The objective of this study was to define the role of ICAP1 in endothelial cells. Loss of ICAP1 in primary human endothelial cells causes excessive angiogenic branching and network formation in vitro (3D sprouting angiogenesis) and in vivo (xenotransplantation of ICAP1-silenced human endothelial cells). ICAP1 increases cell motility and the initial formation of capillary sprouts but prevents vessel outgrowth. ICAP1 inhibits Rho kinase activity and ERK (extracellular signal-regulated kinase) phosphorylation and induces expression of the cell cycle inhibitors p21 and p27, leading to less endothelial proliferation. However, ICAP1 promotes endothelial survival and AKT phosphorylation. Global gene expression analyses revealed that the ICAP1 effects are mediated by strong activation of DELTA-NOTCH signaling. Active NOTCH1 or silencing of the NOTCH ligand DLL4 phenocopy the ICAP1 effects and blockade of NOTCH cleavage rescues the ICAP1-mediated defects in endothelial cells. Both ICAP1 and NOTCH1 reduce the expression of ESM1 (endothelial cell-specific molecule-1), and silencing of ESM1 disturbs vascular endothelial growth factor- or fibroblast growth factor 2-induced sprouting angiogenesis. In this study, we identified ICAP1 as a novel regulator to prevent excessive sprouting angiogenesis.

The Role of Vascular Endothelial Growth Factor Gene as the Genetic Marker of Atherothrombotic Disorders and in the Gene Therapy of Coronary Artery Disease

Many human diseases are characterized by vasculature disorders. Out of the many players in the angiogenic network, the vascular endothelial growth genes are by far the best characterized. The vascular endothelial growth factor (VEGF) has been implicated in the pathogenesis of coronary artery disease (CAD) and in its complication, the acute myocardial infarction (AMI). Several common polymorphisms in the promoter region of the VEGF gene have been reported, but only few single nucleotide polymorphisms (SNPs) have been demonstrated to be associated with variations in VEGF serum concentrations and with a susceptibility to CAD and its complications-acute coronary syndromes. Moreover, the -634 C/G VEGF SNP (rs2010963) has been demonstrated to be associated with AMI and the development of heart failure after AMI. Gene-based therapy for patients with refractory CAD has been the subject of extensive investigation. Preclinical studies have shown promise for the delivery of VEGF gene for treating CAD, whereas the results of randomized placebo-controlled trials have not demonstrated unequivocal evidence of efficacy. To conclude, at present the role of VEGF and VEGF SNPs in pathogenesis of AMI and the development of heart failure after AMI is still uncertain and remains to be determined. Obviously, larger studies as well as functional studies are needed to confirm the role of VEGF SNPs in AMI and its complications after AMI.

Genes Encoding Enzymatic Activities Implicated in the Eicosanoid Cascade of Arachidonic Acid and their Receptors are Expressed at mRNA Levels in Human Meningiomas

In view of the important oncogenic action of lipoxygenase (LOX) and cycloxygenase (COX) enzymatic activities we investigated, by using real time PCR, their presence in human meningiomas. Results indicated the presence of 5-LOX, 12-LOX, 15-LOX1, 15-LOX2, COX-1, COX-2, prostaglandin E (PGE) synthase, prostacyclin (PGI) synthase and thromboxane (TX) synthase transcripts in meningiomas but without relation to the tumor grade, the subtype of meningiomas, the presence of inflammatory infiltrated cells, of an associated edema, mitosis, brain invasion, vascularisation or necrosis. Similar results were found for BLT1 and BLT2 transcripts (encoding LTB4 receptors) and for prostanoid receptor transcripts (EP1-4 for PGE2, IP for PGI2 and TP for TXA2). In conclusion, genes encoding enzymatic activities implicated in the eicosanoid cascade are expressed in meningiomas. LOX- and COX-derived arachidonic acid metabolites might act on tumor growth not only by acting on cell growth but also by altering the local cytokine and/ or angiogenic networks.

The stimulation of angiogenesis and collagen deposition by copper

Copper is known to trigger endothelial cells towards angiogenesis. Different approaches have been investigated to develop vascularisation in biomaterials. The angiogenic and healing potential of copper ions in combination with two major angiogenic factors was examined. A 3D culture system in which, under stimulation by FGF-2 and to a lesser degree with VEGF, endothelial cells assembled into structures resembling to an angiogenic process was used. The combination of CuSO 4 with increasing doses of VEGF or FGF-2 enhanced the complexity of angiogenic networks in a significant manner. In vivo studies were also conducted by incorporating FGF-2 with CuSO 4 in a cylindrical collagen-based scaffold. CuSO 4 enhanced significantly the invasion of microvessel compared to control implants and to 20 ng FGF-2 ± CuSO 4. Vascular infiltration was also significantly improved by combination of CuSO 4 with FGF-2, compared to FGF-2 alone (0.2 and 1 μg). Nevertheless, in comparison with CuSO 4 alone, there was a significant increase only with 1 μg of FGF-2 combined with CuSO 4. Significantly, collagen fiber deposition was enhanced following the combinatory loading in comparison to that with FGF-2 alone but not with CuSO 4 only. Thus, copper associated with growth factors may have synergistic effects which are highly attractive in the fields of tissue engineering (e.g., bone) and biomaterials.

De novo synthesis of estrogen in pregnant uterus is critical for stromal decidualization and angiogenesis

Implantation is initiated when the embryo attaches to the uterine luminal epithelium during early pregnancy. Following this event, uterine stromal cells undergo steroid hormone-dependent transformation into morphologically and functionally distinct decidual cells in a unique process known as decidualization. An angiogenic network is also formed in the uterine stromal bed, critically supporting the early development of the embryo. The steroid-induced mechanisms that promote stromal differentiation and endothelial proliferation during decidualization are not fully understood. Although the role of ovarian progesterone as a key regulator of decidualization is well established, the requirement of ovarian estrogen (E) during this process remains unresolved. Here we show that the expression of P450 aromatase, a key enzyme that converts androgens to E, is markedly induced in mouse uterine stromal cells undergoing decidualization. The aromatase then acts in conjunction with other steroid biosynthetic enzymes present in the decidual tissue to support de novo synthesis of E. This locally produced E is able to support the advancement of the stromal differentiation program even in the absence ovarian E in an ovariectomized, progesterone-supplemented pregnant mouse model. Administration of letrozole, a specific aromatase inhibitor, to these mice blocked the stromal differentiation process. Gene expression profiling further revealed that the intrauterine E induces the expression of several stromal factors that promote neovascularization in the decidual tissue. Collectively, these studies identified the decidual uterus as a novel site of E biosynthesis and uncovered E-regulated maternal signaling pathways that critically control uterine differentiation and angiogenesis during early pregnancy.

Antagonism of the Sphingosine 1-Phosphate (S1P) Signaling Pathway in Pregnant Ewes Alters Placentome Architecture, Amino Acid Transport, and Fetal Growth.

Angiogenesis is a critical factor in the process of placentation. In ruminants, angiogenesis occurs primarily in placentomes, formed by interdigitation and branching morphogenesis of placental cotyledons and maternal endometrial caruncles, which supply hematotrophic nutrition to the developing conceptus. We hypothesized that the lysosphingolipid sphingosine-1-phosphate (S1P) stimulates angiogenesis within developing sheep placentomes through activation of the S1P receptors (S1P1-S1P5) and that their antagonism would alter placentomal angiogenesis and subsequent uteroplacental nutrient exchange during early to mid pregnancy. In Study 1, passage 4-5 primary ovine uterine artery endothelial cells (oUAEC) from 3 late pregnant sheep were seeded as monolayers onto three dimensional (3D) collagen matrices containing angiogenic growth factors and S1P. S1P dose-dependently (1–1000 nM) enhanced endothelial cell invasion compared to VEGF and FGF2 alone. Using RT-PCR to detect S1P1-S1P5, only S1P1 and S1P3 receptors were found to be expressed in oUAEC. Antagonism of these receptors with FTY720 and VPC 23019 blocked S1P-induced endothelial cell invasion in vitro. In Study 2, we examined potential physiological mechanisms for actions of endogenous S1P in the control of ovine placental angiogensis. Ewes with a single fetus were given daily intravenous injections of FTY720 (n=6; 1.5 mg/day) or vehicle (n=5) from Days 30 to 60 prior to collection of tissues on Day 60. FTY720 treatment altered the histoarchitecture of the placentome as evidenced by decreased stromal area of caruncular crypts and diminished thickness of connective tissue forming the placentomal capsule. Concomitant with these effects on interdigitation of uterine and placental tissues, the total volume of cotyledonary tissue per placentome was greater in FTY720-treated ewes. Immunofluorescence localization of the intermediate filament proteins, cytokeratin and vimentin indicated no overt change in synctial epithelia, whereas stromal tissue thickness and density were decreased with FTY720 treatment. Immunofluorescence localization of alpha-smooth muscle actin and the endothelial marker, von Willebrand Factor, in placentomes revealed that FTY720 treatment increased diameter of the lumen, thickness of the tunica muscularis of vessels, but decreased the total number of vessels in caruncles as compared to controls. FTY720 treatment also affected transport of amino acids across the placentae and fetal development as evidenced by decreases in methionine levels in allantoic fluid (P<0.05), aspartate and glutamate levels in amniotic fluid (P<0.005), and crown-rump length of fetuses (P<0.05), however, total fetal weight was not affected (P>0.05). Results of this study indicates that antagonism of the S1P signaling pathway using FTY720 interfered with placentomal development, including angiogenesis, transport of micronutrients from the uterus to the placenta and fetal growth. We conclude that S1P is a key regulatory molecule responsible for mediating development of the angiogenic network in placentomes of sheep that provide hematatrophic support for the developing conceptus. (Research supported by National Research Initiative Competitive Grants Program, Cooperative State Research, Education, and Extension Service, USDA award 2008-35203-18830 to KAD and AHA Scientist Development Grant #053020N to KJB) (poster)

TGF-&amp;beta;1 -Induced Expression of the Anti-Apoptotic PAI-1 Protein Requires EGFR Signaling

TGF-β1 and its target gene encoding plasminogen activator inhibitor-1 (PAI-1) are major regulators of capillary outgrowth, vessel maturation and angiogenic network stability. The increasing realization of the complexity of PAI-1 action in the vascular system requires analysis of specific signaling events that impact its expression in a physiologically-relevant cell system. PAI-1 was required for tubular differentiation and maintenance of cellular survival in complex gels since targeted disruption of PAI-1 synthesis or activity with antisense constructs or function-blocking antibodies resulted in network regression. Indeed, serum-deprivation-induced apoptosis of tubulogenic T2 cells was concentration-dependently inhibited by addition of a stable PAI-1 mutant protein consistent with the established pro-survival role of PAI-1 in vascular endothelial cells. PAI-1 induction and ERK pathway activation in response to TGF-β1 was attenuated by EGFR signaling blockade (with AG1478) or preincubation with the MMP/ADAM inhibitor GM6001. The combination of AG1478 + GM6001 completely ablated both responses suggesting that EGFR transactivation is important in PAI-1 gene control and may, at least partially, involve ligand shedding. TGF-β1-stimulated PAI-1 induction was preceded, in fact, by EGFR phosphorylation on Y845 (a src kinase target residue). EGFR1 knockdown with lentiviral shRNA constructs, moreover, effectively decreased (by >75%) TGF-β1-stimulated PAI-1 expression whereas infection with control (i.e. GFP) viruses had no effect. TGF-β1 failed to induce PAI-1 synthesis in EGFR-deficient fibroblasts while introduction of a wild-type EGFR1 construct in EGFR(-/-) cells rescued the PAI-1 response to TGF-β1 confirming, at a genetic level, the targeted knockdown data. The continued clarification of novel cooperative signaling cascades that impact expression of important angiogenic genes (e.g. PAI-1) may provide therapeutically useful targets to manage the pathophysiology of human neoplastic and vascular diseases.

Role of c‐myc in coronary vascular development and pathology

The formation and patterning of the coronary vasculature is critical to development and pathology in the heart. During cardiac pathology and physiological growth, coordinated interactions between growth of the organ and formation of the vascular bed are present. Studies from our lab have found that c‐myc is a main regulator of the angiogenic network. Despite the importance of c‐myc in the regulation of the angiogenic cascade, little has been done to explore the role of c‐myc in the formation of the coronary vasculature. In this study we utilized c‐myc+/− mice to examine the effects of diminished c‐Myc on the formation of the vascular bed, vascular patterning, intercapillary space and cardiac function. We observed that c‐myc+/− hearts had decreased cardiac output, diminished vessel bed formation and increased innercapillary space. However, an increase in fractal values suggests an increase in vessel patterning. To recover the effects of c‐myc happloinsufficiencies we crossed c‐myc+/− to the APCmin/+ mouse model, a model in which c‐Myc is increased via β‐catenin stabilization, to make compound APCMIN/+ c‐myc+/− mice. These mice were observed to have recovered the cardiac deficiencies and vessel bed attenuation observed in the c‐myc+/− mouse. However these vessel beds were found to more tortuous and leaky. Together these data highlight the importance of c‐Myc levels in cardiac function and vascular formation.

VESGEN 2D: Automated, User‐Interactive Software for Quantification and Mapping of Angiogenic and Lymphangiogenic Trees and Networks

AbstractPseudocolor view of vascular branching generations in the chorioallantoic membrane (CAM) of quail. Vascular architecture was analyzed using the automated, user‐interactive software, VESsel GENeration Analysis (VESGEN). See Vickerman, et al., on page 320, in this issue.

VESGEN 2D: Automated, User‐Interactive Software for Quantification and Mapping of Angiogenic and Lymphangiogenic Trees and Networks

Quantification of microvascular remodeling as a meaningful discovery tool requires mapping and measurement of site-specific changes within vascular trees and networks. Vessel density and other critical vascular parameters are often modulated by molecular regulators as determined by local vascular architecture. For example, enlargement of vessel diameter by vascular endothelial growth factor (VEGF) is restricted to specific generations of vessel branching (Parsons-Wingerter et al., Microvascular Research72: 91, 2006). The averaging of vessel diameter over many successively smaller generations is therefore not particularly useful. The newly automated, user-interactive software VESsel GENeration Analysis (VESGEN) quantifies major vessel parameters within two-dimensional (2D) vascular trees, networks, and tree-network composites. This report reviews application of VESGEN 2D to angiogenic and lymphangiogenic tissues that includes the human and murine retina, embryonic coronary vessels, and avian chorioallantoic membrane. Software output includes colorized image maps with quantification of local vessel diameter, fractal dimension, tortuosity, and avascular spacing. The density of parameters such as vessel area, length, number, and branch point are quantified according to site-specific generational branching within vascular trees. The sole user input requirement is a binary (black/white) vascular image. Future applications of VESGEN will include analysis of 3D vascular architecture and bioinformatic dimensions such as blood flow and receptor localization. Branching analysis by VESGEN has demonstrated that numerous regulators including VEGF(165), basic fibroblast growth factor, transforming growth factor beta-1, angiostatin and the clinical steroid triamcinolone acetonide induce 'fingerprint' or 'signature' changes in vascular patterning that provide unique readouts of dominant molecular signaling.

Genomic response of hypoxic Müller cells involves the very low density lipoprotein receptor as part of an angiogenic network

Müller cells have recently been found to produce select angiogenic substances. In choosing a more comprehensive approach, we wanted to study the genomic response of Müller cells to hypoxia to identify novel angiogenic genes. An established Müller cell line (rMC-1) was exposed to standard or hypoxic conditions. We analyzed gene expression with three independent microarrays and determined differential expression levels compared to normoxia. Selected genes were confirmed by real-time PCR (RTPCR). Subcellular localization of proteins was examined by immunocytochemistry. A network-based pathway analysis was performed to investigate how those genes may contribute to angiogenesis. We found 19 004 of 28 000 known rat genes expressed in Müller cells. 211 genes were upregulated by hypoxia 1.5 to 14.9-fold ( p < 0.001, FDR ≤ 5%) and 220 genes were downregulated 1.5–4.6-fold ( p < 0.001, FDR ≤ 5%). Unexpectedly, expression patterns of cell proliferation, differentiation and organogenesis were increased besides predictable declines in cell function. Very low density lipoprotein receptor (VLDLR) and tribbles 3 (TRIB3) were further analyzed because of recent implication in retinal neovascularization and macular degeneration (VLDLR) and in ocular mesodermal development and differentiation (TRIB3), respectively. VLDLR was upregulated 3.1-fold ( p = 0.001, RTPCR 3.0-fold) and TRIB3 2.8-fold ( p = 0.025, RTPCR 5.1-fold). VEGF was increased 3.1-fold ( p = 0.003, RTPCR 8.3-fold) and apelin, a novel factor of retinal angiogenesis, 5.6-fold ( p = 0.006, RTPCR 8.7-fold). A network of interacting angiogenic genes was identified in silico that included VLDLR as a surface receptor. VLDLR protein localized to the perinucleus, cytoplasm and cell membrane, while TRIB3 was found in nucleoli, the nucleus and cytoplasm. We conclude that hypoxia triggers an angiogenic network response in Müller cells with VLDLR as a novel node and gene expression patterns of proliferation, differentiation and organogenesis.

Angiogenic, hyperpermeability and vasodilator network in utero-placental units along pregnancy in the guinea-pig (Cavia porcellus)

BackgroundThe angiogenic and invasive properties of the cytotrophoblast are crucial to provide an adequate area for feto-maternal exchange. The present study aimed at identifying the localization of interrelated angiogenic, hyperpermeability and vasodilator factors in the feto-maternal interface in pregnant guinea-pigs.MethodsUtero-placental units were collected from early to term pregnancy. VEGF, Flt-1, KDR, B2R and eNOS were analyzed by immunohistochemistry, and the intensity of the signals in placenta and syncytial streamers was digitally analysed. Flt1 and eNOS content of placental homogenates was determined by western blotting. Statistical analysis used one-way analysis of variance and Tukey's Multiple Comparison post-hoc test.ResultsIn the subplacenta, placental interlobium and labyrinth VEGF, Flt-1, KDR, B2R and eNOS were expressed in all stages of pregnancy. Syncytial streamers in all stages of gestation, and cytotrophoblasts surrounding myometrial arteries in early and mid pregnancy – and replacing the smooth muscle at term – displayed immunoreactivity for VEGF, Flt-1, KDR, eNOS and B2R. In partly disrupted mesometrial arteries in late pregnancy cytotrophoblasts and endothelial cells expressed VEGF, Flt-1, KDR, B2R and eNOS. Sections incubated in absence of the first antibody, or in presence of rabbit IgG fraction and mouse IgG serum, yielded no staining. According to the digital analysis, Flt-1 increased in the placental interlobium in days 40 and 60 as compared to day 20 (P = 0.016), and in the labyrinth in day 60 as compared to days 20 and 40 (P = 0.026), while the signals for VEGF, KDR, B2R, and eNOS showed no variations along pregnancy. In syncytial streamers the intensity of VEGF immunoreactivity was increased in day 40 in comparison to day 20 (P = 0.027), while that of B2R decreased in days 40 and 60 as compared to day 20 (P = 0.011); VEGF, Flt-1, KDR, B2R and eNOS expression showed no variations. Western blots for eNOS and Flt-1 in placental homogenates showed no significant temporal differences along pregnancy.ConclusionThe demonstration of different angiogenic, hyperpermeability and vasodilator factors in the same cellular protagonists of angiogenesis and invasion in the pregnant guinea-pig, supports the presence of a functional network, and strengthens the argument that this species provides an adequate model to understand human pregnancy.

Transcriptional Network Governing the Angiogenic Switch in Human Pancreatic Cancer

A shift of the angiogenic balance to the proangiogenic state, termed the “angiogenic switch,” is a hallmark of cancer progression. Here we devise a strategy for identifying genetic participants of the angiogenic switch based on inverse regulation of genes in human endothelial cells in response to key endogenous pro- and antiangiogenic proteins. This approach reveals a global network pattern for vascular homeostasis connecting known angiogenesis-related genes with previously unknown signaling components. We also demonstrate that the angiogenic switch is governed by simultaneous regulations of multiple genes organized as transcriptional circuitries. In pancreatic cancer patients, we validate the transcriptome-derived switch of the identified “angiogenic network:” The angiogenic state in chronic pancreatitis specimens is intermediate between the normal (angiogenesis off) and neoplastic (angiogenesis on) condition, suggesting that aberrant proangiogenic environment contributes to the increased cancer risk in patients with chronic pancreatitis. In knockout experiments in mice, we show that the targeted removal of a hub node (peroxisome proliferative-activated receptor δ) of the angiogenic network markedly impairs angiogenesis and tumor growth. Further, in tumor patients, we show that peroxisome proliferative-activated receptor δ expression levels are correlated with advanced pathological tumor stage, increased risk for tumor recurrence, and distant metastasis. Our results therefore also may contribute to the rational design of antiangiogenic cancer agents; whereas “narrow” targeted cancer drugs may fail to shift the robust angiogenic regulatory network toward antiangiogenesis, the network may be more vulnerable to multiple or broad-spectrum inhibitors or to the targeted removal of the identified angiogenic “hub” nodes.