Endothelial Cell Tubulogenesis Research Articles

Pericytes, Stem‐Cell‐Like Cells, but not Mesenchymal Stem Cells are Recruited to Support Microvascular Tube Stabilization

An experimental model is introduced for the induction of endothelial cell (EC) tubulogenesis after 24 h of incubation on micropatterned polymer surfaces. Pericytes or mesenchymal stem cells are added separately to this system to evaluate their effect on tubular stabilization. In the absence of additional cells, the tubular structures are lost after 36 h. Addition of only pericytes, however, stabilizes the EC vasculogenic tubes.

Abstract 96: Estrogen modulates cellular and molecular markers of hypoxic tumor microenvironment in thyroid cancer.

Abstract Thyroid proliferative diseases are three to four times more prevalent in women than in men. Our laboratory has systemically elucidated the role of estrogen in thyroid cancer as a promoter of increased aggressiveness, however the role of estrogen within the hypoxic environment of thyroid cancer is not known. Based on our observation that estrogen promotes neo-vascularization of thyroid tumors by modulating angiogenic factors, vascular endothelial growth factors (VEGF) and matrix metalloproteinases (MMPs), we hypothesize that tumor hypoxia synergizes with estrogen to produce an inflammatory microenvironment that helps the progression of thyroid cancer. We observed that thyroid cancer cells are responsive to hypoxic conditions and estrogen induces hypoxia inducible factor-1α (HIF-1α) nuclear translocation in these cells. Furthermore, estrogen and hypoxia modulate the HIF-1 signaling in thyroid cancer cells which is abrogated by the anti-estrogen fulvestrant and the HIF-1 inhibitor YC-1 (3-(5’-hydroxymethyl-2’-furyl)-1-benzylindazole). Conditioned medium from estrogen treated thyroid cancer cells lead to enhanced migration and tubulogenesis of human umbilical vein endothelial cells (HUVECs), due to secretion of hypoxia and estrogen responsive proteins by thyroid cancer cells leading to increased angiogenesis of endothelial cells. This phenomenon was abrogated by HIF-1 inhibitor. These results demonstrate for the first time that estrogen signaling pathways mimic the signaling pathways in the hypoxic environment in thyroid cancer cells, thus suggesting that estrogen potentially mimics oxygen deprivation at the molecular level to produce an inflammatory tumor microenvironment thus fostering the progression of thyroid cancer. We conclude that apart from the direct activity of estrogen on thyroid cancer phenotype, estrogen also modulates the tumor microenvironment specifically maintaining hypoxic conditions which may be essential for thyroid cancer progression and/or metastases. Citation Format: Shilpi Rajoria, Elyse Hanly, Andrea George, Robert Suriano, Jan Geliebter, Masoud Saman, Stimson Schantz, Raj K. Tiwari. Estrogen modulates cellular and molecular markers of hypoxic tumor microenvironment in thyroid cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 96. doi:10.1158/1538-7445.AM2013-96

Southern Brazilian autumnal propolis shows anti-angiogenic activity: An in vitro and in vivo study

The present study focuses on the effects of a hydro-alcoholic propolis extract collected in autumn (2010) in Santa Catarina State (Southern Brazil), on the angiogenesis, using in vitro and in vivo models. Cultures of human umbilical vein endothelial cells were used to assess the effects of propolis on viability, proliferation, and cell migration, as well as capillary tube formation. The propolis autumnal extracts significantly decreased the cell viability, based on CC50 values, which decreased (56%) from 297 to 130μg/ml in 24h and 72h of treatment, respectively (cytotoxicity assay). The process of cell proliferation was decreased by 81.7 to 48.4% due to exposure (72h) to 130–180μg/ml of propolis extract, as compared with control (vehicle). In these same concentrations, the cell migration was also reduced by 39.6 to 12.6%, respectively (versus control). Furthermore, autumnal propolis extract (100–200μg/ml) inhibited the tube-like structure formation (tubulogenesis) of endothelial cells on Matrigel™ (16.2–69.9% inhibition). The treatments performed in vivo with administration of 450mg propolis.kg−1 inhibited both angiogenesis and vasculogenesis by 82.3 and 66.5% in the chorioallantoic and yolk-sac membranes of chick embryos. Furthermore, by means of UV–vis-spectrophotometry, reverse phase-high performance liquid chromatography analysis and 1D and 2D-nuclear magnetic resonance experiments reveal higher contents of flavonoids and total phenolic compounds with predominance of the flavonol quercetin and the phenolic acids, e.g., gallic acid, protocatechuic acid and chlorogenic acid in the propolis hydro-alcoholic extract. Our findings related to the anti-proliferative, anti-migration, and anti-tubulogenic actions on human umbilical vein endothelial cell line agree with the inhibitory effects in the in vivo vessel formation exerted by propolis extract under study. The results also suggest that autumnal propolis extract might be potentially instrumental in providing alternative tools for angiogenic disease therapeutics.

Cell‐Laden Hydrogels in Integrated Microfluidic Devices for Long‐Term Cell Culture and Tubulogenesis Assays

A hydrogel biochip combining microfluidic mixing and orthogonal supplementation strategies is developed and validated to allow facile generation of libraries of optically transparent 3D culture microenvironments. Live, on-chip tracing of embryonic stem cell differentiation and endothelial cell tubulogenesis confirms that the platform can be used to both create communities of discrete 3D microenvironments as well as to locally monitor subsequent divergent responses at both single cell and multi-cell scales.

Aquaporin-1 Plays a Crucial Role in Estrogen-Induced Tubulogenesis of Vascular Endothelial Cells

Aquaporin-1 (AQP1) has been proposed as a mediator of estrogen-induced angiogenesis in human breast cancer and endometrial cancer. Elucidation of the molecular mechanisms governing AQP1-mediated, estrogen-induced angiogenesis may contribute to an improved understanding of tumor development. Our objective was to identify the estrogen-response element (ERE) in the promoter of the Aqp1 gene and investigate the effects and mechanisms of AQP1 on estrogen-induced tubulogenesis of vascular endothelial cells. The study was conducted in a university hospital in eastern China. Immunohistological, real-time PCR and Western blot analyses were used to determine the expression AQP1 mRNA and protein in vascular endothelial cells. Chromatin immunoprecipitation analyses and luciferase reporter assays identified ERE-like motif in the promoter of the Aqp1 gene. Expression of AQP1 in blood vessels of human breast and endometrial carcinoma tissues were significantly higher than controls. Estradiol (E2) dose-dependently increased the expression levels of AQP1 mRNA and protein in human umbilical vein endothelial cells (HUVECs). A functional ERE-like motif was identified in the promoter of the Aqp1 gene. AQP1 colocalized with ezrin, a component of the ezrin/radixin/moesin protein complex, and, ezrin colocalized with filamentous actin in HUVECs. Knockdown of AQP1 or ezrin with specific small interfering RNA significantly attenuated the formation of transcytoplasmic filamentous actin stress fibers induced by E2 and inhibited E2-enhanced cell proliferation, migration, invasion, and tubule formation of HUVECs. Estrogen induces AQP1 expression by activating ERE in the promoter of the Aqp1 gene, resulting in tubulogenesis of vascular endothelial cells. These results provide new insights into the molecular mechanisms underpinning the angiogenic effects of estrogen.

Characterisation and therapeutic potential of endothelial progenitor cells

Endothelial progenitor cells (EPCs) may have great potential for use as a cellular therapy for promoting vascular repair of ischaemic tissues, such as complex wounds. The term EPC, however, has been used to describe a diverse variety of cells. This ambiguity has led to conflicting results when comparing results of EPC studies from different research laboratories. Recently, there is consensus that when selective in-vitro cell culture techniques are used, two distinct EPC subpopulations are generated—namely, myeloid angiogenic cells (MACs) and outgrowth endothelial cells (OECs). This study provides a full phenotypic and functional characterisation of MACs and OECs and compares their potential therapeutic role in wound healing.EPC subtypes were isolated from human peripheral blood and umbilical cord blood. MACs appeared early in culture (∼7 days) as elongated cells with multiple fine dendritic processes and low proliferative potential. OEC colonies appeared later in culture (3–5 weeks), and formed a cobblestone-shaped monolayer. Genome-wide transcriptomics demonstrated that MACs and OECs belong to the haemopoietic and endothelial lineages, respectively. MACs cultured in vitro were further analysed, and were found to be phenotypically very similar to M2 alternative activated macrophages.Analysis of functional characteristics showed that MACs have low proliferative potential. However, OECs possess high proliferative potential, enabling them to be single-cell cloned, with an average 60 population doublings in 80 days for cord-derived OECs.Using in-vitro angiogenesis assays, OECs demonstrated de-novo tubulogenesis capacity, and were capable of interacting with mature endothelial cells through adherens and tight junctions. MACs did not form tubes and did not differentiate into endothelial cells, but significantly promoted tubulogenesis of mature endothelial cells by releasing paracrine factors such as interleukin 8 and monocyte chemoattractant protein 1. FundingNorthern Ireland Medical and Dental Training Agency.

Decreased expression of ADAMTS-1 in human breast tumors stimulates migration and invasion

BackgroundADAMTS-1 (a disintegrin and metalloprotease with thrombospondin motifs) is a member of the ADAMTS family of metalloproteases. Here, we investigated mRNA and protein levels of ADAMTS-1 in normal and neoplastic tissues using qPCR, immunohistochemistry and immunoblot analyses, and we addressed the role of ADAMTS-1 in regulating migration, invasion and invadopodia formation in breast tumor cell lines.ResultsIn a series of primary breast tumors, we observed variable levels of ADAMTS-1 mRNA expression but lower levels of ADAMTS-1 protein expression in human breast cancers as compared to normal tissue, with a striking decrease observed in high-malignancy cases (triple-negative for estrogen, progesterone and Her-2). This result prompted us to analyze the effect of ADAMTS-1 knockdown in breast cancer cells in vitro. MDA-MB-231 cells with depleted ADAMTS-1 expression demonstrated increased migration, invasion and invadopodia formation. The regulatory mechanisms underlying the effects of ADAMTS-1 may be related to VEGF, a growth factor involved in migration and invasion. MDA-MB-231 cells with depleted ADAMTS-1 showed increased VEGF concentrations in conditioned medium capable of inducing human endothelial cells (HUVEC) tubulogenesis. Furthermore, expression of the VEGF receptor (VEGFR2) was increased in MDA-MB-231 cells as compared to MCF7 cells. To further determine the relationship between ADAMTS-1 and VEGF regulating breast cancer cells, MDA-MB-231 cells with reduced expression of ADAMTS-1 were pretreated with a function-blocking antibody against VEGF and then tested in migration and invasion assays; both were partially rescued to control levels.ConclusionsADAMTS-1 expression was decreased in human breast tumors, and ADAMTS-1 knockdown stimulated migration, invasion and invadopodia formation in breast cancer cells in vitro. Therefore, this series of experiments suggests that VEGF is involved in the effects mediated by ADAMTS-1 in breast cancer cells.

Computer Aided Design and Manufacturing of Soft, Three-Dimensional, Multi-Layer, Biological Constructs Via Laser Printing Onto Laser Machined Composite Biopapers

Engineering a tissue construct which mimics a native tissue's form and function requires mimicry of the material, chemical, and morphological properties of the target tissue. However, depending on the target tissue, matching these very properties make fabrication of a composite structure challenging. Here we discuss a collection of techniques which allow us to create threedimensional soft tissue constructs comprised of multiple layers of hydrogels with high inter-layer registration. These techniques include a custom bioreactor platform integrated with NRL's Biological Laser Printing (BioLP™) stage and CAM application which allows for quick and reproducible printing, culturing and stacking of individual biopaers. The Bioreactors are designed for culturing and mounting laser machined biopapers, and the laser machined biopaper frames which allow for handling and high resolution (less than 100 micron) registration of otherwise difficult to handle 500Pa – 5kPa hydrogels selected for their mechanical and chemical resemblance to in vivo soft tissues. We demonstrate printing of immortalized endothelial cells (EC) onto biopapers and their subsequent self-assembly into capillary-like structures or tubulogenesis. Our composite stainless steel framed - collagen membrane supported – hydrogel biopapers enable asynchronous printing, cell development and delayed stacking of the soft substrates required for EC tubulogenesis.

Glucose toxic effects on granulation tissue productive cells: the diabetics' impaired healing.

Type 2 diabetes mellitus is a metabolic noncommunicable disease with an expanding pandemic magnitude. Diabetes predisposes to lower extremities ulceration and impairs the healing process leading to wound chronification. Diabetes also dismantles innate immunity favoring wound infection. Amputation is therefore acknowledged as one of the disease's complications. Hyperglycemia is the proximal detonator of systemic and local toxic effectors including proinflammation, acute-phase proteins elevation, and spillover of reactive oxygen and nitrogen species. Insulin axis deficiency weakens wounds' anabolism and predisposes to inflammation. The systemic accumulation of advanced glycation end-products irreversibly impairs the entire physiology from cells-to-organs. These factors in concert hamper fibroblasts and endothelial cells proliferation, migration, homing, secretion, and organization of a productive granulation tissue. Diabetic wound bed may turn chronically inflammed, procatabolic, and an additional source of circulating pro-inflammatory cytokines, establishing a self-perpetuating loop. Diabetic fibroblasts and endothelial cells may bear mitochondrial damages becoming prone to apoptosis, which impairs granulation tissue cellularity and perfusion. Endothelial progenitor cells recruitment and tubulogenesis are also impaired. Failure of wound reepithelialization remains a clinical challenge while it appears to be biologically multifactorial. Ulcer prevention by primary care surveillance, education, and attention programs is of outmost importance to reduce worldwide amputation figures.

Beneficial effects of ginsenoside-Rg1 on ischemia-induced angiogenesis in diabetic mice

Neovascularization and the formation of collateral vessels are often impaired in diabetes mellitus (DM) population compared with non-diabetics. Alterations in vascular endothelial growth factor (VEGF) signaling and endothelial nitric oxide synthase (eNOS) dysfunction have been confirmed to play a crucial role in impaired neovascularization in diabetic mice. Accumulating data have suggested that Rg1, a main component of Panax ginseng, has the ability to promote tubulogenesis of human umbilical vein endothelial cells (HUVECs) in vitro, and that the mechanism involves increased expression level of VEGF as well as increased eNOS activation. Thus, we speculated that Rg1 might also have therapeutic effects on the impairment of neovascularization in diabetic individuals. The aim of the present study was to investigate whether Rg1 could improve angiogenesis in ischemic hindlimb of diabetic mice in vivo. Our data demonstrated that Rg1 treatment resulted in improved angiogenesis in the diabetic ischemic hindlimb, and the potential mechanism might involve increased eNOS activation, upregulated VEGF expression, and inhibited apoptosis. Our results suggest that Rg1 may be used as a novel and useful adjunctive drug for the therapy of peripheral arterial disease in DM.

Adenoviral E4 Gene Stimulates Secretion of PEDF That Maintains Long-Term Survival of Human Glomerulus-Derived Endothelial Cells

AbstractAbstract 5187Renal glomerular endothelial cells are specialized cells with an important role in physiological filtration and glomerular disease. However, maintenance of human primary endothelial cells requires stimulation with serum and growth factors that often results in modification of the cells properties. Previously, expression of early adenovirus region E4 was shown to help maintaining long-term survival of human endothelial cells in serum free media without addition of growth factors. In the current study, we showed that media conditioned with human epithelial cells stably transfected with Ad E4 region supported survival of human glomerulus-derived endothelial cells in serum-free media (Fig. 1 A and B). Mass-spectrometry analysis of the conditioned media identified pigmental epithelium derived factor (PEDF) as a major component of the conditioned media. PEDF expression in 293-E4 cells was validated by RT- PCR (Fig. 1C), Western Blot and ELISA analysis (Fig. 1D). PEDF expression was detected in mouse glomeruli. Supplementation with recombinant PEDF supported survival of primary endothelial cells and the cells transformed with SV40 large T antigen in serum-free media, and extended the life-span of both cell cultures. PEDF did not inhibit FGF-2 stimulated growth and tubulogenesis of endothelial cells. Thus we demonstrated that adenoviral E4 region stimulated expression and secretion of PEDF by human renal epithelial cells that acted as a survival factor for glomerulus-derived endothelial cells. [Display omitted] Acknowledgments:This work was supported NIH Research Grants SC1GM082325, R25 HL003679, 2G12RR003048, 8G12MD007597, K25GM097501 and 1P30HL107253. This study was also supported in part by National Kidney Foundation grant. Disclosures:No relevant conflicts of interest to declare.

Adenoviral E4 Gene Stimulates Secretion of Pigmental Epithelium Derived Factor (PEDF) that Maintains Long-term Survival of Human Glomerulus-derived Endothelial Cells

Renal glomerular endothelial cells are specialized cells with an important role in physiological filtration and glomerular disease. However, maintenance of human primary endothelial cells requires stimulation with serum and growth factors that often results in modification of the cells properties. Previously, expression of early adenovirus region E4 was shown to help maintaining long-term survival of human endothelial cells in serum free media without addition of growth factors. In the current study, we showed that media conditioned with human epithelial cells stably transfected with Ad E4 region also supported survival of human glomerulus-derived endothelial cells in serum-free media. Mass-spectrometry analysis of the conditioned media identified pigmental epithelium derived factor (PEDF) as a major component of the conditioned media. PEDF expression in 293-E4 cells was validated by RT-PCR, Western blot and ELISA analysis. PEDF expression was detected in mouse glomeruli. Supplementation with recombinant PEDF supported survival of primary endothelial cells and the cells transformed with SV40 large T antigen in serum-free media, and extended the life-span of both cell cultures. PEDF did not inhibit FGF-2 stimulated growth and tubulogenesis of endothelial cells. Thus we demonstrated that adenoviral E4 region stimulated expression and secretion of PEDF by human renal epithelial cells that acted as a survival factor for glomerulus-derived endothelial cells.

LGALS3BP, lectin galactoside-binding soluble 3 binding protein, induces vascular endothelial growth factor in human breast cancer cells and promotes angiogenesis

Elevated serum or tissue levels of lectin galactoside-binding soluble 3 binding protein (LGALS3BP) have been associated with short survival and development of metastasis in a variety of human cancers. However, the role of LGALS3BP, particularly in the context of tumor-host relationships, is still missing. Here, we show that LGALS3BP knockdown in MDA-MB-231 human breast cancer cells leads to a decreased adhesion to fibronectin, a reduced transendothelial migration and, more importantly, a reduced expression of vascular endothelial growth factor (VEGF). Production of VEGF, that was restored by exposure of silenced cells to recombinant LGALS3BP, required an intact PI3k/Akt signaling. Furthermore, we show that LGALS3BP was able to directly stimulate HUVEC tubulogenesis in a VEGF-independent, galectin-3-dependent manner. Immunohistochemical analysis of human breast cancer tissues revealed a correlation among LGALS3BP expression, VEGF expression, and blood vessel density. We propose that in addition to its prometastatic role, LGALS3BP secreted by breast cancer cells functions critically as a pro-angiogenic factor through a dual mechanism, i.e by induction of tumor VEGF and stimulation of endothelial cell tubulogenesis.

Targeted Disruption of Heparan Sulfate Interaction with Hepatocyte and Vascular Endothelial Growth Factors Blocks Normal and Oncogenic Signaling

Hepatocyte growth factor (HGF) and vascular endothelial cell growth factor (VEGF) regulate normal development and homeostasis and drive disease progression in many forms of cancer. Both proteins signal by binding to receptor tyrosine kinases and heparan sulfate (HS) proteoglycans on target cell surfaces. Basic residues comprising the primary HS binding sites on HGF and VEGF provide similar surface charge distributions without underlying structural similarity. Combining three acidic amino acid substitutions in these sites in the HGF isoform NK1 or the VEGF isoform VEGF165 transformed each into potent, selective competitive antagonists of their respective normal and oncogenic signaling pathways. Our findings illustrate the importance of HS in growth factor driven cancer progression and reveal an efficient strategy for therapeutic antagonist development.

Geometrical Microfeature Cues for Directing Tubulogenesis of Endothelial Cells

Angiogenesis, the formation of new blood vessels by sprouting from pre-existing ones, is critical for the establishment and maintenance of complex tissues. Angiogenesis is usually triggered by soluble growth factors such as VEGF. However, geometrical cues also play an important role in this process. Here we report the induction of angiogenesis solely by SVVYGLR peptide micropatterning on polymer surfaces. SVVYGLR peptide stripes were micropatterned onto polymer surfaces by photolithography to study their effects on endothelial cell (EC) behavior. Our results showed that the EC behaviors (cell spreading, orientation and migration) were significantly more guided and regulated on narrower SVVYGLR micropatterns (10 and 50 µm) than on larger stripes (100 µm). Also, EC morphogenesis into tube formation was switched on onto the smaller patterns. We illustrated that the central lumen of tubular structures can be formed by only one-to-four cells due to geometrical constraints on the micropatterns which mediated cell-substrate adhesion and generated a correct maturation of adherens junctions. In addition, sprouting of ECs and vascular networks were also induced by geometrical cues on surfaces micropatterned with SVVYGLR peptides. These micropatterned surfaces provide opportunities for mimicking angiogenesis by peptide modification instead of exogenous growth factors. The organization of ECs into tubular structures and the induction of sprouting angiogenesis are important towards the fabrication of vascularized tissues, and this work has great potential applications in tissue engineering and tissue regeneration.

The p53 isoform, Δ133p53α, stimulates angiogenesis and tumour progression

The tumour suppressor p53, involved in DNA repair, cell cycle arrest and apoptosis, also inhibits blood vessel formation, that is, angiogenesis, a process strongly contributing to tumour development. The p53 gene expresses 12 different proteins (isoforms), including TAp53 (p53 (or p53α), p53β and p53γ) and Δ133p53 isoforms (Δ133p53α, Δ133p53β and Δ133p53γ). The Δ133p53α isoform was shown to modulate p53 transcriptional activity and is overexpressed in various human tumours. However, its role in tumour progression is still unexplored. In the present study, we examined the involvement of Δ133p53 isoforms in tumoural angiogenesis and tumour growth in the highly angiogenic human glioblastoma U87. Our data show that conditioned media from U87 cells depleted for Δ133p53 isoforms block endothelial cell migration and tubulogenesis without affecting endothelial cell proliferation in vitro. The Δ133p53 depletion in U2OS osteosarcoma cells resulted in a similar angiogenesis blockade. Furthermore, using conditioned media from U87 cells ectopically expressing each Δ133p53 isoform, we determined that Δ133p53α and Δ133p53γ but not Δ133p53β, stimulate angiogenesis. Our in vivo data using the chicken chorio-allantoic membrane and mice xenografts establish that angiogenesis and growth of glioblastoma U87 tumours are inhibited upon depletion of Δ133p53 isoforms. By TaqMan low-density array, we show that alteration of expression ratio of Δ133p53 and TAp53 isoforms differentially regulates angiogenic gene expression with Δ133p53 isoforms inducing pro-angiogenic gene expression and repressing anti-angiogenic gene expression.

Liver X Receptor Activation Reduces Angiogenesis by Impairing Lipid Raft Localization and Signaling of Vascular Endothelial Growth Factor Receptor-2

Liver X receptors (LXRα, LXRβ) are master regulators of cholesterol homeostasis. In the endothelium, perturbations of cell cholesterol have an impact on fundamental processes. We, therefore, assessed the effects of LXR activation on endothelial functions related to angiogenesis in vitro and in vivo. LXR agonists (T0901317, GW3965) blunted migration, tubulogenesis, and proliferation of human umbilical vein endothelial cells. By affecting endothelial cholesterol homeostasis, LXR activation impaired the compartmentation of vascular endothelial growth factor receptor-2 in lipid rafts/caveolae and led to defective phosphorylation and downstream signaling of vascular endothelial growth factor receptor-2 upon vascular endothelial growth factor-A stimulation. Consistently, the antiangiogenic actions of LXR agonists could be prevented by coadministration of exogenous cholesterol. LXR agonists reduced endothelial sprouting from wild-type but not from LXRα(-/-)/LXRβ(-/-) knockout aortas and blunted the vascularization of implanted angioreactors in vivo. Furthermore, T0901317 reduced the growth of Lewis lung carcinoma grafts in mice by impairing angiogenesis. Pharmacological activation of endothelial LXRs reduces angiogenesis by restraining cholesterol-dependent vascular endothelial growth factor receptor-2 compartmentation and signaling. Thus, administration of LXR agonists could exert therapeutic effects in pathological conditions characterized by uncontrolled angiogenesis.

Molecular target based combinational therapeutic approaches in thyroid cancer

BackgroundThyroid cancer, as with other types of cancer, is dependent on angiogenesis for its continued growth and development. Interestingly, estrogen has been shown to contribute to thyroid cancer aggressiveness in vitro, which is in full support of the observed increased incidence of thyroid cancer in women over men. Provided that estrogen has been observed to contribute to increased angiogenesis of estrogen responsive breast cancer, it is conceivable to speculate that estrogen also contributes to angiogenesis of estrogen responsive thyroid cancer.MethodsIn this study, three human thyroid cancer cells (B-CPAP, CGTH-W-1, ML-1) were treated with estrogen alone or estrogen and anti-estrogens (fulvestrant and 3,3′-diindolylmethane, a natural dietary compound) for 24 hours. The cell culture media was then added to human umbilical vein endothelial cell (HUVECs) and assayed for angiogenesis associated events. Vascular endothelial growth factor (VEGF) levels were also quantified in the conditioned media so as to evaluate if it is a key player involved in these observations.ResultsConditioned medium from estrogen treated thyroid cancer cells enhanced phenotypical changes (proliferation, migration and tubulogenesis) of endothelial cells typically observed during angiogenesis. These phenotypic changes observed in HUVECs were determined to be modulated by estrogen induced secretion of VEGF by the cancer cells. Lastly, we show that VEGF secretion was inhibited by the anti-estrogens, fulvestrant and 3,3′-diindolylmethane, which resulted in diminished angiogenesis associated events in HUVECs.ConclusionOur data establishes estrogen as being a key regulator of VEGF secretion/expression in thyroid cells which enhances the process of angiogenesis in thyroid cancer. These findings also suggest the clinical utility of anti-estrogens as anti-angiogenic compounds to be used as a therapeutic means to treat thyroid cancer. We also observed that 3,3′-diindolylmethane is a promising naturally occurring anti-estrogen which can be used as a part of therapeutic regimen to treat thyroid cancer.

To Enhance or to Inhibit Integrin Function in Angiogenesis, that is the Question

The idea that tumors recruit blood vessels to survive, grow, and metastasize was proposed more than 40 years ago by Dr. Judah Folkman. Since then, studies have been conducted to identify factors able to stimulate endothelial cell functions and to inhibit their pro-angiogenic activity in pathological conditions, such as tumor-associated angiogenesis. The process of angiogenesis requires three major steps, namely endothelial cell proliferation, migration and tubulogenesis. Although release of growth factors, cytokines, and proteases is critical for the regulation of endothelial cell functions, interactions of endothelial cells with the surrounding extracellular matrix initiate intracellular signaling that result in proand/ or anti-angiogenic cues. Interactions of cells with the extracellular matrix are made possible by the transmembrane receptors integrins. Upon ligand activation, these receptors can activate various intracellular signaling, thus regulating processes such as adhesion, migration, proliferation and survival. The finding that these receptors are expressed on vascular endothelial cells and they can either promote or inhibit endothelial cell functions, has initiated studies to determine their role in physiological and pathological angiogenesis. In this review, we will focus on the role of integrins, with emphasis on the collagen binding and the alpha v containing integrins, in angiogenesis and highlight hope and tribulations of targeting these receptors for anti-angiogenic therapy. This review is dedicated to Dr. Judah Folkman who introduced us to the concept of ‘malignant’ angiogenesis.

Factors Secreted by Endothelial Progenitor Cells Promote Brain Endothelial Cell Tubulogenesis through PI3-Kinase

Factors Secreted by Endothelial Progenitor Cells Promote Brain Endothelial Cell Tubulogenesis through PI3-Kinase