Gaoyuan Cao, Rashmin C Savani, Melane Fehrenbach, Chris Lyons, Lin Zhang, George Coukos, Horace M Delisser
Pulmonary, Allergy and Critical Care Division, Department of Medicine, Division of Neonatology, Children's Hospital of Philadelphia, Philadelphia, PA, USA.
CD44, a cell-surface receptor for hyaluronan, has been implicated in endothelial cell functions, but its role in the formation of blood vessels in vivo has not been established. In CD44-null mice, vascularization of Matrigel implants and tumor and wound angiogenesis were inhibited. Leukocyte accumulation during tumor growth and wound healing in wild-type and CD44-null mice were comparable, and reconstitution of CD44-null mice with wild-type bone marrow did not restore the wild-type phenotype, suggesting that impairments in angiogenesis in CD44-deficient mice are due to the loss of endothelial CD44. Although the cell proliferation, survival, and wound-induced migration of CD44-null endothelial cells were intact, these cells were impaired in their in vitro ability to form tubes. Nascent vessels in Matrigel implants from CD44-null mice demonstrated irregular luminal surfaces characterized by retracted cells and thinned endothelia. Further, an anti-CD44 antibody that disrupted in vitro tube formation induced hemorrhage around Matrigel implants, suggesting that antagonism of endothelial CD44 undermined the integrity of the endothelium of nascent vessels. These data establish a role for CD44 during in vivo angiogenesis and suggest that CD44 may contribute to the organization and/or stability of developing endothelial tubular networks.
Angiogenesis-associated crosstalk between collagens, CXC chemokines, and thrombospondin domain-containing proteins.
Department of Biomedical Engineering, School of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA. email@example.com
Excessive vascularization is a hallmark of many diseases including cancer, rheumatoid arthritis, diabetic nephropathy, pathologic obesity, age-related macular degeneration, and asthma. Compounds that inhibit angiogenesis represent potential therapeutics for many diseases. Karagiannis and Popel [Proc. Natl. Acad. Sci. USA 105(37):13775-13780, 2008] used a bioinformatics approach to identify more than 100 peptides with sequence homology to known angiogenesis inhibitors. The peptides could be grouped into families by the conserved domain of the proteins they were derived from. The families included type IV collagen fibrils, CXC chemokine ligands, and type I thrombospondin domain-containing proteins. The relationships between these families have received relatively little attention. To investigate these relationships, we approached the problem by placing the families of proteins in the context of the human interactome including >120,000 physical interactions among proteins, genes, and transcripts. We built on a graph theoretic approach to identify proteins that may represent conduits of crosstalk between protein families. We validated these findings by statistical analysis and analysis of a time series gene expression data set taken during angiogenesis. We identified six proteins at the center of the angiogenesis-associated network including three syndecans, MMP9, CD44, and versican. These findings shed light on the complex signaling networks that govern angiogenesis phenomena.
Stephanie K Seidlits, Charles T Drinnan, Rebecca R Petersen, Jason B Shear, Laura J Suggs, Christine E Schmidt
Department of Biomedical Engineering, University of Texas at Austin, 1 University Station, CO800, Austin, TX 78712, USA.
Biomaterials that actively promote both wound healing and angiogenesis are of critical importance for many biomedical applications, including tissue engineering. In particular, hyaluronic acid (HA) is an important player that has multiple roles throughout the angiogenic process in the body. Previously, our laboratory has developed photocrosslinkable HA-based scaffolds that promote angiogenesis when implanted in vivo. This paper reports the incorporation of a photocrosslinkable fibronectin (FN) conjugate into three-dimensional (3-D) HA hydrogel networks to enhance endothelial cell adhesion and angiogenesis. The results demonstrate significantly better retention of FN that was photocrosslinked within HA hydrogels compared to FN that was physically adsorbed within HA hydrogels. Increased viability of endothelial cells cultured in 3-D HA hydrogels with photoimmobilized FN, compared to adsorbed FN, was also observed. Endothelial cells were cultured within hydrogels for up to 6 days, a period over which cell proliferation, migration and an angiogenic phenotype were influenced by varying the concentration of incorporated FN. The results demonstrate the potential of these composite hydrogels as biomaterial scaffolds capable of promoting wound healing and angiogenesis.
FKBPL and peptide derivatives: novel biological agents that inhibit angiogenesis by a CD44-dependent mechanism.
Andrea Valentine, Martin O'Rourke, Anita Yakkundi, Jenny Worthington, Michelle Hookham, Roy Bicknell, Helen O McCarthy, Keeva McClelland, Lynn McCallum, Hayder Dyer, Hayley McKeen, David J J Waugh, Jennifer Roberts, Joanne McGregor, Graham Cotton, Iain James, Timothy Harrison, David G Hirst, Tracy Robson
School of Pharmacy, Queen's University Belfast, Belfast, United Kingdom.
PURPOSE Antiangiogenic therapies can be an important adjunct to the management of many malignancies. Here we investigated a novel protein, FKBPL, and peptide derivative for their antiangiogenic activity and mechanism of action. EXPERIMENTAL DESIGN Recombinant FKBPL (rFKBPL) and its peptide derivative were assessed in a range of human microvascular endothelial cell (HMEC-1) assays in vitro. Their ability to inhibit proliferation, migration, and Matrigel-dependent tubule formation was determined. They were further evaluated in an ex vivo rat model of neovascularization and in two in vivo mouse models of angiogenesis, that is, the sponge implantation and the intravital microscopy models. Antitumor efficacy was determined in two human tumor xenograft models grown in severe compromised immunodeficient (SCID) mice. Finally, the dependence of peptide on CD44 was determined using a CD44-targeted siRNA approach or in cell lines of differing CD44 status. RESULTS rFKBPL inhibited endothelial cell migration, tubule formation, and microvessel formation in vitro and in vivo. The region responsible for FKBPL's antiangiogenic activity was identified, and a 24-amino acid peptide (AD-01) spanning this sequence was synthesized. It was potently antiangiogenic and inhibited growth in two human tumor xenograft models (DU145 and MDA-231) when administered systemically, either on its own or in combination with docetaxel. The antiangiogenic activity of FKBPL and AD-01 was dependent on the cell-surface receptor CD44, and signaling downstream of this receptor promoted an antimigratory phenotype. CONCLUSION FKBPL and its peptide derivative AD-01 have potent antiangiogenic activity. Thus, these agents offer the potential of an attractive new approach to antiangiogenic therapy.
Hyaluronic acid stimulates neovascularization during the regeneration of bone marrow after ablation.
Andrew L Raines, Moonhae Sunwoo, Arthur A Gertzman, Kipling Thacker, Robert E Guldberg, Zvi Schwartz, Barbara D Boyan
Parker H Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia, USA.
Restoration of vasculature is a critical component for successful integration of implants in musculoskeletal tissue. Sodium hyaluronate (NaHY) has been used as a carrier for demineralized bone matrix (DBM). DBM is osteoinductive and osteoconductive, but whether NaHY by itself has an effect is not known. NaHY has been reported to promote neovascularization, suggesting it may increase neovasculature when used with DBM as well. To test this, we used a rat tibial marrow ablation model to assess neovascularization during bone formation and regeneration of marrow with different combinations of NaHY alone and NaHY+DBM. To assess neovascularization during normal healing, animals were euthanized at 3-, 6-, 14-, 21-, and 28-days post-ablation, and the vasculature perfused using a radio-opaque contrast agent. Vascular morphology was assessed using μCT and histology. Peak vessel volume within the marrow cavity was observed on day-14 post-ablation. Test materials were injected into the ablated marrow space as follows:(A) empty defect controls;(B) high MW (700-800 kDa) NaHY + heat inactivated DBM;(C) DBM in PBS;(D) low MW NaHY (35 kDa)+ DBM;(E) high MW NaHY + DBM;(F) D:E 50:50;(G) low MW NaHY;(H) high MW NaHY; and (I) G:H 50:50. Neovascularization varied with bone substitute formulation. μCT results revealed that addition of NaHY resulted in an increase in vessel number compared to empty defects. Total blood vessel volume in all NaHY only groups were similar to DBM alone. Histomorphometry of sagittal sections showed that all three formulations of NaHY increased blood vessel number within the marrow cavity, confirming that NaHY promotes neovascularization.
Identification of CD44 as a senescence-induced cell adhesion gene responsible for the enhanced monocytes recruitment to senescent endothelial cells.
1Kyungpook National University.
The mechanism that is responsible for progression of atherosclerosis seen with increasing age remains controversial. This issue was addressed in the present study, by searching for genes that are uniquely expressed in senescent endothelial cells and functionally involved in inflammatory leukocytes adhesion recognized as a critical step in the initiation of atherogenesis. Senescent human umbilical vein endothelial cells (HUVECs) prepared by continuous sub-culturing in vitro showed higher binding affinity for monocytes (THP-1 cells, human acute monocytic leukemia cell line) compared with young cells. Gene expression profiles between young and senescent endothelial cells were compared by the cDNA microarray method, and CD44 was identified as one of the 'senescence-induced cell adhesion genes' whose expression was up-regulated in senescent cells and whose gene ontology annotation indicated their role in cell adhesion. The enhanced gene expression of CD44 in senescent endothelial cells was verified both at the mRNA and protein levels. Adhesion of monocytes to senescent endothelial cells was significantly reduced following pre-treatment of endothelial cells with the CD44 antibody or small interfering RNA (siRNA), thus reinforcing the critical role of CD44 in the inflammatory event. Exogenous expression of CD44 in young HUVECs and in human aortic endothelial cells led to increase in monocytes adhesion. CD44 expression levels in the rat aorta endothelium were found to increase in an age-dependent manner, as determined by immunohistochemistry and Western blotting. CD44 and other 'senescence-induced cell adhesion genes' identified in this study may provide the novel targets for the prevention of inflammatory leukocytes adhesion leading to the development atherosclerosis.
Blood. 2009 Dec 10;114 (25):5114-5 20007812
University of Pennsylvania, PA, USA.
Martina Tremmel, Alexandra Matzke, Imke Albrecht, Anna M Laib, Vivienne Olaku, Kurt Ballmer-Hofer, Gerhard Christofori, Melanie Heroult, Hellmut G Augustin, Helmut Ponta, Veronique Orian-Rousseau
Forschungszentrum Karlsruhe, Institute for Toxicology and Genetics, Karlsruhe, Germany.
A specific splice variant of the CD44 cell surface protein family, CD44v6, has been shown to act as a co-receptor for the receptor tyrosine kinase (RTK) c-Met on epithelial cells. Here we show that also on endothelial cells (ECs), the activity of c-Met is dependent on CD44v6. Furthermore, another RTK, VEGFR-2, is also regulated by CD44v6. The CD44v6 ectodomain and a small peptide mimicking a specific extracellular motif of CD44v6 or a CD44v6-specific antibody prevent CD44v6-mediated receptor activation. This indicates that the extracellular part of CD44v6 is required for interaction with c-Met or VEGFR-2. In the cytoplasm signaling by activated c-Met and VEGFR-2 requires association of the CD44 carboxy-terminus with ezrin that couples CD44v6 to the cytoskeleton. CD44v6 controls EC migration, sprouting and tubule formation induced by HGF or VEGF-A. In vivo the development of blood vessels from grafted EC spheroids and angiogenesis in tumors is impaired by CD44v6 blocking reagents suggesting that the co-receptor function of CD44v6 for c-Met and VEGFR-2 is a promising target to block angiogenesis in pathological conditions.
CD44 deficiency improves healing tendon mechanics and increases matrix and cytokine expression in a mouse patellar tendon injury model.
McKay Orthopaedic Research Laboratory, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
CD44 plays an important role in inflammation and healing. Previous studies investigated its role in inflammatory diseases and skin wounds; however, the role of CD44 in tendon healing is unknown. Therefore, we investigated the effect of CD44 in the healing of the patellar tendon in a knockout mouse model. We hypothesized that in comparison to wild-type counterparts, CD44 knockout mice would have decreased material parameters, increased organization, decreased expression of proinflammatory cytokines, and increased expression of matrix components during healing. These hypotheses were tested through an in vivo surgical model and mechanical, organizational, and gene expression analyses. Material strength and tissue organization were significantly improved in the CD44 knockout mouse. This could be attributed to increased expression of cytokines and matrix components that are also elevated in regenerative healing. Our study showed that the absence of CD44 in a mouse patellar tendon injury creates an environment that is conducive to regenerative healing through altered gene expression, resulting in superior material properties and reduced cross-sectional area. Therefore, limiting the role of CD44 may improve healing parameters in adult tendon injury.
Clemson University; Medical University of South Carolina Bioengineering Program; Charleston, South Carolina USA.
Angiogenesis represents the outgrowth of new blood vessels from existing ones, a physiologic process that is vital to supply nourishment to newly forming tissues during development and tissue remodeling and repair (wound healing). Regulation of angiogenesis in the healthy body occurs through a fine balance of angiogenesis-stimulating factors and angiogenesis inhibitors. When this balance is disturbed, excessive or deficient angiogenesis can result and contribute to development of a wide variety of pathological conditions. The therapeutic stimulation or suppression of angiogenesis could be the key to abrogating these diseases. In recent years, tissue engineering has emerged as a promising technology for regenerating tissues or organs that are diseased beyond repair. Among the critical challenges that deter the practical realization of the vision of regenerating functional tissues for clinical implantation, is how tissues of finite size can be regenerated and maintained viable in the long-term. Since the diffusion of nutrients and essential gases to cells, and removal of metabolic wastes is typically limited to a depth of 150-250 microm from a capillary (3-10 cells thick), tissue constructs must mandatorily permit in-growth of a blood capillary network to nourish and sustain the viability of cells within. The purpose of this article is to provide an overview of the role and significance of hyaluronan (HA), a glycosaminoglycan (GAG) component of connective tissues, in physiologic and pathological angiogenesis, its applicability as a therapeutic to stimulate or suppress angiogenesis in situ within necrotic tissues in vivo, and the factors determining its potential utility as a pro-angiogenic stimulus that will enable tissue engineering of neo-vascularized and functional tissue constructs for clinical use.
Pulmonary, Allergy, and Critical Care Division, SVM-Hill Pavilion, Rm. 410B, 380 South Univ. Ave., Philadelphia, PA 19104-4539, USA.
Several protocols for the isolation of endothelial cells (ECs) from murine lung have been described in the literature. We, however, encountered a number of problems while using these procedures that prevented us from consistently or reliably obtaining pure populations of ECs from the lungs of mice. By incorporating specific elements from previously published protocols, as well as adding some novel features, we developed a new strategy for isolating ECs from murine lung. In this approach, a suspension of lung cells is initially prepared from the lungs of 7- to 14-day-old mouse pups using procedures that prevent intravascular clotting and leukocyte activation, minimize mechanical trauma to the lung tissue, and limit exposure to the digesting enzymes. The resulting cell suspension is cultured for 2-3 days, trypsinized to produce a suspension of single cells, and then subjected to fluorescence-activated cell sorting using an anti-ICAM-2 antibody. The sorted cells are then plated and split 1:2 at each passage to maintain a high density of the cells. Using this approach, we have been able to isolate pure populations of ECs that were sustainable for extended periods in culture without the emergence of fibroblast overgrowth or the development of senescence. We believe the success of this approach will provide opportunities to take advantage of the large and growing number of knockout and transgenic mouse lines to investigate the endothelial-specific roles of targeted molecules in the pulmonary vasculature.
Other papers by authors:
Horace M DeLisser, Brian P Helmke, Gaoyuan Cao, Patricia M Egan, Darren Taichman, Melane Fehrenbach, Aisha Zaman, Zheng Cui, Gopi S Mohan, H Scott Baldwin, Peter F Davies, Rashmin C Savani
Pulmonary, Allergy and Critical Care Division, Department of Medicine, Institute for Medicine and Engineering, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-4318, USA.
The final stage of lung development in humans and rodents occurs principally after birth and involves the partitioning of the large primary saccules into smaller air spaces by the inward protrusion of septae derived from the walls of the saccules. Several observations in animal models implicate angiogenesis as critical to this process of alveolarization, but all anti-angiogenic treatments examined to date have resulted in endothelial cell (EC) death. We therefore targeted the function of platelet endothelial cell adhesion molecule,(PECAM-1), an EC surface molecule that promotes EC migration and has been implicated in in vivo angiogenesis. Administration of an anti-PECAM-1 antibody that inhibits EC migration, but not proliferation or survival in vitro, disrupted normal alveolar septation in neonatal rat pups without reducing EC content. Three-dimensional reconstruction of lungs showed that pups treated with a blocking PECAM-1 antibody had remodeling of more proximal branches resulting in large tubular airways. Subsequent studies in PECAM-1-null mice confirmed that the absence of PECAM-1 impaired murine alveolarization, without affecting EC content, proliferation, or survival. Further, cell migration was reduced in lung endothelial cells isolated from these mice. These data suggest that the loss of PECAM-1 function compromises postnatal lung development and provide evidence that inhibition of EC function, in contrast to a loss of viable EC, inhibits alveolarization.
Tumor-derived vascular endothelial growth factor up-regulates angiopoietin-2 in host endothelium and destabilizes host vasculature, supporting angiogenesis in ovarian cancer.
Lin Zhang, Nuo Yang, Jin-Wan Park, Dionyssios Katsaros, Stefano Fracchioli, Gaoyuan Cao, Ann O'Brien-Jenkins, Thomas C Randall, Stephen C Rubin, George Coukos
Abramson Family Cancer Research Institute, Center for Research on Reproduction and Women's Health, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Vascular remodeling in host tissues surrounding growing tumors is implicated in the successful development of tumor neovasculature. Cooperation between vascular endothelial growth factor (VEGF) and angiopoietins (Angs) is considered to be critical in this context. However, the mechanisms regulating the coordinated expression of these molecules remain, to date, elusive. In this study, we used a murine ovarian cancer angiogenesis model induced by overexpression of VEGF, as well as 52 human ovarian cancer specimens and 36 established cancer cell lines to characterize the expression and regulation of Ang-2 in the context of tumor angiogenesis. Using a combination of immunohistochemistry, laser capture microdissection and real-time quantitative reverse transcription-PCR, we showed that tumor-derived VEGF significantly up-regulated the expression of Ang-2 in host stroma endothelial cells, resulting in markedly increased Ang-2/Tie-2 mRNA copy number ratio in vivo. In vitro experiments showed that VEGF directly up-regulated Ang-2, which is mediated via VEGF receptor-2/flk-1/KDR pathway, in cultured endothelial cells through transcriptional activation rather than the enhanced mRNA stability. In human ovarian cancer, Ang-2 was primarily expressed in stroma endothelial cells and detectable in tumor cells of only 12% tumor specimens; however, it was not detected in the majority of established ovarian cancer cell lines. In addition, a significant correlation was observed between VEGF and Ang-2 mRNA expression (P < 0.01) but not between VEGF and Ang-1 or Tie-2 in human ovarian cancer specimens. In the mouse ovarian cancer model, up-regulation of Ang-2 in host stroma endothelial cells was significantly associated with pericyte loss and instability of the host vasculature surrounding the tumor. Our study suggests a novel mechanism by which tumor-derived VEGF interacts with Angs/Tie-2 system in host stroma endothelial cells and induces in a paracrine manner the remodeling of host vasculature to support angiogenesis during tumor growth.
Methods Mol Biol. 2012 ;822 :295-306 22144208
Department of Obstetrics and Gynecology, Center for Research on Early Detection and Cure of Ovarian Cancer, University of Pennsylvania, Philadelphia, PA, USA.
MicroRNAs (miRNAs) are small (∼18-25 nucleotides), endogenous, noncoding RNAs that regulate gene expression in a sequence-specific manner via the degradation of target mRNAs or the inhibition of protein translation. miRNAs are predicted to target up to one-third of all human mRNAs. Each miRNA can target hundreds of transcripts and proteins directly or indirectly, and more than one miRNA can converge on a single target transcript; thus, the potential regulatory circuitry afforded by miRNAs is enormous. Increasing evidence is revealing that the expression of miRNAs is deregulated in cancer. High-throughput miRNA quantification technologies provide powerful tools to study global miRNA profiles. It has become progressively more apparent that, although the number of miRNAs (∼1,000) is much smaller than the number of protein-coding genes (∼22,000), miRNA expression signatures more accurately reflect the developmental lineage and tissue origin of human cancers. Large-scale studies in human cancer have further demonstrated that miRNA expression signatures are associated not only with specific tumor subtypes but also with clinical outcomes.
Cancer Res. 2012 Jan 1;72 (1):154-64 22058146
A combined array-based comparative genomic hybridization and functional library screening approach identifies mir-30d as an oncomir in cancer.
Ning Li, Sippy Kaur, Joel Greshock, Heini Lassus, Xiaomin Zhong, Yanling Wang, Arto Leminen, Zhongjun Shao, Xiaowen Hu, Shun Liang, Dionyssios Katsaros, Qihong Huang, Ralf Bützow, Barbara L Weber, George Coukos, Lin Zhang
Ovarian Cancer Research Center and Department of Obstetrics & Gynecology, University of Pennsylvania, Wistar Institute, Philadelphia, Pennsylvania, USA.
Oncomirs are microRNAs (miRNA) that acts as oncogenes or tumor suppressor genes. Efficient identification of oncomirs remains a challenge. Here we report a novel, clinically guided genetic screening approach for the identification of oncomirs, identifying mir-30d through this strategy. mir-30d regulates tumor cell proliferation, apoptosis, senescence, and migration. The chromosomal locus harboring mir-30d was amplified in more than 30% of multiple types of human solid tumors (n = 1,283). Importantly, higher levels of mir-30d expression were associated significantly with poor clinical outcomes in ovarian cancer patients (n = 330, P = 0.0016). Mechanistic investigations suggested that mir-30d regulates a large number of cancer-associated genes, including the apoptotic caspase CASP3. The guided genetic screening approach validated by this study offers a powerful tool to identify oncomirs that may have utility as biomarkers or targets for drug development.
Tissue-based immune monitoring I: tumor core needle biopsies allow in-depth interrogation of the tumor microenvironment.
Andrea R Hagemann, Mark Cadungog, Ian S Hagemann, Rachel Hammond, Sarah F Adams, Christina S Chu, Steven C Rubin, Lin Zhang, Kathakali Addya, Michael J Birrer, Phyllis A Gimotty, George Coukos
Ovarian Cancer Research Center, Division of Gynecologic Oncology, University of Pennsylvania, Philadelphia, USA.
We sought to assess the feasibility and reproducibility of performing tissue-based immune characterization of the tumor microenvironment using CT-compatible needle biopsy material. Three independent biopsies were obtained intraoperatively from one metastatic epithelial ovarian cancer lesion of 7 consecutive patients undergoing surgical cytoreduction using a 16-gauge core biopsy needle. Core specimens were snap-frozen and subjected to immunohistochemistry (IHC) against human CD3, CD4, CD8, and FoxP3. A portion of the cores was used to isolate RNA for 1) real-time quantitative (q)PCR for CD3, CD4, CD8, FoxP3, IL-10 and TGF-beta, 2) multiplexed PCR-based T cell receptor (TCR) CDR3 Vβ region spectratyping, and 3) gene expression profiling. Pearson's correlations were examined for immunohistochemistry and PCR gene expression, as well as for gene expression array data obtained from different tumor biopsies. Needle biopsy yielded sufficient tissue for all assays in all patients. IHC was highly reproducible and informative. Significant correlations were seen between the frequency of CD3+, CD8+ and FoxP3+ T cells by IHC with CD3ε, CD8A, and FoxP3 gene expression, respectively, by qPCR (r=0.61, 0.86, and 0.89; all p< 0.05). CDR3 spectratyping was feasible and highly reproducible in each tumor, and indicated a restricted repertoire for specific TCR Vβ chains in tumor-infiltrating T cells. Microarray gene expression revealed strong correlation between different biopsies collected from the same tumor. Our results demonstrate a feasible and reproducible method of immune monitoring using CT-compatible needle biopsies from tumor tissue, thereby paving the way for sophisticated translational studies during tumor biological therapy.
Andrea Facciabene, Xiaohui Peng, Ian S Hagemann, Klara Balint, Andrea Barchetti, Li-Ping Wang, Phyllis A Gimotty, C Blake Gilks, Priti Lal, Lin Zhang, George Coukos
Ovarian Cancer Research Center, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Although immune mechanisms can suppress tumour growth, tumours establish potent, overlapping mechanisms that mediate immune evasion. Emerging evidence suggests a link between angiogenesis and the tolerance of tumours to immune mechanisms. Hypoxia, a condition that is known to drive angiogenesis in tumours, results in the release of damage-associated pattern molecules, which can trigger the rejection of tumours by the immune system. Thus, the counter-activation of tolerance mechanisms at the site of tumour hypoxia would be a crucial condition for maintaining the immunological escape of tumours. However, a direct link between tumour hypoxia and tolerance through the recruitment of regulatory cells has not been established. We proposed that tumour hypoxia induces the expression of chemotactic factors that promote tolerance. Here we show that tumour hypoxia promotes the recruitment of regulatory T (T(reg)) cells through induction of expression of the chemokine CC-chemokine ligand 28 (CCL28), which, in turn, promotes tumour tolerance and angiogenesis. Thus, peripheral immune tolerance and angiogenesis programs are closely connected and cooperate to sustain tumour growth.
Frequent genetic abnormalities of the PI3K/AKT pathway in primary ovarian cancer predict patient outcome.
Jia Huang, Lin Zhang, Joel Greshock, Theresa A Colligon, Yan Wang, Renee Ward, Dionyssios Katsaros, Heini Lassus, Ralf Butzow, Andrew K Godwin, Joseph R Testa, Katherine L Nathanson, Phyllis A Gimotty, George Coukos, Barbara L Weber, Yan Degenhardt
Department of Biology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
Identification and characterization of underlying genetic aberrations could facilitate diagnosis and treatment of ovarian cancer. Copy number analysis using array Comparative Genomic Hybridization (aCGH) on 93 primary ovarian tumors identified PI3K/AKT pathway as the most frequently altered cancer related pathway. Furthermore, survival analyses to correlate gene copy number and mutation data with patient outcome showed that copy number gains of PIK3CA, PIK3CB, and PIK3R4 in these tumors were associated with decreased survival. To confirm these findings at the protein level, immunohistochemistry (IHC) for PIK3CA product p110α and p-Akt was performed on tissue microarrays from 522 independent serous ovarian cancers. Overexpression of either of these two proteins was found to be associated with decreased survival. Multivariant analysis from these samples further showed that overexpression of p-AKT and/or p110α is an independent prognostic factor for these tumors. siRNAs targeting altered PI3K/AKT pathway genes inhibited proliferation and induced apoptosis in ovarian cancer cell lines. In addition, the effect of the siRNAs in different cell lines seemed to correlate with the particular genetic alterations that the cell line carries. These results strongly support the utilization of PI3K pathway inhibitors in ovarian cancer. They also suggest identifying the specific component in the PI3K pathway that is genetically altered has the potential to help select the most effective therapy. Both mutation as well as copy number changes can be used as predictive markers for this purpose.
Integrated analysis of multiple microarray datasets identifies a reproducible survival predictor in ovarian cancer.
Panagiotis A Konstantinopoulos, Stephen A Cannistra, Helen Fountzilas, Aedin Culhane, Kamana Pillay, Bo Rueda, Daniel Cramer, Michael Seiden, Michael Birrer, George Coukos, Lin Zhang, John Quackenbush, Dimitrios Spentzos
Division of Hematology/Oncology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, United States of America.
Public data integration may help overcome challenges in clinical implementation of microarray profiles. We integrated several ovarian cancer datasets to identify a reproducible predictor of survival. Four microarray datasets from different institutions comprising 265 advanced stage tumors were uniformly reprocessed into a single training dataset, also adjusting for inter-laboratory variation ("batch-effect"). Supervised principal component survival analysis was employed to identify prognostic models. Models were independently validated in a 61-patient cohort using a custom array genechip and a publicly available 229-array dataset. Molecular correspondence of high- and low-risk outcome groups between training and validation datasets was demonstrated using Subclass Mapping. Previously established molecular phenotypes in the 2(nd) validation set were correlated with high and low-risk outcome groups. Functional representational and pathway analysis was used to explore gene networks associated with high and low risk phenotypes. A 19-gene model showed optimal performance in the training set (median OS 31 and 78 months, p < 0.01), 1(st) validation set (median OS 32 months versus not-yet-reached, p = 0.026) and 2(nd) validation set (median OS 43 versus 61 months, p = 0.013) maintaining independent prognostic power in multivariate analysis. There was strong molecular correspondence of the respective high- and low-risk tumors between training and 1(st) validation set. Low and high-risk tumors were enriched for favorable and unfavorable molecular subtypes and pathways, previously defined in the public 2(nd) validation set. Integration of previously generated cancer microarray datasets may lead to robust and widely applicable survival predictors. These predictors are not simply a compilation of prognostic genes but appear to track true molecular phenotypes of good- and poor-outcome.
Double-negative feedback loop between reprogramming factor LIN28 and microRNA let-7 regulates aldehyde dehydrogenase 1-positive cancer stem cells.
Xiaojun Yang, Xiaojuan Lin, Xiaomin Zhong, Sippy Kaur, Ning Li, Shun Liang, Heini Lassus, Liping Wang, Dionyssios Katsaros, Kathleen Montone, Xia Zhao, Youcheng Zhang, Ralf Bützow, George Coukos, Lin Zhang
Center for Research on Early Detection and Cure of Ovarian Cancer, Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
A relatively rare aldehyde dehydrogenase 1 (ALDH1)-positive "stem cell-like" subpopulation of tumor cells has the unique ability to initiate and perpetuate tumor growth; moreover, it is highly resistant to chemotherapy and significantly associated with poor clinical outcomes. The development of more effective therapies for cancer requires targeting of this cell population. Using cDNA microarray analysis, we identified that the expression of the Caenorhabditis elegans lin-28 homologue (LIN28) was positively correlated with the percentage of ALDH1+ tumor cells; this was further validated in an independent set of tissue arrays (n=197). Both loss-of-function and gain-of-function studies showed that LIN28 plays a critical role in the maintenance of ALDH1+ tumor cells. In addition, we found that there is a double-negative feedback loop between LIN28 and let-7 in tumor cells, and that let-7 negatively regulates ALDH1+ tumor cells. Finally, we report that a LIN28/let-7 loop modulates self-renewal and differentiation of mammary gland epithelial progenitor cells. Our data provide evidence that cancer stem cells may arise through a "reprogramming-like" mechanism. A rebalancing of the LIN28/let-7 regulatory loop could be a novel therapeutic strategy to target ALDH1+ cancer stem cells.
Identification of microRNAs regulating reprogramming factor LIN28 in embryonic stem cells and cancer cells.
Center for Research on Early Detection and Cure of Ovarian Cancer, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
LIN28 (a homologue of the Caenorhabditis elegans lin-28 gene) is an evolutionarily conserved RNA-binding protein and a master regulator controlling the pluripotency of embryonic stem cells. Together with OCT4, SOX2, and NANOG, LIN28 can reprogram somatic cells, producing induced pluripotent stem cells. Expression of LIN28 is highly restricted to embryonic stem cells and developing tissues. In human tumors, LIN28 is up-regulated and functions as an oncogene promoting malignant transformation and tumor progression. However, the mechanisms of transcriptional and post-transcriptional regulation of LIN28 are still largely unknown. To examine microRNAs (miRNAs) that repress LIN28 expression, a combined in silico prediction and miRNA library screening approach was used in the present study. Four miRNAs directly regulating LIN28 (let-7, mir-125, mir-9, and mir-30) were initially identified by this approach and further validated by quantitative RT-PCR, Western blot analysis, and a LIN28 3'-UTR reporter assay. We found that expression levels of these four miRNAs were clustered together and inversely correlated with LIN28 expression during embryonic stem cell differentiation. In addition, the expression of these miRNAs was remarkably lower in LIN28-positive tumor cells compared with LIN28-negative tumor cells. Importantly, we demonstrated that these miRNAs were able to regulate the expression and activity of let-7, mediated by LIN28. Taken together, our studies demonstrate that miRNAs let-7, mir-125, mir-9, and mir-30 directly repress LIN28 expression in embryonic stem and cancer cells. Global down-regulation of these miRNAs may be one of the mechanisms of LIN28 reactivation in human cancers.
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Peptides. 2011 Jul ;32 (7):1469-76 21693141
Reinaldo Ramos, João Pedro Silva, Ana Cristina Rodrigues, Raquel Costa, Luísa Guardão, Fernando Schmitt, Raquel Soares, Manuel Vilanova, Lucília Domingues, Miguel Gama
IBB, Institute of Biotechnology and Bioengineering, Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal.
Antimicrobial peptides (AMPs) are part of the innate immune system and are generally defined as cationic, amphipathic peptides, with less than 50 amino acids, including multiple arginine and lysine residues. The human cathelicidin antimicrobial peptide LL37 can be found at different concentrations in many different cells, tissues and body fluids and has a broad spectrum of antimicrobial and immunomodulatory activities. The healing of wound is a complex process that involves different steps: hemostasis, inflammation, remodeling/granulation tissue formation and re-epithelialization. Inflammation and angiogenesis are two fundamental physiological conditions implicated in this process. We have recently developed a new method for the expression and purification of recombinant LL37. In this work, we show that the recombinant peptide P-LL37 with a N-terminus proline preserves its immunophysiological properties in vitro and in vivo. P-LL37 neutralized the activation of macrophages by lipopolysaccharide (LPS). Besides, the peptide induced proliferation, migration and tubule-like structures formation by endothelial cells. Wound healing experiments were performed in dexamethasone-treated mice to study the effect of LL37 on angiogenesis and wound regeneration. The topical application of synthetic and recombinant LL37 increased vascularization and re-epithelialization. Taken together, these results clearly demonstrate that LL37 may have a key role in wound regeneration through vascularization.
Blood. 2011 May 5;117 (18):4978-87 21378273
Elzbieta Pluskota, James J Dowling, Natalie Gordon, Jeffrey A Golden, Dorota Szpak, Xiaoxia Z West, Carla Nestor, Yan-Qing Ma, Katarzyna Bialkowska, Tatiana Byzova, Edward F Plow
Department of Molecular Cardiology, Cleveland Clinic, Cleveland, OH, USA.
Kindlin-2, a widely distributed cytoskeletal protein, has been implicated in integrin activation, and its absence is embryonically lethal in mice and causes severe developmental defects in zebrafish. Knockdown of kindlin-2 levels in endothelial cells resulted in defective adhesive and migratory responses, suggesting that angiogenesis might be aberrant even with partial reduction of kindlin-2. This hypothesis has now been tested in the kindlin-2(+/-) mice. RM1 prostate tumors grown in kindlin-2(+/-) mice had fewer blood vessels, which were thinner and shorter and supported less tumor growth compared with wild-type littermates. The vessels that did form in the kindlin-2(+/-) mice lacked smooth muscle cells and pericytes and had thinner basement membranes, indicative of immature vessels. VEGF-induced angiogenesis in matrigel implants was also abnormal in the kindlin-2(+/-) mice. Vessels in the kindlin-2(+/-) mice were leaky, and BM transplantation from kindlin-2(+/-) to WT mice did not correct this defect. Endothelial cells derived from kindlin-2(+/-) mice had integrin expression levels similar to WT mice but reduced αVβ3-dependent signaling, migration, adhesion, spreading, and tube formation. Developmental angiogenesis was markedly impaired by kindlin-2 morpholinos in zebrafish. Taken together, kindlin-2 plays an important role in pathologic and developmental angiogenesis, which arises from defective activation of integrin αVβ3.
Trevigen, Gaithersburg, Maryland, USA.
A protocol is presented here for a rapid, quantitative and reliable in vitro angiogenesis assay that can be adapted for high throughput use. Endothelial cells are plated on a gelled basement matrix, their natural substrate, and form capillary-like structures with a lumen. The assay can be used to identify inhibitors or stimulators of angiogenesis, as well as genes and signaling pathways involved in angiogenesis. It has also been used to identify endothelial progenitor cells. This assay involves endothelial cell adhesion, migration, protease activity and tubule formation. This tube formation assay is preferred, as other in vitro assays for angiogenesis, such as cell adhesion, migration and invasion, measure limited steps in the angiogenesis process. The tube formation assay on basement membrane can be completed in a day because transformed endothelial cells form tubes within 3 h, whereas non-transformed endothelial cells form tubes within 6 h.
Oncogene. 2010 Mar 8;: 20208559
DLC2 modulates angiogenic responses in vascular endothelial cells by regulating cell attachment and migration.
 Department of Biochemistry and Molecular Medicine University of California-Davis, Sacramento, CA, USA  Department of Pathology, First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, Guangdong, PR China.
Deleted in liver cancer 1 (DLC1) is a RhoGTPase activation protein-containing tumor suppressor that associates with various types of cancer. Although DLC2 shares a similar domain structure with that of DLC1, the function of DLC2 is not well characterized. Here, we describe the expression and ablation of DLC2 in mice using a reporter-knockout approach. DLC2 is expressed in several tissues and in endothelial cells (ECs) of blood vessels. Although ECs and blood vessels show no histological abnormalities and mice appear overall healthy, DLC2-mutant mice display enhanced angiogenic responses induced by matrigel and by tumor cells. Silencing of DLC2 in human ECs has reduced cell attachment, increased migration, and tube formation. These changes are rescued by silencing of RhoA, suggesting that the process is RhoA pathway dependent. These results indicate that DLC2 is not required for mouse development and normal vessel formation, but may protect mouse from unwanted angiogenesis induced by, for example, tumor cells.Oncogene advance online publication, 8 March 2010; doi:10.1038/onc.2010.54.
Department of Ophthalmology, Peking University People's Hospital, Xizhimen South Street 11, Xi Cheng District, Beijing, China.
PURPOSE Robo1, a member of the roundabout (Robo) family, serves as neuronal guidance receptors and has been reported to mediate tumor angiogenesis. In this study, we investigated the function of Robo1 and its possible role in retinal angiogenesis in vitro and in vivo. METHODS Expression of Robo1 in relation to retinal neovascularization was studied in an animal model of retinopathy of prematurity (ROP). siRNA technology was used to knockdown Robo1 expression to study its effects on monkey choroidal retinal endothelial cells (RF/6A) in vitro. Cell proliferation, migration, spreading, cycling, and apoptosis were assessed with the methyl thiazolyl tetrazolium (MTT), Boyden chamber, immunohistochemistry, and flow cytometry assay. Tube formation by RF/6A on Matrigel was also analyzed. RESULTS Robo1 expression was elevated in the ROP murine eyes (P < 0.01). Knockdown of Robo1 expression inhibited cell proliferation and migration. Tube formation by RF/6A was also disturbed. CONCLUSIONS Our results show that Robo1 inhibits choroidal and retinal angiogenesis in vitro. Further studies are ongoing to evaluate Robo1 as a potential target for the treatment of choroidal or retinal angiogenesis.
Marijana Miljkovic-Licina, Philippe Hammel, Sarah Garrido-Urbani, Paul F Bradfield, Pierre Szepetowski, Beat A Imhof
*Department of Pathology and Immunology, CMU, University of Geneva, Geneva, Switzerland; andINSERM, UMR 901 and UMR 910, Université de la Méditerranée, Marseille, France.
On appropriate stimuli, quiescent endothelial cells start to proliferate and form de novo blood vessels through angiogenesis. To further define molecular mechanisms accompanying the activation of endothelial cells during angiogenesis, we identified genes that were differentially regulated during this process using microarray analyses. In this work, we established a regulatory role for Sushi repeat protein X-linked 2 (Srpx2) in endothelial cell remodeling during angiogenesis. In particular, silencing of Srpx2 using small interfering RNAs (siRNAs) specifically attenuated endothelial cell migration and delayed angiogenic sprout formation. In vivo, Srpx2 expression was detected in de novo formation of blood vessels in angiogenic tissues by in situ mRNA hybridization and immunostaining. Pulldown experiments identified Srpx2 as a ligand for vascular uPAR, a key molecule involved in invasive migration of angiogenic endothelium. Immunostaining revealed coexpression of the Srpx2 and uPAR on vascular endothelium. These findings suggest that Srpx2 regulates endothelial cell migration and tube formation and provides a new target for modulating angiogenesis.-Miljkovic-Licina, M., Hammel, P., Garrido-Urbani, S., Bradfield, P. F., Szepetowski, P., Imhof, B. A. Sushi repeat protein X-linked 2, a novel mediator, of angiogenesis.
The First Faculty of Medicine, Institute of Histology and Embryology, Charles University Prague, Albertov 4, CZ-128 00 Prague, Czech Republic.
Abstract Similar to growing and metabolically active tissues, tumors require a dense vasculature to gain access to oxygen and nutrients. However, blood vessels in tumors differ from vessels in normal tissues in many respects. In particular, the tumor vasculature is in an active state of angiogenesis or vasculogenesis, and it is immature and leaky. Blood vessels are multicellular tubes formed by polarized endothelial cells, which face the patent vascular lumen with their apical cell surface, while their basal cell surface faces extracellular matrix on the outside of the vessels. The same cell polarity can be found in other tubular structures, such as in the bronchial tubes of the lung or the kidney tubules. In contrast, blood vessels in invertebrates often have a vascular lumen lined by basal cell surfaces. These vessels are often formed by a process that we call 'ancestral vascular tube formation'. Here we discuss the hypothesis that the supply of tumors with blood may be achieved by both endothelial cell-lined tubes as well as tubes formed by the tumor cells themselves using the ancestral vascular tube formation mechanism. We discuss this hypothesis with a particular focus on gastrointestinal tumors.
Tomoya Kitamura, Naoya Asai, Atsushi Enomoto, Kengo Maeda, Takuya Kato, Maki Ishida, Ping Jiang, Takashi Watanabe, Jiro Usukura, Takahisa Kondo, Frank Costantini, Toyoaki Murohara, Masahide Takahashi
Department of Cardiology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan.
The serine/threonine protein kinase Akt is involved in a variety of cellular processes including cell proliferation, survival, metabolism and gene expression. It is essential in vascular endothelial growth factor (VEGF)-mediated angiogenesis; however, it is not known how Akt regulates the migration of endothelial cells, a crucial process for vessel sprouting, branching and the formation of networks during angiogenesis. Here we report that Akt-mediated phosphorylation of Girdin, an actin-binding protein, promotes VEGF-dependent migration of endothelial cells and tube formation by these cells. We found that exogenously delivered adenovirus harbouring Girdin short interfering RNA in Matrigel embedded in mice, markedly inhibited VEGF-mediated angiogenesis. Targeted disruption of the Girdin gene in mice impaired vessel remodelling in the retina and angiogenesis from aortic rings, whereas Girdin was dispensable for embryonic vasculogenesis. These findings demonstrate that the Akt/Girdin signalling pathway is essential in VEGF-mediated postneonatal angiogenesis.
The Wilms' tumour suppressor WT1 is involved in endothelial cell proliferation and migration: expression in tumour vessels in vivo.
INSERM U907, Nice, France.
Vascularization is an important step in tumour growth. Although a variety of molecules, for example, VEGF, ETS-1 or nestin have been implicated in tumour angiogenesis, the molecular mechanisms of vessel formation are not fully characterized. We showed that the Wilms' tumour suppressor WT1 activates nestin during development. Here we tested whether WT1 might also be involved in tumour angiogenesis. Endothelial WT1 expression was detected in 95% of 113 tumours of different origin. To analyse the function of WT1 in endothelial cells, we used an RNAi approach in vitro and showed that inhibition of WT1 reduces cell proliferation, migration and endothelial tube formation. On a molecular level, WT1 silencing diminished expression of the ETS-1 transcription factor. WT1 and ETS-1 shared an overlapping expression in tumour endothelia. The ETS-1 promoter was stimulated approximately 10-fold by transient co-transfection of a WT1 expression construct and WT1 bound to the ETS-1 promoter in chromatin immunoprecipitation and electrophoretic mobility shift assays. Deletion of the identified WT1-binding site abolished stimulation of the ETS-1 promoter by WT1. These findings suggest that transcriptional activation of ETS-1 by the Wilms' tumour suppressor WT1 is a crucial step in tumour vascularization via regulation of endothelial cell proliferation and migration.
Inhibition of telomerase in the endothelial cells disrupts tumor angiogenesis in glioblastoma xenografts.
Maria Laura Falchetti, Maria Patrizia Mongiardi, Paolo Fiorenzo, Giovanna Petrucci, Francesco Pierconti, Igea D'Agnano, Giorgio D'Alessandris, Giulio Alessandri, Maurizio Gelati, Lucia Ricci-Vitiani, Giulio Maira, Luigi Maria Larocca, Andrea Levi, Roberto Pallini
Institute of Neurobiology and Molecular Medicine, CNR, Rome, Italy. firstname.lastname@example.org
Tumor angiogenesis is a complex process that involves a series of interactions between tumor cells and endothelial cells (ECs). In vitro, glioblastoma multiforme (GBM) cells are known to induce an increase in proliferation, migration and tube formation by the ECs. We have previously shown that in human GBM specimens the proliferating ECs of the tumor vasculature express the catalytic component of telomerase, hTERT, and that telomerase can be upregulated in human ECs by exposing these cells to GBM in vitro. Here, we developed a controlled in vivo assay of tumor angiogenesis in which primary human umbilical vascular endothelial cells (HUVECs) were subcutaneously grafted with or without human GBM cells in immunocompromised mice as Matrigel implants. We found that primary HUVECs did not survive in Matrigel implants, and that telomerase upregulation had little effect on HUVEC survival. In the presence of GBM cells, however, the grafted HUVECs not only survived in Matrigel implants but developed tubule structures that integrated with murine microvessels. Telomerase upregulation in HUVECs enhanced such effect. More importantly, inhibition of telomerase in HUVECs completely abolished tubule formation and greatly reduced survival of these cells in the tumor xenografts. Our data demonstrate that telomerase upregulation by the ECs is a key requisite for GBM tumor angiogenesis.