scholarly journals P192Opposing effects of cAMP/PKA and cAMP/Epac signalling on in vitro angiogenesis: role of Rho gtpases

2014 ◽  
Vol 103 (suppl 1) ◽  
pp. S34.2-S34
Author(s):  
M Aslam ◽  
H Nef ◽  
C Troidl ◽  
C Hamm ◽  
D Guenduez
Keyword(s):  
Rho Gtpases ◽  
In Vitro Angiogenesis

scholarly journals Dual Effects ofβ3Integrin Subunit Expression on Human Pancreatic Cancer Models

2010 ◽  
Vol 33 (5-6) ◽  
pp. 191-205 ◽  
Author(s):  
S. Marchán ◽  
S. Pérez-Torras ◽  
A. Vidal ◽  
J. Adan ◽  
F. Mitjans ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Rho Gtpases ◽  
Cell Attachment ◽  
Human Pancreatic Cancer ◽  
Cell Growth And Migration ◽  
Subunit Expression ◽  
And Migration

Background: Pancreatic cancer, the fifth leading cause of adult cancer death in Western countries, lacks early detection, and displays significant dissemination ability. Accumulating evidence shows that integrin-mediated cell attachment to the extracellular matrix induces phenotypes and signaling pathways that regulate tumor cell growth and migration.Methods: In view of these findings, we examined the role ofβ3in pancreatic cancer by generating two stableβ3-expressing pancreatic human cell lines and characterizing their behavior in vitro and in vivo.Results: Transduction ofβ3selectively augmented the functional membraneαvβ3integrin levels, as evident from the enhanced adhesion and migration abilities related to active Rho GTPases. No effects on in vitro anchorage-dependent growth, but higher anoikis were detected inβ3-overexpressing cells. Moreover, tumors expressingβ3displayed reduced growth. Interestingly, treatment of mice with anαv-blocking antibody inhibited the growth ofβ3-expressing tumors to a higher extent.Conclusion: Our results collectively support the hypothesis thatαvβ3integrin has dual actions depending on the cell environment, and provide additional evidence on the role of integrins in pancreatic cancer, which should eventually aid in improving prediction of the effects of therapies addressed to modulate integrin activities in these tumors.

Three-Dimensional Simulation of In Vitro Angiogenesis: Effects of Extracellular Matrix Structure and Density

2010 ◽  
Author(s):  
Lowell Taylor Edgar ◽  
James E. Guilkey ◽  
Clayton J. Underwood ◽  
Brenda Baggett ◽  
Urs Utzinger ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Three Dimensional ◽  
Vascular Endothelial ◽  
Chemical Factors ◽  
Dimensional Simulation ◽  
Endothelial Growth Factor ◽  
In Vitro Angiogenesis

The process of angiogenesis is regulated by both chemical and mechanical signaling. While the role of chemical factors such as vascular endothelial growth factor (VEGF) during angiogenesis has been extensively studied, the influence of the mechanostructural environment on new vessel generation has received significantly less attention. During angiogenesis, endothelial cells in the existing vasculature detach and migrate out into the surrounding extracellular matrix (ECM), forming tubular structures that eventually mature into new blood vessels. This process is modulated by the structure and composition of the ECM [1]. The ECM is then remodeled by endothelial cells in the elongating neovessel tip, resulting in matrix condensation and changes in fiber orientation [2]. The mechanism as to how angiogenic vasculature and the ECM influence each other is poorly understood.

Key role of endothelial importin-α in VEGF expression and gastric angiogenesis: novel insight into aging gastropathy

2014 ◽  
Vol 306 (4) ◽  
pp. G338-G345 ◽  
Author(s):  
Amrita Ahluwalia ◽  
Michael K. Jones ◽  
Andrzej S. Tarnawski
Keyword(s):  
Nuclear Transport ◽  
Gene Activation ◽  
In Vivo Studies ◽  
Vegf Gene ◽  
Vegf Expression ◽  
Importin Α ◽  
In Vitro Angiogenesis ◽  
Impaired Angiogenesis

Recent in vivo studies demonstrated that aging gastric mucosa has impaired angiogenesis and reduced expression of vascular endothelial growth factor (VEGF). Angiogenesis is triggered by hypoxia and VEGF gene activation, and the latter requires transport of transcription factor(s) into endothelial cell nuclei. We focused on gastric mucosal endothelial cells (GMEC), which are key targets and effectors of gastric angiogenesis, and determined whether and to what extent importin-α, a nuclear transport protein, regulates VEGF gene activation and gastric angiogenesis and the possible role of importin-α in aging gastropathy. GMEC were isolated from rats 3 and 24 mo of age, young (YGEC) and aging (AGEC), respectively. We examined in these cells 1) in vitro angiogenesis, 2) expression of VEGF and importin-α, 3) nuclear transport of hypoxia-inducible factor (HIF)-1α by importin-α, 4) binding of HIF-1α to the VEGF gene promoter, and 5) effects of importin-α silencing in YGEC and its upregulation in AGEC on angiogenesis and VEGF expression. AGEC exhibited significantly impaired in vitro angiogenesis by fourfold and decreased expression of VEGF, importin-α, and nuclear HIF-1α by 1.4-fold, 1.6-fold, and 2.9-fold, respectively, vs. YGEC. Upregulation of importin-α in AGEC significantly reversed all these abnormalities. In YGEC, knockdown of importins-α1 and -α3 significantly reduced in vitro angiogenesis by 93% and 73% and VEGF expression by 48% and 52%, respectively. The above findings demonstrate that importin-α is a novel and critical regulator of gastric angiogenesis. Its reduced expression in AGEC is the key mechanism for impaired angiogenesis and reduced VEGF.

scholarly journals Multiple Myeloma Pathogenesis: The Role of Junb in Bone Marrow Angiogenesis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4341-4341
Author(s):  
Fengjuan Fan ◽  
Stefano Malvestiti ◽  
Yujia Shen ◽  
Eugenio Morelli ◽  
Yuji Mishima ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Board Of Directors ◽  
Research Funding ◽  
Angiogenic Factors ◽  
University Research ◽  
Advisory Committees ◽  
Cell Clones ◽  
In Vitro Angiogenesis

A significant increase in bone marrow (BM) angiogenesis represents a key event in early, microenvironment-dependent, multiple myeloma (MM). Angiogenic growth factor- and cytokine- production and secretion is a complex process regulated by a plethora of transcription factors (TFs). Over the past years, members of the AP-1 family of TFs have emerged as potential new therapeutic targets. Our recent work demonstrated for the first time a pivotal role for the AP-1 family member JunB in MM pathogenesis (Fan et al., 2017). Whether JunB also contributes to MM BM angiogenesis is currently unknown. In silico and immunohistochemical analyses revealed a correlative increase of JunB and angiogenic growth factors in samples isolated from healthy donors to MGUS and MM patients; and a decrease in samples isolated from patients with plasma cell leukemia. These data were supported by the utilization of an innovative in vivo MM model of clonal evolution. Specifically, JunB as well as selected angiogenic factors were significantly increased in tumor cell clones at primary sites (bone chips) versus tumor cell clones at metastatic (distant BM) sites, as evidenced by whole exome and RNA sequencing. Functionally, doxycyclin- induced inhibition of stroma cell: MM cell co-culture- as well as of IL-6- mediated JunB upregulation in TetR-shJunB/ MM.1S cells significantly reduced production and secretion of angiogenic factors; and consequently inhibited in vitro angiogenesis. Conversely, 4-hydroxytamoxifen (4-OHT)-mediated upregulation of JUNB activity in JUNB-ER/MM cells strongly increased the expression and secretion of angiogenic factors and in vitro angiogenesis. The interaction of JunB with angiogenic factor- encoding DNA in MM cells was further confirmed utilizing chromatin immunoprecipitation (ChIP)- sequencing. Finally, treatment with doxycycline effectively inhibited JunB levels and consistently reduced microvessel density in immunodeficient NSG mice inoculated with TetR-shJUNB/ MM.1S, but not TetR-SCR/ MM.1S. In conclusion, our findings demonstrate a pivotal role of JUNB in MM BM angiogenesis; they thereby provide further evidence that JUNB is a promising therapeutic target particularly in early MM. Disclosures Vallet: Pfizer: Honoraria; Roche Pharmaceuticals: Consultancy; MSD: Honoraria. Roccaro:Associazione Italiana per al Ricerca sul Cancro (AIRC): Research Funding; Associazione Italiana per al Ricerca sul Cancro (AIRC): Research Funding; AstraZeneca: Research Funding; Transcan2-ERANET: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Transcan2-ERANET: Research Funding; AstraZeneca: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; European Hematology Association: Research Funding; European Hematology Association: Research Funding. Goldschmidt:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; John-Hopkins University: Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; MSD: Research Funding; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive Biotechnology: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Research Funding; Dietmar-Hopp-Stiftung: Research Funding; John-Hopkins University: Research Funding; Chugai: Honoraria, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Research Funding; Molecular Partners: Research Funding. Podar:Takeda: Consultancy; Celgene: Consultancy, Honoraria; Amgen Inc.: Honoraria; Janssen Pharmaceuticals: Consultancy, Honoraria; Roche Pharmaceuticals: Research Funding.

Identification of EpHA3 As a New Potential Molecular Target in Multiple Myeloma.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2926-2926
Author(s):  
Antonella Caivano ◽  
Francesco La Rocca ◽  
Alessandra Favole ◽  
Sonia Carturan ◽  
Enrico Bracco ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Monoclonal Antibody ◽  
Cell Lines ◽  
Plasma Cells ◽  
Molecular Target ◽  
Over Expression ◽  
In Vitro Angiogenesis ◽  
Novel Therapeutic

Abstract Abstract 2926 Introduction Angiogenesis plays a central role in the progression of both solid and hematological tumors. In particular, in multiple myeloma (MM) the critical role of bone marrow (BM) microenvironment and angiogenesis has been well documented. The past decade has witnessed a dramatic improvement in the therapeutic options in MM. However, the disease remains incurable, underscoring the need for continued efforts towards understanding MM biology and exploitation of novel therapeutic approaches. In this setting, monoclonal antibodies against myeloma-specific cell surface antigens represent a promising therapeutic approach, which is however hampered by a lack of appropriate target structures expressed across all pathogenic myeloma cells. The Eph receptors, a large family of receptor tyrosine kinases (RTKs) activated by ephrins binding, have been implicated in many processes involved in malignancy, including alteration of the tumor microenvironment and in angiogenesis, in both of which EpHA3 likely plays an active role. Aberrant expression of EpHA3 is seen in many types of hematolologic malignancies (some leukemic cell lines, T-cell lymphoma, acute lymphoblastic leukemia, myeloproliferative neoplasms) although it is not expressed ubiquitously. Finally, the over-expression of Eph is believed to be sufficient to confer tumorigenic potential although probably further mechanisms can occur to abnormally activate the receptor. Basing on the role of EpHA3 in haematological malignancies, a first-in-class engineered IgG1 antibody targeting the EpHA (KB004) was developed and it is now under phase I clinical trials in USA and Australia for the treatment of EpHA3 overexpressing hematological myeloid malignancies refractory to conventional treatment. We investigated the EpHA3 role and its preferential membrane–bound by GPI linker ligand EFNA5, in MM patients in order to define EpHA3 as new molecular target for a novel therapeutic approach with a specific anti EpHA3 monoclonal antibody. The EpHA3 expression has been studied through a comparative proteomic analysis between BM endothelial cells (ECs) of patients with MM (MMECs) or with monoclonal gammopathy of undetermined significance (MGECs), of control subjects (normal ECs) and in MM cell lines. Methods After written informed consent, BM aspirates have been collected from 20 MM and 4 MGUS patients. Normal ECs were derived from 3 BM aspirates of subjects with anemia due to iron or vitamin B12 deficiency. We analyzed the expression levels of EpHA3 in normal ECs, MGECs and MMECs and MM cell lines evaluating the mRNA and protein levels by RT-qPCR and by WB coupled to ImmunoFluorescence analysis. The biological effects of EpHA3 targeting in MMECs have been studied silencing the EpHA3 mRNA in MMECs and testing them at 72h after silencing in series of functinal assays including viability assay by trypan blue exclusion staining and by in vitro angiogenesis assay followed by measurement of mesh areas and vessel length. Moreover, we studied EFNA5 mRNA expression levels in Normal ECs, MGECs and MMECs and in MM cell lines by PCR. Results Our data showed that EpHA3 mRNA levels are progressively increased from ECs to MGECs reaching the highest values in MMECs. Subsequent analysis by WB and immunofluorescence confirmed EpHA3 protein upregulation among the different EC types. The MMECs in which EpHA3 has been silenced revealed a protein level reduction of approximately 60% when compared to the control. We could not detect major viability defects. Furthermore, in vitro angiogenesis inhibition was marginal when compared to the not silenced counterpart. To know whether EpHA3 may impact not only MM angiogenesis but also plasma cells, three MM cell lines were studied for the EpHA3 expression. We found the plasma cell lines gave constant over expression of EpHA3. Finally, the preliminary data regarding EFNA5 mRNA expression level showed it is expressed in either MMECs and MM plasma cell lines. The evaluation of KB004 effect on MMECs in term of apoptosis induction and in vitro tube formation inhibition, as well as the analysis of EpHA3 levels in primary MM plasma cells are in progress. Conclusions From this study we expect to characterize the role of the EpHA3in MM patients and to provide experimental evidences supporting the possibility of using EpHA3 as a new molecular target for MM by proving the in vitro efficacy of a monoclonal antibody to target the angiogenesis of MM. Disclosures: No relevant conflicts of interest to declare.

PC4 THE ROLE OF THE TIE-2 RECEPTOR AND ITS LIGANDS, THE ANGIOPOIETINS, IN A NEW 3D IN VITRO ANGIOGENESIS MODEL

2004 ◽  
Vol 11 (6) ◽  
pp. 540-540
Author(s):  
J. A. Gillard ◽  
H. R. Pearce ◽  
J. L. Burn ◽  
I. D. Kumar ◽  
M. W. R. Reed ◽  
...  
Keyword(s):  
Angiogenesis Model ◽  
In Vitro Angiogenesis

scholarly journals Autocrine Role for Interleukin-8 in Bartonella henselae-Induced Angiogenesis

2006 ◽  
Vol 74 (9) ◽  
pp. 5185-5190 ◽  
Author(s):  
Amy M. McCord ◽  
Sandra I. Resto-Ruiz ◽  
Burt E. Anderson
Keyword(s):  
Endothelial Cells ◽  
Capillary Tube ◽  
Bartonella Henselae ◽  
Tube Formation ◽  
Interleukin 8 ◽  
Endothelial Cell Proliferation ◽  
Chemokine Receptor Cxcr2 ◽  
In Vitro Angiogenesis

ABSTRACT The gram-negative bacterium Bartonella henselae is capable of causing angiogenic lesions as a result of infection. Previously, it has been shown that B. henselae infection can result in production of the chemokine interleukin-8 (IL-8). In this study, we demonstrated that monocytes, endothelial cells, and hepatocytes produce IL-8 in response to B. henselae infection. We also investigated the role of IL-8 in B. henselae-induced endothelial cell proliferation and capillary tube formation. Both in vitro angiogenesis assays were IL-8 dependent. B. henselae-mediated inhibition of apoptosis, as indicated by gene expression of Bax and Bcl-2, was also shown to be IL-8 dependent in endothelial cells. Furthermore, infection of endothelial cells with B. henselae stimulated upregulation of the IL-8 chemokine receptor CXCR2. Infection of human endothelial cells by B. henselae resulting in IL-8 production likely plays a central role in the ability of this organism to cause angiogenesis during infection.

Rho GTPase CDC42 Signals Separately Regulate Directed Migration Versus Random Movement in Neutrophil.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 636-636
Author(s):  
Marie-Dominique Filippi ◽  
Haiming Xu ◽  
Kathleen Szczur ◽  
Yi Zheng ◽  
David A. Williams
Keyword(s):  
Rho Gtpases ◽  
Cell Movement ◽  
Leading Edge ◽  
Actin Assembly ◽  
Directed Migration ◽  
Attachment Structures ◽  
Pmn Functions

Abstract Neutrophils (PMN) are a critical cell in inflammation. In response to external stimuli, they activate various signaling pathways to move rapidly to a site of microbial invasion and perform phagocytosis, cytokine and reactive oxygen species release. Rho GTPases, Rac1, Rac2, CDC42 and Rho, are central regulators of cell movement via actin rearrangement. We have shown the specific role of Rac1 and Rac2 in PMN functions (Gu, Science 2003; Filippi, Nat Immunol, 2004) which raises the question of the role of other Rho GTPases in PMN functions. CDC42 primarily regulates filopodia formation and controls cell polarity and migration in non-hematopoietic cells and some hematopoietic cell lines. Most of previous studies have used dominant active or negative mutants which lack specificity and cannot be used to define in vivo cell biology. Here, we used mice genetically deficient in the CDC42 negative regulator CDC42 GTPase Activating Protein (GAP) to study the role of CDC42 in PMN functions in vitro and in vivo. PMN deficient in CDC42GAP (CDC42GAP−/−) displayed a 2-fold increase in CDC42 activity. In vivo recruitment of PMN in peritoneal cavities was significantly higher in CDC42GAP−/− animals than in WT mice (4.5 ± 0.3x106 vs 3.4 ± 0.2x106, p<0.05) indicating that CDC42 plays a physiological role in neutrophil migration. We examined F-actin assembly upon integrin ligation. Podosome-like structures identified by a vinculin ring surrounding F-actin that are present at the leading edge in WT PMN were significantly reduced in frequency in the mutant cells (15% vs 3%). In addition, CDC42GAP−/− PMN showed increased lateral filopodia-like formation and abnormally elongated uropod with tail filopodia. Thus, CDC42GAP−/− PMN appeared less polarized than WT PMN (50% vs 16%). This abnormal F-actin assembly was associated with abnormal cell motility. In vitro, CDC42GAP−/− PMN showed increase random movement (chemokinesis) compared with WT PMN. By contrast but similar to the loss of CDC42 activity, CDC42GAP−/− PMN displayed defective directed migration towards fMLP suggesting that CDC42 activity plays a critical role in both chemokinesis and directed migration. These functions may be regulated by podosome-like and filopodia formation respectively. To further understand this correlation at a mechanistic level, we examined MAPK signaling. CDC42GAP−/− PMN showed sustained ERK phosphorylation at 15min compared to WT PMN. By contrast, p38MAPK was significantly decreased in CDC42GAP−/− PMN compared to WT at both 5 and 15min. Pharmacological inhibition of ERK activity in CDC42GAP−/− PMN using U0126 rescued the abnormal increased chemokinesis to level similar to WT and was associated with partial rescue of podosome-like formation at the leading edge of the cells. Inhibition of p38MAPK activity in WT PMN using SB203580 reduced directed migration and was associated with increased tail filopodia that mimicked CDC42GAP−/− PMN. Taken together, these results suggest that CDC42GAP plays an important role in PMN chemokinesis and directed migration likely via distinct signaling pathways. CDC42GAP may control chemokinesis via ERK-mediated podosome-like turnover at the leading edge. CDC42GAP may regulate directed migration by inhibiting filopodia at the uropod via p38MAPK and subsequently by restraining filopodia to the leading edge. This reinforces the importance of turnover of attachment structures during cell movement and suggests a new role for CDC42 in attachment structures in neutrophils and for p38MAPK in CDC42-mediated directed migration.

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