Pre-clinical evidence for an anti-tumor activity of defibrotide, a DNA-based, endothelium stabilizing drug

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 14144-14144
Author(s):  
C. Echart ◽  
M. Iacobelli ◽  
P. Richardson ◽  
C. Mitsiades ◽  
T. Ignoni ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tumor Cells ◽  
Degradation Products ◽  
Microvascular Endothelial Cells ◽  
Human Gastric Cancer ◽  
Western Blots ◽  
Microvascular Endothelial ◽  
Anti Tumor Activity

14144 Background: Defibrotide (DF) is a mixture of polydeoxyribonucleotides with anti-thrombotic activity. Next to endothelium stabilization, recent data suggest anti-neoplastic properties of DF modulating interactions of tumor cells with their microenvironment. We investigated whether DF regulates expression and activity of heparanase, an enzyme critically involved in breaking down extracellular barriers and releasing growth factors linked to tumor invasion and angiogenesis. Methods: Heparanase expression was tested by RT-PCR and flow cytometry with multiple myeloma (MM) and microvascular endothelial cells. Heparanase activity was measured in cellular extracts with a heparan-degrading enzymatic assay. Serum degradation products of DF were identified by SEC-HPLC. The anti-angiogenic potential of DF was tested in vitro using a kit with human microvascular endothelial cells forming tubes across a layer of fibroblasts. In vivo, DF was tested in the dorsal skin-fold chamber assay in mice after inoculation of human gastric cancer cells. Proliferation was assessed by trypan blue exclusion. Results: We demonstrate a striking downregulation of expression and enzymatic activity of heparanase in endothelial as well as MM cells. In contrast, the degradation products of DF failed to exert any biological activity, suggesting that the intact mixture of deoxyoligonucleotides is responsible for the anti-tumor effect. We could also show that DF prevents (tumor) angiogenesis in vitro and in vivo. Western blots suggest that DF reduces phosphorylation-activation of p70S6 kinase, a key target in the mTOR pathway linked to angiogenesis. In addition, DF does not influence proliferation of vascular or tumor cells, rather acts via selective inhibition of tube formation of endothelial cells. Conclusion: In the present report we provide evidence for an anti-tumor activity of DF. DF inhibits (tumor) vessel formation and heparanase activity, and thus should be considered as an anti-cancer agent. [Table: see text]

scholarly journals Tranilast inhibits the proliferation, chemotaxis and tube formation of human microvascular endothelial cells in vitro and angiogenesis in vivo

1997 ◽  
Vol 122 (6) ◽  
pp. 1061-1066 ◽  
Author(s):  
Masayuki Isaji ◽  
Hiroshi Miyata ◽  
Yoshiyuki Ajisawa ◽  
Yasuo Takehana ◽  
Nagahisa Yoshimura
Keyword(s):  
Endothelial Cells ◽  
Tube Formation ◽  
Microvascular Endothelial Cells ◽  
Microvascular Endothelial

scholarly journals A Genomic Comparison of in vivo and in vitro Brain Microvascular Endothelial Cells

2007 ◽  
Vol 28 (1) ◽  
pp. 135-148 ◽  
Author(s):  
Anthony R Calabria ◽  
Eric V Shusta
Keyword(s):  
Endothelial Cells ◽  
Quantitative Polymerase Chain Reaction ◽  
Genomic Analysis ◽  
Subtractive Hybridization ◽  
Gene Panel ◽  
Microvascular Endothelial Cells ◽  
Brain Microvascular Endothelial Cells ◽  
Microvascular Endothelial

The blood—brain barrier (BBB) is composed of uniquely differentiated brain microvascular endothelial cells (BMEC). Often, it is of interest to replicate these attributes in the form of an in vitro model, and such models are widely used in the research community. However, the BMEC used to create in vitro BBB models de-differentiate in culture and lose many specialized characteristics. These changes are poorly understood at a molecular level, and little is known regarding the consequences of removing BMEC from their local in vivo microenvironment. To address these issues, suppression subtractive hybridization (SSH) was used to identify 25 gene transcripts that were differentially expressed between in vivo and in vitro BMEC. Genes affected included those involved in angiogenesis, transport and neurogenesis, and real-time quantitative polymerase chain reaction (qPCR) verified transcripts were primarily and significantly downregulated. Since this quantitative gene panel represented those BMEC characteristics lost upon culture, we used it to assess how culture manipulation, specifically BMEC purification and barrier induction by hydrocortisone, influenced the quality of in vitro models. Puromycin purification of BMEC elicited minimal differences compared with untreated BMEC, as assessed by qPCR. In contrast, qPCR-based gene panel analysis after induction with hydrocortisone indicated a modest shift of 10 of the 23 genes toward a more ‘ in vivo-like’ gene expression profile, which correlated with improved barrier phenotype. Genomic analysis of BMEC de-differentiation in culture has thus yielded a functionally diverse set of genes useful for comparing the in vitro and in vivo BBB.

scholarly journals Decrease of miR-19b-3p in Brain Microvascular Endothelial Cells Attenuates Meningitic Escherichia coli-Induced Neuroinflammation via TNFAIP3-Mediated NF-κB Inhibition

Pathogens ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 268 ◽  
Author(s):  
Amjad ◽  
Yang ◽  
Li ◽  
Fu ◽  
Yang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Endothelial Cells ◽  
Negative Regulator ◽  
Microvascular Endothelial Cells ◽  
Brain Microvascular Endothelial Cells ◽  
E Coli ◽  
Inflammatory Damage ◽  
Microvascular Endothelial

Meningitic Escherichia coli can traverse the host’s blood–brain barrier (BBB) and induce severe neuroinflammatory damage to the central nervous system (CNS). During this process, the host needs to reasonably balance the battle between bacteria and brain microvascular endothelial cells (BMECs) to minimize inflammatory damage, but this quenching of neuroinflammatory responses at the BBB is unclear. MicroRNAs (miRNAs) are widely recognized as key negative regulators in many pathophysiological processes, including inflammatory responses. Our previous transcriptome sequencing revealed numbers of differential miRNAs in BMECs upon meningitic E. coli infection; we next sought to explore whether and how these miRNAs worked to modulate neuroinflammatory responses at meningitic E. coli entry of the BBB. Here, we demonstrated in vivo and in vitro that meningitic E. coli infection of BMECs significantly downregulated miR-19b-3p, which led to attenuated production of proinflammatory cytokines and chemokines via increasing the expression of TNFAIP3, a negative regulator of NF-κB signaling. Moreover, in vivo injection of miR-19b-3p mimics during meningitic E. coli challenge further aggravated the inflammatory damage to mice brains. These in vivo and in vitro findings indicate a novel quenching mechanism of the host by attenuating miR-19b-3p/TNFAIP3/NF-κB signaling in BMECs in response to meningitic E. coli, thus preventing CNS from further neuroinflammatory damage.

scholarly journals Mucosal angiogenesis regulation by CXCR4 and its ligand CXCL12 expressed by human intestinal microvascular endothelial cells

2004 ◽  
Vol 286 (6) ◽  
pp. G1059-G1068 ◽  
Author(s):  
Jan Heidemann ◽  
Hitoshi Ogawa ◽  
Parvaneh Rafiee ◽  
Norbert Lügering ◽  
Christian Maaser ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Primary Cultures ◽  
Tube Formation ◽  
Microvascular Endothelial Cells ◽  
Endothelial Tube Formation ◽  
Microvascular Endothelial ◽  
Stromal Cell Derived Factor ◽  
Phosphoinositide 3 Kinase

Mice genetically deficient in the chemokine receptor CXCR4 or its ligand stromal cell-derived factor (SDF)-1/CXCL12 die perinatally with marked defects in vascularization of the gastrointestinal tract. The aim of this study was to define the expression and angiogenic functions of microvascular CXCR4 and SDF-1/CXCL12 in the human intestinal tract. Studies of human colonic mucosa in vivo and primary cultures of human intestinal microvascular endothelial cells (HIMEC) in vitro showed that the intestinal microvasculature expresses CXCR4 and its cognate ligand SDF-1/CXCL12. Moreover, SDF-1/CXCL12 stimulation of HIMEC triggers CXCR4-linked G proteins, phosphorylates ERK1/2, and activates proliferative and chemotactic responses. Pharmacological studies indicate SDF-1/CXCL12 evokes HIMEC chemotaxis via activation of ERK1/2 and phosphoinositide 3-kinase signaling pathways. Consistent with chemotaxis and proliferation, endothelial tube formation was inhibited by neutralizing CXCR4 or SDF-1/CXCL12 antibodies, as well as the ERK1/2 inhibitor PD-98059. Taken together, these data demonstrate an important mechanistic role for CXCR4 and SDF-1/CXCL12 in regulating angiogenesis within the human intestinal mucosa.

scholarly journals Type 5 phosphodiesterase expression is a critical determinant of the endothelial cell angiogenic phenotype

2009 ◽  
Vol 296 (2) ◽  
pp. L220-L228 ◽  
Author(s):  
Bing Zhu ◽  
Li Zhang ◽  
Mikhail Alexeyev ◽  
Diego F. Alvarez ◽  
Samuel J. Strada ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Network Formation ◽  
Microvascular Endothelial Cells ◽  
Critical Determinant ◽  
Angiogenic Potential ◽  
Matrigel Plug ◽  
Microvascular Endothelial

Type 5 phosphodiesterase (PDE5) inhibitors increase endothelial cell cGMP and promote angiogenesis. However, not all endothelial cell phenotypes express PDE5. Indeed, whereas conduit endothelial cells express PDE5, microvascular endothelial cells do not express this enzyme, and they are rapidly angiogenic. These findings bring into question whether PDE5 activity is a critical determinant of the endothelial cell angiogenic potential. To address this question, human full-length PDE5A1 was stably expressed in pulmonary microvascular endothelial cells. hPDE5A1 expression reduced the basal and atrial natriuretic peptide (ANP)-stimulated cGMP concentrations in these cells. hPDE5A1-expressing cells displayed attenuated network formation on Matrigel in vitro and also produced fewer blood vessels in Matrigel plug assays in vivo; the inhibitory actions of hPDE5A1 were reversed using sildenafil. To examine whether endogenous PDE5 activity suppresses endothelial cell angiogenic potential, small interfering RNA (siRNA) constructs were stably expressed in pulmonary artery endothelial cells. siRNA selectively decreased PDE5 expression and increased basal and ANP-stimulated cGMP concentrations in these conduit cells. PDE5 downregulation increased network formation on Matrigel in vitro and increased blood vessel formation in Matrigel plug assays in vivo. Collectively, our results indicate that PDE5 activity is an essential determinant of angiogenesis and suggest that PDE5 downregulation in microvascular endothelium imparts a stable, enhanced angiogenic potential to this cell type.

Pro-angiogenic effects of Carthami Flos whole extract in human microvascular endothelial cells in vitro and in zebrafish in vivo

Phytomedicine ◽  
2014 ◽  
Vol 21 (11) ◽  
pp. 1256-1263 ◽  
Author(s):  
Xuelin Zhou ◽  
Wing-Sum Siu ◽  
Chak-Hei Fung ◽  
Ling Cheng ◽  
Chun-Wai Wong ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Microvascular Endothelial Cells ◽  
Microvascular Endothelial

EDRF released from microvascular endothelial cells dilates arterioles in vivo

1991 ◽  
Vol 261 (1) ◽  
pp. H128-H133 ◽  
Author(s):  
A. Koller ◽  
N. Seyedi ◽  
M. E. Gerritsen ◽  
G. Kaley
Keyword(s):  
Endothelial Cells ◽  
Microvascular Endothelial Cells ◽  
Cremaster Muscle ◽  
Microcarrier Beads ◽  
Cytodex 3 ◽  
Microvascular Endothelial ◽  
Prior Administration ◽  
Arteriolar Diameter

Microvascular endothelial cells (MECs) from rat epididymal fat pad were isolated and cultured in vitro on Cytodex 3 microcarrier beads. In Krebs-suffused cremaster muscle of pentobarbital-anesthetized rats arteriolar diameters (mean control diam 20.9 +/- 0.9 micron) were measured using image shearing video microscopy. Two lines of suffusate (1.5 ml/min each) were established; one contained a column of microcarrier beads only (no cells in line; NC) the other contained a 1-ml column of MECs grown on beads (through cells; TC). The muscle preparation and the MECs were first treated with indomethacin (Indo; 28 microM). Indo treatment blocked arteriolar dilation to A23187 (1 microM) and arachidonic acid (AA; 0.25 microM) administered into the NC line. A 4.0 +/- 0.6 micron increase in arteriolar diameter was observed, however, when A23187 (but not AA) was infused through the TC line containing Indotreated MECs on beads. The A23187-elicited dilation was abolished by the introduction of NG-monomethyl-L-arginine (L-NMMA; 200 microM) into the TC line. Administration of atropine (2 microM) onto the cremaster muscle via the NC line inhibited the dilations in response to acetylcholine (ACh; 2.7 microM) given through the NC line. Infusion of ACh through the TC line onto the atropine-treated cremaster muscle, however, elicited a 5.8 +/- 1.3 micron increase in arteriolar diameter, a response that was blocked by prior administration of L-NMMA into the TC line. Arteriolar dilation induced by adenosine (0.5 microM) or sodium nitroprusside (0.5 microM) applied via the NC or TC line was unaffected by L-NMMA.(ABSTRACT TRUNCATED AT 250 WORDS)

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