Vascular permeability and drug delivery in cancers

The endothelial barrier strictly maintains vascular and tissue homeostasis, and therefore modulates many physiological processes such as angiogenesis, immune responses, and dynamic exchanges throughout organs. Consequently, alteration of this finely tuned function may have devastating consequences for the organism. This is particularly obvious in cancers, where a disorganized and leaky blood vessel network irrigates solid tumors. In this context, vascular permeability drives tumor-induced angiogenesis, blood flow disturbances, inflammatory cell infiltration, and tumor cell extravasation. This can directly restrain the efficacy of conventional therapies by limiting intravenous drug delivery. Indeed, for more effective anti-angiogenic therapies, it is now accepted that not only should excessive angiogenesis be alleviated, but also that the tumor vasculature needs to be normalized. Recovery of normal state vasculature requires diminishing hyperpermeability, increasing pericyte coverage, and restoring the basement membrane, to subsequently reduce hypoxia and interstitial fluid pressure. In this review, we will introduce how vascular permeability accompanies tumor progression and, as a collateral damage, impacts on efficient drug delivery. The molecular mechanisms involved in tumor-driven vascular permeability will next be detailed, with a particular focus on the main factors produced by tumor cells, especially the emblematic vascular endothelial growth factor (VEGF). Finally, new perspectives in cancer therapy will be presented, centered on the use of anti-permeability factors and normalization agents

SHORT REPORT Open Access

The C-terminus region of β-arrestin1 modulates VE-cadherin expression and endothelial cell permeabilit

Glioblastoma cell-secreted interleukin-8 induces brain endothelial cell permeability via CXCR2.

Glioblastoma constitutes the most aggressive and deadly of brain tumors. As yet, both conventional and molecular-based therapies have met with limited success in treatment of this cancer. Among other explanations, the heterogeneity of glioblastoma and the associated microenvironment contribute to its development, as well as resistance and recurrence in response to treatments. Increased vascularity suggests that tumor angiogenesis plays an important role in glioblastoma progression. However, the molecular crosstalk between endothelial and glioblastoma cells requires further investigation. To examine the effects of glioblastoma-derived signals on endothelial homeostasis, glioblastoma cell secretions were collected and used to treat brain endothelial cells. Here, we present evidence that the glioblastoma secretome provides pro-angiogenic signals sufficient to disrupt VE-cadherin-mediated cell-cell junctions and promote endothelial permeability in brain microvascular endothelial cells. An unbiased angiogenesis-specific antibody array screen identified the chemokine, interleukin-8, which was further demonstrated to function as a key factor involved in glioblastoma-induced permeability, mediated through its receptor CXCR2 on brain endothelia. This underappreciated interface between glioblastoma cells and associated endothelium may inspire the development of novel therapeutic strategies to induce tumor regression by preventing vascular permeability and inhibiting angiogenesis

Semaphorin 3A elevates endothelial cell permeability through PP2A inactivation

International audienceVE-cadherin-mediated cell-cell junction weakening increases paracellular permeability in response to both angiogenic and inflammatory stimuli. Although Semaphorin 3A has emerged as one of the few known anti-angiogenic factors to exhibit pro-permeability activity, little is known about how it triggers vascular leakage. Here we report that Semaphorin 3A induced VE-cadherin serine phosphorylation and internalisation, cell-cell junction destabilisation, and loss of barrier integrity in brain endothelial cells. In addition, high-grade glioma-isolated tumour-initiating cells were found to secrete Semaphorin 3A, which promoted brain endothelial monolayer permeability. From a mechanistic standpoint, Semaphorin 3A impinged upon the basal activity of the serine phosphatase PP2A and disrupted PP2A interaction with VE-cadherin, leading to cell-cell junction disorganization and increased permeability. Accordingly, both pharmacological inhibition and siRNA-based knockdown of PP2A mimicked Semaphorin 3A effects on VE-cadherin. Hence, local Semaphorin 3A production impacts on the PP2A/VE-cadherin equilibrium and contributes to elevated vascular permeability

Glioblastoma cell-secreted factors induce loss of brain endothelial monolayer integrity.

<p>A) Starved human cerebral microvascular endothelial cells (hCMEC/D3) were incubated for 5 min with serum-free media (Neg) or U87-conditioned media (CM), diluted as indicated. Protein lysates were examined by western-blot for phosphorylated (p) ERK1/2 and ERK2. Ratios between pERK1/2 and ERK2 intensities are indicated below scans. B) Similarly, ERK1/2 phosphorylation was assessed in hCMEC/D3 stimulated with CM derived from glioblastoma cell lines (GBM, U87, U138, U251 and LN229), where hCMEC/D3-CM (D3) served as control. C–E) <i>In vitro</i> tubulogenesis of hCMEC/D3 was scored after incubation in matrigel with serum free-media (Neg), 50 ng/ml each VEGF/bFGF (Pos) or U87-CM (U87). After 8 h of incubation, cells were photographed and both tube length (C) and number of branch points per field of view (FOV) (D) were quantified. Representative images are shown. F) Using similar conditions, permeability to FITC-dextran was measured after 1 h incubation on hCMEC/D3 monolayers. Graph shows the mean fold-increase normalized to Neg conditions. G–H) Localization of VE-cadherin in response to stimulation with U87-CM at indicated time points was analyzed by confocal microscopy (G), and three-dimensional (3D) images were reconstructed using z-stacks (H). Scale bars: 5 µm (G) and 20 µm (H). I) VE-cadherin phosphorylation on S665 (pVEC) was analyzed by western-blot in hCMEC/D3 stimulated with U87-CM for the indicated times. Total VEC served as a loading control. One out of three independent experiments is shown. Ratios between pVEC and VEC intensities are indicated below scans. T test on 3 independent experiments: *p<0.05; **p<0.01; ***p<0.001.</p

Glioblastoma cell-secreted IL-8 modulates brain endothelial cell properties.

<p>A–B) IL-8 secretion was decreased in U87 using three independent IL-8-targeting siRNA (si-1, si-2, and si-3) with the non-silencing sequence (SIC) serving as control. IL-8 siRNA efficiency was confirmed by ELISA (A) and RT-PCR (B). C–E) Three day-old starved human cerebral microvascular endothelial cells (hCMEC/D3) were stimulated with U87 conditioned medium (CM, 1/20 diluted) collected 72 h after transfection with IL-8 siRNAs, and analyzed for ERK activation (pERK1/2) by western-blot (C), permeability to FITC-dextran (D), and VE-cadherin (VEC) immunolocalization (E). Ratios between pERK1/2 and ERK2 intensities are indicated below scans. Scale bar: 10 µm. One out of three independent experiments is shown. T test on 3 independent experiments: **p<0.01.</p

Expression of IL-8, but not CXCR2, is elevated in human glioblastoma.

<p>A–D) Retrospective gene array analysis was performed using the REMBRANDT (Repository of Molecular Brain Neoplasia Data) collection from National Cancer Institute (NCI) and the National Institute of Neurological Disorders and Stroke (NINDS). Median expression intensities are reported as a graph for IL-8 and CXCR2 genes in (A) and for VEGF-A, MCP-1 (Monocyte Chemotactic Protein), Ang-1 (angiopoeitin) and FGF-2 (fibroblast growth factor) genes in (B), on Non T: non tumor tissue; Astro: astrocytoma; GBM: glioblastoma multiforme; Oligo: oligodendroglioma. C–D) Probability of survival is shown for GBM divided into groups with either high (3-fold increase) or low (3-fold decrease) gene expression of IL-8 (C) and CXCR2 (D). p values (p) are provided. N indicates sample number in each category. All data were obtained using the REMBRANDT database accessed on June 11^th 2012.</p

The glioblastoma cell secretome contains high levels of IL-8.

<p>A) The status of the NF-κB (pIκBα and IκBα) and MAPK (pERK1/2, ERK2, pp38, pJNK, and JNK) signaling pathways were assessed in lysates from serum-starved hCMEC/D3 (D3) and U87 by western-blot. B) Promoter activity of both AP-1 and NF-κB in D3 and U87 were determined by luciferase reporter assays. C) Immunofluorescent staining for NF-κB p65 (green) and c-Jun (green) was performed as readout of NF-κB and AP-1 signaling activity, respectively, in serum-starved D3 and U87. DAPI (red) was used to stain nuclei, scale bars: 10 µm. D) The angiogenic secretome profile was determined in U87 conditioned medium (CM) using antibody array, IL-8 and its closely related family member, Gro-α, are indicated. E) The mRNA levels of IL-8, CXCR1 and CXCR2 cells were determined in U87 and D3 by RT-PCR. β-actin was used as a control for loading and PCR efficacy. F) IL-8 secretion in conditioned media from U87 and D3 was examined through ELISA. G) Expression of the IL-8 transcript was examined in D3, U87 and additional glioblastoma cell lines (GBM, U138, U251 and LN229) by RT-PCR. H) Similarly, IL-8 secretion was measured in these cell lines by ELISA. One out of three independent experiments is shown. T test on 3 independent experiments: *p<0.05; **p<0.01.</p