Newly identified biologically active and proteolysis-resistant VEGF-A isoform VEGF111 is induced by genotoxic agents

Ultraviolet B and genotoxic drugs induce the expression of a vascular endothelial growth factor A (VEGF-A) splice variant (VEGF111) encoded by exons 1–4 and 8 in many cultured cells. Although not detected in a series of normal human and mouse tissue, VEGF111 expression is induced in MCF-7 xenografts in nude mice upon treatment by camptothecin. The skipping of exons that contain proteolytic cleavage sites and extracellular matrix–binding domains makes VEGF111 diffusible and resistant to proteolysis. Recombinant VEGF111 activates VEGF receptor 2 (VEGF-R2) and extracellularly regulated kinase 1/2 in human umbilical vascular endothelial cells and porcine aortic endothelial cells expressing VEGF-R2. The mitogenic and chemotactic activity and VEGF111's ability to promote vascular network formation during embyonic stem cell differentiation are similar to those of VEGF121 and 165. Tumors in nude mice formed by HEK293 cells expressing VEGF111 develop a more widespread network of numerous small vessels in the peritumoral tissue than those expressing other isoforms. Its potent angiogenic activity and remarkable resistance to proteolysis makes VEGF111 a potential adverse factor during chemotherapy but a beneficial therapeutic tool for ischemic diseases

Distinct pathways in the over-expression of matrix metalloproteinases in human fibroblasts by relaxation of mechanical tension.

peer reviewedaudience: researcherThe aim of the work was to analyze, on a comparative basis, the signaling pathways operating in the regulation of a panel of matrix metalloproteinases (MMP) expressed by human dermal fibroblasts submitted to mechanical stress relaxation by cytochalasin D (CD) and in a retracting collagen gel (RCG). The mRNA steady-state level of MMPs was measured by a quantitative RT-PCR procedure using a synthetic RNA as internal standard. In monolayer, most MMPs were barely detected, except MMP-2. Disruption of the actin stress fibers by CD induced a moderate increase of MMP-2 mRNA and a much larger stimulation of MMP-3, -9, -13 and -14 mRNAs. In RCG, a significant up-regulation of these MMPs was also observed although to a lower extent than in CD-treated monolayers. Among the investigated MMPs, the MMP-8 and -11 were not reproducibly detected. MMP-2 was processed to its active form both by CD and in RCG. The CD-induced up-regulation of gene expression was largely repressed by blocking protein synthesis by cycloheximide for all the MMPs, by inhibiting the tyrosine-kinases of the src family by herbimycin A for all MMPs, except MMP-2, and by inhibiting the TPA-inducible PKC isoforms by bisindoyl maleimide for all MMPs, except MMP-14. The up-regulation induced by stress relaxation in RCG was protein synthesis-dependent for MMP-2 and MMP-13, tyrosine kinases-dependent for MMP-3 and MMP-13, as previously described for MMP-1. Inhibiting TPA-inducible PKC did not affect any MMP in RCG except MMP-13, which was strongly induced. The processing of MMP-2 was tyrosine kinases-dependent but PKC-independent. Inhibitors of the ERK1,2 and p38 MAP kinases pathways diversely affected the MMPs expression. Inhibiting the Rho-kinase activity by Y-27632 was inactive. These results point to the potent regulation operated by the status of the cytoskeleton on the cell phenotype, and to distinct regulatory pathways involved in the control of different MMPs expression

Rewarming-induced ROS production and <b>ROS-dependant apoptosis.</b>

<p>(A) ROS were measured in WI26 cells cultured at 25°C for 5 days and then warmed-up at 37°C for 2 to 24h in absence (-) and in presence (+) of 15mM N-Acetylcysteine (NAC). Data are expressed in arbitrary units taking T0 as 1. Significant inhibition by NAC versus condition without NAC is indicated # # # (p<0.001). (B) Cell viability was measured by FACS analysis after labeling with FITC-annexin V (FITC-A, X-axes) and propidium iodide (PI, Y-axes) of cells maintained 5 days at 25°C and then warmed-up at 37°C for 4h (4h) in the presence or in the absence of 15mM of NAC added at T0. (C) Percentage of apoptotic cells in the indicated culture conditions.</p

Cold shock and rewarming induce autophagy and a cellular stress response.

<p>The level of LC3 I and II (A), phospho-JNK (P-JNK) and total JNK (D) was analyzed by western blot in cells cultured at 25°C for 1 and 5 days and then warmed-up at 37°C for 1 to 24h. The levels of ERK1/2 were taken as calibrator and used to monitor protein loading. Results are expressed as the mean ratio of LC3 II/I (B) and of P-JNK/ total JNK (D) taking T0 as 1. (C) Immunostaining of LC3 was performed in WI26 cells maintained at 37°C in the presence or in the absence of serum or at various time points during the cold shock and rewarming. Actin stress fibers appear in red, LC3 in green and nucleus in blue. Bar: 100µm.</p

Phase contrast micrographs of WI26 cells.

<p>Cells were continuously cultured at 37°C (A), at 25°C for 5 days (B) or at 25°C for 5 days followed by a rewarming to 37°C for 24h (C). Arrows point to refringent, apoptotic-like cells. Bar: 100µm.</p

Cold shock and rewarming induce H2AX phosphorylation through ROS production.

<p>The levels of phosphorylated H2AX (γH2AX) were analyzed in WI26 cells cultured at 25°C for 1 and 5 days and then warmed-up at 37°C for 1 to 24h. (A) Representative western blots. (B) γH2AX was quantified by western blotting. Data are expressed in arbitrary units after normalization by ERK1/2, taking T0 as 1. (C) γH2AX in WI26 cells in normal conditions (1D37°C) and during cold shock for 5 days and rewarming for 2 to 24h was evidenced by immunostaining. Actin stress fibers are stained in red and γH2AX in green. Bar: 100µm. (D) γH2AX was measured by western blot in WI26 cells cultured at 25°C for 5 days and then warmed-up at 37°C for 2 to 24h in absence (-) and in presence (+) of NAC 15mM added at T0. After normalization by GAPDH, results are expressed in arbitrary units taking 5D as 1.</p

Return to normal temperature after a cold shock affects the phosphorylation of p53.

<p>Total and phosphorylated p53 (P-p53) were analyzed in WI26 cells cultured in normal conditions or at 25°C for 1 and 5 days and then warmed-up at 37°C for 1 to 24h. (A) Representative western blot. (B) Quantifications of the western-blots are expressed as the mean ratio P-p53/total p53, taking T0 as 1. Total protein loading was monitored by ERK1/2.</p

Cold shock and rewarming affect cell viability.

<p>WI26 cells were cultured at 25°C for 5 days and then warmed-up at 37°C for 2 to 24h. Cells kept at 37°C were used as control (T0). FACS analysis was performed after labeling with FITC-annexin V (FITC-A, X-axes) and propidium iodide (PI, Y-axes). 12.000 to 18.000 events were collected for each experiment. (A) Example of dots graphs (I), contour graphs (II) and annexin V curves (III) of control cells and cells maintained 5 days at 25°C and then warmed-up at 37°C for 4h (4h). Alive cells [A] (double negative staining), cells in early apoptosis [EA] (annexin V positive, PI negative), in late apoptosis [LA] (double positive) and necrotic [N] (annexin V negative, PI positive) are indicated on the graphs. Annexin V curves (III) were used to define the gating allowing to discriminate the populations. (B) Percentage of apoptotic cells as measured by FACS analysis. Cells were cultured at 37°C (T0) or 25°C for 5 days and subsequent warming-up at 37°C for 2 to 24h. (C) Percentage of dead cells as measured by trypan blue exclusion assay. Cells were cultured in duplicate for 5 days at 25°C and then rewarmed for 2 to 24h at 37°C.</p