Paradoxic effects of metformin on endothelial cells and angiogenesis

Abstract

The biguanide metformin is used in type 2 diabetes management and has gained significant attention as a potential cancer preventive agent. Angioprevention represents a mechanism of chemoprevention, yet conflicting data concerning the antiangiogenic action of metformin have emerged. Here, we clarify some of the contradictory effects of metformin on endothelial cells and angiogenesis, using in vitro and in vivo assays combined with transcriptomic and protein array approaches. Metformin inhibits formation of capillary-like networks by endothelial cells; this effect is partially dependent on the energy sensor adenosine-monophosphate-activated protein kinase (AMPK) as shown by small interfering RNA knockdown. Gene expression profiling of human umbilical vein endothelial cells revealed a paradoxical modulation of several angiogenesis-associated genes and proteins by metformin, with short-term induction of vascular endothelial growth factor (VEGF), cyclooxygenase 2 and CXC chemokine receptor 4 at the messenger RNA level and downregulation of ADAMTS1. Antibody array analysis shows an essentially opposite regulation of numerous angiogenesis-associated proteins in endothelial and breast cancer cells including interleukin-8, angiogenin and TIMP-1, as well as selective regulation of angiopioetin-1, -2, endoglin and others. Endothelial cell production of the cytochrome P450 member CYP1B1 is upregulated by tumor cell supernatants in an AMPK-dependent manner, metformin blocks this effect. Metformin inhibits VEGF-dependent activation of extracellular signal-regulated kinase 1/2, and the inhibition of AMPK activity abrogates this event. Metformin hinders angiogenesis in matrigel pellets in vivo, prevents the microvessel density increase observed in obese mice on a high-fat diet, downregulating the number of white adipose tissue endothelial precursor cells. Our data show that metformin has an antiangiogenic activity in vitro and in vivo associated with a contradictory short-term enhancement of pro-angiogenic mediators, as well as with a differential regulation in endothelial and breast cancer cells.

Figures

Fig. 1.

Metformin inhibits endothelial cell morphogenesis. HUVE cells were treated with metformin at the indicated concentrations and seeded on the top of the matrigel layer previously solidified on 24-well plates. The formation of capillary-like networks was documented by photography after 6h (×5 magnification). (A) Metformin (Metf) inhibited morphogenesis in a dose-dependent manner. (B) Quantification of capillary-like structures (both number of segments and segments length) using the Angiogenesis Analyzer ImageJ toolkit showed significant differences in both parameters, P-values are shown, C− is the negative control. (C) HUVE cells were transfected with an AMPKα1-specific (siRNAα1 or α1) control (siCTR) siRNA, cell lysates were harvested and analyzed by western blotting for AMPKα1 expression; AMPKα1 was successfully downregulated in HUVE transfected cells as compared with control. (D) Transfected HUVE cells were seeded on top of a matrigel layer in presence or absence of metformin and analyzed at 6h as in (A). Metformin-mediated inhibition of capillary-like structures formation was reverted when AMPKα1 expression was downregulated. (E) Quantification of capillary-like structures as described previously, P-values are shown.

Fig. 2.

Metformin modulates several angiogenesis-related genes. Microarray analysis was performed on total RNAs isolated from HUVE cells from three different donors that were treated with either vehicle alone (CTR) or 6 or 24h with 10mM metformin (Metf). Data from the scans were normalized and analyzed for relative intensity of regulation of genes with metformin treatment. (A) Hierarchical clustering of human endothelial cells from the three different donors shows clear division into control, 6 and 24h metformin treatment, genes showing statistically significant regulation in all donors are shown. The genes are as follows: VEGF-A; PTGS2 (COX2); F3 (coagulation factor III, thromboplastin); ERG (ETS-related gene); FLT1 (VEGF receptor 1); SAT1 (spermidine/spermine N1-acetyltransferase 1); PRKD1 (protein kinase D1) CYP1B1 (cytochrome P450, family 1, subfamily B, polypeptide 1); WARS (tryptophanyl-tRNA synthetase); ADAMTS1; CXCR4; and CCL2 (CC chemokine ligand 2). (B) Real-time PCR validation of gene expression of the statistically significant angiogenesis-related genes up or downregulated by metformin in microarrays. Relative expression values are indicated as fold change over untreated HUVE cells (=1) assessed after normalization on glyceraldehyde 3-phosphate dehydrogenase, RNA polymerase II and glucose-6-phosphate dehydrogenase expression data obtained from reactions run in parallel. All amplifications were performed in triplicate. (C) Analysis of CXCR4, CYP1B1, COX2, VEGF-A and ADAMTS1 protein expression on HUVE cells, by western blotting. A table summarizing protein changes relative to control (Ctrl) is also shown (upregulated proteins are shown in red; downregulated proteins are shown in green). β-Actin was used as loading control.

Fig. 3.

Tumor cell supernatants upregulate CYP1B1 expression in HUVE cells, metformin abrogates this effect. HUVE cells were incubated with serum-free unconditioned medium (NC) or conditioned supernatants (conditioned medium, CM) from tumor cells (MDA-MB-231 for breast cancer; PC3 and DU-145 for prostate cancer), all harvested after 24h. (A) Western blotting shows a significant upregulation of CYP1B1 in HUVE cells cultured in presence of conditioned supernatants as compared with those treated with unconditioned medium. Addition of metformin significantly abrogates this effect. (B) HUVE cells were transfected with an AMPKα-specific or control (SiCTR) siRNAs, treated with or without 10mM metformin, lysed and analyzed by western blotting for CYP1B1 expression. CYP1B1 is significantly downregulated in AMPKα siRNA-transfected cells as compared with control siRNA. Metformin upregulates CYP1B1 levels in AMPKα siRNA-transfected HUVE cells, suggesting AMPK-dependent CYP1B1 expression in these cells. β-Actin was used as a loading control. The values in the western blot panels represented the ratio of the indicated protein and actin.

Fig. 4.

Expression of angiogenesis-related proteins in HUVE and breast cancer cells treated with metformin. HUVE and MCF-7 (breast cancer) cells were treated with vehicle or 10mM metformin for 24h. Cell culture medium were collected, centrifuged and applied to a human angiogenesis antibody array as described in Materials and methods section. Control proteins are located in duplicate in three corners of the arrays, whereas negative control is indicated as an empty box (lower right corner) in the array. The experiment was repeated in duplicate cell lysates. Modulated proteins in treated cells as compared with control are highlighted with squares and indicated by numbers. Each spot is spotted on the array membrane in duplicate. (A) Metformin modulates 13 angiogenesis-related proteins in HUVE cells. (B) Relative amounts of proteins in selected spots indicated in (A) showing mean and standard error of the three experiments in duplicate. (C) In MCF-7, metformin downregulates 19 angiogenesis protein with either antiangiogenic or pro-angiogenic function. (D) Relative amounts of proteins in selected spots indicated in (B) showing mean and standard error of the duplicate. (E) When we compare the modulation of several factors by metformin in the two cell types, we observed different effects. The phenomenon is summarized in the table: in green, downregulated protein; in red: upregulated protein; in white: no significant modulation.

Fig. 5.

Metformin inhibits VEGF-induced ERK1/2 activation in an AMPK-dependent manner. HUVE cells were plated onto six-well plates, treated with or without VEGF in presence or absence of 10mM metformin and/or 10 µM compound C and analyzed at different time points by western blotting. ERK1/2 phosphorylation was measured at 5, 15, 30min or 6h after treatment. (A) ERK1/2 activation was reduced in metformin-treated cells, starting at 15min after metformin addition to the medium. (B) Metformin inhibited phosphorylation of ERK1/2 and inhibited VEGF-induced ERK1/2 phosphorylation. When compound C (cC) was added to the medium, the effects of metformin inhibition and induction of phosphorylation by VEGF alone were counteracted suggesting an involvement of AMPK in ERK1/2 signaling. The experiments were performed twice in triplicate with similar results. The values in the western blot panels represented the ratio of the indicated protein and actin; β-actin was used as loading control.

Fig. 6.

Metformin inhibits angiogenesis in vivo in normal and obese mice. A cocktail of pro-angiogenic factors (VEGF-A, TNFα and heparin), either alone (Ctrl) or in combination with different concentrations of metformin as indicated was added into matrigel and injected subcutaneously into C57/BL6 mice. (A) Measurement of the hemoglobin content of the matrigel sponges (*P < 0.05, Mann–Whitney). Inset: matrigel pellets photographed 4 days postinjection. Six-week-old C57 mice were fed with either a control ND or a HFD. After 30 days of the respective diet, mice then received the same diet with metformin (0.5mg metformin/ml drinking water, leading to 2mg metformin/mouse/day) or without (control vehicle in drinking water) for further 60 consecutive days (n = 10 per study arm). On day 90, mice were killed. (B) Measurement of the weight of the WAT shows that the mice on the HFD were clearly obese (P < 0.0001, Mann–Whitney). Mice fed with ND or HFD treated with metformin or vehicle were injected with matrigel as described. (C) Immunofluorescence analysis of vascularization by staining matrigel plug sections with anti-CD31 antibody for endothelial cells in mice either on ND or HFD, with or without oral metformin. (D) Quantification of MVD in the sponges based on CD31 staining (shown as CD31-positive cells/field normalized to control). HFD increased the number of CD31-positive cells as compared with ND, whereas metformin abrogated this effect. (E and F) Blood and visceral WAT were collected for the enumeration of CD45−Sca1+CD34+CD31+ progenitor cells by flow cytometry as described previously (29). (E) Obesity was associated with higher numbers of CD45−Sca1+CD34+CD31+ progenitor cells in the WAT (P = 0.0009, Mann–Whitney). Metformin treatment of the obese mice drastically lowered the CD45−Sca1+CD34+CD31+ progenitor cells in the WAT (P < 0.0001, Mann–Whitney), yet had no effect on the numbers of these cells in mice on a ND. (F) Obesity and/or metformin treatment did not significantly alter the number of CD45−Sca1+CD34+CD31+ progenitor cells in the peripheral blood.