Igor Hrgovic

Suppression of VEGFR2 expression in human endothelial cells by dimethylfumarate treatment: evidence for anti-angiogenic action.

The association between angiogenesis and chronic inflammatory diseases, such as psoriasis, seems to be an important phenomenon implicated in the pathogenesis of these medical conditions. Recent studies provide evidence that dimethylfumarate (DMF) has a profound anti-inflammatory as well as anti-tumorigenic action, although the effect of DMF on angiogenesis is unknown. Signaling via the vascular endothelial growth factor receptor-2 (VEGFR2) pathway is critical for angiogenic responses. Therefore, we explored whether the known anti-inflammatory and anti-tumorigenic properties of DMF might be mediated in part by anti-angiogenic effects through the reduction in VEGFR2 expression. In this study, DMF was found to inhibit endothelial VEGFR2 expression; time- and concentration-dependent inhibition was demonstrated both at the level of protein and mRNA expression. This blockade was coincident with the inhibition of the formation of capillary-like structures. The DMF-dependent inhibition of VEGFR2 transcription was found to be mediated by an element located between base pairs -60 and -37, which contains two adjacent, consensus Sp1 transcription factor-binding sites, and the constitutive formation of complexes containing Sp1 at this site is decreased by DMF treatment. Inhibition of VEGFR-2 is shown to be one critical aspect in DMF-mediated anti-angiogenic effects.

PPARδ agonists suppress angiogenesis in a VEGFR2-dependent manner

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that have a pleiotropic impact on the regulation of differentiation, cell growth, and the metabolism of lipids and glucose. PPARδ agonists display a variety of effects on pro- and anti-tumor processes, and seem to have pro-angiogenic activity at very low concentrations. We analyzed the influence of higher concentrations of PPARδ agonists on angiogenesis and its underlying mechanisms. We found that treatment with PPARδ agonists inhibited the formation of capillary-like structures and endothelial cell migration. Since signaling via the vascular endothelial growth factor receptor-2 (VEGFR2) pathway is critical for angiogenic responses during chronic inflammation and tumor development, we explored whether PPARδ agonist inhibition acted by diminishing VEGFR2 expression. PPARδ agonists inhibited endothelial VEGFR2 protein expression in a time- and concentration-dependent manner. In contrast, neither tie-2, neuropilin-1 nor VEGFR1 expression was significantly affected by PPARδ agonist treatment. We also demonstrated that PPARδ agonists significantly suppressed accumulation of VEGFR2 mRNA. Consistent with these results, promoter luciferase assays showed that the inhibitory effects of PPAR agonists occur through suppression of VEGFR2 promoter activity. Hence, VEGFR-2 expression may be a critical molecular target of PPAR δ agonists, which may be responsible for their anti-angiogenic effects. These results may help to define the optimal therapeutic doses of PPARδ agonists in prospective therapeutic applications.

Peroxisome Proliferator-activated Receptor δ Activators Induce IL-8 Expression in Nonstimulated Endothelial Cells in a Transcriptional and Posttranscriptional Manner

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that are implicated in the regulation of lipid and glucose homeostasis. PPAR agonists have been shown to control inflammatory processes, in part by inhibiting distinct proinflammatory genes (e.g. Il-1β and IFN-γ). IL-8 is a member of the proinflammatory chemokine family that is important for various functions, such as mediating the adhesion of eosinophilic granulocytes onto endothelial cells. The influence of PPARδ activators on the expression of IL-8 in noninduced quiescent endothelial cells is unclear. Therefore, we explored the influence of PPARδ activators on the expression of IL-8 in nonstimulated endothelial cells. PPARδ agonists induce IL-8 expression in human umbilical vein endothelial cells. This induction is demonstrated at the level of both protein and mRNA expression. Transcriptional activation studies using IL-8 reporter gene constructs and DNA binding assays revealed that PPARδ agonists mediated their effects via an NFκB binding site. It is well known that IL-8 is also regulated by mRNA stability. To provide further evidence for this concept, we performed mRNA stability assays and found that PPARδ agonists induce the mRNA stability of IL-8. In addition, we showed that PPARδ agonists induce the phosphorylation of ERK1/2 and p38, which are known to be involved in the increase of mRNA stability. The inhibition of these MAPK signaling pathways resulted in a significant suppression of the induced IL-8 expression and the reduced mRNA stability. Therefore, our data provide the first evidence that PPARδ induces IL-8 expression in nonstimulated endothelial cells via transcriptional as well as posttranscriptional mechanisms.

Inhibition of Rac1 GTPase downregulates vascular endothelial growth factor receptor-2 expression by suppressing Sp1-dependent DNA binding in human endothelial cells.

Abstract:  RhoA, Rac1 and CDC42 are small GTP‐binding proteins of the Rho family that play a crucial role in regulation of the actin‐based cytoskeleton. In addition to cell growth regulation, they are implicated in transcriptional activation, oncogenic transformation and angiogenesis. The small Rho‐GTPases have been linked to vascular endothelial growth factor (VEGF)‐induced signalling pathways, but their role has not yet been elucidated. As signalling via the VEGF receptor‐2 (VEGFR2) pathway is critical for angiogenic responses in cancer, wound repair and ischaemic and inflammatory diseases, we investigated whether the small Rho‐GTPase Rac1 influences VEGFR2 expression in human endothelial cells. In this study, we show that a dominant negative Rac1 expression vector led to a pronounced decrease in VEGFR2 mRNA and protein expression. To identify minimal promoter requirements and potential applications of the small Rho‐GTPases, we used VEGFR2 promoter–reporter gene constructs containing various deletions. The inhibitory effects of dominant negative Rac1 on the transcriptional activity of the VEGFR2 promoter localized to an element between −77 and −60 that contains an Sp1 transcription factor binding site. Electrophoretic mobility shift assays demonstrated that constitutive Sp1‐dependent DNA binding decreased with Rac1 inhibition. Hence, repression of the small Rho GTPase Rac1 seems to be an additional critical molecular mechanism in the regulation of VEGFR2 expression.

AP1-dependent repression of TGFα-mediated MMP9 upregulation by PPARδ agonists in keratinocytes

Abstract:  Peroxisome proliferator‐activated receptors (PPARs) are ligand‐activated transcription factors that function mainly in the regulation of glucose and lipid homeostasis. PPAR agonists have been shown to control inflammation by inhibition of distinct proinflammatory genes. Aberrant activation of the epidermal growth factor receptor and/or overexpression of its ligand, transforming growth factor‐α (TGFα), are key features of both neoplastic and inflammatory hyperproliferative epithelia. Matrix metalloproteinase 9 (MMP9) belongs to the set of genes that are effectively induced by TGFα in keratinocytes. Induction of MMP9 expression is strongly linked to regenerative skin repair mechanisms, inflammatory skin diseases and tumor metastasis. We explored whether the known anti‐inflammatory effects of PPARδ ligands involve inhibiting the TGFα‐mediated upregulation of MMP9. The PPARδ agonists potently inhibited TGFα‐induced MMP9 expression in human keratinocytes. This inhibition was observed at both the protein and mRNA levels. Transcriptional activation studies with deletion constructs of a reporter gene revealed that PPARδ agonists mediate their inhibitory effects via an AP1‐binding site. Electromobility shift assay analysis indicated that MMP9 gene expression is inhibited by repressing site‐dependent DNA binding and transactivation by c‐fos. In conclusion, our data provide the first evidence that MMP9 expression induced by TGFα is a valid target of PPARδ ligands in keratinocytes.

Dimethylfumarate inhibits melanoma cell proliferation via p21 and p53 induction and bcl-2 and cyclin B1 downregulation

Recent evidence suggests that dimethylfumarate (DMF), known as a highly potent anti-psoriatic agent, might have anti-tumorigenic properties in melanoma. It has recently been demonstrated that DMF inhibits melanoma proliferation by apoptosis and cell cycle inhibition and therefore inhibits melanoma metastasis. Nonetheless, the underlying mechanisms remain to be evaluated. To elucidate the effects of DMF on melanoma cell lines (A375, SK-Mel), we first performed cytotoxicity assays. No significant lactatedehydogenase (LDH) release could be found. In further analysis, we showed that DMF suppresses melanoma cell proliferation in a concentration-dependent manner. To examine whether these effects are conveyed by apoptotic mechanisms, we studied the amount of apoptotic nucleosomes and caspase 3/7 activity using ELISA analysis. Significant apoptosis was induced by DMF in both cell lines, and this could be paralleled with bcl-2 downregulation and PARP-1 cleavage. We also performed cell cycle analysis and found that DMF induced concentration-dependent arrests of G0/G1 as well as G2/M. To examine the underlying mechanisms of cell cycle arrest, we analyzed the expression profiles of important cell cycle regulator proteins such as p53, p21, cyclins A, B1, and D1, and CDKs 3, 4, and 6. Interestingly, DMF induced p53 and p21 yet inhibited cyclin B1 expression in a concentration-dependent manner. Other cell cycle regulators were not influenced by DMF. The knockdown of DMF induced p53 via siRNA led to significantly reduced apoptosis but had no influence on cell cycle arrest. We examined the adhesion of melanoma cells on lymphendothelial cells during DMF treatment and found a significant reduction in interaction. These data provide evidence that DMF inhibits melanoma proliferation by reinduction of important cell cycle inhibitors leading to a concentration-dependent G0/G1 or G2/M cell cycle arrest and induction of apoptosis via downregulation of bcl-2 and induction of p53 and PARP-1 cleavage. Hence, DMF might be an interesting agent in the treatment of melanoma and is worth further investigation in vivo.

Dimethylfumarate protects against TNF-α-induced secretion of inflammatory cytokines in human endothelial cells

BackgroundInflammation, angiogenesis and oxidative stress have been implicated in the pathogenesis of various vascular diseases. Recent evidence suggests that dimethylfumarate (DMF), an antiposriatic and anti-multiple sclerosis agent, possesses anti-inflammatory, anti-oxidative and anti-angiogenic properties. Here, we analyze the influence of DMF on TNF-α-induced expression of the important pro-inflammatory and pro-atherogenic chemokine MCP-1 and investigate the underlying mechanisms of this expression.FindingsWe analyzed constitutive and TNF-α-induced expression of MCP-1 in human umbilical vascular endothelial cells (HUVEC) +/− DMF treatment via enzyme-linkes immunosorbent assay (ELISA). DMF significantly inhibited the protein expression levels in a time- and concentration-dependent manner. Furthermore, MCP-1 mRNA expression was also reduced in response to DMF, as demonstrated by RT-PCR. Thus, the regulation occurs at the transcriptional level. Interestingly, DMF prolonged the TNF-α-induced p38 and JNK phosphorylation in HUVEC, as demonstrated by Western blot analysis; however, the p38 and JNK inhibitor SB203580 did not affect the DMF-conveyed suppression of TNF-α-induced MCP-1 expression. DMF suppressed the TNF-α-induced nuclear translocation and phosphorylation (Serine 536) of p65 in these cells. These results were additionally approved by p65 luciferase promoter assays. Furthermore, we found that DMF slightly inhibited the early degradation of IκBα. In addition, we verified our results using other important inflammatory cytokines such as CCL-5, PDGF-BB, GM-CSF and IL-6.ConclusionDMF suppresses various TNF-α-induced pro-inflammatory and pro-atherogenic cytokines/chemokines in human endothelial cells. This action is regulated by reduced p65 activity and nuclear translocation, which can be explained in part by the reduced early degradation of IκBα and more important the reduced phosphorylation of p65 at Serine 536. These effects were independent of the p38, PI3K and p42/44 signaling pathways. As a result, DMF might be suitable for treating patients with vascular diseases.

Ligand activation of peroxisome proliferator-activated receptor delta suppresses cathepsin B expression in human endothelial cells in a posttranslational manner.

Peroxisome proliferator‐activated receptor (PPAR) delta agonists are known to have distinct anti‐inflammatory and antitumor effects; though, the knowledge regarding their mode of action has thus far been limited. Different cathepsins have been shown to be upregulated in a broad range of pathological events, such as rheumatoid arthritis, psoriasis, atherosclerosis and diverse tumor entities, for example melanoma. Recent work demonstrated that cathepsin B in particular is an important pro‐angiogenic protease in various pathological conditions. We therefore analysed whether cathepsins are a valid target for PPARδ agonists. This study reveals an inhibitory effect of two commonly used PPARδ agonists, GW501516 and L‐165,041, on the protein expression and enzyme activity of cathepsin B in human endothelial cells. In contrast, no inhibitory effects were observed on cathepsin L and cathepsin D protein expression after treatment with PPARδ agonists. Furthermore, the results substantiate that PPARδ activators mediate their inhibitory action in a PPARδ‐dependent manner and that the underlying regulatory mechanism is not based on a transcriptional but rather on a posttranslational mode of action, via the reduction in the cathepsin B protein half‐life. Mechanisms conveying the suppressive effect by 5′‐alternative splicing, a 3′‐UTR‐dependent way or by miRNA could be excluded. The data of this study explore cathepsin B as a new valid target for PPARδ agonists in endothelial cells. The results bolster other studies demonstrating PPARδ agonists as anti‐inflammatory and anticarcinogenic agents and thus might have the potential to help to develop new pharmaceutical drugs.

Histone deacetylase inhibitors interfere with angiogenesis by decreasing endothelial VEGFR-2 protein half-life in part via a VE-cadherin-dependent mechanism

Recent evidence suggests that histone deacetylase inhibitors (HDACi) may mediate part of their antitumor effects by interfering with tumor angiogenesis. As signalling via the vascular endothelial growth factor receptor‐2 (VEGFR‐2) pathway is critical for angiogenic responses during tumor progression, we explored whether established antitumor effects of HDACi are partly mediated through diminished endothelial VEGFR‐2 expression. We therefore examined the potential impact of three different HDACi, trichostatin A (TSA), sodium butyrate (But) and valproic acid (VPA), on VEGFR‐2 protein expression. TSA, VPA and But significantly inhibit VEGFR‐2 protein expression in endothelial cells. Pertinent to these data, VEGFR‐2 protein half‐life is shown to be decreased in response to HDACi. Recently, it could be demonstrated that expression of VE‐cadherin influences VEGFR‐2 protein half‐life. In our experiments, VEGFR‐2 downregulation was accompanied by HDACi‐induced VE‐cadherin suppression. Interestingly, siRNA‐mediated knockdown of VE‐cadherin led to a pronounced loss of VEGFR‐2 expression on the protein as well as on the mRNA level, implicating that VE‐cadherin not only influences VEGFR‐2 protein half‐life but also the transcriptional level. To further distinguish which of the eight different histone deacetylases are responsible for the regulation of VEGFR‐2 expression, specific HDAC genes were silenced by transfecting respective siRNAs. These studies revealed that HDACs 1, 4, 5 and 6 are preferentially involved in VEGFR‐2 expression. Therefore, these results provide an explanation for the anti‐angiogenic action of HDAC inhibitors via a VE‐cadherin, HDAC 1 and HDACs 4–6‐mediated suppression of VEGFR‐2 expression and might be of importance in the development of new anti‐angiogenic drugs.

Dimethylfumarate effectively inhibits lymphangiogenesis via p21 induction and G1 cell cycle arrest

Different pathologies, such as lymphoedema, cancer or psoriasis, are associated with abnormal lymphatic vessel formation. Therefore, influencing lymphangiogenesis is an interesting target. Recent evidence suggests that dimethylfumarate (DMF), an antipsoriatic agent, might have antitumorigenic and antilymphangiogenic properties. To prove this assumption, we performed proliferation and functional assays with primary human dermal lymphendothelial cells (DLEC). We could demonstrated that DMF suppresses DLEC proliferation and formation of capillary‐like structures. Underlying apoptotic mechanisms could be ruled out. Cell cycle analysis demonstrated a pronounced G1‐arrest. Further evaluations revealed increases in p21 expression. In addition, DMF suppressed Cyclin D1 and Cyclin A expression in a concentration‐dependent manner. p21 knockdown experiments demonstrated a p21‐dependent mechanism of regulation. Further analysis showed an increased p21 mRNA expression after DMF treatment. This transcriptional regulation was enforced by post‐transcriptional and post‐translational mechanisms. In addition, we could demonstrate that the combination of a proteasomal inhibitor and DMF superinduced the p21 expression. Hence, DMF is a new antilymphangiogenic compound and might be used in various illnesses associated with increased lymphangiogenesis.