Arsenic Trioxide Decreases Lymphangiogenesis by Inducing Apoptotic Pathways and Inhibition of Important Endothelial Cell Receptors.

Tumor-induced lymphangiogenesis is strongly associated with the formation of tumor metastasis. Therefore, the regulation of lymphangiogenesis offers a promising target in cancer therapy. Arsenic trioxide (ATO) is highly effective in the treatment of patients with acute promyelocytic leukemia (APL). As ATO mediates anti-angiogenic effects on endothelial and tumor cells, we aimed to explore the impact of ATO on lymphangiogenesis in human lymphatic endothelial cells (LEC). The BrdU assay and flow cytometry analysis were used to evaluate the influence of ATO on the proliferation and cell cycle distribution of LECs. The lymphatic suppression effects of ATO were investigated in vitro using the lymphatic tube formation assay. The effects of ATO on apoptosis, mitochondrial membrane potential and endothelial cell receptors were investigated by Western blotting, ELISA, flow cytometry and qRT-PCR. The treatment of LECs with ATO attenuated cell proliferation, blocked tube formation and induced subG0/G1 arrest in LECs, thus suggesting enhanced apoptosis. Although subG0/G1 arrest was accompanied by the upregulation of p21 and p53, ATO treatment did not lead to visible cell cycle arrest in LECs. In addition, ATO caused apoptosis via the release of cytochrome c from mitochondria, activating caspases 3, 8 and 9; downregulating the anti-apoptotic proteins survivin, XIAP and cIAP-2; and upregulating the pro-apoptotic protein Fas. Furthermore, we observed that ATO inhibited the VEGF-induced proliferation of LECs, indicating that pro-survival VEGF/VEGFR signaling was affected by ATO treatment. Finally, we found that ATO inhibited the expression of the important endothelial cell receptors VEGFR-2, VEGFR-3, Tie-2 and Lyve-1. In conclusion, we demonstrate that ATO inhibits lymphangiogenesis by activating apoptotic pathways and inhibiting important endothelial cell receptors, which suggests that this drug should be further evaluated in the treatment of tumor-associated lymphangiogenesis.

Evaluation of 2-methoxyestradiol serum levels as a potential prognostic marker in malignant melanoma.

Experimental findings indicated that 2-methoxyestradiol (2-ME), an endogenous metabolite of 17ß-estradiol, may exhibit anti-tumorigenic properties in various types of tumour, such as melanoma and endometrial carcinoma. In patients with endometrial cancer, the serum levels of 2-ME are decreased compared with those in healthy controls, and this finding has been associated with a poor outcome. The aim of the present study was to examine whether the serum levels of 2-ME are decreased in patients with melanoma, and whether this decrease may be correlated with disease stage and, therefore, serve as a prognostic indicator. ELISA was used to detect serum levels of 2-ME in patients with stage I-IV malignant melanoma (MM). A cohort of 78 patients with MM was analysed, along with 25 healthy controls, among whom 15 were women in the second trimester of pregnancy (positive control). As expected, significantly elevated levels of serum 2-ME were observed in pregnant control patients compared with those in patients with MM and healthy controls. There was no observed correlation between 2-ME serum levels in patients with MM and disease stage, tumour thickness, lactate dehydrogenase or S100 calcium-binding protein B levels. In addition, the 2-ME levels of patients with MM did not differ significantly from those of normal healthy controls. Overall, the findings of the present study indicated that the 2-ME serum levels in patients with MM were not decreased, and there was no correlation with early- or advanced-stage disease. Therefore, in contrast to published results on endometrial cancer, endogenous serum 2-ME levels in MM were not found to be correlated with tumour stage and did not appear to be a suitable prognostic factor in MM.

Tests on asymmetry for ordered categorical variables.

Skewness is a well-established statistical concept for continuous and, to a lesser extent, for discrete quantitative statistical variables. However, for ordered categorical variables, limited literature concerning skewness exists, although this type of variables is common for behavioral, educational, and social sciences. Suitable measures of skewness for ordered categorical variables have to be invariant with respect to the group of strictly increasing, continuous transformations. Therefore, they have to depend on the corresponding maximal-invariants. Based on these maximal-invariants, we propose a new class of skewness functionals, show that members of this class preserve a suitable ordering of skewness and derive the asymptotic distribution of the corresponding skewness statistic. Finally, we show the good power behavior of the corresponding skewness tests and illustrate these tests by applying real data examples.

(Generalized) Maximum Cumulative Direct, Residual, and Paired Φ Entropy Approach.

A distribution that maximizes an entropy can be found by applying two different principles. On the one hand, Jaynes (1957a,b) formulated the maximum entropy principle (MaxEnt) as the search for a distribution maximizing a given entropy under some given constraints. On the other hand, Kapur (1994) and Kesavan and Kapur (1989) introduced the generalized maximum entropy principle (GMaxEnt) as the derivation of an entropy for which a given distribution has the maximum entropy property under some given constraints. In this paper, both principles were considered for cumulative entropies. Such entropies depend either on the distribution function (direct), on the survival function (residual) or on both (paired). We incorporate cumulative direct, residual, and paired entropies in one approach called cumulative Φ entropies. Maximizing this entropy without any constraints produces an extremely U-shaped (=bipolar) distribution. Maximizing the cumulative entropy under the constraints of fixed mean and variance tries to transform a distribution in the direction of a bipolar distribution, as far as it is allowed by the constraints. A bipolar distribution represents so-called contradictory information, which is in contrast to minimum or no information. In the literature, to date, only a few maximum entropy distributions for cumulative entropies have been derived. In this paper, we extended the results to well known flexible distributions (like the generalized logistic distribution) and derived some special distributions (like the skewed logistic, the skewed Tukey λ and the extended Burr XII distribution). The generalized maximum entropy principle was applied to the generalized Tukey λ distribution and the Fechner family of skewed distributions. Finally, cumulative entropies were estimated such that the data was drawn from a maximum entropy distribution. This estimator will be applied to the daily S&P500 returns and time durations between mine explosions.

Dimethylfumarate Inhibits Colorectal Carcinoma Cell Proliferation: Evidence for Cell Cycle Arrest, Apoptosis and Autophagy.

Recent studies have proven that Dimethylfumarate (DMF) has a marked anti-proliferative impact on diverse cancer entities e.g., on malignant melanoma. To explore its anti-tumorigenic potential, we examined the effects of DMF on human colon carcinoma cell lines and the underlying mechanisms of action. Human colon cancer cell line HT-29 and human colorectal carcinoma cell line T84 were treated with or without DMF. Effects of DMF on proliferation, cell cycle progression, and apoptosis were analyzed mainly by Bromodeoxyuridine (BrdU)- and Lactatdehydrogenase (LDH)assays, caspase activation, flowcytometry, immunofluorescence, and immunoblotting. In addition, combinational treatments with radiation and chemotherapy were performed. DMF inhibits cell proliferation in both cell lines. It was shown that DMF induces a cell cycle arrest in G0/G1 phase, which is accompanied by upregulation of p21 and downregulation of cyclin D1 and Cyclin dependent kinase (CDK)4. Furthermore, upregulation of autophagy associated proteins suggests that autophagy is involved. In addition, the activation of apoptotic markers provides evidence that apoptosis is involved. Our results show that DMF supports the action of oxaliplatin in a synergetic manner and failed synergy with radiation. We demonstrated that DMF has distinct antitumorigenic, cell dependent effects on colon cancer cells by arresting cell cycle in G0/G1 phase as well as activating both the autophagic and apoptotic pathways and synergizes with chemotherapy.

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.

The histone deacetylase inhibitor trichostatin a decreases lymphangiogenesis by inducing apoptosis and cell cycle arrest via p21-dependent pathways.

BACKGROUND: The formation of new lymphatic vessels provides an additional route for tumour cells to metastasize. Therefore, inhibiting lymphangiogenesis represents an interesting target in cancer therapy. First evidence suggests that histone deacetylase inhibitors (HDACi) may mediate part of their antitumor effects by interfering with lymphangiogenesis. However, the underlying mechanisms of HDACi induced anti-lymphangiogenic properties are not fully investigated so far and in part remain unknown. METHODS: Human lymphatic endothelial cells (LEC) were cultured in vitro and treated with or without HDACi. Effects of HDACi on proliferation and cell cycle progress were analysed by BrdU assay and flow cytometry. Apoptosis was measured by quantifying mono- and oligonucleosomes in the cytoplasmic fraction of cell lysates. In vitro lymphangiogenesis was investigated using the Matrigel short term lymphangiogenesis assay. The effects of TSA on cell cycle regulatory proteins and apoptosis-related proteins were examined by western blotting, immunofluorescence staining and semi-quantitative RT-PCR. Protein- and mRNA half-life of p21 were analysed by western blotting and quantitative RT-PCR. The activity of the p21 promoter was determined using a dual luciferase assay and DNA-binding activity of Sp1/3 was investigated using EMSA. Furthermore, siRNA assays were performed to analyse the role of p21 and p53 on TSA-mediated anti-lymphangiogenic effects. RESULTS: We found that HDACi inhibited cell proliferation and that the pan-HDACi TSA induced G0/G1 arrest in LEC. Cell cycle arrest was accompanied by up-regulation of p21, p27 and p53. Additionally, we observed that p21 protein accumulated in cellular nuclei after treatment with TSA. Moreover, we found that p21 mRNA was significantly up-regulated by TSA, while the protein and mRNA half-life remained largely unaffected. The promoter activity of p21 was enhanced by TSA indicating a transcriptional mechanism. Subsequent EMSA analyses showed increased constitutive Sp1/3-dependent DNA binding in response to HDACi. We demonstrated that p53 was not required for TSA induced p21 expression and growth inhibition of LECs. Interestingly, siRNA-mediated p21 depletion almost completely reversed the anti-proliferative effects of TSA in LEC. In addition, TSA induced apoptosis by cytochrome c release contributed to activating caspases-9, -7 and -3 and downregulating the anti-apoptotic proteins cIAP-1 and -2. CONCLUSIONS: In conclusion, we demonstrate that TSA - a pan-HDACi - has distinct anti-lymphangiogenic effects in primary human lymphatic endothelial cells by activating intrinsic apoptotic pathway and cell cycle arrest via p21-dependent pathways.

Peroxisome proliferator-activated receptor (PPAR) α and δ activators induce ICAM-1 expression in quiescent non stimulated endothelial cells.

BACKGROUND: 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 the expression of distinct proinflammatory genes such as vascular cell adhesion molecule-1 (VCAM-1), IL-8, and intercellular adhesion molecule-1 (ICAM-1). ICAM-1 is an important endothelial membrane receptor that facilitates the transmigration of leukocytes across the endothelium. To date, the influence of PPARα and δ activators on the expression of ICAM-1 in non-induced, quiescent endothelial cells has been unclear. Therefore, we examined the effects of various PPARα and δ agonists on the expression of ICAM-1 in non-stimulated primary human endothelial cells. RESULTS: We found that PPARα and PPARδ agonists significantly induced ICAM-1 surface, intracellular protein, and mRNA expression in a time and concentration-dependent manner. The PPARδ induced ICAM-1 expression could be paralleled with a significantly increased T-cell adherence to the endothelial cells whereas PPARα failed to do so. Transcriptional activity studies using an ICAM-1 reporter gene constructs revealed that PPARδ, but not PPARα agonists induced gene expression by stimulating ICAM-1 promoter activity via an Sp1 transcription factor binding site and inhibit the binding of the transcription factors Sp1 and Sp3. Furthermore, we performed mRNA stability assays and found that PPARα and PPARδ agonists increased ICAM-1 mRNA stability. CONCLUSION: Therefore, our data provide the first evidence that PPARα and PPARδ agonists induce ICAM-1 expression in non-stimulated endothelial cells via transcriptional and posttranscriptional mechanisms.

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 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.

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

BACKGROUND: Inflammation, 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. FINDINGS: We 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. CONCLUSION: DMF 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.

PPARα agonist Wy14643 suppresses cathepsin B in human endothelial cells via transcriptional, post-transcriptional and post-translational mechanisms.

Cathepsin B has been shown to be important in angiogenesis; therefore, understanding its regulation in endothelial cells should provide fundamental information that will aid in the development of new treatment options. Peroxisome proliferator-activated receptors (PPARs) have been shown to have anti-inflammatory, anti-angiogenic and anti-tumorigenic properties. We explored the influence of a PPARα agonist on cathepsin B expression in human endothelial cells. The PPARα agonist, Wy14643, was found to inhibit cathepsin B protein expression. Further studies demonstrated the Wy14643-dependent but PPARα-independent suppression of cathepsin B. This has been previously described for other PPAR agonists. Wy14643 suppressed the accumulation of cathepsin B mRNA, which was accompanied by the selective suppression of a 5'-alternative splice variant. Consistent with these results, luciferase promoter assays and electrophoretic mobility shift analysis demonstrated that the suppression was facilitated by reduced binding of the transcription factors USF1/2 to an E-box within the cathepsin B promoter. Additionally, Wy14643 treatment resulted in a reduction in cathepsin B half-life, suggesting a posttranslational regulatory mechanism. Overall, our results suggest that the PPARα-dependent anti-angiogenic action of Wy14643 seems to be mediated, in part, by Wy14643-dependent but PPARα-independent regulation of cathepsin B expression.

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.

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

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.

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 {delta} 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-1ß 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.