Vascular PPARβ/δ Promotes Tumor Angiogenesis and Progression

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors, which function as transcription factors. Among them, PPARβ/δ is highly expressed in endothelial cells. Pharmacological activation with PPARβ/δ agonists had been shown to increase their angiogenic properties. PPARβ/δ has been suggested to be involved in the regulation of the angiogenic switch in tumor progression. However, until now, it is not clear to what extent the expression of PPARβ/δ in tumor endothelium influences tumor progression and metastasis formation. We addressed this question using transgenic mice with an inducible conditional vascular-specific overexpression of PPARβ/δ. Following specific over-expression of PPARβ/δ in endothelial cells, we induced syngenic tumors. We observed an enhanced tumor growth, a higher vessel density, and enhanced metastasis formation in the tumors of animals with vessel-specific overexpression of PPARβ/δ. In order to identify molecular downstream targets of PPARβ/δ in the tumor endothelium, we sorted endothelial cells from the tumors and performed RNA sequencing. We identified platelet-derived growth factor receptor beta (Pdgfrb), platelet-derived growth factor subunit B (Pdgfb), and the tyrosinkinase KIT (c-Kit) as new PPARβ/δ -dependent molecules. We show here that PPARβ/δ activation, regardless of its action on different cancer cell types, leads to a higher tumor vascularization which favors tumor growth and metastasis formation.


Introduction
Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors. They function as ligand activated transcription factors. They exist in three isoforms, PPARα, PPARδ (formerly PPARβ), and PPARγ. For all PPARs, lipids are endogenous ligands, linking them directly to metabolism. PPARs are considered as important transcriptional regulators of genes involved in lipid metabolism and cardiac energy production [1]. PPARs form heterodimers with retinoic X receptors, and, upon ligand binding, modulate gene expression of downstream target genes dependent on the presence of co-repressors or co-activators. This results in cell-type specific complex regulations of proliferation, differentiation, and cell survival. Specific synthetic agonists for all PPARs are available. PPARα and PPARγ agonists are already in clinical use for the treatment of hyperlipidemia and type 2 diabetes, respectively. More recently, PPARβ/δ activation came into focus as an interesting novel approach for the treatment of metabolic syndrome and associated cardiovascular diseases.
cell-specific upregulation of PPARβ/δ. Furthermore, we identify platelet-derived growth factor receptor beta (Pdgfrb), platelet-derived growth factor subunit B (Pdgfb), and the tyrosinkinase KIT (c-Kit) as new PPARβ/δ -dependent molecules involved in tumor vessel formation based on RNA sequencing and subsequent verification applying a variety of molecular biology approaches.

Apoptosis Assays
Apoptotic cells were detected by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining of HUVECs, 48 h after treatment with DMSO, GW0742, or GSK3787 using the In Situ Cell Death Detection Kit (Roche Molecular Biochemicals, Meylan, France) according to the manufacturer's instructions. LLC1 cells were incubated with APC-conjugated annexin V (Roche, Meylan, France) and counterstained with propidium iodide to distinguish necrotic from apoptotic cell death. LLC1 cells treated with 100 nmol/L Staurosporine (Sigma, St. Louis, MO, USA) overnight served as positive controls.

Endothelial Cell Isolation
Mouse tumor endothelial cells (EC) were isolated from Tie2-CreERT2;PPARβ/δ mice treated with Tamoxifen or vehicle as previously described [10,17,18]. Tie2-CreERT2 animals injected with Tamoxifen served as an additional control. Briefly, tumor tissues were cut into small fragments and digested with 1 mg/mL collagenase A and 100 IU/mL type I DNase (Roche Diagnostics, Meylan, France) for 45 min at 37 • C. ECs were then purified from the cell suspension using CD31 MicroBeads followed by magnetic separation in LS columns (Miltenyi Biotec SAS, Paris, France).

RT-PCR and Quantitative RT-PCR
Total RNA was isolated using the Trizol reagent (Invitrogen). First-strand cDNA synthesis was performed with 0.5 µg of total RNA using the Thermo Scientific Maxima First Strand cDNA synthesis kit (Thermo Scientific). The reaction product was diluted to 200 µL and 1 µL of the diluted reaction product was taken for real time RT-PCR amplification (StepOne plus, Applied Biosystems) using the SYBR ® Select Master Mix (Applied Biosystems). Expression of each gene was normalized to the respective arithmetic means of Gapdh, Actnb, and Rplp0 expression.
Primer sequences for mouse genes are given in Table 1 and for human genes in Table 2.

mRNA Sequencing
For sequencing, RNAs from tumor sorted endothelial cells from Tie2-CreERT2;PPARβ/δ mice treated with Tamoxifen or vehicle were used (n = 4 each). RNA sequencing and data analysis was performed by Novogene, Beijing, China. Briefly, RNA quality was monitored on 1% agarose gels. RNA purity was checked using a NanoPhotometer. RNA concentration was measured using the Qubit ® RNA Assay Kit in a Qubit ® 2.0 Flurometer (Life Technologies, CA, USA). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). A total amount of 1 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext ® UltraTM RNA Library Prep Kit for Illumina ® (NEB, USA) following manufacturer's instructions. Library quality was assessed on the Agilent Bioanalyzer 2100 system. Library preparations were sequenced on an Illumina Hiseq platform and 125 bp/150 bp paired-end reads were generated. Clean reads were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. HTSeq v0.6.1 was used to count the reads numbers mapped to each gene. Fragments per kilobase of transcript sequence per millions base pairs sequenced (FPKM) of each gene was calculated based on the length of the gene and reads count mapped to this gene. Differential expression analysis of the two groups was performed using the DESeq R package (1.18.0). Resulting p-values were adjusted using the Benjamini and Hochberg's approach. Genes with an adjusted p-value < 0.05 were assigned as differentially expressed. The RNA sequencing data have been deposited in NCBI's Gene Expression Omnibus [19,20] and are accessible through GEO Series accession number GSE140513 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE140513).

Tissue Samples and Immunohistology
The study adheres to the principles of the Declaration of Helsinki and to Title 45 of the U.S. Code of Federal Regulations (Part 46, Protection of Human Subjects). Paraffin-embedded samples, cut at 3µm, were used for immunohistochemical detection of PPARβ/δ, CD31, and PCNA. In total, 35 paraffin-embedded human tumor samples (7 liver carcinomas, 7 melanomas, 7 pancreas carcinomas, 7 ovary carcinomas, and 7 prostate cancers) were used for this study. For immunofluorescence double-labeling of human tumor samples, anti-CD31 mouse monoclonal antibody from Dako (Trappes, France, clone JC70A) was combined with the anti-PPARβ/δ antibody (ThermoFisher Scientific), using Dylight 488 donkey anti rabbit and Dylight 594 donkey anti mouse secondary antibodies. Negative controls were obtained by omission of first antibodies. Images were taken using a confocal ZEISS LSM Exciter microscope (Zeiss, Jena, Germany).
Mouse tissue sections were in routine stained with hematoxylin-eosin. After heat-mediated antigen retrieval and quenching of endogenous peroxidase activity, PPARβ/δ (ThermoFisher Scientific) or CD31 (rabbit polyclonal, 1:50, Abcam) were detected using the EnVisionTM Peroxidase/DAB Detection System from Dako. Negative controls were obtained by incubation of samples with a rabbit IgG Control (Abcam). Sections were counterstained with hematoxylin (Dako) and analyzed by two independent investigators, one of them an experienced pathologist. Slides were viewed under an epifluorescence microscope (DMLB, Leica, Germany) connected to a digital camera (Spot RT Slider, Diagnostic Instruments, Scotland).
As vector backbone, pGl3 basic (Promega) was used for all constructs.

Statistical Analysis
Data are expressed as mean ± SEM. Student's t-tests (Instat, GraphPad) were performed to determine statistical significance. A p-value of less than 0.05 was considered significant.

The PPARβ/δ Agonist GW0742 Decreases LLC1 Lewis Lung Cancer Cell Proliferation In Vitro
As a prerequisite before in vivo treatment of tumor-bearing mice with a PPARβ/δ agonist, we investigated the in vitro effects on syngenic tumor cells, which we aimed to inject into animals. LLC1 cells were treated for 24 h with the PPARβ/δ agonist GW0742 or the PPARβ/δ antagonist GSK3787 at a concentration of 1 µmol/L. Thereafter, we performed bromodeoxyuridine (BrdU) incorporation assays and immunostaining for proliferating cell nuclear antigen (PCNA), with subsequent quantification of PCNA-positive cells relative to the total cell number. GW0742 decreased BrdU incorporation and the fraction of PCNA-positive cells significantly, GSK3787 had the opposite effects (Figure 1a-c). These findings are in line with the effects of PPARβ/δ modulation on the proliferation of melanoma cells reported by our group [5] and recently confirmed in a study where a PPARβ/δ antagonist enhanced melanoma progression [28]. To determine whether apoptosis might contribute to the observed effects on cell growth upon GW0742 treatment, we used Annexin V/propidium iodide (PI) labeling to detect apoptotic events followed by fluorescence-activated cell sorting (FACS) analysis. Apoptosis of LLC1 cells was neither influenced by the PPARβ/δ agonist GW0742, nor the antagonist GSK3787 ( Figure 1d).
As mentioned before, the role of PPARβ/δ in cancer is highly controversial. Concerning lung cancer, two studies reported an increased expression of PPARβ/δ in human non-small cell lung cancer (NSCLC) compared to normal lung and observed in NSCLC cell lines that PPARβ/δ activation promoted proliferation [29,30]. However, an earlier report observed an inhibition of proliferation in a NSCLC cell line upon PPARβ/δ activation [31] and finally, neither growth promoting nor inhibiting effects were observed in another study [32]. Thus, it is difficult to draw a definitive conclusion regarding the biological effect of PPARβ/δ in human lung cancer. There is also a high variability in the kind and concentration of PPARβ/δ agonists used, which might further contribute to these conflicting results. Nevertheless, the aim of our study was not to determine the effects of PPARβ/δ modulation in one cancer cell line, but to clarify the in vivo relevance of PPARβ/δ for tumor vessel formation and cancer growth, which necessitated to distinguish between direct effects on tumor cells and effects on the tumor microenvironment, especially tumor vessels. As mentioned before, the role of PPARβ/δ in cancer is highly controversial. Concerning lung cancer, two studies reported an increased expression of PPARβ/δ in human non-small cell lung cancer (NSCLC) compared to normal lung and observed in NSCLC cell lines that PPARβ/δ activation

The PPARβ/δ Agonist GW0742 Increases LLC1 Lewis Lung Cancer Progression In Vivo
We investigated the effect of the PPARβ/δ agonist GW0742 on tumor growth by subcutaneous implantation of LLC1 tumor cells and subsequent injections of GW0742. Control animals received DMSO injections in the same frequency. Tumor growth was comparable between the two groups in the first days and started to increase in the animals receiving the PPARβ/δ agonist in the second week (Figure 2a). After three weeks, we determined tumor-to-body weight ratios in both groups and observed significantly higher tumor weights in GW0742 treated animals (Figure 2b,c). Next, we analyzed whether treatment with a PPARβ/δ agonist might modify metastasis occurrence. We investigated serial lung and liver sections of both groups for metastases formation. All animals, which had received the PPARβ/δ agonist displayed lung metastases formation, whereas only 30% of the animals in the control group had metastases occurrence in the lung. Lung metastases were not only more frequent in the treated group, but also of a bigger size than in controls. Liver metastases could be observed in 16% of the GW0742 treated mice, whereas none of the control animals had liver metastases (Figure 2d). Vessel density in tumors was determined by immunostaining for Cd31. Strikingly, in tumors of GW0742 treated mice, vessel densities were more than doubled compared to controls (Figure 2e). Double-staining for Cd31 and PCNA suggested a higher number of PCNA-positive endothelial cells in the tumors of PPARβ/δ agonist treated animals (Figure 2f).
Although the PPARβ/δ agonist GW0742 inhibited tumor cell proliferation in vitro, we could observe a strikingly different situation in vivo, where tumor growth and metastasis formation were enhanced. Tumor vascularization was strongly increased, which is in line with previous studies reporting a rapid boost of vascularization after pharmacological PPARβ/δ activation [8][9][10]. The increased tumor vascularization upon PPARβ/δ activation seems therefore to be sufficient to dominate over the anti-proliferative effect of the PPARβ/δ agonist on tumor cells. Increasing neovascularization is not only required for further expansion of the tumor-cell population, but also correlates with a rising rate of metastasis. In agreement with our findings, a recent study observed a marked inhibition of tumor angiogenesis and growth in PPARβ/δ −/− mouse models of subcutaneous Lewis lung carcinoma and B16 melanoma, with occurrence of immature, leaky microvascular structures [33]. However, another group demonstrated enhanced extravasation of B16 melanoma cells in PPARβ/δ −/− mice using an experimental model of metastases formation by injecting the tumor cells in the tail vein of animals, resulting in enhanced pulmonary metastases as compared to wildtype animals [28]. In contrast to the patho-physiologically occurring spontaneous metastasis formation, this assay does not require the formation of functional blood vessels necessary for the propagation of the primary tumor cells to other organs. The enhanced pulmonary metastases formation observed in the PPARβ/δ −/− animals could therefore simply be due to the presence of non-functional leaky microvessels [33], facilitating the anchorage of the tumor cells in the lung.

PPARβ/δ Is Expressed in the Vasculature of Human Tumors and PPARβ/δ Modulation Impacts Proliferation and Expression of Pro-Angiogenic Factors in Human Endothelial Cells
To confirm a relevance for human pathophysiology, we investigated human tumor samples (liver, pancreas, ovary, and prostate carcinoma, as well as melanoma) for expression of PPARβ/δ in the tumor vasculature. Confocal microscopy of tumor samples labeled for PPARβ/δ and CD31 confirmed co-localization of PPARβ/δ in endothelial cells ( Figure 3). Furthermore, PPARβ/δ could be detected in tumor cells at various degrees in different tumor types.  Treatment of human umbilical vein endothelial cells (HUVECs) with GW0742 increased proliferation, while the PPARβ/δ antagonist GSK3787 tended to reduce HUVEC proliferation ( Figure  4a). This is in agreement with previous works reporting enhanced endothelial cell proliferation [34] and activation of angiogenesis upon PPARβ/δ activation [7] and the general view of PPARβ/δ as a pro-angiogenic factor [4]. As an initial study suggested that PPARβ/δ is involved in endothelial cell apoptosis [35], we tested for differences in apoptosis upon modulation of PPARβ/δ. Rarely, TUNELpositive endothelial cells were observed under any of the culture conditions (Figure 4b), suggesting that PPARβ/δ modulation does not influence apoptosis in vascular cells.
To identify molecular events in the observed pro-angiogenic effect of the PPAR agonist GW0742 on HUVECs, we performed quantitative RT-PCRs. First, we confirmed up-regulation of established PPARβ/δ target genes as Angiopoietin-like 4 (ANGPTL4) [36], Fatty acid binding protein 4 (FABP4) [37], Calcineurin (CNA) [8], and Pyruvate dehydrogenase kinase isoform 4 (PDK4) [38]. Next, we evaluated the expression of genes known to be involved in angiogenesis and found many of them to be up-regulated upon agonist treatment: Vascular endothelial growth factor receptor 1 (Vegfr1), Treatment of human umbilical vein endothelial cells (HUVECs) with GW0742 increased proliferation, while the PPARβ/δ antagonist GSK3787 tended to reduce HUVEC proliferation (Figure 4a). This is in agreement with previous works reporting enhanced endothelial cell proliferation [34] and activation of angiogenesis upon PPARβ/δ activation [7] and the general view of PPARβ/δ as a pro-angiogenic factor [4]. As an initial study suggested that PPARβ/δ is involved in endothelial cell apoptosis [35], we tested for differences in apoptosis upon modulation of PPARβ/δ. Rarely, TUNEL-positive endothelial cells were observed under any of the culture conditions (Figure 4b), suggesting that PPARβ/δ modulation does not influence apoptosis in vascular cells.  To identify molecular events in the observed pro-angiogenic effect of the PPAR agonist GW0742 on HUVECs, we performed quantitative RT-PCRs. First, we confirmed up-regulation of established PPARβ/δ target genes as Angiopoietin-like 4 (ANGPTL4) [36], Fatty acid binding protein 4 (FABP4) [37], Calcineurin (CNA) [8], and Pyruvate dehydrogenase kinase isoform 4 (PDK4) [38]. Next, we evaluated the expression of genes known to be involved in angiogenesis and found many of them to be up-regulated upon agonist treatment: Vascular endothelial growth factor receptor 1 (Vegfr1), which is critical for endothelial cell survival [39], and platelet/endothelial cell adhesion molecule-1 (CD31), regulating the endothelial cell vascular permeability barrier [40], Platelet-derived growth factor subunit B (PDGFB) and PDGF beta-receptor (PDGFRb), enhancing angiogenesis [17,41], Tyrosinekinase Kit (c-KIT), expressed on endothelial cells [42], implicated in tumor angiogenesis [43], and increasing migration and tube formation of endothelial cells [15,44], and also SRY-related HMG-box transcription factor 18 (SOX18), mediating physiologic and pathologic angiogenesis [45]. Although it has been suggested that Vascular endothelial growth factor (VEGF) is regulated by PPARβ/δ [7,46], we did not observe a statistically significant increase in expression, and no changes in Vascular endothelial growth factor receptor 2 (VEGFR2) expression and Von Willebrand factor (VWF), controlling blood vessel formation [47] (Figure 4c, upper panel). However, in HUVECs treated with the PPARβ/δ antagonist GSK3787, all of the mentioned genes were downregulated (Figure 4c, lower panel) supporting a pro-angiogenic function of PPARβ/δ.

Inducible Vascular-Specific Overexpression of PPARβ/δ Promotes Tumor Angiogenesis, Growth, and Spontaneous Metastases Formation In Vivo
To determine further the functional relevance of PPARβ/δ for tumor vessel formation and tumorigenesis, we used PPARβ/δ-flox +/− mice [12] crossed with Tamoxifen-inducible Tie2-CreERT2 animals. This Cre becomes activated in endothelial cells upon Tamoxifen induction [13]. Using this strategy, we obtained Tie2-CreERT2;PPARβ/δ animals with an inducible conditional vascular-specific overexpression of PPARβ/δ [10]. In adult Tie2-CreERT2;PPARβ/δ, Cre was activated by tamoxifen injection. Tie2-CreERT2;PPARβ/δ injected with vehicle and Tie2-CreERT2 mice injected with tamoxifen served as controls. We investigated the effects of vascular specific PPARβ/δ overexpression on tumor growth by overexpressing PPARβ/δ in Tie2 + cells, followed by subcutaneous implantation of LLC1 tumor cells. Tumor growth curves revealed increased tumor growth rates in mice with vessel-specific PPARβ/δ overexpression (Figure 5a). Three weeks after tumor cell injection, we determined tumor/body weight ratios. Body weights were comparable in all groups of mice (CreERT2;PPARβ/δ + vehicle 36.57 g ± 0.44 g, Tie2-CreERT2 + Tamoxifen 38.75 g ± 0.12 g, Tie2-CreERT2;PPARβ/δ + Tamoxifen 36.77 g ± 0.27 g). Of note, tumor weights were nearly doubled in the mice with vascular PPARβ/δ overexpression (Figure 5b,c). Next, we analyzed whether conditional overexpression of PPARβ/δ in vessels might modify metastasis occurrence. In this respect, the LLC1 model is very useful as it forms spontaneous metastases [15]. We investigated serial lung and liver sections of Tie2-CreERT2;PPARβ/δ + Tamoxifen mice and the respective control groups for metastases. All mice with vascular PPARβ/δ overexpression had lung metastases compared to 30% in the control groups (Figure 5d), and 50% displayed liver metastases whereas in the controls no liver metastases formation could be observed (Figure 5e). These observations strengthen the hypothesis that vascular PPARβ/δ enhances tumor growth and spontaneous metastatic spreading; and they are in perfect agreement with tumor growth inhibition and prolonged survival observed in PPARβ/δ -/mice [33]. We analyzed next PPARβ/δ and Cd31 expression in the tumors by immunostaining. Tie2-CreERT2;PPARβ/δ + Tamoxifen tumors displayed much higher PPARβ/δ and Cd31 positive vessel- Note that in Tie2-CreERT2;PPARβ/δ + Tamoxifen animals, metastases were so considerable in size that the whole photomicrograph shows a metastasis only. The plot on the right shows quantification of the percentage of animals with lung metastases. (e) Representative photomicrographs of liver sections from the three groups of animals. Metastases are indicated with a dotted green circle. In the two control groups, no liver metastases were detectable. Quantification of the percentage of animals with liver metastases is shown on the right. Scale bars indicate 50 µm. Data are mean ± SEM. * p < 0.05, * * * p < 0.001.
We analyzed next PPARβ/δ and Cd31 expression in the tumors by immunostaining. Tie2-CreERT2;PPARβ/δ + Tamoxifen tumors displayed much higher PPARβ/δ and Cd31 positive vessel-and area-densities than both control groups (Figure 6a). To confirm the endothelial PPARβ/δ overexpression on the RNA level, we sorted endothelial cells from the tumors and performed quantitative RT-PCRs. PPARβ/δ expression was nearly doubled in the tumor-derived endothelial cells from Tie2-CreERT2;PPARβ/δ + Tamoxifen mice compared to both controls (Figure 6b). Co-immunostaining for PCNA and Cd31 demonstrated more PCNA-positive endothelial cells in the tumors from animals with vessel-specific overexpression of PPARβ/δ than in the controls, indicating higher proliferation rates of endothelial cells, like it had been the case in the tumors of GW0742 agonist treated mice (Figure 6c).

PPARβ/δ Overexpression Upregulates Angiogenic and Metastases Promoting Molecules in Endothelial Cells In Vivo
To confirm the differential expression of genes we had observed upon pharmacological PPARβ/δ modulation in human endothelial cells and to elucidate further the molecular mechanisms upon PPARβ/δ overexpression in vascular cells in vivo, we performed quantitative RT-PCRs from tumor-sorted endothelial cells (Figure 7). Again, established PPARβ/δ target genes were upregulated except Pdk4, as likewise genes with established angiogenic functions: Vegf, Vegfr1 and 2, Cd31, vwf, Pdgfb, Pdgfrα, and β, Sox18, and c-kit. Furthermore, we found an increase of tumor-angiogenesis related genes such as E26 transformation specific factor 1 (Ets-1) [48,49], and Wt1 (Wilms' tumor suppressor 1) [15,48]. Interestingly, we could demonstrate that PPARβ/δ represses WT1 in human melanoma cells leading to decreased melanoma cell proliferation [5]. The upregulation of Wt1 upon overexpression of PPARβ/δ in endothelial cells observed here, could be due to cell type-specific expression of co-activators/co-repressors, which requires further studies. We additionally analyzed expression of matrix metalloproteinases 1, 8, and 9. It has been observed that pharmacological activation of PPARβ/δ in vascular cells increased MMP9 mRNA levels [7]. However, we could only find an increase for MMP8, which is known to positively regulate angiogenesis [50] in our model. Interleukin 1 and 6 (Il 1 and Il 6) expression was increased, pointing to an inflammatory state of endothelial cells; yet, Interleukin 18 (Il 18) and Tumor necrosis factor alpha (TNFα) expression were unchanged. Sirt1 and Vcam-1 were not differentially expressed. The mRNA expression of anti-angiogenic Thrombospondin -1 and its receptor Cd36 [51] was unaffected by PPARβ/δ overexpression. It seems that vascular overexpression of PPARβ/δ promotes exclusively a hyper-angiogenic phenotype rather than repressing anti-angiogenic pathways. This might contribute to the observed relatively weak phenotype of PPARβ/δ knockout animals, which are viable, develop normally, are fertile, and display only light growth, skin, adipose tissue, and myelinization alterations [52]. Finally, we observed upregulation of the metastases promoting [53,54] CC-chemokine ligands 2 and 5 (Ccl 2 and 5) in tumor-derived PPARβ/δ overexpressing endothelial cells (Figure 7).
In summary of these findings, the use of PPARβ/δ agonists, initially developed to treat hyperlipidemia or cardiovascular diseases, as anti-cancer drugs, even in the setting of an antiproliferative effect on the tumor cells, seems irresponsible. Although the PPARβ/δ agonist 501,516 entered clinical trials for the treatment of metabolic syndrome and diabetes in the beginning of 2000, these trials were stopped in 2007 due to multiple appearance of cancers in mice and rats (cited in [55]. Unfortunately, upon publication of the beneficial effects of PPARβ/δ activation against obesity and on exercise endurance in mice [12,56,57], the PPARβ/δ agonist GW501516 became very popular in the athletes community. overexpression. It seems that vascular overexpression of PPARβ/δ promotes exclusively a hyperangiogenic phenotype rather than repressing anti-angiogenic pathways. This might contribute to the observed relatively weak phenotype of PPARβ/δ knockout animals, which are viable, develop normally, are fertile, and display only light growth, skin, adipose tissue, and myelinization alterations [52]. Finally, we observed upregulation of the metastases promoting [53,54] CCchemokine ligands 2 and 5 (Ccl 2 and 5) in tumor-derived PPARβ/δ overexpressing endothelial cells (Figure 7). Quantitative RT-PCR of known PPARβ/δ target genes, inflammation, and angiogenesis markers in tumor-sorted endothelial cells from LLC1 bearing Tie2-CreERT2;PPARβ/δ + Tamoxifen animals and Tie2-CreERT2;PPARβ/δ +vehicle mice as controls (n = 6 each). Data are mean ± SEM. * P < 0.05, * * P < 0.01, * * * P < 0.001.

RNA Sequencing Further Certifies the Acquisition of a Highly angiogenic Endothelial Phenotype Upon PPARβ/δ Overexpression and Identifies Pro-Tumor-Angiogenic Signaling Networks
To analyze the transcriptome of gene expression patterns in PPARβ/δ overexpressing tumorderived endothelial cells, we sorted endothelial cells from the tumors of Tie2-CreERT2;PPARβ/δ injected with Tamoxifen, or vehicle as controls. mRNAs were after purity and quality controls submitted to sequencing. 283 genes were found to be differentially expressed in vascular cells overexpressing PPARβ/δ, most of them were up-regulated (Figure 8a and Supplementary Table S1). Cluster analysis of differentially expressed genes confirms mostly up-regulation of genes upon vascular overexpression of PPARβ/δ (Figure 8b). Again, this is in line with our observation in the quantitative PCRs, which indicate an angiogenesis boosting effect rather than a repression of anti-angiogenic molecules to enhance angiogenesis. Interestingly, gene ontology term analysis identified angiogenesis as the most upregulated biological process upon PPARβ/δ overexpression, followed by cell adhesion (Figure 8c). Top ten network analysis combined with a search for PPAR responsive elements (PPREs) identified in total six genes with potential PPREs: the three Vegf receptors 1 (Flt1), 2 (Kdr), and 3 (Flt4), all of them known to be implicated in the promotion of tumor angiogenesis [58,59], and Pdgfrβ, Pdgfb, and c-kit, which we had already found to be upregulated upon PPARβ/δ overexpression (Figure 7). Initial clinical efforts to inhibit tumor angiogenesis mainly focused on inhibition of the VEGF/VEGFR signaling, but often further disease progression could be observed. Angiogenesis involves multiple pathways, and it became clear that inhibition of only one pathway could be compensated by the others resulting in tumor progression. One of these compensatory pathways is platelet-derived growth factor PDGF and PDGFR signaling which led to the development of new anti-angiogenic therapies in the treatment of cancer [60]. Additionally, c-Kit belongs to the group of tumor angiogenesis-promoting molecules and new tyrosine kinase receptor inhibitors have been developed which target also c-Kit [61]. : diamonds and rectangles represent the top ten network; but only diamonds exhibited PPRE prediction. As the Vascular endothelial growth factor (VEGF) pathway has been suggested already to be regulated by PPARβ/δ [7,46], we decided to investigate further a potential regulation of Pdgfrβ, Pdgfb, and c-kit.  Figure 12a). Deletion of each of the three identified PPREs in the PDGFRβ promoter was sufficient to abolish transactivation by PPARβ/δ (Figure 9c). Regarding the PDGFB promoter, only the combined deletion of the three identified PPREs abrogated activation upon co-transfection of the PPARβ/δ construct, indicating a collaborative interaction between these PPREs (Figure 9f). Finally, in the c-Kit promoter, each deletion for the two identified binding regions was sufficient to cancel activation by PPARβ/δ (Figure 9i).
Binding of PPARβ/δ to the predicted PPREs was confirmed by chromatin immunoprecipitation (ChIP) assays. An antibody against acetylated histone H3 was used to check for nucleosome integrity. Specificity of the interaction of PPARβ/δ with the PPAR responsive elements in the PDGFRβ promoter is indicated by the lack of a PCR product when the same samples were amplified with primers specific for the 3 UTR. We confirmed binding to the identified PPREs in the PDGFRβ (Figure 10b,c), PDGFB (Figure 11b,c), and the c-Kit promoter (Figure 12b,c).   CHIP analyses are indicated. For promoter-deletion constructs, the indicated PPREs were removed from the promoter reporter constructs. Chromatin immunoprecipitation (ChIP, n = 4) was performed using polyclonal antibodies against PPARβ/δ or anti-acetyl-histone H3 antibody. Input DNA was used as a positive control for quantitative PCRs (b) or semiquantitative PCRs ((c), representative agarose gels) for the PDGFRβ promoter and respective 3 UTR sequences. Data are mean ± SEM. * p < 0.05. For promoter-deletion constructs, the indicated PPREs were removed from the promoter reporter constructs. Chromatin immunoprecipitation (ChIP, n = 3) was performed using polyclonal antibodies against PPARβ/δ or anti-acetyl-histone H3 antibody. Input DNA was used as a positive control for quantitative PCRs (b) or semiquantitative PCRs ((c), representative agarose gels) for the PDGFRβ promoter and respective 3 UTR sequences. Data are mean ± SEM. * p < 0.05, * * p < 0.01. positive control for quantitative PCRs (b) or semiquantitative PCRs ((c), representative agarose gels) for the PDGFRβ promoter and respective 3′UTR sequences. Data are mean ± SEM. * p < 0.05, * * p < 0.01. For promoter-deletion constructs, the indicated PPREs were excluded from the promoter reporter constructs. Chromatin immunoprecipitation (ChIP, n = 3) was performed using polyclonal antibodies against PPARβ/δ or anti-acetyl-histone H3 antibody. Input DNA was used as a positive control for quantitative PCRs (b) or semiquantitative PCRs ((c), representative agarose gels) for the PDGFRβ promoter and respective 3 UTR sequences. Data are mean ± SEM. * p < 0.05, * * p < 0.01, * * * p < 0.001.

Conclusions
We show here that pharmacological PPARβ/δ activation increases endothelial cell proliferation in vitro and intensifies tumor angiogenesis in vivo, enhancing tumor progression and metastases formation. Conditional vascular specific overexpression of PPARβ/δ also resulted in enhanced tumor angiogenesis, growth, and metastases formation, suggesting an endothelial cell-specific mechanism for PPARβ/δ function in tumor progression, independent from its contrasting effects on specific tumor cell types.
Tumor-angiogenesis promoting effects of PPARβ/δ are mediated via activation of the PDGF/PDGFR pathway, c-Kit, and probably the VEGF/VEGFR pathway. The pharmacological use of PPARβ/δ agonists should be considered as dangerous regarding its consistent consequences on tumor angiogenesis. Consideration of PPARβ/δ antagonists for inhibition of tumor angiogenesis and cancer growth is unlikely to represent a novel direction due to their controversial effects on the proliferation of different tumor cell types.