Background

Lung cancer (LC) with disappointing survival statistics is a leading cause of morbidity in both genders worldwide [1, 2]. The two main types of LC-s are small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) where the latter can be further classified into adeno- (AC), squamous cell- (SCC), large cell (LCC) and various mixed types carcinomas accounting all together for approximately 85% of all LC cases [3]. As the majority of patients are diagnosed at an advanced stage of the disease, the outcome is poor and the overall 5-year survival rarely exceeds 15% [4]. Naturally, earlier recognition would improve the outcome, but currently only a few treatment options are available to lung cancer sufferers [5] and the more specific treatments are largely based on identified driver mutations [6]. Unfortunately, only a small percentage of NSCLC patients have such characteristic mutations therefore the majority cannot benefit from targeted therapy [7]. Recognition that new blood vessel formation is important to tumor growth lead to development of angiogenesis inhibitors [8] to block tumor growth and disease progression. The first monoclonal antibody –bevacizumab- was approved against the human vascular endothelial growth factor A (VEGF-A) a key regulator of angiogenesis [9]. As VEGF-A promotes endothelial cell survival, migration, proliferation and vascular permeability it appeared an ideal target to “starve” the tumor and lead to tumor regression. Although VEGF-A is the main signaling molecule in pathological angiogenesis and is upregulated in many tumors [10] including in NSCLC-s [11] success of anti-angiogenic therapy in human cancers remained far from impressive.

Despite some positive results using bevacizumab mostly in combination therapy [12], patients mainly with squamous histology were excluded from treatment as increased risk of fatal side effects were observed [13]. The reasons for limited responsiveness or increased hemorrhage are still unknown, but several ideas have come to light. For example, the two types of NSCLC-s not only differ in genomic mutations [14], but AC and SCC also possess different intra-tumoral blood vessel formations. Kojima et al. have reported that microvessel density is higher in AC than SCC [15], while Yazdani et al. have hypothesized that intratumoral vessels are less covered by pericytes in SCC than AC, leading to more vulnerable and fragile vascular wall with increased necrosis in newly formed vessels in SCC [16]. As alternative signaling pathways, such as basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF) as well as miRNAs, especially the pro-angiogenic miR-27b and the miR-200 family [17, 18] also play a significant role in the regulation of angiogenesis; solely blocking VEGF-A simply cannot provide a therapeutic solution in NSCLCs [19]. Additionally, the underlying signaling mechanisms have not been fully elucidated that would also be essential to stratify the patient population subjected to anti-angiogenic therapies.

One of the controversial regulators of VEGF-A is peroxisome proliferator-activated receptor gamma (PPARgamma) that has been reported to inhibit endothelial cell function [1. PCR conditions were set as follows: one cycle 95 °C for 2min, 40 cycles at 95 °C for 5 s and 60 °C for 30 s. Changes in gene expression were calculated according to the 2-ddCt method.

Table 1 Quantitative real-time PCR primers
Full size table

TaqMan microRNA assay

Reverse transcription reaction was set up with ABI TaqMan microRNA assay kit (Thermo Fisher Scientific, Waltham, USA) using 100 ng of total RNA according to manufacturer’s recommendations. Each reaction contains specific miR-27b, miR-200b and U6 primers. PCR reaction was performed using TaqMan MicroRNA Assay (20x), TaqMan Universal Master Mix (2x) (Thermo Fisher Scientific, Waltham, USA) and product from reverse transcriptase reaction. TaqMan PCR reaction was performed using ABI StepOnePlus system and data were analyzed with StepOne software. PCR conditions set as follows: one cycle 95 °C for 10min, 40 cycles at 95 °C for 15 s and 60 °C for 60 s. MicroRNA expression was normalized to U6 expression.

Sections

Mice were anaesthetized with sodium pentobarbital intraperitoneally and lungs were filled up with 1:1 ratio of PBS:cryostate embedding media (TissueTek, Alphen aan den Rijn, Netherland), and frozen down at −80 °C. Human samples were collected in PBS containing 1% of FBS and then were filled up with PBS:cryostate embedding media and kept at −80 °C until processing. The 3D lung aggregates were carefully removed from the 96-well plates and embedded into TissueTek embedding media and immediately frozen down at −80 °C. For histological staining 8 μm thick cryostat sections were cut and fixed in 4% PFA for 20min.

Hematoxylin eosin staining

Eight micrometers thick cryostat sections or Transwell inserts (Corning, New York, USA) were cut and stained in Mayer’s hematoxylin solution (Sigma-Aldrich, St. Louis, USA) for 10min. Sections were washed in running tap water for 10min, then differentiated with 0.25% acetic acid (Sigma Aldrich, St. Louis, USA) for 1min. After the differentiation step, slides were washed with tap water and stained in eosin solution for 2min, then washed. Sections were mounted using Vectashield mounting medium (Vector Laboratories, Burlingame, USA). Images were taken using Nikon Eclipse Ti-U inverted microscope (Tokyo, Japan).

Antibodies, fluorescent and immunohistochemical staining

Paraffin embedded lung AC and SCC samples were stained with a routine IHC staining procedure using Vision Biosystems bondTM automated immunostainer (Leica, Wetzlar, Germany). Primary antibody of rat monoclonal anti-human Wnt5a (Clone 442625, R&D Systems, Minneapolis, USA) was used in 1:100 dilution. Cryostat sections were fixed. Fixed slides were rehydrated and blocked for 20min in 5% BSA (Sigma Aldrich, St. Louis, USA) in PBS. For mouse sections, Alexa Fluor 594 conjugated monoclonal anti-mouse CD31 (1:100, Clone MEC13.3, BioLegend, San Diego, USA), Alexa Fluor 488 conjugated monoclonal anti-mouse CD105 (1:100, Clone MJ7/18, BioLegend, San Diego, USA), purified monoclonal anti-mouse VEGF-A (1:100, Clone 1 F07-2C01, BioLegend, San Diego, USA) and purified monoclonal anti-mouse Wnt5a (1:50, Clone 442625, R&D Systems, Minneapolis, USA) primary antibodies were applied. For 3D lung tissue samples, the sections were stained with purified monoclonal anti-human CD31 antibody (1:100, Clone WM59, BioLegend, San Diego, USA). To detect VEGF-A expression in A549 and F11 cell line, purified monoclonal anti-human VEGF-A antibody (1:100, Clone 26503, R&D Systems, Minneapolis, USA) was used. The secondary antibodies were Alexa Fluor 488 or 555 conjugated anti-mouse IgG antibodies (1:200, Thermo Fisher Scientific, Waltham, USA), respectively. The nuclei were counterstained with TO-PRO-3 (1:1000, Thermo Fisher Scientific, Waltham, USA) and showed in blue as pseudo-color blue. Pictures were captured using Zeiss LSM 710 microscope (Zeiss, Oberkochen, Germany) equipped with analysis software. Images and fluorescent intensity were measured with Fiji software [46]. Intensity of two groups was analyzed with the independent samples t-test.

Statistical analysis

Statistical analysis was performed with SPSS version 20 software. Data are presented as mean ± standard error of mean (SEM), and statistical analysis was performed using the independent samples t-test and one-way ANOVA with Bonferroni correction. p < 0.05 was considered as significant.

Results

PPARgamma regulates VEGF-A expression

To clarify the role of PPARgamma in regulation of VEGF-A expression, lungs of PPARgamma knock-out mice were studied. Fluorescent immunohistochemistry of VEGF-A protein revealed a significantly higher expression of the VEGF-A protein in the lungs of PPARgamma KO mice than in their wild-type litter mates (Fig. 1a), indicating that PPARgamma inactivation is required for VEGF-A production. Emphasizing the initial observation, significantly (p < 0.05) increased VEGF-A expression was detected in primary clinical samples (Additional file 2: Table S1) of both AC and SCC (Fig. 1b), while PPARgamma levels were reduced (p < 0.05 and p < 0.01, respectively) in both tumor types compared to normal, non-diseased, primary lung controls. Additionally, comparative analysis of primary AC and SCC samples highlighted existing differences in the two NSCLC subtypes. While lower PPARgamma mRNA levels characterized significantly higher VEGF-A expression in AC, higher PPARgamma and lower VEGF-A mRNA described SCC (Fig. 1b).

Fig. 1
figure 1

VEGF-A expression in PPARgamma KO mice and human lung tumors. a, Hematoxylin eosin staining and immunofluorescent staining of the lung of wild type C57BL/6 and PPARgamma knock-out (KO) mice show a significantly increased level of mVEGF-A in KO lung tissues. Scale bars, 200 μm and 50 μm. Intensity data are representation of three independent experiments as mean ± SEM. b, Resected human lung AC and SCC revealed significant PPARgamma decrease and VEGF-A expression increase. Data are presented as mean ± SEM. One-way ANOVA, post hoc Bonferroni; n = 11 and n = 12 per groups. P < 0.05 was considered as significant, * p < 0.05, ** p < 0.01, *** p < 0.001

Full size image

To prove that canonical Wnt signaling induced downregulation of PPARgamma triggers VEGF-A expression, A549 lung adenocarcinoma cell line was treated with the chemical activator of the canonical Wnt signaling pathway, the beta-catenin activator LiCl [41]. Inhibition of PPAR reporter activity (Fig. 2a) by LiCl (10 mM) lead to increased VEGF-A mRNA (Fig. 2b) and protein expression (Fig. 2c). Inhibition of PPARgamma by LiCl did not affect PPARgamma mRNA expression suggesting that PPARgamma activity is the key factor in VEGF-A regulation.

Fig. 2
figure 2

VEGF-A expression following modification of PPARgamma activity in A549 lung adenocarcinoma cell line tranfected with PPRE control or reporter plasmid. a, Mimicking beta-catenin dependent canonical Wnt pathway activation using 10 mM LiCl led to significant decrease in PPRE reporter activity. b, 10 mM LiCl treatment induced VEGF-A mRNA expression compared to PPRE control cells and also c, 10 mM LiCl increased VEGF-A protein levels. Error bars, SEM. One-way ANOVA, post hoc Bonferroni; n = 4. Scale bars, 20 μm. d, VEGF-A mRNA expression decreased after 10 μM PPARgamma agonist treatment (RSG), while 10 μm PPARgamma specific antagonist (GW9662) increased VEGF-A transcript levels. Independent samples t-test, n = 3. e, VEGF-A protein level shows similar pattern after 10 μm RSG and 10 μm GW9662 treatment. Fluorescence intensity are representations of three different experiments as mean ± SEM. One-way ANOVA, post hoc Bonferroni; n = 3. Scale bars, 20 μm. P < 0.05 was considered as significant, * p < 0.05, ** p < 0.01, *** p < 0.001

Full size image

To support this observation and as the reporter system is not specific to PPARgamma, we used direct PPARgamma agonist and antagonist treatment of human fibroblast cells to determine the role of PPARgamma in VEGF-A regulation. Inhibition of PPARgamma upon GW9662 antagonist treatment increased VEGF-A levels, while the presence of a PPARgamma agonist resulted in decreased VEGF-A mRNA and protein levels (Fig. 2d and e) supporting the theory that VEGF-A upregulation in AC and SCC lung cancers are a direct consequence of PPARgamma reduction.

Wnt5a induces miR-27b a regulator of both PPARgamma expression and blood vessel branching

As only the beta-catenin dependent canonical Wnt signaling has been reported repeatedly to down-regulate PPARs [30, 44] and consequently up-regulate VEGF-A [47], it was not clear how SCC samples with high levels of non-canonical Wnt ligands (Fig. 3a and b) can trigger the same mechanism? In the literature PPARgamma has been described to have a highly conserved binding site in its 3′UTR that is a direct target of miR-27b [48]. Binding of miR-27b to its target sequence can suppress PPARgamma expression [49] and augment VEGF-A induced angiogenesis [50]. Importantly, both miR-27b as well as another miRNA, miR-200b can inhibit blood vessel branching during angiogenesis [51, 52]. Based on the above studies we theorized that differences in formation of blood vessel networks in AC and SCC are due to variations in microRNAs and VEGF-A levels. To investigate, both miR-27b and miR-200b levels were measured in primary human AC and SCC samples. Significantly higher expression levels were determined for both miRNA in primary SCC tissues compared to AC (Fig. 3c) indicating existence of miRNA dependent differential regulation of angiogenesis in the two NSCLC subtypes. To test whether the different molecular microenvironment characterized by increased Wnt5a levels in SCC would explain variations in miRNA expression, 3D lung aggregate cultures were exposed to non-canonical Wnt-s; rhWnt5a and rhWnt11. Wnt5a and Wnt11 were selected in the experiments as they were both reported to have higher levels in SCC than in AC [37]. Interestingly, while rhWnt11 had no effect on either miRNAs (Fig. 3d), miR-27b expression was significantly increased by rhWnt5a treatment (Fig. 3d), while miR-200b levels were unaffected. Remarkably, rhWnt5a could not up-regulate miR-27b in the presence of a PPARgamma agonist, only if the antagonist was present (Fig. 3e). The actual VEGF-A expression was, however, lower (Fig. 3e) than induced by the PPARgamma antagonist on its own. Additionally, while rhWnt5a treatment on its own did not increase VEGF-A levels at the time point analyzed (48 h treatment, data not shown) implicating a parallel mechanism that is required for PPARgamma inhibition even in the presence of the non-canonical Wnt pathway dominated microenvironment.

Fig. 3
figure 3

Transcript analysis of Wnt5a, miRNA and VEGF-A in primary human lung cancer samples of AC and SCC and 3D in vitro lung aggregate cultures. a, Wnt5a mRNA is significantly upregulated in SCC compared to both normal lung and AC specimens. Error bars, SEM. One-way ANOVA, post hoc Bonferroni; n = 11 and n = 12 per groups. b, Immunohistochemical staining of Wnt5a in primary resected AC and SCC samples, n = 3 per groups. c, miR-27b and miR-200b expression levels are significantly lower in AC compared to SCC. Error bars; SEM. Independent samples t-test, n = 5 per groups. d, miR-27b is up-regulated by rhWnt5a in 3D lung aggregate cultures, while neither miR-27b nor miR-200b was affected by rhWnt11. Error bars; SEM. One-way ANOVA, post hoc Bonferroni, n = 6. e, VEGF-A and miR-27b expression levels after 10 μM RSG (PPARgamma agonist) and 10 μM GW9662 (PPARgamma antagonist) and combination treatment with rhWnt5a. Error bars; SEM. Independent samples t-test n = 3. P < 0.05 was considered as significant, * p < 0.05, ** p < 0.01, *** p < 0.001

Full size image

Wnt5 regulates VEGF-A induced endothelial cell motility

As VEGF-A has previously been determined to stimulate endothelial cell proliferation and migration [53], expression of endoglin (Eng or CD105) a transmembrane auxillary receptor for transforming growth factor-beta (TGF-beta) that is predominantly expressed on proliferating endothelial cells was also tested [54]. Analysis of primary SCC and AC tissue samples revealed that the endothelial cell proliferation marker Eng (CD105) was significantly lower in SCC compared to AC (Fig. 4a and b). Although VEGF-A was significantly higher than normal in both cancer subtypes, VEGF-A levels in SCC were lower than in AC (Fig. 1b). To be able to study the molecular effects of high VEGF-A levels on endothelial cells, in vitro studies were performed using a three dimensional (3D) human lung aggregate model tissue consisting of primary human small airway epithelial cells (SAEC), microvascular lung endothelial cells (HMVEC-L), and human fibroblasts (NHLF and F11) (Additional file 2: Figure S1). The 3D aggregate culture conditions provided close to natural, yet defined, cellular environment for molecular studies. qRT-PCR analysis of the lung model tissue aggregates revealed highly similar expression levels of angiogenic stimulators including VEGF-A, IL-1beta and HIF-1alpha to primary human lungs (Additional file 3: Figure S2) indicating that the model is suitable to study pro- and/or anti-angiogenic mechanisms. To recreate the VEGF-A high tumor-like microenvironment VEGF-A165 was cloned into human fibroblast cells (Additional file 4: Figure S3). The effects of VEGF-A were investigated by comparing aggregates with VEGF-Anormal and VEGF-A over-expressing (VEGF-Ahigh) fibroblasts (Additional file 5: Figure S4). Flow cytometric analysis revealed that high levels of VEGF-A lead to elevated Eng (CD105) expression on CD31+ endothelial cells (Fig. 4c, Additional file 6: Figure S5). To test whether Wnt5a can modulate VEGF-Ahigh microenvironment, 3D lung tissue aggregates containing VEGF-Anormal and VEGF-Ahigh fibroblasts were exposed to rhWnt5a. Flow cytometric analysis revealed that rhWnt5a did not block proliferation marker Eng (CD105) expression (Fig. 4c, Additional file 6: Figure S5) indicating that Eng (CD105) is not under Wnt5a control.

Fig. 4
figure 4

The effect of Wnt5a on VEGF-A induced endothelial cell activation and motility. a, CD105 mRNA expression is significantly higher in primary AC compared to SCC samples. Error bars, SEM. One-way ANOVA, post hoc Bonferroni; n = 11 and n = 12 per groups. b, Flow cytometric analysis of CD105 protein expression in CD31 positive endothelial cells in primary AC and SCC samples. n = 6 per groups. c, Flow cytometric analysis of CD105 levels in normal and high VEGF-A microenvironment in 3D lung aggregate tissues has also shown an increase of activation marker CD105 in VEGF-Ahigh tissues. The double positive (CD105/CD31) cell population was considered as activated endothelial cells. Independent samples t-test, n = 6. 1 μg/ml rhWnt5a treatment had no effect on the VEGF-A induced endothelial cell activation measured by the double positive (CD105/CD31) cell population identified by flow cytometric analysis. Independent samples t-test, n = 6. d, Localization of endothelial cells was identified by immunoflurescent staining of CD31 and analyzed by confocal microscopy in 3D lung tissue aggregates. In VEGF-Anormal microenvironment endothelial cells remained diffuse in the tissue. Under VEGF-A excess endothelial cell migrated towards the source (VEGF-Ahigh fibroblasts) of the signal in the center of the aggregate tissue. 1 μg/ml rhWnt5a treatment of VEGF-Ahigh tissue aggregates inhibited endothelial cell accumulation in the center of the aggregate. Bar chart represents the quantification of endothelial cell distribution. Relative area of CD31+ endothelial cells are compared to total field. Percentages were calculated as relative area of endothelial cells/area of total field *100. Error bars; SEM. Independent samples t-test n = 3. Representative images of three independent experiments are shown. Scale bars, 50 μm. e, HMVEC-L transwell migration assay. Endothelial cells migrate significantly faster towards VEGF-Ahigh fibroblast, while 1 μg/ml rhWnt5a can reverse the effect of elevated VEGF-A level. Scale bar 100 μm. One-way ANOVA, n = 3. P < 0.05 was considered as significant, * p < 0.05, ** p < 0.01, *** p < 0.001

Full size image

This result was surprising. Because the endothelial cell proliferation marker Eng (CD105) expression was lower in SCC compared to AC, and VEGF-A levels were similarly elevated in both cancer types, endothelial cell proliferation would be expected. To investigate, lung tissues of control and PPARgamma KO mice were stained for CD105. Significantly lower levels of CD105 was detected in PPARgamma KO animals than in their wild type controls (Additional file 7: Figure S6) indicating that PPARgamma associated events are responsible for the reduction but not Wnt5a. As Wnt5a levels are not affected in lungs of the PPARgamma KO animals (Additional file 8: Figure S7), our findings further suggest that CD105 expression is not under Wnt5a control. To clarify the role of Wnt5a, cellular motility was investigated in the model cultures. All these findings led us to the hypothesis that endothelial activity cannot be the only answer to the different therapeutic response. As SCC possesses more vulnerable capillaries [16] and lower microvessel density [15], it might be an indication of an altered endothelial behavior, especially in migration and motility.

In the VEGF-Ahigh microenvironment endothelial cells migrated towards the source of VEGF-A, the VEGF-Ahigh fibroblasts (Fig. 4d and Additional file 5: Figure S4). As fibroblasts naturally provide the core of the 3D lung tissue aggregate co-cultures, endothelial cells concentrated in the center of the tissue aggregate. In the aggregate tissues with normal VEGF-A levels the endothelial cells remained evenly distributed (Fig. 4d). To investigate whether Wnt5a affects endothelial cell motility, aggregate cultures were treated with rhWnt5a. This inhibited endothelial cell migration towards VEGF-Ahigh fibroblasts, and endothelial cells remained scattered amongst other cell types in the aggregate lung tissue. The inhibitory effect of Wnt5a on endothelial cell motility was tested using migration assays in transwell chambers. HMVEC-L cells migrated significantly faster towards VEGF-Ahigh fibroblasts, while addition of recombinant human Wnt5a significantly inhibited VEGF-A induced endothelial cell migration (Fig. 4e).

Discussion

While cancer progression is frequently attributed to increased angiogenesis, molecular regulation that can lead to significant differences in anti-angiogenic therapy responses have not been reported. As AC and SCC have characteristic molecular differences in their Wnt microenvironment [37] and differential treatment responses to anti-angiogenic therapy [13] including increased necrosis and pulmonary hemorrhage in SCC, comparative studies of the two NSCLC subtypes provided a promising platform for molecular studies of blood vessel formation. Recent evidence suggested that adverse reactions in SCC patients might be due to malfunction of endothelial repair processes [55], tumor type specific erosion of blood vessels [56] or involvement of major vascular structures in SCC [57]. Our studies provided an additional dimension to the potential mechanism by making extensive use of primary human lung tissues both of normal lung and resected lung AC and SCC tumors, knock-out mice as well as an in vitro 3D human lung tissues model assembled to assess the role of Wnt5a in angiogenesis. Based on our study, we postulate that decreased expression of PPARgamma is primarily responsible for induction of increased VEGF-A levels detected both in AC and SCC. However, the molecular mechanism that leads to PPARgamma down-regulation is different in AC and SCC, and it is this molecular mechanism rather than tumor localization that leads to more serious adverse reactions in SCC patients.

This conclusion is based on several pieces of evidence presented here. Down-regulation of PPARgamma was demonstrated to be essential for up-regulation of the angiogenesis stimulator factor VEGF-A using PPARgamma KO animals, cell lines and a fully human in vitro lung tissue aggregate system where the PPRE reporter system, application of PPARgamma agonist and antagonist supported direct regulatory interactions between the two molecules. Consequently, the significantly reduced PPARgamma and increased VEGF-A levels in both primary AC and SCC explained increased vascularization in both tumors. Differential therapeutic response, however, could not be explained by the very similar molecular microenvironments of the two tumor subtypes.

For better understanding of the process of PPARgamma down-regulation we considered that down-regulation of PPARgamma transcription is a beta-catenin-dependent and therefore canonical Wnt pathway dependent process [44]. As lung ACs are characterized by increased canonical and therefore beta-catenin dependent Wnt signal activity [

Abbreviations

AC:

Adenocarcinoma

APC:

Allophycocyanine

bFGF:

Basic fibroblast growth factor

EGM-2 MV:

Microvascular endothelial cell growth medium

FGM-2:

Fibroblast growth medium

HMVEC-L:

Human lung microvascular endothelial cell

LC:

Lung cancer

NHLF:

Normal human lung fibroblast

NSCLC:

Non small cell lung cancer

PBS:

Phosphate buffered saline

PCP:

Planar cell polarity

PDGF:

Platelet derived growth factor

PFA:

Paraformaldehyde

PPAR:

Peroxisome proliferator-activated receptor

PPARgamma KO:

PPARgamma knock-out

PPRE:

PPAR response element

qRT-PCR:

Quantitative real-time polymerase chain reaction

RSG:

Rosiglitazone

SAEC:

Small airway epithelial cell

SAGM:

Small airway cell growth medium

SCC:

Squamous cell carcinoma

SCLC:

Small cell lung cancer

VEGF:

Vascular endothelial growth factor

References

  1. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ. Cancer statistics. CA Cancer J Clin [Internet]. 2009;59:225–49. [cited 2015 Jul 16] Available from: http://www.ncbi.nlm.nih.gov/pubmed/19474385.

    Article  PubMed  Google Scholar 

  2. Ettinger DS, Akerley W, Borghaei H, Chang AC, Cheney RT, Chirieac LR, et al. Non-small cell lung cancer. J Natl Compr Canc Netw. [Internet] 2012;10:1236–71. Available from: https://www.ncbi.nlm.nih.gov/pubmed/23054877.

  3. Molina JR, Yang P, Cassivi SD, Schild SE, Adjei AA. Non-small cell lung cancer: epidemiology, risk factors, treatment, and survivorship. Mayo Clin Proc [Internet]. 2008;83:584–94. [cited 2014 Nov 16] Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2718421&tool=pmcentrez&rendertype=abstract.

    Article  Google Scholar 

  4. D’Addario G, Früh M, Reck M, Baumann P, Klepetko W, Felip E. Metastatic non-small-cell lung cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol [Internet]. 2010;21 Suppl 5:v116–9. [cited 2015 Sep 12] Available from: http://annonc.oxfordjournals.org/content/21/suppl_5/v116.full.

    Article  Google Scholar 

  5. Stahel R, Peters S, Baas P, Brambilla E, Cappuzzo F, De Ruysscher D, et al. Strategies for improving outcomes in NSCLC: a look to the future. Lung Cancer [Internet]. 2013;82:375–82. [cited 2015 Oct 16] Available from: http://www.sciencedirect.com/science/article/pii/S0169500213003905.

    Article  Google Scholar 

  6. Vogelstein B, Papadopoulos N, Velculescu VE, Zhou S, Diaz LA, Kinzler KW. Cancer genome landscapes. Science [Internet]. 2013;339:1546–58. [cited 2014 Jul 9] Available from: http://www.sciencemag.org/content/339/6127/1546.abstract.

    CAS  Google Scholar 

  7. Boolell V, Alamgeer M, Watkins D, Ganju V. The evolution of therapies in non-small cell lung cancer. Cancers (Basel) [Internet]. 2015;7:1815–46. [cited 2015 Oct 16] Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=4586797&tool=pmcentrez&rendertype=abstract.

    Article  Google Scholar 

  8. Korpanty G, Smyth E, Sullivan LA, Brekken RA, Carney DN. Antiangiogenic therapy in lung cancer: focus on vascular endothelial growth factor pathway. Exp Biol Med (Maywood) [Internet]. 2010;235:3–9. SAGE Publications. [cited 2015 Nov 13] Available from: http://ebm.sagepub.com/content/235/1/3.full.

    Article  CAS  Google Scholar 

  9. Moens S, Goveia J, Stapor PC, Cantelmo AR, Carmeliet P. The multifaceted activity of VEGF in angiogenesis - Implications for therapy responses. Cytokine Growth Factor Rev [Internet]. 2014;25:473–82. [cited 2015 Sep 21] Available from: http://www.ncbi.nlm.nih.gov/pubmed/25169850.

    Article  CAS  Google Scholar 

  10. Ranieri G, Patruno R, Ruggieri E, Montemurro S, Valerio P, Ribatti D. Vascular endothelial growth factor (VEGF) as a target of bevacizumab in cancer: from the biology to the clinic. Curr Med Chem [Internet]. 2006;13:1845–57. [cited 2015 Oct 21] Available from: http://www.ncbi.nlm.nih.gov/pubmed/16842197.

    Article  CAS  Google Scholar 

  11. Zhan P, Wang J, Lv X, Wang Q, Qiu L, Lin X, et al. Prognostic value of vascular endothelial growth factor expression in patients with lung cancer: a systematic review with meta-analysis. J Thorac Oncol [Internet]. 2009;4:1094–103. [cited 2015 Oct 20] Available from: http://www.ncbi.nlm.nih.gov/pubmed/19687765.

    Article  Google Scholar 

  12. Sandler A, Gray R, Perry MC, Brahmer J, Schiller JH, Dowlati A, et al. Paclitaxel-carboplatin alone or with bevacizumab for non-small-cell lung cancer. N Engl J Med [Internet]. 2006;355:2542–50. [cited 2015 Sep 30] Available from: http://www.ncbi.nlm.nih.gov/pubmed/17167137.

    Article  CAS  PubMed  Google Scholar 

  13. Piperdi B, Merla A, Perez-Soler R. Targeting angiogenesis in squamous non-small cell lung cancer. Drugs [Internet]. 2014;74:403–13. [cited 2015 Sep 8] Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=4014113&tool=pmcentrez&rendertype=abstract.

    Article  CAS  Google Scholar 

  14. Oliver TG, Patel J, Akerley W. Squamous non-small cell lung cancer as a distinct clinical entity. Am J Clin Oncol [Internet]. 2015;38:220–6. [cited 2015 Oct 21] Available from: http://www.ncbi.nlm.nih.gov/pubmed/25806712.

    Article  CAS  Google Scholar 

  15. Kojima H, Shijubo N, Abe S. Thymidine phosphorylase and vascular endothelial growth factor in patients with Stage I lung adenocarcinoma. Cancer [Internet]. 2002;94:1083–93. [cited 2015 Oct 21] Available from: http://www.ncbi.nlm.nih.gov/pubmed/11920479.

    Article  CAS  Google Scholar 

  16. Yazdani S, Miki Y, Tamaki K, Ono K, Iwabuchi E, Abe K, et al. Proliferation and maturation of intratumoral blood vessels in non-small cell lung cancer. Hum Pathol [Internet]. 2013;44:1586–96. [cited 2015 Oct 20] Available from: http://www.ncbi.nlm.nih.gov/pubmed/23522064.

    Article  PubMed  Google Scholar 

  17. Pan H, Clarke B, Wickline SA. Macrophage pro-angiogenic miRNA, miR-27b, is under NF-kB transcription regulation. FASEB J. 2012;26:1120.9–1120.9. Federation of American Societies for Experimental Biology.

  18. Pecot CV, Rupaimoole R, Yang D, Akbani R, Ivan C, Lu C, et al. Tumour angiogenesis regulation by the miR-200 family. Nat Commun [Internet]. 2013;4:2427. [cited 2016 Sep 6] Available from: http://www.ncbi.nlm.nih.gov/pubmed/24018975.

    Google Scholar 

  19. Vasudev NS, Reynolds AR. Anti-angiogenic therapy for cancer: current progress, unresolved questions and future directions. Angiogenesis [Internet]. 2014;17:471–94. [cited 2015 Sep 8] Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=4061466&tool=pmcentrez&rendertype=abstract.

    Article  CAS  Google Scholar 

  20. **n X, Yang S, Kowalski J, Gerritsen ME. Peroxisome proliferator-activated receptor gamma ligands are potent inhibitors of angiogenesis in vitro and in vivo. J Biol Chem [Internet]. 1999;274:9116–21. [cited 2015 Jul 20] Available from: http://www.ncbi.nlm.nih.gov/pubmed/10085162.

    Article  CAS  PubMed  Google Scholar 

  21. Jiménez R, Sánchez M, Zarzuelo MJ, Romero M, Quintela AM, López-Sepúlveda R, et al. Endothelium-dependent vasodilator effects of peroxisome proliferator-activated receptor beta agonists via the phosphatidyl-inositol-3 kinase-Akt pathway. J Pharmacol Exp Ther [Internet]. 2010;332:554–61. [cited 2015 Jul 20] Available from: http://www.ncbi.nlm.nih.gov/pubmed/19906781.

    Article  Google Scholar 

  22. Peeters LLH, Vigne J-L, Tee MK, Zhao D, Waite LL, Taylor RN. PPAR $\upgamma$ represses VEGF expression in human endometrial cells: Implications for uterine angiogenesis. Angiogenesis [Internet]. 2006;8:373–9. [cited 2015 Oct 21] Available from: http://www.ncbi.nlm.nih.gov/pubmed/16400517.

    Article  Google Scholar 

  23. Gacche RN. Compensatory angiogenesis and tumor refractoriness. Oncogenesis [Internet]. 2015;4:e153. Macmillan Publishers Limited. [cited 2015 Nov 13] Available from: http://dx.doi.org/10.1038/oncsis.2015.14.

    Article  CAS  Google Scholar 

  24. Margeli A, Kouraklis G, Theocharis S. Peroxisome proliferator activated receptor-gamma (PPAR-gamma) ligands and angiogenesis. Angiogenesis [Internet]. 2003;6:165–9. [cited 2015 Oct 20] Available from: http://www.ncbi.nlm.nih.gov/pubmed/15041792.

    Article  CAS  Google Scholar 

  25. Jerkic M, Rivas-Elena JV, Prieto M, Carrón R, Sanz-Rodríguez F, Pérez-Barriocanal F, et al. Endoglin regulates nitric oxide-dependent vasodilatation. FASEB J [Internet]. 2004;18:609–11. [cited 2015 Jul 20] Available from: http://www.ncbi.nlm.nih.gov/pubmed/14734648.

    CAS  Google Scholar 

  26. Lebrin F, Goumans M-J, Jonker L, Carvalho RLC, Valdimarsdottir G, Thorikay M, et al. Endoglin promotes endothelial cell proliferation and TGF-beta/ALK1 signal transduction. EMBO J [Internet]. 2004;23:4018–28. [cited 2015 Jul 20] Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=524335&tool=pmcentrez&rendertype=abstract.

    Article  CAS  Google Scholar 

  27. Salomone S, Drago F. Effects of PPARγ ligands on vascular tone. Curr Mol Pharmacol [Internet]. 2012;5:282–91. [cited 2015 Oct 20] Available from: http://www.ncbi.nlm.nih.gov/pubmed/22122458.

    Article  CAS  Google Scholar 

  28. Toporsian M, Gros R, Kabir MG, Vera S, Govindaraju K, Eidelman DH, et al. A role for endoglin in coupling eNOS activity and regulating vascular tone revealed in hereditary hemorrhagic telangiectasia. Circ Res [Internet]. 2005;96:684–92. [cited 2015 Oct 20] Available from: http://www.ncbi.nlm.nih.gov/pubmed/15718503.

    Article  CAS  Google Scholar 

  29. Albrecht EWJA, Stegeman CA, Heeringa P, Henning RH, van Goor H. Protective role of endothelial nitric oxide synthase. J Pathol [Internet]. 2003;199:8–17. [cited 2015 Oct 29] Available from: http://www.ncbi.nlm.nih.gov/pubmed/12474221.

    Article  Google Scholar 

  30. Takada I, Kouzmenko AP, Kato S. Wnt and PPARgamma signaling in osteoblastogenesis and adipogenesis. Nat Rev Rheumatol [Internet]. 2009;5:442–7. [cited 2015 Sep 9] Available from: http://www.ncbi.nlm.nih.gov/pubmed/19581903.

    Article  CAS  Google Scholar 

  31. Lecarpentier Y, Claes V, Duthoit G, Hébert J-L. Circadian rhythms, Wnt/beta-catenin pathway and PPAR alpha/gamma profiles in diseases with primary or secondary cardiac dysfunction. Front Physiol [Internet]. 2014;5:429. [cited 2016 Feb 25] Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=4220097&tool=pmcentrez&rendertype=abstract.

    PubMed  PubMed Central  Google Scholar 

  32. Dejana E. The role of wnt signaling in physiological and pathological angiogenesis. Circ Res [Internet]. 2010;107:943–52. [cited 2015 Oct 21] Available from: http://www.ncbi.nlm.nih.gov/pubmed/20947863.

    Article  CAS  Google Scholar 

  33. He B, Barg RN, You L, Xu Z, Reguart N, Mikami I, et al. Wnt signaling in stem cells and non-small-cell lung cancer. Clin Lung Cancer [Internet]. 2005;7:54–60. [cited 2015 Oct 21] Available from: http://www.ncbi.nlm.nih.gov/pubmed/16098245.

    Article  CAS  Google Scholar 

  34. Pongracz JE, Stockley RA. Wnt signalling in lung development and diseases. Respir Res [Internet]. 2006;7:15. [cited 2015 Sep 9] Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1397816&tool=pmcentrez&rendertype=abstract.

    Article  Google Scholar 

  35. Rao TP, Kühl M. An updated overview on Wnt signaling pathways: a prelude for more. Circ Res [Internet]. 2010;106:1798–806. [cited 2015 Jun 1] Available from: http://circres.ahajournals.org/content/106/12/1798.full.

    Article  CAS  Google Scholar 

  36. Stewart DJ. Wnt signaling pathway in non-small cell lung cancer. J Natl Cancer Inst [Internet]. 2014;106:djt356. [cited 2015 Jun 17] Available from: http://www.ncbi.nlm.nih.gov/pubmed/24309006.

    Article  Google Scholar 

  37. Bartis D, Csongei V, Weich A, Kiss E, Barko S, Kovacs T, et al. Down-regulation of canonical and up-regulation of non-canonical Wnt signalling in the carcinogenic process of squamous cell lung carcinoma. PLoS One [Internet]. 2013;8:e57393. [cited 2015 Jul 20] Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3591434&tool=pmcentrez&rendertype=abstract.

    Article  CAS  Google Scholar 

  38. Cheng C, Yeh J, Fan T-P, Smith SK, Charnock-Jones DS. Wnt5a-mediated non-canonical Wnt signalling regulates human endothelial cell proliferation and migration. Biochem Biophys Res Commun [Internet]. 2008;365:285–90. [cited 2015 Oct 21] Available from: http://www.ncbi.nlm.nih.gov/pubmed/17986384.

    Article  CAS  PubMed  Google Scholar 

  39. Nadra K, Quignodon L, Sardella C, Joye E, Mucciolo A, Chrast R, et al. PPARgamma in placental angiogenesis. Endocrinology [Internet]. 2010;151:4969–81. [cited 2016 Mar 2] Available from: http://www.ncbi.nlm.nih.gov/pubmed/20810566.

    Article  CAS  Google Scholar 

  40. Geraghty RJ, Capes-Davis A, Davis JM, Downward J, Freshney RI, Knezevic I, et al. Guidelines for the use of cell lines in biomedical research. Br J Cancer [Internet]. 2014;111:1021–46. [cited 2016 Sep 6] Available from: http://www.ncbi.nlm.nih.gov/pubmed/25117809.

    Article  PubMed  PubMed Central  Google Scholar 

  41. Haq S, Michael A, Andreucci M, Bhattacharya K, Dotto P, Walters B, et al. Stabilization of beta-catenin by a Wnt-independent mechanism regulates cardiomyocyte growth. Proc Natl Acad Sci U S A [Internet]. 2003;100:4610–5. National Academy of Sciences; [cited 2016 Sep 7] Available from: http://www.ncbi.nlm.nih.gov/pubmed/12668767.

    Article  CAS  Google Scholar 

  42. Whiteside C, Wang H, **a L, Munk S, Goldberg HJ, Fantus IG, et al. Rosiglitazone prevents high glucose-induced vascular endothelial growth factor and collagen IV expression in cultured mesangial cells. Exp Diabetes Res [Internet]. 2009;2009:1–11. Hindawi Publishing Corporation. [cited 2016 Aug 8] Available from: http://www.hindawi.com/journals/jdr/2009/910783/.

    Article  Google Scholar 

  43. Seargent JM, Yates EA, Gill JH. GW9662, a potent antagonist of PPARgamma, inhibits growth of breast tumour cells and promotes the anticancer effects of the PPARgamma agonist rosiglitazone, independently of PPARgamma activation. Br J Pharmacol [Internet]. 2004;143:933–7. Wiley-Blackwell. [cited 2016 Aug 8] Available from: http://www.ncbi.nlm.nih.gov/pubmed/15533890.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Kovacs T, Csongei V, Feller D, Ernszt D, Smuk G, Sarosi V, et al. Alteration in the Wnt microenvironment directly regulates molecular events leading to pulmonary senescence. Aging Cell [Internet]. 2014;13:838–49. [cited 2015 Jul 20] Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=4331750&tool=pmcentrez&rendertype=abstract.

    Article  CAS  Google Scholar 

  45. Bovia F. Efficient transduction of primary human B lymphocytes and nondividing myeloma B cells with HIV-1-derived lentiviral vectors. Blood [Internet]. 2002;101:1727–33. [cited 2015 Sep 23] Available from: http://www.ncbi.nlm.nih.gov/pubmed/12406892.

    Article  Google Scholar 

  46. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods [Internet]. 2012;9:676–82. [cited 2016 Aug 8] Available from: http://www.ncbi.nlm.nih.gov/pubmed/22743772.

    Article  CAS  Google Scholar 

  47. Chintalgattu V, Harris GS, Akula SM, Katwa LC. PPAR-gamma agonists induce the expression of VEGF and its receptors in cultured cardiac myofibroblasts. Cardiovasc Res [Internet]. 2007;74:140–50. [cited 2016 Aug 8] Available from: http://www.ncbi.nlm.nih.gov/pubmed/17320065.

    Article  CAS  PubMed  Google Scholar 

  48. Karbiener M, Fischer C, Nowitsch S, Opriessnig P, Papak C, Ailhaud G, et al. microRNA miR-27b impairs human adipocyte differentiation and targets PPARgamma. Biochem Biophys Res Commun [Internet]. 2009;390:247–51. [cited 2015 Oct 30] Available from: http://www.ncbi.nlm.nih.gov/pubmed/19800867.

    Article  CAS  PubMed  Google Scholar 

  49. Jennewein C, von Knethen A, Schmid T, Brüne B. MicroRNA-27b contributes to lipopolysaccharide-mediated peroxisome proliferator-activated receptor gamma (PPARgamma) mRNA destabilization. J Biol Chem [Internet]. 2010;285:11846–53. [cited 2015 Sep 8] Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2852921&tool=pmcentrez&rendertype=abstract.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Veliceasa D, Biyashev D, Qin G, Misener S, Mackie AR, Kishore R, et al. Therapeutic manipulation of angiogenesis with miR-27b. Vasc Cell [Internet]. 2015;7:6. [cited 2015 Nov 13] Available from: http://www.vascularcell.com/content/7/1/6.

    Article  Google Scholar 

  51. Hannafon BN, Carpenter KJ, Berry WL, Janknecht R, Dooley WC, Ding W-Q. Exosome-mediated microRNA signaling from breast cancer cells is altered by the anti-angiogenesis agent docosahexaenoic acid (DHA). Mol Cancer [Internet]. 2015;14:133. [cited 2015 Jul 18] Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=4504101&tool=pmcentrez&rendertype=abstract.

    Article  PubMed  PubMed Central  Google Scholar 

  52. Choi Y-C, Yoon S, Jeong Y, Yoon J, Baek K. Regulation of vascular endothelial growth factor signaling by miR-200b. Mol Cells [Internet]. 2011;32:77–82. Korean Society for Molecular and Cellular Biology. [cited 2016 Sep 6] Available from: http://springer.longhoe.net/10.1007/s10059-011-1042-2.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Hoeben A, Landuyt B, Highley MS, Wildiers H, Van Oosterom AT, De Bruijn EA. Vascular endothelial growth factor and angiogenesis. Pharmacol Rev [Internet]. 2004;56:549–80. [cited 2015 Jan 26] Available from: http://www.ncbi.nlm.nih.gov/pubmed/15602010.

    Article  CAS  PubMed  Google Scholar 

  54. Liu Z, Lebrin F, Maring JA, van den Driesche S, van der Brink S, van Dinther M, et al. ENDOGLIN is dispensable for vasculogenesis, but required for vascular endothelial growth factor-induced angiogenesis. PLoS One [Internet]. 2014;9:e86273. Public Library of Science. [cited 2015 Oct 23] Available from: http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0086273.

    Article  Google Scholar 

  55. Schmidinger M, Bellmunt J. Plethora of agents, plethora of targets, plethora of side effects in metastatic renal cell carcinoma. Cancer Treat Rev [Internet]. 2010;36:416–24. [cited 2015 Sep 9] Available from: http://www.ncbi.nlm.nih.gov/pubmed/20163917.

    Article  CAS  Google Scholar 

  56. Kamba T, McDonald DM. Mechanisms of adverse effects of anti-VEGF therapy for cancer. Br J Cancer [Internet]. 2007;96:1788–95. [cited 2015 Aug 31] Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2359962&tool=pmcentrez&rendertype=abstract.

    Article  CAS  Google Scholar 

  57. Reck M, Barlesi F, Crinò L, Henschke CI, Isla D, Stiebeler S, et al. Predicting and managing the risk of pulmonary haemorrhage in patients with NSCLC treated with bevacizumab: a consensus report from a panel of experts. Ann Oncol [Internet]. 2012;23:1111–20. [cited 2015 Sep 8] Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3335247&tool=pmcentrez&rendertype=abstract.

    Article  CAS  Google Scholar 

  58. Li X-Q, Yang X-L, Zhang G, Wu S-P, Deng X-B, **ao S-J, et al. Nuclear β-catenin accumulation is associated with increased expression of Nanog protein and predicts poor prognosis of non-small cell lung cancer. J Transl Med [Internet]. 2013;11:114. [cited 2015 Nov 13] Available from: http://www.translational-medicine.com/content/11/1/114.

    Article  CAS  Google Scholar 

  59. Yao L, Sun B, Zhao X, Zhao X, Gu Q, Dong X, et al. Overexpression of Wnt5a promotes angiogenesis in NSCLC. Biomed Res Int [Internet]. 2014;2014:832562. Available from: [cited 2015 Oct 27] http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=4066942&tool=pmcentrez&rendertype=abstract.

    Google Scholar 

  60. Biselli-Chicote PM, Oliveira ARCP, Pavarino EC, Goloni-Bertollo EM. VEGF gene alternative splicing: pro- and anti-angiogenic isoforms in cancer. J Cancer Res Clin Oncol [Internet]. 2012;138:363–70. Springer-Verlag. [cited 2016 Sep 6] Available from: http://springer.longhoe.net/10.1007/s00432-011-1073-2.

    Article  CAS  PubMed  Google Scholar 

  61. Lee A, Frischer J, Serur A, Huang J, Bae J-O, Kornfield ZN, et al. Inhibition of cyclooxygenase-2 disrupts tumor vascular mural cell recruitment and survival signaling. Cancer Res [Internet]. 2006;66:4378–84. [cited 2016 Sep 7] Available from: http://www.ncbi.nlm.nih.gov/pubmed/16618763.

    Article  CAS  PubMed  Google Scholar 

  62. Bishop-Bailey D, Swales KE, Bishop-Bailey D, Swales KE. The Role of PPARs in the Endothelium: Implications for Cancer Therapy. PPAR Res [Internet]. 2008;2008:1–12. [cited 2016 Sep 7] Hindawi Publishing Corporation Available from: http://www.hindawi.com/journals/ppar/2008/904251/.

    Google Scholar 

  63. McArthur K, Feng B, Wu Y, Chen S, Chakrabarti S. MicroRNA-200b regulates vascular endothelial growth factor-mediated alterations in diabetic retinopathy. Diabetes [Internet]. 2011;60:1314–23. [cited 2015 Sep 8] Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3064105&tool=pmcentrez&rendertype=abstract.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Thulasingam S, Massilamany C, Gangaplara A, Dai H, Yarbaeva S, Subramaniam S, et al. miR-27b*, an oxidative stress-responsive microRNA modulates nuclear factor-kB pathway in RAW 264.7 cells. Mol Cell Biochem [Internet]. 2011;352:181–8. [cited 2015 Sep 8] Available from: http://www.ncbi.nlm.nih.gov/pubmed/21350856.

    Article  CAS  Google Scholar 

  65. Graham JR, Williams CMM, Yang Z. MicroRNA-27b targets gremlin 1 to modulate fibrotic responses in pulmonary cells. J Cell Biochem [Internet]. 2014;115:1539–48. [cited 2015 Sep 10] Available from: http://doi.wiley.com/10.1002/jcb.24809.

    Article  CAS  Google Scholar 

  66. Tabruyn SP, Griffioen AW. A new role for NF-κB in angiogenesis inhibition. Cell Death Differ [Internet]. 2007;14:1393–7. [cited 2015 Nov 13] Available from: http://dx.doi.org/10.1038/sj.cdd.4402156.

    Article  CAS  Google Scholar 

  67. Mitola S, Ravelli C, Moroni E, Salvi V, Leali D, Ballmer-Hofer K, et al. Gremlin is a novel agonist of the major proangiogenic receptor VEGFR2. Blood [Internet]. 2010;116:3677–80.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The present scientific contribution is dedicated to the 650th anniversary of the foundation of the University of Pécs, Hungary. The authors are grateful to Attila Pap for breeding the PPARgamma null mouse strain in the Laboratory Animal Core Facility of the University of Debrecen. The authors would like to thank Prof M Nyitrai Department of Biophysics, University of Pecs for consultations during confocal microscopic analysis. The authors are also indebted to Prof Mary Keen, Department of Pharmacy and Therapeutics, School of Clinical and Experimental Medicine, College of Medical and Dental Sciences, University of Birmingham for discussions and language editing.

Funding

JEP was supported by the European Union and the State of Hungary, co-financed by the European Social Fund in the framework of TÁMOP-4.2.4.A/ 2-11/1-2012-0001 ‘National Excellence Program’.

Availability of data and materials

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Authors’ contributions

JR: performed the experiments, EK, DF: isolated RNA from lung tissues, MM: performed flow cytometry, SZME: confocal microscopic analysis, KK: prepared recombinant constructs. TFM, VS, TL, GS: provided the lung tissue and performed staining and analysis of primary human lung tissues, JEP designed the studies, JR and JEP have written the manuscript. All authors read and approved the final manuscript.

Competing interests

JR, EK, MM, DF, salary from Humeltis Ltd; JEP, shareholder in Humeltis Ltd.

Consent for publication

Not applicable.

Ethics approval and consent to participate

Lung tissue samples were collected during lung resections at the Department of Surgery, University of Pecs, Hungary. The project was approved by the Ethical Committee of the University of Pecs (ETT-TUKEB 366/2015). Patients had given written consent to provide samples for research purposes. Ethics approval obtained for the patient samples covered the use of the cells obtained from patients.

Animals were kept at the Laboratory Animal Core Facility of the University of Debrecen that is registered to breed genetically-modified mouse strains (reg. no.: TMF/82-10/2015). Formal ethics approval was not required for our study, as no direct experimentation was performed on the mouse strain otherwise euthanized in the Animal Core Facility.

Author information

Authors and Affiliations

  1. Department of Pharmaceutical Biotechnology, School of Pharmacy, University of Pecs, 2 Rokus Str, Pecs, 7624, Hungary

    J. Rapp, E. Kiss, D. Feller, K. Kvell & J. E. Pongracz

  2. Medical Microbiology and Immunity, University of Pécs, 12 Szigeti Str, Pécs, 7624, Hungary

    M. Meggyes

  3. Biophysics, University of Pécs, 12 Szigeti Str, Pécs, 7624, Hungary

    E. Szabo-Meleg

  4. Pathology, University of Pécs, 12 Szigeti Str, Pécs, 7624, Hungary

    G. Smuk & T. Laszlo

  5. Internal Medicine, Pulmonology, University of Pécs, 2 Rakoczi Str, Pécs, 7623, Hungary

    V. Sarosi

  6. Operational Medicine, University of Pécs, 12 Szigeti Str, Pécs, 7624, Hungary

    T. F. Molnar

  7. János Szentágothai Research Centre, University of Pécs, 20 Ifjúság Str, Pecs, 7622, Hungary

    J. Rapp, E. Kiss, E. Szabo-Meleg, D. Feller, K. Kvell & J. E. Pongracz

  8. Department of Surgery, Thoracic Surgery Unit, Petz A Hospital, 2-4 Vasvari Str, Győr, 9023, Hungary

    T. F. Molnar

  9. Humeltis Ltd, János Szentágothai Research Center, University of Pécs, 20 Ifjúság Str, Pécs, 7622, Hungary

    J. Rapp, E. Kiss, M. Meggyes, D. Feller & J. E. Pongracz

Authors
  1. J. Rapp
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. E. Kiss
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M. Meggyes
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. E. Szabo-Meleg
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. D. Feller
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. G. Smuk
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. T. Laszlo
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. V. Sarosi
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. T. F. Molnar
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. K. Kvell
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. J. E. Pongracz
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to J. E. Pongracz.

Additional files

Additional file 1:

Table S1. Characteristics of patients. The table contains number, histological types, sex, age and pathological TNM status of all patients. (DOCX 14 kb)

Additional file 2:

Figure S1. 3D human lung tissue aggregates to study angiogenesis. To investigate the molecular background of angiogenesis, a three dimensional human lung tissue model was set up using three characteristic cell types of the lung. Namely, primary normal small airway epithelial cells (SAEC), normal human lung fibroblast (NHLF) or VEGF-A-GFP overexpressing human fibroblast (F11) and human microvascular endothelial cells lung subtype (HMVEC-L). GFP aided the visualization of VEGF-A overexpressing fibroblasts in the core of the lung model. Actin was labeled by Alexa Fluor 568 conjugated phalloidin (Thermo Fisher Scientific, Waltham, USA) to detect all cells in the tissue. Scale bar, 200 μm. (TIF 1897 kb)

Additional file 3:

Figure S2. Analysis of expression of angiogenic genes in 3D lung aggregates and normal human lung. Comparing the in vitro model to normal human lung tissue revealed that co-culture expresses VEGF-A, IL-1beta and HIF-1alpha angiogenic factors at the same level. qRT-PCR data are normalized to beta-actin housekee** gene. Error bars, SEM. Independent samples t-test, n = 3. P < 0.05 was considered as significant, ns: not significant. (TIF 90 kb)

Additional file 4:

Figure S3. Overexpression of VEGF-A in F11 cell line. Human VEGF-A-IRES-GFP was cloned into human F11 cell line. Overexpression of hVEGF-A protein was analyzed by fluorescent staining using anti-human VEGF-A as primary antibody. The secondary antibody was Northern Light anti-mouse NL-557. As VEGF-A is a secreted, soluble factor, cells were treated with brefeldin-A at 10 μg/ml for 4 h to inhibit protein transport. Accumulation of the protein was detected after brefeldin-A treatment. Images were captured using Olympus IXB1 fluorescence microscopy equipped with CCD camera. Scale bars, 50 μm. (TIF 3030 kb)

Additional file 5

Figure S4. Area of sprout outgrowth from 3D lung aggregates in normal and high VEGF-A microenvironment. Endothelial cells are evenly distributed in VEGF-Anormal microenvironment, while in VEGF-Ahigh microenvironment the endothelial cells remained close to the VEGF-A source. Data are representation of three independent experiments. Independent samples t-test. Scale bars, 200 μm. (TIF 1597 kb)

Additional file 6:

Figure S5. Flow cytometric analysis of VEGF-A overexpressing 3D lung aggregates in the absence or presence of rhWnt5a. Representative dot plots of flow cytometric analysis of five independent experiments. 3D tissue cultures were created using normal VEGF-A and VEGF-Ahigh fibroblasts, respectively. Then the cultures were incubated in the presence or absence of rhWnt5a, dissociated and stained with anti-CD105 and anti-CD31 antibodies and analyzed in a flow cytometer. (TIF 469 kb)

Additional file 7:

Figure S6. Endoglin (CD105) expression of CD31+ endothelial cells in the lung of WT and PPARgamma KO mice. Immunfluorescence staining of wild type and PPARgamma KO mice showed decreased level of CD105 protein expression in PPARgamma KO lung tissues. Intensity data are representation of three individual experiments. Scale bars 50 μm. Staining intensity was quantified and presented in a bar chart. Error bars, SEM. Independent samples t-test, n = 3. P < 0.05 was considered as significant, P < 0.001. (TIF 3853 kb)

Additional file 8:

Figure S7. Wnt5a expression in the lung of WT and PPARgamma KO mice. No differences were detected in Wnt5a protein expression in wild type and PPARgamma KO mice. (Scale bars 50 μm). The stainings of lung tissue sections are representatives of three individual experiments. Fluorescence intensity was quantified and presented in a bar chart. Error bars, SEM. Independent samples t-test, n = 3. P < 0.05 was considered as significant, ns: not significant. (TIF 769 kb)

Rights and permissions

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Rapp, J., Kiss, E., Meggyes, M. et al. Increased Wnt5a in squamous cell lung carcinoma inhibits endothelial cell motility. BMC Cancer 16, 915 (2016). https://doi.org/10.1186/s12885-016-2943-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12885-016-2943-4

Keywords