Anti-angiogenic effects of testis-specific gene antigen 10 on primary endothe‐ lial cells

Elahe Valipour, Vajihe Taghdiri Nooshabadi, Shadi Mahdipour, Sasan Shabani, Leila Farhady-Tooli, Sina Majidian, Zahra Noroozi, Kamran Mansouri, Elaheh Motevaseli, Mohammad Hossein Modarressi

PII: S0378-1119(20)30525-4
DOI: https://doi.org/10.1016/j.gene.2020.144856
Reference: GENE 144856

To appear in: Gene Gene

Received Date: 19 December 2019
Revised Date: 24 May 2020
Accepted Date: 4 June 2020

Please cite this article as: E. Valipour, V.T. Nooshabadi, S. Mahdipour, S. Shabani, L. Farhady-Tooli, S. Majidian, Z. Noroozi, K. Mansouri, E. Motevaseli, M.H. Modarressi, Anti-angiogenic effects of testis-specific gene antigen 10 on primary endothelial cells, Gene Gene (2020), doi: https://doi.org/10.1016/j.gene.2020.144856

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.Anti-angiogenic effects of testis-specific gene antigen 10 on primary endothelial cells

Elahe Valipour a. Vajihe Taghdiri Nooshabadi b. Shadi Mahdipour a. Sasan Shabani a. Leila Farhady-Tooli
Imagec. Sina Majidian d. Zahra Noroozi e. Kamran Mansouri f. Elaheh Motevaseli d. Mohammad Hossein Modarressi a *

aDepartment of Medical Genetics, Faculty of Medicine, Tehran University of Medical Sciences, Tehran,
Iran

b Department of Tissue Engineering and Applied Cell Sciences, Faculty of Medicine, Semnan University of medical sciences, Semnan, Iran

c Department of Microbiology, School of Biology, College of Science, Tehran University, Tehran, Iran

dSchool of Electrical Engineering, Iran University of Science and Technology, Tehran, Iran

e Department of Molecular Medicine, School of Advanced Medical Technologies, Tehran University of Medical Sciences, Tehran, Iran

fMedical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran

*Corresponding Authors:

Mohammad Hossein Modarressi, [email protected] Phone: 0098 21 64053209, ORCID: 0000-0003-2763-1964

Abbreviations:
TSGA10, testis-specific gene antigen 10; ECs, endothelial cells; VEGF, Vascular endothelial growth factor; SDF-1a, stromal-derived factor-1a; HUVECs, human umbilical vein endothelial cells; ERK, extracellular signal-regulated kinase; Akt, protein kinase B

Abstract: Growing evidence indicates the antitumor and antiangiogenesis activities of testis-specific gene antigen 10 (TSGA10). However, the underlying mechanisms and precise role of TSGA10 in angiogenesis are still elusive. In this study, we isolated human umbilical cord vein endothelial cells (HUVECs) and stably transfected with pcDNA3.1 carrying TSGA10 coding sequence. We demonstrated that TSGA10 over- expression significantly decreases HUVEC tubulogenesis and interconnected capillary network formation.
HUVECs over-expressing TSGA10 exhibited a significant decrease in migration and proliferation rates. TSGA10 over-expression markedly decreased expression of angiogenesis-related genes, including VEGF-A, VEGFR-2, Ang-1, Ang-2, and Tie-2. Our ELISA results showed the decrease in VEGF-A mRNA expression level is associated with a significant decrease in its protein secretion. Additionally, over-expressing TSGA10 decreased expression levels of marker genes of cell migration (MMP-2, MMP-9, and SDF-1a) and proliferation (PCNA and Ki-67. Furthermore, ERK-1 and AKT phosphorylation significantly reduced in HUVECs over-expressing TSGA10. Our findings suggest a potent anti-angiogenesis activity of TSGA10 in HUVECs through down-regulation of ERK and AKT signalling pathways, and may provide therapeutic benefits for the management of different pathological angiogenesis.

Keywords: TSGA10, VEGF-A, Cell migration, Angiogenesis, ERK/AKT pathway, GEO database

1. Introduction

Angiogenesis is a sophisticated multi-step physiological process that leads to the formation of new vessels from pre-existing ones. Apart from normal angiogenesis, aberrant angiogenesis is the primary trigger of specific pathological conditions, including malignant tumors. During the angiogenic switch, endothelial cells (ECs) are activated and then start to secrete a variety of growth, migration, and angiogenesis-related factors, leading to migration, proliferation, remodeling, and transforming of ECs into capillary tube structures and developing into novel basement membranes.Various intracellular signaling pathways mediate these cellular events. Vascular endothelial growth factor-A (VEGF-A), through type II VEGF receptor tyrosine kinase (VEGFR-2), is themost potent mediator of physiological and pathological angiogenesis (Fang et al., 2019). VEGF- A activates the early responsive intracellular signaling molecules ERK1/2 MAP Kinase and Akt and thereby leads to the new vessel formation (Abhinand et al., 2016; Strieter, 2005). In addition to intracellular signaling molecules, there are various secreted factors with prominent roles in angiogenesis. During directional migration of ECs for angiogenesis, chemokine stromal cell- derived factor-1a (SDF-1a) plays a vital role by activating its cell surface receptor, C-X-C motif chemokine receptor 4 (CXCR4). SDF-1a activity increases secretion of matrix metalloproteinases (MMPs) and thereby supports survival, cytoskeletal rearrangement, chemotaxis and migration of ECs (Petit et al., 2007; Tseng et al., 2011). Moreover, numerous studies indicate that signaling involving angiopoietins (e.g., Ang-1 and Ang-2) and receptor tyrosine kinase Tie-2 are essential for the stability of new forming vessels (Thomas and Augustin, 2009).

Oxygen deprivation appears to be the principal trigger of the angiogenic switch during tumor progression (Liekens et al., 2001). Hypoxia-inducible factor 1a (HIF-1a) acts as the master regulator of intracellular oxygen homeostasis and angiogenesis (Hirota and Semenza, 2006).A healthy adult testis tissue is an extremely hypoxic environment with the high rates of cell proliferation and differentiation, and high HIF-1a expression. Nevertheless, there is no tumorogenesis in the testis tissue. Thus, there must be inhibitory factors with the ability to prevent testis tissue cells from shifting towards cancerous cells. TSGA10 has been suggested among factors suppressing tumor development in the testis by hindering HIF localization into nucleus (Hägele et al., 2006).

TSGA10 gene encodes an 82 KDa protein (Hossein Modarressi et al., 2001) with predominant expression in the testis and widespread distribution in other healthy tissues (Behnam et al., 2009, 2006; Mobasheri et al., 2006; Roghanian et al., 2010). There are various reports that TSGA10 exerts essential roles in normal spermatogenesis, active cell division, differentiation, and proliferation (Asgharzadeh et al., 2019; Miryounesi et al., 2014; Sha et al., 2018). Further, recent studies indicate that a wide variety of tumors decrease TSGA10 expression during progression to advanced grades and stages, like nasopharyngeal carcinoma and esophageal squamous cellcarcinomas (Bao et al., 2018; Yuan et al., 2013; Zhang et al., 2019), suggesting the involvement of TSGA10 in controlling malignancy and clinical features of tumors.

ImageDespite growing studies on the bioactivity of TSGA10, the exact effects of TSGA10 on angiogenesis and the functional mechanisms underlying its involvement in the regulation of angiogenesis remains elusive. In the present study, ECs were isolated from the vein of the human umbilical cord to investigate the effects of TSGA10 on the angiogenesis ability of the cells. The results of this study can provide information for future research to clarify the functional mechanisms of TSGA10 and its involvement in angiogenesis.

2. Materials and methods
2.1. Reagents and antibodies

Endothelial Cell Medium (ECM), Endothelial Cell growth Supplement (EGCS), M199 medium, low glucose DMEM, fetal bovine serum (FBS), 0.25% trypsin-EDTA (1X) and penicillin- streptomycin (100X) were purchased from BioIdea Company (Iran). Amphotericin B (DNA Biotech brand), cell culture flasks and plates (SPL Life Sciences Co, Korea), and 1X phosphate- buffered saline (PBS) were purchased from KalaZist company (Iran). RIPA buffer was from KIAZIST life sciences (Hamadan, Iran). Lipofectamine 2000 was purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). Antibodies used in the present study are as follows: Anti-TSGA10 antibody (MBS7005955), Goat Anti-Rabbit IgG (HRP) (ab205718), Anti- phosphorylated ERK-1 (ab24157), ERK-1 Antibody (AF1879), Anti-AKT Antibody (Biolegend 680302), Goat Anti-Mouse IgG H&L (HRP) (ab97023), Anti-phosphorylated AKT (Biolegend 649002), Anti-beta Actin Antibody (ab8227). All other reagents were purchased from Merck (Germany) and Sigma-Aldrich (Taufkirchen, Germany) unless otherwise stated in the text.
2.2. Plasmid preparation

The recombinant expression pcDNA3.1 plasmid carrying the coding sequence (CDS) of the TSGA10 gene was constructed in our previous study (Mansouri et al., 2015). Briefly, RNA was extracted from 10 mg of human testis tissue and TSGA10 CDS was obtained by quantitative RT- PCR to produce the final fragment 2115 bp of TSGA10 CDS containing a BamH1 site in the 5’end and an EcoR1 site in the 3’ end and ligate into a 5.4 kb pcDNA3.1 plasmid (Invitrogen,USA). Midi preparation of the recombinant plasmids amplified in DH5α cells was performed using the QIAGEN Midiprep Kit according to the supplier’s guide.

2.3. HUVEC isolation

ImageHUVECs were isolated from the vein of human umbilical cord tissue obtained from a female newborn delivered by cesarean in the Gynecology Clinic of Valiasr Hospital, Imam Medical Complex, in Tehran. The protocols in this study were approved by the Ethics Committee of Tehran University of Medical Science (TUMS). HUVECs were isolated according to the protocol reported by Bruno Larrivee (Larrivée and Karsan, 2005). The isolated cells were cultured with complete ECM (ECM containing 40 µg/ml ECGS and 20% FBS) and 1% antibiotic solution in the T25 flasks pre-coated with 1% gelatin A (Sigma) solution. Cells after passage 3 were stained though Immunocytochemistry (ICC) to study the expression of CD31 marker as an EC adhesion molecule.

2.4. Immunocytochemistry

The cells were permeabilized in pre-cold methanol for 1 min, blocked with 4% FBS in 1X PBS containing 0.1% Triton X-100 for 10 min, and then incubated with anti-CD31 primary antibody (Biorbyt company, Cambridge, cat no. orb10314) for 1 h. Then the cells were washed and incubated with FITC-conjugated Goat Anti-Rabbit IgG (Abcam, cat no. ab6717) in the dark for 45 min. Nuclei were counterstained with DAPI (Invitrogen Life Science Technologies) for 2 min.

2.5. Stable transfection

First, 400 µg/ml G418 (Gibco) was selected as the minimum concentration that could kill all the cells for 14 days. HUVECs were starved (0% FBS) overnight before being transfected using lipofectamine 2000 and then incubated with transfection solution (ECM with 2% FBS, 5 μg plasmid, 5μL lipofectamine) for 5 h. The medium was then replaced with fresh complete ECM. After two days, the cells were subjected to 400μg/mL G418 for 6 weeks. G418-resistant cell clones (HUVECs-TSGA10 cells) were picked out using a cell scraper and expanded.

2.6. Matrigel tube formation assay

ImageFirst, 50 μL/well of pre-thawed Matrigel (Corning, cat no. 354234) was coated into a 96-well plate. Serum-starved HUVECs and HUVECs-TSGA10 were seeded onto Matrigel-coated wells at a density of 1×104 cells and incubated with M199 containing 50 ng/ml VEGF-A and 1% FBS at 37oc, 5% co2 for 24 h. Three wells were used for each cell. Photos were taken under a digital camera, inverted microscope and analyzed using ImageJ Ver. 1.44p (NIH, Bethesda, MD, USA) for colony scoring and measuring the tube length.

2.7. Wound (scratch) closure assay in cell culture

2×105 cells/well of HUVECs and HUVECs-TSGA10 were seeded in a 6-well plate. A monolayer confluent culture of the cells was scratched using a sterilized 1000 μL pipette tip. After washing the cells twice with pre-warmed PBS, the cells were incubated for 48 h with ECM containing 2% FBS and stained at 0 and 48 h of scratching using 0.2% crystal violet in 2% aqueous ethanol. Wound gaps were photographed using a phase-contrast microscope.
2.8. Transwell migration assay

A total of 1×104 cells were added on top of the 8 µm pore polycarbonate membrane transwell inserts with or without 30 μL Matrigel in 24-well plates containing 500 μL M199 with 1% FBS. At the bottom well, 750 μL M199 supplemented with 50 ng/ml VEGF-A was added, and the cells were incubated for 12 h at 37oc /5% co2. The migrated cells were fixed with 200 μL methanol for 1 min, stained with 200 μL Giemsa for 2 min, and then pictures were taken per high power field under a phase-contrast microscope. The migration activity of HUVEC and HUVEC- TSGA10 cells was calculated as the average number of migrated cells in three experiments.

2.9. Growth curves and doubling time

First, 2× 104 cells/well of HUVECs and HUVECs-TSGA10 were seeded and starved in serum- free medium for 48 h to be synchronized. The cells were then incubated with complete ECM. Growth curves were plotted after cell counting in duplicate every 24 h. Doubling time (DT) was calculated using the following formula: DT=[(t×log2)/log(nt/n0)], t: the day in hour where the maximum number of cells counted, nt: the number of cells counted at time t, and n0: the number of cells seeded.

2.10. Cell proliferation (MTT) assay

5×103 cells/well of serum-starved HUVECs and HUVECs-TSGA10 were seeded in a 96-well plate in triplicates and incubated with complete ECM for 48 h. The cells were treated with 50 µl/well (5 mg/ml in PBS) of 3-(4,5-dimethyl thiazolyl-2)-2, 5-diphenyltetrazolium bromide solution (MTT, Sigma, USA). The precipitated formazan crystals (purple-blue) were dissolved in 150 µl DMSO. Optical density was measured using ELISA reader 450/630 nm.

2.11. ImageEnzyme-linked immunosorbent assay (ELISA)

1×1o6 cells of HUVECs and HUVECs-TSGA10 were plated in 70-mm gelatin A-coated dishes in complete ECM and allowed cells to grow until they were approximately 80% confluent. Then, cells were washed twice with pre-warmed PBS and incubated with 4 ml of low glucose DMEM supplemented with 2% FBS for 72 h. The conditioned media (CM) were collected and concentrated using a BCA protein assay kit (Thermo Scientific). Concentrations of VEGF-A were quantified using a human VEGF Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

2.12. Western blot

Protein extraction from HUVECs and HUVECs-TSGA10 was performed using RIPA buffer. 30 µg/ml of each protein sample was separated on 10% SDS-PAGE gel and then transferred to PVDF membranes (Millipore, MA, USA). The membranes were blocked (PBS containing 5% BSA), incubated with a primary antibody, and then washed three times. Subsequently, the membranes were incubated with corresponding HRP-conjugated secondary antibody. The resulting immunoreactive bands were visualized with the enhanced chemiluminescence (ECL) reagent kit (Amersham Pharmacia Crop, Piscataway, USA) according to the manufacturer’s directions. The intensity of the bands was calculated using the ImageJ and GelAnalyzer software and then normalized against beta-actin.

2.13. RNA extraction, cDNA synthesis, and qRT-PCR

Total RNA of the synchronized cells was extracted using the RiboEX reagent (GeneAll, Korea) according to the manufacture’s protocol. CDNA was synthesized from 1 μg total RNA using BioFact™ cDNA synthesis Kit as recommended by the supplier. QRT- PCR was performed using BioFact™ 2X Real-Time PCR Smart mix Sybergreen by Rotor-Gene 6000 system(Corbett Research, Australia). The sequence of primers and the corresponding Tm are shown in table 1. The Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) gene was used as a reference gene. The fold change was calculated using the following formula, 2-ΔΔCt = [(2- (Ct target - Ct ref.) in HUVEC-TSGA10 cells) / (2 (Ct target - Ct ref.) in HUVEC cells)].

2.14. ImageEvaluation of gene expression in GEO datasets

To further investigate the relationship between TSGA10 and angiogenesis, the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/) was used. After looking for a dataset including gene expression profiles of all stages and grades of particular cancer, dataset GSE136661 was used for the expression analysis of TSGA10 in all three grades (I-III) of meningiomas and evaluation of how the TSGA10 expression levels change with tumor progression. Also, the gene expression profiles of GSE38408, GSE62947, and GSE114040 were applied to investigate the changes in the TSGA10 expression associated with the use of antiangiogenic therapeutics. In addition, expression fold changes of angiogenesis-related genes were calculated for the mentioned GEO datasets. The GEO dataset GSE136661 was also used for evaluating the correlations of TSGA10 with angiogenesis-related genes due to the high number of samples. Gene expression analysis of GEO database data (RNA-seq and expression array) was performed using R software (version 3.6.1) and related packages, the “DESeq” (Anders and Huber, 2010) and “Limma” (Ritchie et al., 2015).

2.15. Statistical analysis

All data were presented as mean ± SD. Relative mRNA expression levels were normalized to GAPDH mRNA levels. Quantification analysis of relative gene expression ratios was performed using the Relative Expression Software Tool (REST) and Graph Pad Prism 8.0.2. In order to show expression fold changes, number 1 was considered as the baseline (gene expression levels in control cells) in all qRT-PCR. The intensity of bands of western blot was calculated using the GelAnalyzer2010a and ImageJ (NIH, Bethesda, MD, USA) and normalized with the intensity of beta-actin. All charts were plotted using the GraphPad Prism 8.0.2. Statistically significant differences were calculated after analysis of one-way ANOVA followed by Tukey’s posthoc test. Values ≤ 0.05 were considered statistically significant. Each point or column represents the mean
± SEM. (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

3. Results
3.1. Isolation and stable transfection of primary HUVEC cells

ImageIn the present study, primary ECs were successfully isolated from the human umbilical vein. Data from ICC staining showed that 75% of the isolated cells were positive for CD31 marker (Figure 1A). The cells were cultured under ECM containing ECGS to prevent cell differentiation and senescence. A homogeneous cell population of the isolated cells with a cobblestone appearance is shown in Figure 1B.
HUVECs with the stable over-expression of TSGA10 were successfully established to investigate the effects of TSGA10 on the angiogenic behavior of the ECs. The efficiency of transfection was verified by qRT-PCR and western blot. Our results showed that the mRNA expression level of TSGA10 increased 4.9 fold (****p<0.0001) in the transfected cells compared to untransfected cells. Nevertheless, the TSGA10 protein expression level revealed 2.27 fold change (*p=0.015) (Figure 1C, D).

3.2. TSGA10 can inhibit in vitro tubulogenesis and migration of HUVECs

In order to explore the effects of TSGA10 on the ability of the ECs for angiogenesis, the tube formation assay on Matrigel was assessed. It is known that angiogenic factors stimulate morphological changes of ECs to form capillary-like structures on Matrigel. Therefore, colony score and tube length were measured under 50 ng/ml VEGF-A as an angiogenic factor.
The architecture of the endothelial tubular network of the untransfected HUVECs is shown in Figure 2A. Statistical analysis revealed the inhibitory effects of TSGA10 over-expression on the HUVEC tubulogenesis scoring (*p<0.05) as well as on the tube length (**p <0.01). Further, a remarkable difference was observed between the diameter of the tubes formed by HUVECs and HUVECs-TSGA10. As shown in Figure 2A, the tubes formed by HUVECs were much thicker than the tubes formed by HUVECs-TSGA10. Accordingly, TSGA10 over-expression was able to exert an inhibitory effect on VEGF-A induced tubulogenesis.In addition, the wound closure assay in culture and transwell migration assay were employed to investigate the effects of TSGA10 over-expression on the migration of HUVECs. In the gap area analysis of scratching after 48 h a markeable decrease was found in the migration of HUVECs-

TSGA10 (Figure 2B). Although the untransfected HUVECs approximately filled the cell-free area after 48 h, HUVECs-TSGA10 showed a considerable decrease in motility and migration. This observation was similar to the result obtained from the transwell migration assay. The number of the migrated HUVECs-TSGA10 in the presence or absence of Matrigel was significantly lower than HUVECs (***p<0.001) (Figure 2C). Further, our transwell migration assay showed that HUVECs aggregated after migrating into the media containing VEGF-A. Nevertheless, the cell migration and aggregation in HUVECs-TSGA10 were significantly lower, that indicate TSGA10 over-expression significantly decreases the tendency of HUVECs to VEGF-A.
The qRT-PCR analysis showed that the expression levels of migratory factors MMP-2, MMP-9 (***p<0.001), and SDF-1a (*p<0.05) significantly decreased in HUVECs-TSGA10 (Figure 2D). Collectively, these results suggest that TSGA10 over-expression could decrease HUVEC migration in vitro.
3.3. TSGA10 decreases the proliferation rate of HUVECs

To investigate the effect of TSGA10 on the EC growth, the MTT assay was performed. Our data showed that TSGA10 over-expression significantly decreased (***p<0.001) the proliferation rate of HUVECs (Figure 3A). in order to be ensure that this observation is due only to TSGA10 over- expression and not to vector components, the proliferation rates of the untransfected HUVECs and transfected HUVECs with an empty vector (HUVECs-pcDNA3.1) were compared in the MTT assay. No significant difference was observed between HUVECs and HUVECs- pcDNA3.1.
To further investigate, doubling time (DT) of the cells was calculated. Cell cycles first were synchronized at the G0/G1 stage by serum-starvation and then restarted with serum stimulation. Subsequently, the cells were counted at specified time intervals, and cell growth curves were plotted. The calculated DTs for HUVECs and HUVECs-TSGA10 were 35.47 h and 46.18 h, respectively (Figure 3B).
The proliferation-related markers Ki-67 and PCNA were also examined using qRT-PCR. Data showed a significant decrease in the Ki-67 and PCNA mRNA expression levels (***p<0.001)(Figure 3C). According to these results, increased expression of TSGA10 can decrease the proliferation rate of HUVECs.

3.4. TSGA10 over-expression results in a decrease in proangiogenic factors

ImageDue to the importance of signaling molecules VEGF-A and angiopoietins in inducing the EC proliferation, migration, angiogenic response and stability of new forming tubes, the expression levels of VEGF-A, Ang-1, and Ang-2 as well as underlying receptors VEGFR-2 and Tie-2 were examined using qRT-PCR. Data indicated that there is a significant decrease in the expression of these angiogenic markers in HUVECs over-expressing TSGA10 (Figure 4A). ELISA results showed that the reduced VEGF-A mRNA level in HUVECs-TSGA10 results in a significant decrease in VEGF-A protein secreted from the cells (***p<0.001) (Figure 4B).

3.5. TSGA10 decreases phosphorylation of ERK-1 and AKT in HUVECs

ERK-1 and AKT participate in regulating the early angiogenic response of ECs as well as in controlling the cell proliferation and migration. Thus, the phosphorylation level of ERK-1 and AKT in HUVECs over-expressing TSGA10 was examined. The immunoblots in our study revealed that TSGA10 over-expression significantly decreased the ERK-1 and AKT phosphorylation, without affecting the un-phosphorylated ERK-1 and AKT (Figure 5A, B). These data showed that TSGA10 has potential to decrease the pho-ERK/ERK (***p<0.001) and pho-AKT/AKT (*p<0.01) ratios in primary HUVECs in vitro.

3.6. Gene expression analysis in GEO datasets

Evaluation of the TSGA10 expression changes in the online gene expression profiles obtained from the GEO database is shown in Figure 6. As can be seen in the GSE136661 dataset, more reduced expression of TSGA10 has been detected in more advanced grades of the tumor (Figure 6A). In addition, the use of antiangiogenic therapeutics (TIMP-2, SRPIN803, Tamoxifen, and Ginsenoside Rg3) is associated with the increased expression of TSGA10. Additive antitumor effects of antiangiogenic therapeutics (Ginsenoside Rg3 compared to tamoxifen) were resulted in a further increase in the TSGA10 expression levels (GSE114040) (Figure 6B). Furthermore, most angiogenesis-related genes showed different expression fold changes in the studied GEO datasets than expected (Tables 2 and 3). Nevertheless, negative correlations with TSGA10 wereImageobserved for MMP2, SDF-1a, Ki-67, Tie-2, and VEGFR-2 at different grades of meningiomas of the GEO dataset GSE136661 (Table 2). Also, MMP2 was the only gene that showed decreased expression in the all studied antiangiogenic therapeutics-related datasets. Other genes showed increased or decreased expression fold changes in the use of different antiangiogenic therapeutics (Table 3).

Discussion

Angiogenesis is a critical step in tumor progression and metastasis. The function of ECs during angiogenesis can be modeled in vitro using the tube formation assay where ECs migrate to aggregate and then morphologically change, and sprout out into the surrounding matrix to organize into hollow tubes and subsequently form interconnected spider web-like networks (DeCicco-Skinner et al., 2014; Ponce, 2009). Cell migration for sprouting angiogenesis is a directed and guided process in response to angiogenic stimulators, which is supported by the local environment (Auerbach et al., 2003). In the present study, Matrigel was used as a basement membrane matrix to create a local supportive environment that permits to the migration of ECs for angiogenesis (Ponce, 2009). VEGF-A was also used as a migratory and angiogenic factor.

VEGF-A is a potent inducer of angiogenesis both in vitro and in vivo by binding to its receptor, VEGFR-2. The VEGF-A/VEGFR-2 activity triggers cascades of downstream signaling events for regulation of the EC permeability, focal adhesion turnover, migration and sprouting (Carmeliet and Jain, 2011; Sitohy et al., 2012; Sun et al., 2019). Signaling pathways including EC surface receptor Tie-2 and its ligands (Ang-1, and Ang-2) support the stabilization and maturation of the newly formed capillary buds into vascular tubes. Tie-2 is strongly expressed in ECs where its expression, cell surface localization, and activation/phosphorylation are required to govern vascular stabilization and growth (Eklund et al., 2016; Fagiani and Christofori, 2013; Jeansson et al., 2011). Ang-1 is agonistic/activating ligand for Tie-2 in the presence or absence of VEGF-A. Ang-2 acts as a competitive antagonist of the Ang-1/Tie-2 axis in the absence of angiogenic stimulators such as VEGF-A, which in turn leads to the death of EC and disruption of vascularization (Hansen et al., 2010). However, Ang-2 and Ang-1 collaborate in the presence of VEGF-A to enhance cell proliferation, migration, and tubulogenesis (Eklund et al., 2016; Lim etal., 2004; Singh et al., 2009). Therefore, increased expression and secretion of Ang-1 and Ang-2 result in the phosphorylation of Tie-2 and activation of downstream signaling pathways such as the PI3K/AKT axis that promote cell proliferation and migration as well as vascular stability (DeBusk et al., 2004; Ye et al., 2018).

ImageOur results showed that TSGA10 over-expression results in a significant decrease in the VEGF- A induced migration and angiogenesis of HUVECs. The dynamic of the cell behavior on Matrigel was different between HUVECs and HUVECs over-expressing TSGA10. Interestingly, HUVECs started to sprout in response to VEGF-A immediately after seeding on Matrigel and formed interconnected tubular networks. Nevertheless, increased expression of TSGA10 resulted in a decrease in cell aggregation rate and budding of HUVECs, preventing the interconnection of tubular sprouts, and hence; the functional capillary network is formed. Lack of the HUVEC tubular organization associated with increased TSGA10 may be due to decreased ability of cell sprouting or decreased tubular stability and regression of developing tubes. Our results demonstrate that TSGA10 over-expression decreases the expression and secretion level of VEGF-A, expression of VEGFR-2 as well as the expression of Tie2 and its ligands (Ang1 and Ang2), resulting in the inhibition of HUVEC sprouting and tubulogenesis.

In addition, cell migration is a vital process for neo-angiogenesis and neovascularization (Lamalice et al., 2007). Accumulating reports indicate that EC migration, tube formation, and long-term stable new forming tubes in response to VEGF-A are highly dependent on chemokine SDF-1a/CXCR4 signaling (Jin et al., 2012). Increased VEGF-A and activation of SDF- 1a/CXCR4 signaling exert a positive feedback effect on each other (Fang et al., 2019; Petit et al., 2007), leading to the cytoskeletal rearrangement and cell migration by activating the ERK and AKT signaling and incrased expression of matrix metalloproteinases MMP-2 and MMP-9 (Meng et al., 2018; Tang et al., 2007; Wang et al., 2005). Also, increased secretion of MMP-2 and MMP-9 promotes VEGF-A release, which induces angiogenesis. Our results revealed that TSGA10 over-expression results in a prominent decrease in the expression of MMP-2 and MMP-9 as well as SDF-1a, leading to a decrease in VEGF-A induced migration response of HUVECs.

ImageThe secretion of many angiogenic molecules such as angiopoietins, matrix metalloproteinases, and VEGF-A is regulated by intracellular signaling pathways ERK and AKT (Agarwal et al., 2013; Chin and Toker, 2009; Gentile et al., 2018; Namkoong et al., 2009; Tan et al., 2013). Our results showed that HUVECs over-expressing TSGA10 exhibit decreased phosphorylation levels of the early responsive intracellular signaling molecules ERK-1 and AKT under normoxic conditions and VEGF-A stimulation. Although the down-regulation of the both pathways can also explain the decrease in angiogenesis-related activities of HUVECs, other intracellular signaling mechanisms still need to be investigated to determine if they can mediate the effects of TSGA10 on angiogenesis-related activities in vitro or not. All of the genes studied in this study were of HIF target genes, and our expression analysis data showed TSGA10 over-expression results in a change in HIF transcriptional activity. Also, in our previous studies, it was reported that the TSGA10 effects are HIF dependent in both hypoxic and normoxic conditions (Hägele et al., 2006; Mansouri et al., 2015), especially in endothelial cells (Amoorahim et al., 2020). Further, Roghanian et al. (Roghanian et al., 2010) reported the possibility of TSGA10 involvement in cell migration based on its interaction with Vimentin, which is directly related to tumor growth and invasion.
Our observations are consistent with the previous evidence indicating the involvement of TSGA10 in cell migration and ciliary structure (Behnam et al., 2015; Carvalho-Santos et al., 2012; Wu et al., 2019) as well as studies suggesting the potential inhibitory effects of TSGA10 on tumorogenesis, angiogenesis, and metastasis. Bao et al. (Bao et al., 2018) reported that metastasis-associated miR-23a mediates angiogenesis in nasopharyngeal carcinoma through directly repressing TSGA10. Interestingly, increased miR-23a and thereby decreased TSGA10 expression levels were associated with microvessel density and pre-metastatic and metastatic stages.
Further, our experiments indicated that the increased TSGA10 exerts the same biological effect on the proliferation of HUVECs. Thus, HUVECs over-expressing TSGA10 exhibited reduced proliferation rate as well as reduced expression levels of the cell proliferation markers Ki-67 and PCNA. Our data were in line with the previous studies suggesting TSGA10 as one of the factors involved in cell cycle inactivation. Despite lower expression levels of TSGA10 in a wide variety of normal adult tissues, considerably increased TSGA10 expression is found in post-meiotic

Imageevents of spermatogenesis and in several adult tissues like brain, kidney, and retina where most cells are differentiated and non-proliferating and the cell cycle is retained at G0 phase (Behnam et al., 2009, 2006; Hughes et al., 2010; Stroka et al., 2001). Miryounesi’s and Behnam’s studies showed that undifferentiated embryonic stem cells express TSGA10 at a very low level. But the expression of TSGA10 increases during the transition of the cells from the proliferation phase to the differentiation phase (Behnam et al., 2006; Miryounesi et al., 2014). Xiang Yuan et al. (Yuan et al., 2013) also indicated that down-regulated TSGA10 expression is associated with the G1/S phase transition, high cell proliferation ability, and thereby increased malignancy and clinical features of esophageal squamous cell carcinomas.
To date, the GEO database includes the most array- and sequence-based gene expression profiles that can be freely used to gain insights about differences in gene expression patterns. Expression analysis using GEO datasets demonstrated that TSGA10 is down-regulated in advanced grades and stages of tumors. It is well documented that tumors are not able to grow larger than the microscopic size of 2-3 mm3 without angiogenesis and may become apoptotic or even necrotic due to the lack of continuous recruitment of adequate blood supply (Holmgren et al., 1995; Nishida et al., 2006; Parangi et al., 1996). The deprivation of oxygen leads to the stabilization of HIF-1a in tumor cells. HIF-1a activates the expression of VEGF and its receptor as well as more than 40 other genes to induce neo-angiogenesis of surrounding capillaries (Benita et al., 2009). Numerous studies reported the over-expression of TSGA10 in the early stages of a variety of tumors (Behnam et al., 2009; Dianatpour et al., 2012; Hoseinkhani et al., 2019; Mobasheri et al., 2006; Tanaka et al., 2004). Therefore, it appears that the TSGA10 up- regulation in cancerous cells of small size tumors can be a defense and control mechanism against cell proliferation and tumor growth and angiogenesis. However, after disrupting the balance between negative and positive regulators in favor of angiogenic switch, tumors start to grow toward more advanced stages where the importance of HIF-1a is diminished. Thus, the expression level of TSGA10 is reduced in the undifferentiated and invasive cells of late stages of cancers as an escape way to gain uncontrolled proliferation as well as migration, invasion, and metastasis ability.
Interestingly, the expression analysis of TSGA10 in the GEO datasets showed that the expression levels of TSGA10 are increased using antiangiogenic therapeutics. Bourboulia et al.

Image(Bourboulia et al., 2013) reported that A549 cells over-expressing tissue inhibitor of metalloproteinase-2 (TIMP-2) displayed reduced tumor growth and angiogenesis in vivo by providing microarray-based GSE38408 gene expression profile. TIMP-2 is an endogenous inhibitor of angiogenesis and metastasis. The expression analysis of TSGA10 of the dataset showed that the TIMP-2 over-expression results in an increase in the TSGA10 expression. Also, gene expression profiles of microarray-based GSE62947 and high throughput sequencing-based GSE114040 showed the similar results. The GSE62947 dataset contains the gene expression profiles of ARPE-19 cells treated with the antiangiogenic therapeutics SRPIN803, which is a small molecule that acts as a dual inhibitor of serine-arginine protein kinase-1 (SRPK1) and casein kinase-2 (CK2), attenuating pathological angiogenesis of macular degeneration (Morooka et al., 2015). The GSE114040 dataset includes gene expression profiles of PANC-1 cells treated with Tamoxifen or Ginsenoside Rg3 in combination with cantharidin, one of the antitumor components of mylabris (Xu et al., 2018). Ginsenoside Rg3 is an active component of ginseng root with the inhibition potent of tumor angiogenesis through attenuating the VEGF- dependent ERK/p38 and AKT/eNOS signaling pathways (Xu et al., 2018). Interestingly, the expression analysis of TSGA10 in the GSE114040 dataset showed that the expression level of TSGA10 is more increased after treating PANC-1 cells with cantharidin/Ginsenoside Rg3 than cantharidin/Tamoxifen. Although these observations can suggest antitumor effects for TSGA10, the changes in TSGA10 expression may be a secondary effect of an initial event or signaling pathway or may as well be two independent events. Unlike consistent expression changes of TSGA10 in the GEO datasets with our in vitro experiments, most angiogenesis-related genes included in our study showed inconsistent expression changes with what was expected. Nevertheless, MMP2 was the only gene that showed decreased expression consistent with increased expression of TSGA10 in the all studied antiangiogenic therapeutics-related datasets as well as a negative expression correlation with TSGA10 in the dataset GSE136661. Therefore, further investigation and finding the relationship between TSGA10 and MMP2 as well as other key angiogenesis-related genes are suggested.

It is noteworthy that the biological effects of TSGA10 found in HUVECs may not be universal and be different regarding cell type, different cellular contexts and corresponding signaling in tumorigenesis, as there are various reports about TSGA10 functions in AML (Hoseinkhani et al., 2019), bladder (Wu et al., 2019), brain (Behnam et al., 2009) and breast (Dianatpour et al., 2012)cancers. Nevertheless, gene augmentation strategies for TSGA10 over-expression in endothelial cells surrounding tumors are suggested to be investigated in animal models, leading to provide more precise knowledge of anti-tumorogenesis functions of TSGA10 and hope to shed new light on cancer treatment.

4. ImageConclusions

Tumor growth and metastasis are strongly dependent on neo-angiogenesis, which is provoked by a wide variety of angiogenic stimulators. Although a large number of angiogenic mediators have been identified, it is entirely clear that the inhibition of pathological angiogenesis is very complicated. Therefore, deciphering the kinetics of different genes affecting angiogenesis enables us to manage the phenomenon in different tissue pathologies. Our results provide the insights that TSGA10 performs negative influences on various aspects of HUVECs activities. HUVECs with the stable over-expression of TSGA10 underwent a slower proliferation and migration. TSGA10 over-expression decreased the ability of HUVECs for VEGF-induced capillary tubular network formation. Decreased expression of the genes related to these processes and also the down-regulation of ERK and AKT signaling pathways could be an explanation for our observations. Our observations open a new perspective for the therapeutic application of TSGA10 in tumor anti-proliferative and anti-angiogenesis.

Acknowledgments
We are grateful to Dr. Sedigheh Hantoushzadeh for coordinating the Gynecology Clinic of Valiasr Hospital and Dr. Mahdieh Shirzad for excellent technical assistance and helpful comments.

Compliance with ethical standards Funding
This study was supported by Tehran University of medical sciences [Grant number 98-02-30- 42453].

Declaration of interest
None

Informed consent
ImageInformed written consent was obtained from all participants.

Ethical approval
This study was approved by Ethical Committee of Tehran University of Medical Sciences.

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