Shinako Masuda,Katsuhisa Matsuura,Tatsuya Shimizu
GATA6 regulates anti-angiogenic properties in human cardiac fibroblasts via modulating LYPD1 expression
Cardiac fibroblasts,Angiogenesis,Tissue engineering,GATA6,LYPD1
Introduction Fibroblasts contribute to the structure and function of tissue and organs; however, their properties differ in each organ given the topographic variation in gene expression among tissues. We previously reported that LYPD1, which is expressed in cardiac fibroblasts, has the capacity to inhibit sprouting of vascular endothelial cells. LYPD1 has been shown to be highly expressed in the human brain and heart, but the regulation of LYPD1 expression in cardiac fibroblasts has not been elucidated in detail. Methods To identify the LYPD1-modulating transcription factor, motif enrichment analysis and differential expressed gene analysis using microarray data were performed. Quantitative real-time PCR was used to evaluate gene expression. Gene silencing were performed by transfection of siRNA. Western blot analyzed protein expression in NHCF-a. To assess the effect of GATA6 on the regulation of LYPD1 gene expression, dual-luciferase reporter assay was performed. Co-culture and rescue experiments were performed to evaluate endothelial network formation. Results Motif enrichment analysis and differential expressed gene analysis using microarray data and quantitative real-time PCR revealed that CUX1, GATA6, and MAFK were candidate transcription factors. Of these, the inhibition of GATA6 expression using siRNA decreased LYPD1 gene expression and co-expression of GATA6 with a reporter vector containing the upstream sequence of the LYPD1 gene resulted in increased reporter activity. Endothelial cell network formation was attenuated when co-cultured with cardiac fibroblasts, but it was significantly restored when co-cultured with cardiac fibroblasts wherein the expression of GATA6 was knocked down with siRNA. Conclusion GATA6 regulate the anti-angiogenic properties of cardiac fibroblasts by modulating LYPD1 expression.
GATA6 regulates anti-angiogenic properties in human cardiac fibroblasts via modulating LYPD1 expression

Fibroblasts contribute to the structure and function of tissue and organs; however, their properties differ in each organ given the topographic variation in gene expression among tissues. We previously reported that LYPD1, which is expressed in cardiac fibroblasts, has the capacity to inhibit sprouting of vascular endothelial cells. LYPD1 has been shown to be highly expressed in the human brain and heart, but the regulation of LYPD1 expression in cardiac fibroblasts has not been elucidated in detail.

To identify the LYPD1-modulating transcription factor, motif enrichment analysis and differential expressed gene analysis using microarray data were performed. Quantitative real-time PCR was used to evaluate gene expression. Gene silencing were performed by transfection of siRNA. Western blot analyzed protein expression in NHCF-a. To assess the effect of GATA6 on the regulation of LYPD1 gene expression, dual-luciferase reporter assay was performed. Co-culture and rescue experiments were performed to evaluate endothelial network formation.

Motif enrichment analysis and differential expressed gene analysis using microarray data and quantitative real-time PCR revealed that CUX1, GATA6, and MAFK were candidate transcription factors. Of these, the inhibition of GATA6 expression using siRNA decreased LYPD1 gene expression and co-expression of GATA6 with a reporter vector containing the upstream sequence of the LYPD1 gene resulted in increased reporter activity. Endothelial cell network formation was attenuated when co-cultured with cardiac fibroblasts, but it was significantly restored when co-cultured with cardiac fibroblasts wherein the expression of GATA6 was knocked down with siRNA.

GATA6 regulate the anti-angiogenic properties of cardiac fibroblasts by modulating LYPD1 expression.

It has been strongly desired to construct three-dimensional (3D) tissues with vascular networks for regenerative medicine and drug discovery research. Non-parenchymal cells, such as fibroblasts and vascular endothelial cells, in various organs serve not only as structural support components of interstitial tissues but also influence parenchymal cells by providing a physiological and pathological tissue environment. In accordance, we previously reported that cardiac fibroblasts are indispensable for fabricating bioengineered cardiac tissue [1] and vascular endothelial cells within the tissue promote early 3D tissue engraftment during transplantation [2,3]. Moreover, we and others found that there are different effects on angiogenesis among some stromal cells when co-cultured with human endothelial cells. For example, atrial fibroblasts, ventricular fibroblasts, and iPS cell-derived fibroblasts are anti-angiogenic, whereas dermal fibroblasts and mesenchymal stem cells (MSC) are pro-angiogenic [[4], [5], [6]]. Furthermore, we identified Ly6/PLAUR domain containing 1 (LYPD1) as a gene responsible for the anti-angiogenic property of cardiac fibroblasts using a microarray analysis of anti- and pro-angiogenic fibroblasts in conjunction with siRNA screening experiments [4]. Despite reports on the construction of various bioengineered tissues, there have been few reports showing sufficient vascular network formation using cardiac fibroblasts in bioengineered cardiac tissue, and dermal fibroblasts or mesenchymal cells have been used as alternatives to cardiac fibroblasts. For example, dermal fibroblasts with low LYPD1 expression have a pro-angiogenic effect, but are not suitable for the fabrication of cardiac cell sheets because they are inferior to cardiac fibroblasts in terms of cardiomyocyte proliferation [7]. On the other hand, cardiac fibroblasts express VCAM-1 and have positive effects on the proliferation and survival of cardiomyocytes, suggesting that cardiac fibroblasts are suitable for the construction of cardiac tissue. However, since cardiac fibroblasts also express anti-angiogenic LYPD1, we hypothesized that down-regulation of LYPD1 expression in cardiac fibroblasts would induce endothelial network in cardiac tissue. LYPD1, also known as LYNX2 and PHTS, is a member of the Ly6 (lymphocyte antigen-6)/uPAR (urokinase-type plasminogen activator receptor) superfamily [[8], [9], [10]] containing a three-fingered structural motif constructed by a disulfide-bridge pattern formed between 10 cysteine residues [8]. LYPD1 is presumed to be a membrane-bound GPI-anchored protein and play a role in controlling anxiety by binding and modulating neuronal nicotinic acetylcholine receptors [9,11]. We previously reported that LYPD1 protein directly inhibits endothelial cell tube formation [4]. Consistent with these functions, LYPD1 has been shown to be highly expressed in the human brain and heart [12]. Moreover, the regulation of LYPD1 expression has been reported in some cancer cell lines. The expression of the LYPD1 gene is specifically up-regulated by NFkB in breast cancer brain-metastasis variants [13]. LYPD1 expression is regulated by HULC, which is a long non-coding RNA, through the sponging of miR-6754–5p that affects the progression of breast cancer cells [14]. However, the regulation of LYPD1 expression in cardiac fibroblasts has not been elucidated in detail. In the present study, we identified the transcription factors in cardiac fibroblasts involved in LYPD1 gene expression. Using an integrated bioinformatics approach of differentially expressed genes and siRNA experiments, GATA6 was identified as a transcription factor that positively controls the expression of the LYPD1 gene and modulates the angiogenesis inhibitory activity of cardiac fibroblasts.

Microarray data reported previously (GEO Accession: GSE202991) [4] was used to analyze differentially expressed genes between atrial fibroblasts, ventricular fibroblasts, dermal fibroblasts, MSCs and iPS cell-derived fibroblasts. To investigate gene expression differences, we selected genes that were Welch's t-test with non-corrected p-value <0.05 and up- or down-regulated by setting a threshold of 2-fold. Differentially expressed genes between atrial fibroblasts and dermal fibroblasts were designated as atrial fibroblast differentially expressed genes (CFa-DEGs). The UCSC Genome Browser (https://genome.ucsc.edu) was used to search CFa-specific promoter regions in which H3K4me3 peaks (GEO Accession: GSM945312) overlapping with the upstream regions from TSSs of CFa-DEGs. Then, de novo TF motif enrichment analysis for CFa-specific promoter regions was performed using the findMotifsGenome.pl script of the HOMER version 4.10 software. GO annotation for molecular function of differentially expressed genes was performed using DAVID 6.8 (https://david.ncifcrf.gov). Venn diagrams were constructed using VENNY 2.1 (https://bioinfogp.cnb.csic.es/tools/venny/).

NHDFs (Lot no. 0000247101, 0000254465, 0000214247, 0000471665, 0000472033, 0000477954, 0000661761 and 0000664504), NHCF-a (Lot no. 0000205381, 0000214476, 0000228094, 0000662121 and 18TL241906), HUVECs (Lot no. 0000119548) were purchased from Lonza Group, Ltd (Basel, Switzerland). These primary cells were maintained in commercially available growth media: FGM-2 for NHDF, FGM-3 for NHCF-a, and EGM-2 for HUVECs. Cells were cultured at 37 °C with 5% CO2 and used in all experiments at passages 5–7. The 293 FT cells were maintained in DMEM supplemented with 10% FBS, 1 mM MEM sodium pyruvate, 0.1 mM MEM non-essential amino acids, 6 mM l-glutamine, 1% pen-strep at 37 °C with 5% CO2.

Total RNA extraction was performed using the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany). RNA quantity was determined using a Nanodrop One spectrophotometer (Thermo Fisher Scientific, Inc.). First-strand cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (ABI) using isolated total RNA. First-strand cDNA synthesis was performed with a T3000 ThermoCycler (Biometra). TaqMan-probe based quantitative real-time polymerase chain reaction (qRT-PCR) was carried out using a StepOnePlus Real-Time PCR system (ABI), in accordance with the manufacturer's instructions, and relative expression was determined by the delta-delta Ct method. Gene expression levels were measured using TaqMan gene expression assays (ABI) for cut like homeobox 1 (CUX1; Hs00738851_m1), Friend leukemia integration 1 (FLI1; Hs00956709_m1), GATA binding protein 6 (GATA6; Hs00232018_m1), MAF bZIP transcription factor K (MAFK; Hs00242747_m1), and LYPD1 (Hs00375991_m1). Gene expression was normalized to endogenous β-actin (Hs99999903_m1).

NHCF-a were transfected with either Silencer select pre-designed siRNAs (Thermo) against CUX1 (siRNA ID no. s3768, s3769, and s3770), GATA6 (siRNA ID no. s5606, s5607 and s223692), and MAFK (siRNA ID no. s15504, s194858, and s194859) or Silencer select negative control siRNA2 (4390846, Thermo). siRNAs were transfected at a final concentration of 1 nM using Lipofectamine RNAiMAX reagent (Thermo). The medium was replaced after 24 h, and siRNA-treated NHCF-a cells were co-cultured with HUVECs to assess endothelial cell network structure after two days of transfection.

Cells were harvested and lysed in Laemmli buffer with sonication on ice and boiled at 95 °C for 5 min. The lysate was subjected to SDS-PAGE (12.5% gel) and blotted onto Immobilon-P membranes (Merck, Germany). Western blotting was carried out using the following antibodies: rabbit anti-GATA6 (D61E4) XP antibody (Cell Signaling Technology (CST), MA, USA), rabbit anti β-actin (13E5) antibody (CST) and Peroxidase AffiniPure Goat Anti-Rabbit IgG (H + L) (Wako, Japan). Bands were visualized using ECL Prime Western Blotting Detection Reagent (Cytiva, MA, USA), according to the manufacturer's instructions, and captured by ChemiDoc Touch MP Imaging System (BIO-RAD, CA, USA).

The LYPD1-5′ untranslated region (from −1395 to +273) was amplified from human genomic DNA as a template using PCR and inserted into the pNL2.2 vector (Promega, Madison, USA) to produce LYPD1-Luc. The cells were co-transfected with LYPD1-Luc and the firefly luciferase pGL4.53 vector using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) To analyze the transcriptional activity of the GATA6 protein, 293FT cells were transfected with the LYPD1-Luc vector, pcDNA3.1-GATA6 vector, and pGL4.53 vector (Promega). After 48 h of transfection, the luciferase activity was measured in triplicate for each group, using the Nano-Glo® Dual-Luciferase® Reporter Assay System (Promega), in accordance with the manufacturer's instructions. The relative luciferase activity was calculated by dividing the value of NanoLuc luciferase by the value of the internal control firefly luciferase.

Recombinant LYPD1 was prepared using a modification from previously described methods [4]. Briefly, human LYPD1 gene with a FLAG sequence was inserted after the signal sequence and the deleted sequence of the C-terminal GPI-anchored region was inserted into the pcDNA3.1 vector (pFLAG-LYPD1). After a 48-h transfection with pFLAG-LYPD1, 293FT cells were lysed and FLAG-LYPD1 was immunoprecipitated using anti-DYKDDDDK tag antibody magnetic beads (Wako). Proteins were quantitated using a Pierce Microplate BCA Protein Assay Kit - Reducing Agent Compatible (Thermo Scientific) according to the manufacturer's instructions.

Co-culture and rescue experiments were performed as previously described [4]. Briefly, siRNA-treated NHCF-a were mixed with HUVECs at a ratio of 12:1 and seeded at a cell density of 2.6 × 105 cells/cm2 in the presence or absence of FLAG-LYPD1 (1 μg/ml). The cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C with 5% CO2. After three days of co-culture, the cells were fixed with 5% dimethyl sulfoxide in methanol and blocked with 5% FBS in PBS. The fixed cells were stained with a phycoerythrin-conjugated monoclonal antibody against human CD31 (R&D Systems) overnight at 4 °C. Hoechst 33342 was used to visualize the nuclei. Images of endothelial cell network structures were captured using the ImageXpress Ultra confocal high content screening system (Molecular Devices, LLC, Sunnyvale, CA, USA) as previously described [4]. The computer systems and hardware were operated by MetaXpress software (Molecular Devices, LLC). The total length of endothelial network was scored using MetaXpress software.

Three or more biological replicates per sample condition were used to generate the represented data. Data are presented as means ± standard deviation. Comparison between two groups was conducted using a two-tailed unpaired Student's t-test following by an F test to compare variances. A Welch's correction for unequal variances was applied when appropriate. The total length of the endothelial network formed in the absence of recombinant LYPD1 was analyzed using Dunnett's test between the control (NC2) and respective treatment. Differences were considered statistically significant at P < 0.05.

We previously reported that there was a significant difference in the effect of cardiac fibroblasts and dermal fibroblasts on the endothelial network formation when co-cultured with HUVECs, and LYPD1 is a molecule that is highly expressed in cardiac fibroblasts and possesses anti-angiogenic effects. A clustering analysis of the microarray data (GEO Accession: GSE202991) derived from several stromal cells, including cardiac fibroblasts and dermal fibroblasts, revealed that these cells are clustered into two groups based on the degree of LYPD1 expression [4]. iPS-derived fibroblasts, which were clustered in the same group as cardiac fibroblasts exhibiting high LYPD1 expression, also suppress endothelial cell network formation when co-cultured with HUVECs [4]. On the other hand, an abundance of HUVEC network formation was observed upon co-culturing with dermal fibroblasts exhibiting low LYPD1 expression. Similarly, the promotion of sprouting of coexisting vascular endothelial cells was observed in both organoid formation and tri-culture with MSCs [5,6], which were clustered in the same group as dermal fibroblasts. In addition, HUVEC sprouting was also observed when co-cultured with lung fibroblasts exhibiting low LYPD1 expression (data not shown). These results suggest that LYPD1 may have an influence on the angiogenic effects of stromal cells in various organs; however, details regarding the regulation of LYPD1 expression have not been determined. Therefore, to clarify the regulation of LYPD1 expression, we identified the transcription factors that control the expression of LYPD1 (Fig. 1). First, we hypothesized that the transcription factors regulating LYPD1 would be among the transcription factors regulating the genes that are significantly expressed in cells with high LYPD1 expression, and selected the transcription factors specifically expressed in NHCF-a using microarray data (GEO Accession: GSE202991) [4] and the HOMER program (version 4.10) [15] (Fig. 1). Microarray data revealed that 2036 genes were defined as atrial fibroblast differentially expressed genes (CFa-DEGs) in which the expression difference between atrial fibroblasts and dermal fibroblasts was greater than 2-fold (p < 0.05) (Supplementary data S1). The transcription start sites (TSSs) of these 2036 CFa-DEGs were extracted based on the hg19 human reference genome. The regions where the H3K4me3 peaks (GEO Accession: GSM945312) overlap with the TSSs were defined as CFa-specific promoter regions and used for motif analysis to predict the transcription factors regulating CFa-DEGs. Transcription factor binding motifs (n = 393) predicted to bind to the CFa-specific promoter regions were identified using HOMER (Supplementary data S2). On the other hand, Gene Ontology (GO) analysis revealed that 66 up-regulated genes among the CFa-DEGs were shown to be associated with the GO term, “sequence-specific DNA binding”. 12 genes that overlapped between the 393 transcription factor binding motifs predicted using HOMER and the 66 sequence-specific DNA binding were identified as the CFa-specific transcription factors (Supplementary figure a and Supplementary data S2). To further validate the candidates, we compared the gene expression profiles using microarray data (GEO Accession: GSE202991) [4] from ventricular fibroblasts, MSCs, and iPS cell-derived fibroblasts in addition to atrial fibroblasts and dermal fibroblasts. Differentially expressed genes whose expression difference was greater than 2-fold (p < 0.05) were identified between the cell types with low LYPD1 expression that promote angiogenesis (dermal fibroblasts, MSC) and the cell types with high LYPD1 expression that suppress angiogenesis (atrial fibroblasts, ventricular fibroblasts, iPS cell-derived fibroblasts). Expression differences (>2-fold, p < 0.05) were observed for 1544 genes between NHCF-a and MSC, in 1921 genes between NHCF-v and NHDF, in 1574 genes between NHCF-v and MSC, in 3279 genes between iPS cell-derived fibroblasts and NHDF, and in 2873 genes between iPS cell-derived fibroblasts and MSC (Supplementary data S1). Venn diagrams were constructed to show the overlap of the differentially expressed genes in NHCF-a, NHCF-v, and iPS cell-derived fibroblasts against NHDF (Supplementary figure b, upper left) and in NHCF-a, NHCF-v, and iPS cell-derived fibroblasts against MSC (Supplementary figure b, upper right). There were 825 and 652 common sets of genes among NHCF-a, NHCF-v, and iPS cell-derived fibroblasts relative to NHDF and among NHCF-a, NHCF-v, and iPS cell-derived fibroblasts relative to MSC (Supplementary data S3). Of these, 246 genes were common to each comparative analysis (Supplementary figure b, lower) and 4 genes (CUX1, FLI1, GATA6, and MAFK) overlapping with the 12 CFa-specific transcription factors were identified as candidate transcription factors that modulate the expression of the LYPD1 gene (Supplementary figure c). Next, we used qRT-PCR to measure the expression of CUX1, FLI1, GATA6, and MAFK in NHCF-a and NHDF (Fig. 2). There were no significant differences in the expression levels of FLI1; however, the expression of CUX1, GATA6, and MAFK were significantly higher in NHCF-a compared with NHDF.

To assess the effect of the candidate genes, we used qRT-PCR to evaluate the expression of LYPD1 in NHCF-a in which CUX1, GATA6, and MAFK was knocked down by siRNA. To evaluate the potential off-target effects of the siRNAs, three different siRNA molecules were used to knockdown each gene. The knockdown efficiency was determined by qRT-PCR at 48 h following siRNA treatment. The expression levels of the three genes in the siRNA-treated cells were significantly decreased compared with the cells treated with negative control siRNA [CUX1: 9.2% ± 3.3% (s3768), 8.9% ± 2.6% (s3769), and 13.1% ± 2.9% (s3770); GATA6: 34.3% ± 0.9% (s5606), 18.1% ± 1.8% (s5607), and 25.9% ± 2.0% (s223692); MAFK: 44.2% ± 9.6% (s15504), 35.9% ± 2.4% (s194858), and 38.3% ± 11.3% (s194859)] (Fig. 3a, b, c). There was no significant difference in the expression of the LYPD1 gene in NHCF-a by CUX1 or MAFK gene knockdown using each of the three siRNA molecules compared with NHCF-a treated with negative control siRNA (Fig. 3d, f). In contrast, the expression of LYPD1 was significantly decreased in NHCF-a following GATA6 knockdown using each of three siRNA (Fig. 3e). Protein expression of GATA6 was also reduced by siRNA treatment against GATA6, suggesting that the GATA6 protein positively regulates LYPD1 gene expression (Fig. 3g). Based on these results, we performed a dual-luciferase reporter assay to examine the regulation of LYPD1 gene expression by GATA6. The reporter vector incorporating the 5’ sequence of the LYPD1 gene was prepared and transfected into 293FT cells. To assess the effect of GATA6 on the regulation of LYPD1 gene expression, GATA6 was co-expressed with the reporter vector in 293FT cells (Fig. 3h). Following the co-transfection of the GATA6 expression vector and reporter vector into 293FT cells, the expression of the reporter gene was significantly increased, suggesting that GATA6 positively regulates LYPD1 expression.

We previously reported that endothelial cell network formation was markedly inhibited in co-cultures with NHCF-a, but was significantly restored in co-cultures of NHCF-a wherein the expression of LYPD1 was knocked down by siRNA [4]. Next, we investigated whether the regulation of LYPD1 expression by GATA6 is involved in the anti-angiogenic function of LYPD1. When co-cultured with NHCF-a in which the expression of GATA6 was knocked down by any of the three siRNAs, HUVEC sprouting was significantly restored (Fig. 4, upper panel). This suggests that the recovery of the endothelial network formation resulted from the reduction of LYPD1 expression induced by knockdown of GATA6. Thus, GATA6 is involved in the inhibitory effect on endothelial cell network formation by positively regulating LYPD1 expression. In addition, the recovery of endothelial network formation by GATA6 knockdown was significantly attenuated in the presence of recombinant FLAG-LYPD1 (Fig. 4, lower panel). Thus, treatment with recombinant LYPD1 attenuated the recovery of endothelial network formation in HUVEC cells co-cultured with GATA6 knockdown–NHCF–a, suggesting that GATA6 regulates the anti-angiogenic effects of LYPD1.

Stromal cells, such as fibroblasts and vascular endothelial cells, support parenchymal cells, produce extracellular matrix, and contribute to organogenesis, homeostasis and other pathological states, such as tumor formation and fibrosis. The gene expression profiles of stromal cells vary greatly from tissue to tissue and subtypes are observed even within the same tissue [16,17]. Endothelial cells may contribute to early angiogenesis in 3D regenerative tissues and organoids constructed from various cell types [2,3,6]. We have demonstrated that fibroblasts exhibit either pro- or anti-angiogenesis effects depending on the tissue of origin and LYPD1 is an anti-angiogenic factor expressed in cardiac fibroblasts [4]. Considering these effects of stromal cells on the tissue environment, we hypothesized that effective and functional tissue regeneration may be achieved by controlling the properties of stromal cells. We demonstrated previously the reconstruction of the endothelial network in co-cultures with HUVECs by suppressing the expression of LYPD1 in cardiac fibroblasts [4]. To efficiently construct tissue by regulating the properties of stromal cells, we identified GATA6 as a putative transcription factor that regulates the expression of LYPD1 and showed that endothelial network formation can be attenuated by GATA6. This provides a new method to construct vascularized regenerative tissues that is different from promoting vascular network structure using angiogenic factors directly. Cardiac fibroblasts contribute to inflammation, myofibroblast differentiation, and cardiac hypertrophy in response to multiple stimuli, in addition to maintaining the turnover of the ECM [18,19]. We reported previously and here that the anti-angiogenic property of cardiac fibroblasts is modulated by either LYPD1 [4] or GATA6. Various molecules have been reported as markers for cardiac fibroblasts. For example, platelet-derived growth factor receptor alpha is the most widely used cell surface marker for fibroblasts [20], vimentin is expressed in both quiescent and myofibroblast differentiation states [21,22], and the expression of both periostin and alpha smooth muscle actin is higher during myofibroblast differentiation [23,24]. Some transcription factors, such as Tcf21, WT1, and Tbx18, have been used in lineage-tracing studies for cardiac fibroblasts [25,26]. Recently, it was reported that there are various subtypes of cardiac fibroblasts using single cell analysis [16,27]. Therefore, further analysis regarding the heterogeneity of cardiac fibroblasts is required to assess the potential of LYPD1 or GATA6 as novel markers for the anti-angiogenic subtype of cardiac fibroblasts. By comparing differentially expressed genes in stromal cells with different LYPD1 expression ratios and evaluating the effects on LYPD1 expression through siRNA experiments, we identified GATA6 as a transcription factor that regulates LYPD1 gene expression. The GATA zinc finger transcription factor family plays a pleiotropic role in cell fate decision and tissue morphogenesis in various organs [28,29]. The GATA family is classified into two groups based on their temporal and spatial expression patterns. GATA1/2/3 are primarily expressed in the hematopoietic system, whereas GATA4/5/6 are found in endoderm- and mesoderm-derived tissues. GATA6 is a direct target of the heart-specific transcription factor, Nkx2.5, and is mainly expressed in mesodermal tissues and plays a role during cardiac development [29,30]. GATA6 mutations in patients with persistent truncus arteriosus attenuate the transcriptional activity of downstream target genes, such as semaphorin 3C (SEMA3C) and plexin A2 [31]. GATA6 deletion in neural crest cells resulted in abnormalities including the patterning of the pharengeal arch arteries and attenuated expression of SEMA3C in neural crest-derived smooth muscle cells [32]. SEMA3C suppresses pathological retinal angiogenesis through the neuropilin1 and plexin D1 receptors [33], suggesting transcriptional regulation by GATA6 in suppressing angiogenic signals. Here, we show that GATA6 positively regulates the expression of LYPD1, which has anti-angiogenic effects; however, there are some controversial results associated with the role of GATA6 in angiogenesis. In vascular endothelial cells, GATA6 inhibited the Alk5 signal through suppression of TGF beta 1/2 expression, resulting in the promotion of angiogenesis and cell survival [34]. In cholangiocarcinoma, VEGFA expression was regulated by the interaction between GATA6 and lysyl oxidase-like 2 to promote angiogenesis and tumor growth [35]. These contrasting effects of GATA6 on angiogenesis may result, in part, from the complementarity between GATA families. The function of GATA4 and GATA6 has been reported to be partially or completely compensated for each other in several organs [36]. GATA4 and GATA6 are essential for the expression of cardiac genes, for instance, deletion of both factors prevent cardiomyocyte differentiation resulting in acardia [37], and cardiomyocyte-specific deletion of GATA4 or GATA6 in adulthood resulted in heart failure during pressure overload suggesting they may act redundantly for maintaining normal cardiac function [38,39]. GATA4 and GATA6 are also highly expressed in the fibroblast fraction of the heart, and double deletion of GATA4 and GATA6 in activated fibroblasts resulted in reduction of cardiac function and decrease in the number of capillaries after pressure overload in contrast to the unaffected fibrotic and hypertrophic response, whereas single deletion of one of these factors did not significantly affect cardiac response [40]. GATA4 and GATA6 have some redundant functions in activated fibroblasts, but the capillary density was significantly increased in GATA6 single deleted mice, suggesting that the contribution to capillary formation is not identical between GATA4 and GATA6 in cardiac fibroblasts. We reported here GATA6 positively regulates LYPD1 and acts in anti-angiogenic manner. The effect of GATA4 on the regulation of LYPD1 gene expression has not been investigated, but it should be examined in the future in terms of their redundancy between GATA4 and GATA6. The discrepancy in views on the role of GATA6 in angiogenesis as mentioned above may be due to heterogeneity in the cardiac fibroblast population. Cardiac fibroblasts are composed of different subtypes of cells, and their states, such as homeostatic and activated states, change in response to stimuli. Differences in experimental conditions, such as the method of selection by specific markers and the stimuli given to the cells, may also be responsible for the different results. It should be examined what subtypes of cardiac fibroblasts express GATA6 and LYPD1, and whether their expression changes depending on the state of the cell in the future. We present here positive regulation of LYPD1 expression by GATA6 through knockdown and forced expression of GATA6. However, the predicted GATA6 binding site is not found in the putative LYPD1 promoter region (from −1395 to +273). The fact that LYPD1 expression was not completely suppressed by GATA6 knockdown (about 40–50%, Fig. 3e) and that the luciferase activity was increased by about 2-fold by GATA6 forced expression (Fig. 3h) also suggests that GATA6 does not bind to the promoter region of LYPD1 to regulate its transcription. GATA proteins also share the property that their binding sites are primarily localized in distal regulatory regions [41], therefore LYPD1 expression may also be regulated by GATA6 binding to a distal regulatory region. It should be examined the molecular mechanism of the regulation of LYPD1 expression by GATA6 in detail in future studies.

We demonstrated that GATA6 regulate the anti-angiogenic properties of cardiac fibroblasts by modulating LYPD1 expression. Attenuating GATA6 in cardiac fibroblasts may be important in modulating vascularization during myocardial tissue fabrication.

S.M. conceived the project, designed and performed the experiments, analyzed the data, and wrote the manuscript. K.M. conceived and supervised the project, designed the experiments, and edited the manuscript. T.S. supervised the project.

The datasets generated during and/or analyzed during the current study are available in GEO repository (GEO Accession: GSE202991).

The authors declare that they have no conflict of interest.