Cardiac Endothelial Cell Transcriptome | Arteriosclerosis, Thrombosis, and Vascular Biology / 开普饭

Introduction

Endothelial cells (ECs) form the inner layer of blood and lymphatic vessels. They are involved in a variety of physiological processes including the control of vasomotor tone and blood flow, vascular permeability, leukocyte trafficking, and angiogenesis.¹ Between different and even within 1 organ, ECs show marked cellular diversity.^1,2 This includes the specification into arterial, venous, capillary, or lymphatic ECs from different vascular beds and a large number of functionally specialized endothelium such as fenestrated glomerular endothelium or the ECs forming the blood–brain barrier.² In addition to their own function, ECs interact with different other cell types in an organ-specific manner.³ In the heart, ECs are one of the most abundant cell types,^4,5 and there is a growing body of evidence for their central role in cardiovascular disease. There is extensive EC to cardiomyocyte paracrine communication modulating hypertrophic growth.^6,7 Vice versa, cardiomyocytes can promote angiogenic signaling in ECs and thereby vascularization of the heart and nutrition supply.⁸ ECs interact with immune cells and fibroblasts to control cardiac inflammation and extracellular matrix composition.^6,7 This ascribes them a particular role in the translation of cardiovascular risk factors into disease.⁹

Different studies using RNAseq gene expression data of ECs from brain,¹⁰ kidney,¹¹ aorta,¹² or peripheral vasculature¹³ provide evidence that the endothelial transcriptome is both cell type and organ specific. This reflects the distinct physiological roles of ECs from different organs but might also explain their specific response to stimulus during disease. A recently launched database provides an overview of available data sets¹⁴; however, a comparable resource for cardiac ECs is lacking. Thus, the aim of the present study was to describe and analyze the transcriptome of cardiac ECs as 1 integral cell type involved in cardiac disease.

Materials and Methods

Materials and Methods are available in the online-only Data Supplement.

Results

Isolation of Cardiac ECs

To study the cardiac EC transcriptome, we created endothelial lineage reporter mice with cell type–specific expression of GFP (green fluorescent protein; Cdh5 (cadherin 5)–GFP; Figure 1A and 1B). Transgenic GFP expression in ECs did not alter cardiac morphology, function, or Cdh5 mRNA expression (Figure I in the online-only Data Supplement).

**Figure 1.** Isolation of cardiac endothelial cells (ECs). Reporter mice expressing GFP (green fluorescent protein) in ECs were created using a tamoxifen-inducible Cdh5 (cadherin 5)-driven Cre/loxP system (A). Validation of GFP expression by fluorescence microscopy (B, arrow: arteriole; arrowheads: capillaries). EC RNA for sequencing was isolated after enzymatic and mechanical dissection of the heart and fluorescence-assisted cell sorting (FACS; C and D). ECs contained in the DRAQ5-positive nonmyocytes fraction were identified by size (C) and GFP labeling (D) and separated from myocytes and other cardiac cell types. mRNA expression of marker genes for ECs (*Cdh5*, *Pecam1*, *Vwf, Tek*, *Kdr* [vascular endothelial growth factor receptor type 2]) for cardiomyocytes (*Myh6*, *Pln, Atp2a2, Ryr2, Tnnt2*), fibroblasts (*Col1a1*), myeloid cells (*Lyz2*, *Emr1*) and smooth muscle cells (*Myh11, Acta2*) in ECs when compared with heart tissue was derived from RNAseq (E, representative traces F) and validated by quantitative reverse transcriptase PCR (G). Heart, n=4, ECs, n=3. *P<0.05. FPKM indicates fragments per kilobase of transcript per million fragments mapped; FSC, forward scatter; pA, poly(A); pCA, cytomegaly virus enhancer; and Tam, tamoxifen.

After enzymatic and mechanical tissue dissection, 151 000±41 000 GFP-positive ECs per heart were obtained by fluorescence-assisted cell sorting (n=3; Figure 1C and 1D). To validate the purity of the EC fraction, we assessed the expression levels of cell type–specific marker genes (Figure 1E through 1G). Typical EC marker genes (Cdh5, Pecam1, Vwf, Tek, Kdr [vascular endothelial growth factor receptor type 2]) were enriched in purified ECs versus heart tissue, whereas we observed a depletion of markers for cardiac myocytes (Myh6, Pln, Atp2a2, Ryr2, Tnnt2), fibroblasts (Col1a1), macrophages (Lyz2, Emr1), or vascular smooth muscle cells (Myh11, Acta2; Figure 1E).

We found 14 065 genes expressed (>1 fragments per kilobase of transcript per million fragments mapped) in heart tissue, isolated ECs, or both (Figure 2A). Expression levels of all expressed genes showed little correlation between ECs and heart tissue (Figure 2A). However, correlation significantly increased for genes with higher expression in isolated ECs (Figure 2B). Expression of genes that are specifically expressed in 1 cell type is linearly correlated to their expression in the surrounding tissue.¹⁵ Accordingly, heart tissue expression of EC-typical genes corresponded to their expression in isolated ECs. Genes with >2³-fold higher expression in ECs were considered to be typical for ECs (Spearman correlation coefficient R²=98.5 versus heart tissue; Figure 2B and 2D). Genes with >2-fold higher expression in ECs are termed EC enriched in this article (Figure 2B through 2D).

**Figure 2.** Endothelial cell (EC)–enriched gene expression. Fourteen thousand sixty-five ENSMBL annotated genes were found expressed (>1 fragments per kilobase of transcript per million fragments mapped [FPKM]) in isolated ECs, heart tissue, or both (R², Spearman correlation coefficient, A). Gene count (open squares) and Spearman correlation coefficient R² of endothelial cell vs heart tissue RNA expression (black squares). Genes >2¹-fold or >2³-fold (q<0.05) higher expressed in ECs vs heart tissue were considered to be EC enriched (light green dotted line) or EC typical (dark green dotted line, B), respectively. Read count (% FPKM) from EC-enriched (light and dark green) or EC-typical (dark green) genes was analyzed in RNAseq data from heart tissue or isolated ECs (C). Gene expression in isolated ECs or heart tissue clustered into 5 groups (D). Heart, n=4, ECs, n=3.

Reads from EC-enriched or EC-typical genes contributed to 15.5% of all mapped reads from heart tissue but 64.2% of reads from isolated ECs (Figure 2C). EC-typical genes are mostly classified as protein coding but contain also a proportion of 22 noncoding RNAs including Malat1 or Neat1 (Table II in the online-only Data Supplement).

Signaling Pathway Analysis

Expression of lineage marker genes displayed a typical EC pattern with low expression of mesodermal progenitor markers such as Mesp1 and high expression of VEGF receptor type 2 (Kdr), Cdh5, or Pecam1 (Figure 3A). Transcription factors promoting arterial (Foxc, Hey, Notch) or venous (Sox7, Nr2f2) EC specification were enriched, whereas transcription factors determining lymphatic specification (Prox1, Pdgfrb) were similarly expressed in ECs and heart tissue (Figure 3A).^16,17

**Figure 3.** Molecular pathway analysis of endothelial genes. Isolated cardiac endothelial cells (ECs) expressed marker genes or transcription factors determining EC lineage (A). Enrichment (P<0.001) of molecular pathways derived from gene ontology (GO) or Kyoto Encyclopedia of Genes and Genomes (KEGG) among EC-typical genes was analyzed using ClueGO (B). Heart, n=4, ECs, n=3.

To describe the molecular function of EC-typical genes in more detail, we performed gene expression network analysis using ClueGO (Figure 3B; Figure II in the online-only Data Supplement). We found single signaling pathways or groups of signaling pathways from gene ontology or Kyoto Encyclopedia of Genes and Genomes database significantly (P<0.001) overrepresented among EC-typical genes, including typical pathways reflecting EC function such as blood vessel morphogenesis, vasculogenesis, EC development, or regulation of EC differentiation (Figure 3B). In addition, network analysis revealed an overrepresentation of small GTPase signaling including Rap1 or Ras signaling (Figure 3B).

Genes depleted in ECs belonged to gene ontology terms related to muscle cell development, muscle contraction, and oxidative or metabolic processes indicating that they derive predominantly from cardiac myocytes (Figure II in the online-only Data Supplement).

Transcriptional Regulation of the EC Transcriptome

We aimed to identify transcription factors regulating EC transcription. Eighty-one (6.3%) of the EC-typical genes were classified as transcription factors (gene ontology term GO:0003700, transcription factor activity, sequence-specific DNA binding; Figure 4A and 4B). These included members of the ETS (E26 transformation-specific) transcription factor family, the KLF (Kruppel-like factor) family, or signal transducer and activator of transcription 3 (Stat3; Figure 4A through 4C; Tables II and III in the online-only Data Supplement). To estimate their significance for EC gene expression, we analyzed the 1100 bp promotor region of EC-enriched genes for the presence of transcription factor binding motifs (Figure 4D). Among others we found a significant overrepresentation of the ETS transcription factor family, KLF family, and interferon regulatory factor family binding motif (q<0.001; Figure 4D). A closer analysis of the binding motif density within the promotor region revealed an accumulation in close proximity to the transcription start site (Figure 4D). In line with this, reanalysis of published transcriptome data revealed that the deletion of the EC-enriched transcription factors stem cell leukemia protein (Tal1 [T-cell acute lymphocytic leukemia protein 1]),¹⁸Klf2,¹⁹ or Klf4¹⁹ leads to the dysregulation of EC-expressed genes, including other transcription factors such as Sox7 or Ets2 (Figure III in the online-only Data Supplement).

**Figure 4.** Transcription factor expression and binding motif enrichment. A subset of endothelial cell (EC)–typical genes (A) were classified as transcription factors defined by gene ontology term GO:0003700 (B). mRNA expression of representative transcription factors was validated by quantitative reverse transcriptase PCR (C). Analysis of the promotor region (−1000 bp to +100 bp from transcription start site [TSS]) of EC-enriched genes revealed a significant accumulation (q<0.0001) of distinct transcription factor binding motifs (D). Histograms represent transcription factor binding motif densities as fold of mean binding motifs per bp (D, −1000 bp to +1000 bp from TSS). Heart, n=4, ECs, n=3. *P<0.05. ETS indicates erythroblast transformation specific; IRF, interferon regulatory factor; KLF, Kruppel-like factor; Sp1, specificity protein 1; and YY1, yin yang 1.

Comparing the Cardiac to Other Organ EC Transcriptomes

To identify characteristics of ECs from different tissues, we isolated ECs from brain, kidney, lung, and skeletal muscle (Figure 5A through 5D) and compared their gene expression to cardiac ECs (Figure 5E and 5F). Spearman correlation (Figure 5E) and principal component analysis proofed clustering of the replicates from 1 organ (Figure 5F).

**Figure 5.** The transcriptome of cardiac endothelial cells (ECs) compared with ECs from other organs. ECs from brain (A), kidney (B), lung (C), or skeletal muscle (D) were isolated by fluorescence-assisted cell sorting (FACS). Spearman correlation (E) and principal component analysis (PCA, F) of mRNA expression in cardiac ECs (n=3) compared with renal (n=3), cerebral (n=2), pulmonary (n=1), and skeletal muscle (n=1) ECs. GFP indicates green fluorescent protein.

We found 1126 genes differentially regulated in cardiac versus cerebral ECs, 1058 genes in cardiac versus renal ECs, and 738 genes in cardiac versus pulmonary ECs (q<0.05; Figure 6A through 6C) including several genes with known biological function in ECs. For example, we found vascular cellular adhesion molecule 1 (Vcam1) enriched in cardiac versus renal and pulmonary ECs or G-protein–coupled receptor 124 (Gpr124) enriched in brain versus cardiac ECs (Table IV in the online-only Data Supplement).

**Figure 6.** Organ-specific gene expression in cardiac endothelial cells (ECs). Differential mRNA expression was determined in cardiac vs cerebral (A), renal (B), or pulmonary (C) ECs (q<0.05). Venn diagram showing the overlap of genes with concordant up- or downregulation in cardiac vs renal, cerebral, or pulmonary ECs (q<0.05, D). Enrichment (P<0.05) of molecular pathways derived from Kyoto Encyclopedia of Genes and Genomes (KEGG) among genes enriched in cardiac ECs vs cerebral, renal, and pulmonary ECs (E). mRNA expression of genes with higher expression in cardiac ECs vs their mean expression in renal (n=3), cerebral (n=2) and pulmonary (n=1) ECs (F). Representative trace from RNAseq showing the *Tcf15* locus (G). mRNA expression as determined by quantitative reverse transcriptase PCR in heart, cardiac, or other ECs (H–K). *P<0.05, **P<0.01. ECM indicates extra-cellular matrix; FPKM, fragments per kilobase of transcript per million fragments mapped; and PPAR, peroxisome proliferator-activated receptor.

We analyzed the overlap of genes enriched in cardiac ECs and considered 143 genes that were concordantly upregulated versus other organs as typical for cardiac ECs (Figure 6D). Gene network analysis of typical cardiac EC genes revealed an enrichment (P<0.05) of genes related to cytokine–cytokine receptor interaction, extracellular matrix–receptor interaction, PPAR (peroxisome proliferator-activated receptor) signaling, and others (Figure 6E). Members of the PPAR signaling pathway such as the fatty acid transporter CD36 or fatty acid binding protein 4 (Fabp4) were among the genes with highest expression in cardiac ECs (Figure 6F) and gained our particular interest. mRNA expression of upstream regulatory factors of Cd36 and Fabp4 expression, transcription factor 15 (Tcf15; q<0.05; Figure 6F and 6G), and mesenchyme homeobox 2 (Meox2; q=0.07 versus pulmonary, q<0.05 versus cerebral or renal ECs, Table IV in the online-only Data Supplement), was enriched in cardiac versus other organ ECs. Expression of Cd36, Fabp4, Tcf15, and Meox2 was validated by quantitative reverse transcriptase PCR (Figure 6H through 6K).

To evaluate whether these differences were particular for ECs from heart versus other muscular tissue, we isolated ECs from hindlimb skeletal muscle (Figure 5D; Figure IVA in the online-only Data Supplement). Gene expression in skeletal muscle ECs showed high correlation (Spearman correlation coefficient R²=0.92–0.93; Figure 5E) and clustering with cardiac ECs (Figure 5F). Expression of Cd36, Fabp4, Tcf15, and Meox2 was not different between cardiac and skeletal muscle ECs (Table V in the online-only Data Supplement; Figure IVB in the online-only Data Supplement). Among the 192 genes which we found higher expressed in cardiac versus skeletal muscle ECs, gene network analysis revealed an overrepresentation of genes related to complement and coagulation system (P<0.05; Figure IVC in the online-only Data Supplement).

Discussion

ECs play an important role in cardiac physiology and disease. We provide here a comprehensive data set and in-depth analysis of the cardiac EC transcriptome and compare it to the transcriptome of heart tissue and ECs from other organs.

Comparative analysis of EC versus heart tissue gene expression revealed 9.2% of expressed genes to be EC typical. This fits well with the results from previous studies in which the number of genes considered to be specific for 1 cell type varied from 10% to 34% depending on the tissue, cell isolation method, and cutoff applied.^20–22 Though ECs are highly abundant in the heart,⁵ mapped reads from EC-enriched genes constitute only a minority of the heart tissue transcriptome. mRNA amount increases with cell size²³ and cardiac myocytes, the largest cell type of the heart, dominate the cardiac mRNA expression profile. Accordingly, genes depleted in ECs are related to muscle cell function and energy metabolism. This implies that changes in gene expression in smaller cell types might be masked, and it is required to perform specific analysis on isolated cells instead of tissue. However, cardiac ECs express both markers of arterial or venous specification¹⁶ with higher expression of venous markers. Expression of these marker genes reflects the composition of the cardiac EC population and to assess gene expression in cardiac EC subtypes with low abundance, for example, valvular ECs might require additional sorting.

The differentiation of cardiac mesodermal progenitors into the different cell types of the heart and the specification of ECs is tightly regulated by transcription factors.^16,17 The upregulation of the ETS transcription factor Etv2 plays a central role during differentiation of Mesp1-positive mesodermal progenitors toward myocyte²⁴ (Isl1 or Tbx-5 positive) or EC (Flk1 positive) fate.^16,17 Importantly, Etv2 expression is restricted to a narrow time frame during differentiation²⁵ and is not detectable in mature ECs isolated from adult hearts. In contrast, other transcription factors that are involved in maintenance of cellular identity such as inhibitor of differentiation (Id1)²⁶ or stem cell leukemia protein (Tal1)^18,27 are continuously expressed. In line with this, deletion of Tal1 leads to dysregulation of EC-expressed genes.

We identified several transcription factors expressed that are typical for ECs within the heart. Transcription factor activity requires a corresponding binding site in the regulatory region of its target gene. Binding motifs of endothelial typical transcription factor families were overrepresented in the promotor region of EC-enriched genes. Some of these transcription factors have been described as key regulators of EC function,^16,25 including the ETS transcription factor family or the KLF family. Deletion of either Klf2 or Klf4 in mice alters gene expression in cardiac ECs.¹⁹ However, this effect is markedly aggravated in mice lacking both Klf2 and Klf4, leading to a severe dysregulation of EC gene expression, loss of EC function, and death.¹⁹ Other transcription factors, such as myeloid nuclear differentiation antigen like (Mndal) or PAX binding protein (Gcfc1 [GC-rich sequence DNA-binding factor candidate]), are enriched but of unknown function in ECs. Access of a transcription factor to its corresponding binding site is regulated by coregulatory transcription factors and epigenetic modifications including chromatin state or DNA methylation. The results from the present study provide a basis for future studies on EC epigenetics.

The cardiac EC transcriptome shows marked differences when compared with ECs from other organs. These differences reflect the specialized function of ECs in different organs. In a previous study, we had identified Vcam1 to play an important role in mineralocorticoid-induced cardiac but not renal remodeling.²⁸ We show here that this is related to a 4-fold higher expression of Vcam1 in cardiac versus renal ECs. Vice versa, Gpr124, a G-protein–coupled receptor, is expressed in cerebral but not cardiac ECs and crucial for the maintenance of the blood–brain barrier as recently shown.²⁹

Heart contraction and ion homeostasis are highly energy consuming and up to 90% of cardiac ATP derives from mitochondrial fatty acid oxidation.³⁰ Cardiac ECs in contrast to other organs need to provide continuous fatty acid supply for muscle contraction. When comparing cardiac versus renal, cerebral, or pulmonary ECs, we found genes upregulated that are linked to PPAR signaling, including Fabp4 and Cd36. Both FABP4 and CD36 facilitate fatty acid uptake from the circulation via the endothelial barrier.^31–33 This finding confirms a report comparing the microarray-based gene expression profile of cardiac versus brain or liver ECs.³² In contrast, the transcriptome of ECs from heart and skeletal muscle showed high similarity, including comparable expression of Fabp4 and CD36, suggesting that ECs have similar functions in different muscular tissues.

We found the transcription factors TCF15 and MEOX2 enriched in cardiac ECs versus other tissues. TCF15 and MEOX2 cooperatively regulate the expression of CD36 and Fabp4 and other genes involved in fatty acid uptake.³² Heterozygous deletion of TCF15 and MEOX2 in mice impairs fatty acid uptake and left ventricular function.³² However, CD36 also plays a role in the development of atherosclerosis,³⁴ and genetic variations in the MEOX2 gene have been associated with an increased risk of coronary artery disease in humans.³⁵ Comparison of data from genome-wide association studies on patients with cardiovascular disease with cell type–specific gene expression data might be a promising approach to identify new disease-associated genes.

In conclusion, the results from this study provide a comprehensive resource of gene expression and transcriptional control in cardiac ECs. The cardiac EC transcriptome exhibits distinct differences in gene expression compared with other cardiac cell types and ECs from other organs. We found several transcription factors that control endothelial gene expression including transcription factors with well-known function in physiology and pathophysiology. We also propose new candidate genes with high expression levels and enrichment in cardiac ECs that have not been investigated yet as promising targets for future evaluation.

Nonstandard Abbreviations and Acronyms

Cdh5	cadherin 5
EC	endothelial cell
ETS	E26 transformation specific
FABP4	fatty acid binding protein 4
GFP	green fluorescent protein
Gpr124	G-protein–coupled receptor 124
Id1	inhibitor of differentiation
KLF	Kruppel-like factor
MEOX2	mesenchyme homeobox 2
Mndal	myeloid nuclear differentiation antigen like
Stat3	signal transducer and activator of transcription 3
TCF15	transcription factor 15
Vcam1	vascular cellular adhesion molecule 1

Acknowledgments

We thank the Deep Sequencing Facility, Max Planck Institute of Immunobiology and Epigenetics (Freiburg, Germany) and the Freiburg Galaxy Team, Björn Grüning and Rolf Backofen, Bioinformatics, University of Freiburg, Germany, funded by Collaborative Research Centre 992 Medical Epigenetics (DFG grant SFB 992/2 2016) and German Federal Ministry of Education and Research (BMBF grant 031 A538A RBC [de.NBI]).

Sources of Funding

This study was supported by the Else-Kröner-Fresenius-Stiftung (2016_A163 to A. Lother), the Deutsche Forschungsgemeinschaft (Collaborative Research Centre 992 to L. Hein), the Innovationsfonds des Landes Baden-Württemberg (to L. Hein), and the BIOSS Centre for Biological Signalling Studies, Freiburg, Germany (to L. Hein).

Disclosures

None.

Footnotes

*Both authors contributed equally.

The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.117.310549/-/DC1.

Correspondence to Achim Lother, MD, Heart Center Freiburg University, Department of Cardiology and Angiology I, Hugstetter Straße 55, 79106 Freiburg, Germany. E-mail achim.lother@universitaets-herzzentrum.de

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Cardiac Endothelial Cell Transcriptome | Arteriosclerosis, Thrombosis, and Vascular Biology