Thrombus leukocytes exhibit more endothelial cell-specific angiogenic markers than peripheral blood leukocytes do in acute coronary syndrome patients, suggesting a possibility of trans-differentiation: a comprehensive database mining study
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Current angiogenic therapies for cancers and cardiovascular diseases have not yet achieved expected benefits, which reflects the need for improved understanding of angiogenesis. In this study, we focused on solving the problem of whether tissues have different angiogenic potentials (APs) in physiological conditions and how angiogenesis is regulated in various disease conditions.
In healthy and diseased human and mouse tissues, we profiled the expression of 163 angiogenic genes, including transcription regulators (TRs), growth factors and receptors (GF/Rs), cytokines and chemokines (C/Cs), and proteases and inhibitors (P/Is). TRs were categorized as inflammatory, homeostatic, and endothelial cell-specific TRs, and C/Cs were categorized as pro-angiogenic, anti-angiogenic, and bi-functional C/Cs.
We made the following findings: (1) the human heart, muscle, eye, pancreas, and lymph node are among the tissues with the highest APs; (2) tissues with high APs have more active angiogenic pathways and angiogenic C/C responses; (3) inflammatory TRs dominate regulation of all angiogenic C/Cs; homeostatic TRs regulate all to a lower extent, while endothelial cell-specific TRs mainly regulate pro-angiogenic and bi-functional C/Cs; (4) tissue AP is positively correlated with the expression of oxygen sensors PHD2 and HIF1B, VEGF pathway gene VEGFB, and stem cell gene SOX2; (5) cancers of the digestive system tend to have increased angiogenesis dominated by endothelial cell-specific pro-angiogenic pathways, while lung cancer and prostate cancer have significantly decreased angiogenesis; and (6) endothelial cell-specific pro-angiogenic pathways are significantly increased in thrombus-derived leukocytes in patients with acute coronary artery disease.
Our results demonstrate that thrombus-derived leukocytes express more endothelial cell-specific angiogenic markers to directly promote angiogenesis after myocardial infarction and that certain solid tumors may be more sensitive to anti-angiogenic therapies than others.
KeywordsAngiogenic genes Tissue expression of genes Pathological modulation of angiogenesis Immune regulation of angiogenesis Angiogenic leukocytes
Acute coronary syndrome
- AKT (PKB)
Protein kinase B
Cytokine and chemokine
Endothelial progenitor cell
Expressed sequence tag
Growth factor and receptor
Ingenuity pathway analysis
Induced pluripotent stem
Mitogen-activated protein kinase
National Center of Biotechnology Information
Nuclear factor kappa B
Protease, inhibitor and other
Peripheral artery disease
Peripheral blood mononuclear cell
Hypoxia-inducible factor prolyl hydroxylase
Partial oxygen pressure
Signal transducer and activator of transcription
ST segment elevation myocardial infarction
Type 1 diabetes
Type 2 diabetes
Tyrosine kinase with immunoglobulin-like and EGF-like domains
Transcripts per million
Regulatory T cell
Vascular endothelial growth factor
Vascular smooth muscle cell
Atherosclerosis and its complications, such as myocardial infarction, stroke, and peripheral artery disease (PAD), are the leading cause of morbidity and mortality in the world . Tissue damage induced by ischemia and major artery blockage is one of the major pathological mechanisms underlying these diseases. Thus, development of novel therapeutics for re-gaining efficient blood supply of oxygen and nutrients by regenerating and remodeling vascular system in the ischemic tissues hold great promise for treating these life-threatening diseases . In post-ischemic re-vascularization, the vascular system develops via the coordinated actions of several cell types for vasculogenesis , angiogenesis , arteriogenesis, and collateral growth . In this study, we focus on angiogenesis, which is the process of sprouting new vessels from pre-existing ones . Physiological angiogenesis occurs during development stages and wound healing, which consists of vessel destabilization, endothelial cell (EC) migration and proliferation, and sprouting. This is followed by the resolution phase, which is characterized by reduced EC proliferation and stabilization of the new vessel .
In ischemic diseases, inflammatory factors and cells play an important role in the mobilization of angiogenic cells , and the angiogenic process cross-talks with the inflammation process in many different ways . To determine whether inflammatory factors assist angiogenic cells in recognizing cardiovascular disease risk factors such as hyperlipidemia, hyperglycemia, obesity, metabolic syndrome, hypertension, smoke, and hyperhomocystemia , we recently reported a series of findings on the expression and roles of innate immune/inflammation sensor caspase-1/inflammasome [8, 9, 10] in regulating angiogenic cells, including endothelial cell [11, 12, 13, 14, 15], Sca-1+ progenitor cell [16, 17]. We also found that CD4+Foxp3+ regulatory T cells (Tregs) inhibit vascular inflammation [18, 19, 20] and that Treg-generated newly classified responsive immunosuppressive cytokine interleukin-35 (IL-35)  suppresses EC activation . Also, several angiogenic signaling pathways such as mitogen-activated protein kinase (MAPK) , phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)-protein kinase B (PKB, AKT) , NOTCH receptors (NOTCH1, 2, 3, and 4), NF-κB (nuclear factor kappa B), and Janus kinase (JAK)-signal transducer and activator of transcription (STAT) (JAK-STAT) have been identified in inflammatory conditions . In addition, the characterization of the roles of a variety of angiogenic mediators in re-vascularizing the ischemic myocardium has been the focus of two decades of preclinical research, including vascular endothelial growth factor (VEGF), fibroblast growth factor, hepatocyte growth factor.
Although the reports on this front are very encouraging, the development of efficiently functioning collateral vessels in the ischemic heart still faces a multifaceted challenge, including choosing route of delivery, dosing levels, relevant animal models, duration of treatment, and assessment of phenotype for efficacy , which reflect the urgent need to improve our understanding of the expression and regulation of angiogenic factors in cells and tissues in physiological and pathological settings. In this study, we address several fundamental questions including, first, whether tissues have different angiogenic potentials (APs) under physiological conditions; second, what the determining factors for the tissue AP are; and third, how angiogenesis is regulated in different diseases, especially in cancer and myocardial infarction. In this study, we hypothesized that in physiological conditions, tissues have different APs and programs, and in pathological situations, inflammation and immunosuppression regulate ischemic angiogenesis depending on disease/inflammation settings. To examine this hypothesis, we took a panoramic profiling on the expression of 163 angiogenic genes in healthy and diseased human and mouse tissues including 26 transcription factors (TRs), 64 growth factors and receptors (G/Rs), 27 cytokines and chemokines (C/Cs), and 46 proteases and inhibitors (P/Is). We found that, first, the human heart, muscle, eye, lymph node, and pancreas have the highest tissue APs; second, tissue APs may be regulated by cell oxygen sensors PHD2 and HIF1B, VEGF pathway component VEGFB, and stem cell master gene SOX2; third, angiogenesis is differentially regulated in various cancers, chronic metabolic diseases, and autoimmune diseases; and fourth, thrombus-derived leukocytes exhibit more EC-specific angiogenic makers than peripheral blood leukocytes do in acute coronary syndrome patients, which suggests thrombus-derived leukocytes may phenotypically switch into EC-like angiogenic cells to directly promote angiogenesis. Our new findings will improve the current understanding of angiogenesis in healthy and diseased settings and will eventually lead to novel therapeutics for ischemic diseases and cancers.
Tissue expression profiles of angiogenic genes in physiological conditions
Expression changes of angiogenic genes in pathological conditions
Gene expression profiles were collected from microarray datasets in NIH-GEO database (Fig. 1b). Specific samples were chosen as disease or treatment groups to compare with their parallel controls by GEO2R . The number of samples was always greater than three. By searching for the gene symbols from the microarray data, we selected the genes in our list of 163 angiogenic genes with significant expression changes (p < 0.05). The genes of which the expression changes were greater than or equal to 2-fold were defined as the upregulated genes, while genes with their expression changes less than or equal to 0.5-fold were defined as downregulated ones.
Ingenuity pathway analysis (IPA)
Molecular function types of 163 angiogenic genes were categorized based on IPA database  (Additional file 1: Table S1). Functional interactions and potential physical interactions between molecules were illustrated using IPA Path Designer (Fig. 4a and Fig. 6b).
Protein subcellular location
The subcellular localization of protein was determined using two widely used protein intracellular localization databases , namely COMPARTMENTS subcellular location database  and UniProtKB/Swiss-Prot location database (European Bioinformatics Institute). Details can be found in our previous publication .
The heart, muscle, eye, lymph node, and pancreas are among the tissues with the highest APs in humans
Tissues with high angiogenic potentials are substantiated with more active angiogenic pathways and angiogenic cytokine/chemokine responses under physiological conditions
Seven pro-angiogenic pathways are included in this study
Number of genes
ETS1 JUN MAPK1 MAPK8 MAPK14 MAP4K4
AKT1 FOXO1 FOXO3 PIK3CA PIK3CB PIK3CD PIK3CG PTEN
DLL1 DLL3 DLL4 JAG1 JAG2 NOTCH1 NOTCH2 NOTCH3 NOTCH4 RBPJ
NFKB1 NFKB2 REL RELA RELB
CISH JAK2 STAT1 STAT3
ARNT EGLN1 EPAS1 FIGF FLT1 FLT4 HIF1A KDR NRP1 NRP2 PGF VEGFA VEGFB VEGFC
ANGPT1 ANGPT2 ANGPTL3 ANGPTL4 TEK TIE1
Muscle and lymph node have more active angiogenic pathways in humans: angiogenic pathway expression profiles in 18 common human and mouse tissues
Cytokines/chemokines expression profile in human tissues
Cytokines/chemokines expression profile in mouse tissues
Taken together, these results suggest that the tissues with high APs based on tissue expression of 163 angiogenic genes also have more active pro-angiogenic pathways and stronger C/Cs responses; these angiogenic C/Cs probably are the mediators signaled via the seven angiogenic pathways.
Inflammatory transcription regulators (TRs) dominate regulation of all angiogenic cytokines/chemokines (C/Cs); homeostatic TRs regulate all to a lower extent, while endothelial cell-specific TRs mainly regulate pro-angiogenic and bi-functional C/Cs
APs among human tissues are positively correlated with tissue expression levels of cell oxygen sensors PHD2 and HIF1B, VEGF pathway component VEGFB, and stem cell master gene SOX2
To further understand how these angiogenic regulators indicate tissue APs, we categorized 163 genes into four groups of angiogenic genes (TRs, GF/Rs, C/Cs, and P/Is) and found that (1) the expression of VEGFB positively correlates with APs of all four groups of genes; (2) the expression levels of HIF1B and PHD2 positively correlate with APs of TRs, GF/Rs, and P/Is; and (3) the expression level of SOX2 positively correlates with APs of TRs and GF/Rs (Additional file 1: Figure S1). These results suggest that different AP master genes are associated with the expression of specific groups of angiogenic genes in regulating tissue angiogenesis.
Cancers in digestive system tend to have increased angiogenesis dominated by EC-specific pro-angiogenic pathways, while lung cancer and prostate cancer have significantly decreased angiogenesis
Gene expression changes of pro-angiogenic pathways in cancers
Gene expression changes of cytokines/chemokines in cancers
Pro-angiogenic pathways are significantly upregulated in thrombus-derived leukocytes in patients with acute coronary syndrome, retinopathy, and rheumatoid arthritis; various subcellular-localized upregulated angiogenic proteins in thrombus leukocytes are endothelial cell-specific
Gene expression changes of pro-angiogenic pathways in vascular diseases, metabolic diseases, and autoimmune diseases
Gene expression changes of cytokines/chemokines in vascular diseases, metabolic diseases, and autoimmune diseases
We also found that retinopathy and rheumatoid arthritis (RA) have significant changes in many pro-angiogenic pathways. However, in the conditions of chronic inflammation, atherosclerosis, PAD, metabolic syndrome (MS), type 1 diabetes (T1D), and type 2 diabetes (T2D), there are no significant changes in any of these seven pathways (Table 7), suggesting that low-dose chronic inflammation does not always have a strong systemic angiogenic stimulation. We also examined the C/C expression changes among these diseases. We found that a significant number of C/Cs is upregulated in retinopathy and RA. However, in the chronic inflammatory diseases, there are little to no changes in C/C expression (Table 8). Taken together, these results suggest that diseases with active angiogenic pathways also have highly active C/C responses for cell-cell communication.
Significant progress has been made in characterizing factors regulating angiogenic pathways related to physiological and pathological processes , which has led to the development of preclinical assessment of anti-angiogenic therapies for cancers and other diseases . However, angiogenic therapies have been less than satisfactory . Thus, the development of efficient therapeutics for various clinical settings for inhibiting cancer/tumor growth and improving angiogenesis for re-gaining blood supply in ischemic heart and ischemic brain areas after stroke still faces multifaceted challenges , which reflect the urgent need for improving our understanding of the expression and regulation of angiogenic factors in cells and tissues in physiological and pathological settings. In this study, we examined the expression of 163 angiogenesis-regulatory genes in various tissues in humans and mice in physiological settings and in various pathological processes. We have made the following significant findings: (1) the heart, muscle, eye, lymph node, and pancreas are among the tissues with the highest APs in humans; (2) tissues with high angiogenic potentials have more active angiogenic pathways and angiogenic C/C responses under physiological conditions; (3) inflammatory TRs dominate regulation of all angiogenic C/Cs, and homeostatic TRs regulate all to a lower extent, while endothelial cell-specific TRs mainly regulate pro-angiogenic and bi-functional C/Cs; (4) APs among human tissues are positively correlated (r 2 > 0.4) with tissue expression levels of cell oxygen sensors PHD2 and HIF1B, VEGF pathway component VEGFB, and stem cell master gene SOX2; (5) cancers in digestive system tend to have increased angiogenesis dominated by EC-specific pro-angiogenic pathways, while lung cancer and prostate cancer have significantly decreased angiogenesis; and (6) pro-angiogenic pathways are significantly upregulated in thrombus-derived leukocytes in patients with acute coronary syndrome, retinopathy, and rheumatoid arthritis, and various subcellular-localized upregulated angiogenic proteins in thrombus leukocytes are EC-specific. Our novel findings have provided significant insights into molecular mechanisms in regulating angiogenesis in various tissue physiological and pathological processes.
In determining the master regulators for tissue angiogenesis, two important issues, which are also related to tissue regenerative potential, have never been addressed: first, whether various tissues have differences in angiogenesis and second, what determines tissue AP if there are differences. As we know, all eukaryotic organisms rely on oxygen to support oxidative phosphorylation for efficient adenosine triphosphate (ATP) production. Therefore, a constant O2 supply, maintained by the vascular system in mammals, is critical for proper tissue development, homeostasis, and function. However, the normal physiological O2 concentration varies greatly among tissues. For example, arterial blood has a normal partial oxygen pressure (pO2) of 13%, the myocardium has a pO2 of 10%, and most tissues, including the brain, lung, liver, muscle, and bone marrow, have a pO2 of ~5% . Thus, tissue pO2 levels cannot directly serve as master regulator of the expression of angiogenic genes and therefore tissue AP. To address these questions, we examined the gene expression levels of 163 angiogenic genes in 22 human tissues and 18 mouse tissues. We have found that in physiological conditions, significant positive correlations exist between APs in various tissues and the tissue expression levels of oxygen sensors PHD2 and HIF1B, VEGF pathway component VEGFB, and stem-ness gene SOX2 (Fig. 5b). Our results on tissue APs support current understanding that PHD2, rather than PHD1 or PHD3, is the critical oxygen sensor in normoxia . HIF1B is constitutively expressed, which binds to HIF1A or HIF2A to activate transcription of hypoxia-responsive genes . When tissues with high APs encounter ischemic conditions, significant downregulation of PHD2 leads to de-suppression of HIF1A and HIF2A, which can bind to highly stably expressed HIF1B, and facilitate these tissues’ initiation of quick and strong angiogenic response. It has been reported that VEGFB binds specifically to VEGFR1, which is complex and context-dependent; and that increased VEGFB expression correlates with cancer stage, tumor multiplicity, and vascular invasion in multiple cancers . Thus, our new finding suggests that VEGFB fits well for the angiogenesis indicator. Of note, SOX2 is one of the IPS cell transcription factors or Yamanaka’s re-programming transcription factors  and can be induced in angiogenic pericytes in response to hypoxia . In addition, SOX2 functions with VEGF and drives cancer-initiating stem cells [56, 57]. Our results are the first report showing that tissue expression level of SOX2, but not of other Yamanaka’s re-programming transcription factors including MYC, KLF4, and OCT4, is significantly correlated with tissue APs.
One of the most promising and best studied of anti-angiogenic therapies utilized to date is bevacizumab, a humanized monoclonal antibody against VEGFA. Bevacizumab increases survival when added to chemotherapy in first-line treatment of patients with previously untreated metastatic colorectal cancer. However, the addition of bevacizumab has failed to generate survival advantages for metastatic disease when added to first-line chemotherapy at other sites, including metastatic breast, pancreatic, or ovarian cancer [58, 59]. These results are consistent with our findings that (1) pro-angiogenic pathways in colorectal cancer are more EC-specific, while pro-angiogenic pathways in breast or ovarian cancers are not and (2) pancreatic cancer patients show little to no changes in pro-angiogenic pathways. However, our findings are based on limited datasets in different cancer types. To confirm that EC-specific pathway dominating pro-angiogenic cancers will have better response to anti-VEGF therapy, more robust metadata analyses and experimental studies are needed.
Arterial thrombosis leads to acute ischemic diseases, including myocardial infarction, stroke, and PAD. Thrombus angiogenesis has been shown to play significant roles in promoting thrombus resolution, which may be a potential therapeutic strategy . In addition, anti-angiogenic cancer therapies have been shown to be associated with a significant increase in the risk of arterial thromboembolic events  and venous thromboembolic events . A meta-analysis of randomized controlled trials showed that treatment with anti-angiogenic drug bevacizumab might significantly increase the risk of cardiac ischemic events in cancer patients; however, the risk of ischemic stroke with bevacizumab was not significantly different from controls . Our recent reports have shown that inhibition of innate immune sensor caspase-1 improves VEGFR signaling, angiogenesis , and neovascularization of progenitor cells after myocardial infarction . Inflammatory and innate immune cells migrated to atherogenic arteries promote the formation of plaques and thrombi or limit tissue damage and facilitate resolution  while atherothrombosis, subsequent to plaque rupture or erosion, has prominent features of inflammation in patients with acute coronary syndromes . It was reported that monocytes/macrophages are the most abundant inflammatory cell types of innate immunity in coronary atherothrombosis co-expressing Toll-like receptor 4 . Immune cells, including Tregs, macrophages, monocytes and dendritic cells, have been shown to regulate angiogenesis in different disease settings by providing pro-angiogenic C/Cs and pro-angiogenic niches . However, other roles of thrombus leukocytes, outside of the several reported roles such as triggering thrombosis, causing unstable angina and myocardial infarction, remain unknown. Thus, we attempted to address an important question of whether immune cells can phenotypically switch into EC-like angiogenic cells. We compared the angiogenic gene expression in leukocytes (mainly CD14+ monocytes and CD66B+ granulocytes)  isolated from thrombi from the plaque of ACS patients to that in circulating blood leukocytes. We found the exciting results that as many as 15 important angiogenic regulators and 14 angiogenic C/Cs are significantly upregulated in thrombus leukocytes in comparison to those in peripheral blood leukocytes. The 15 upregulated genes include seven EC-surface receptors for angiogenesis, three hypoxia-response transcription factors, and other important angiogenic secreting regulators. Our results have demonstrated for the first time that thrombus-derived leukocytes can promote angiogenesis not only through secreting angiogenic C/Cs and angiogenic factors but also via phenotypical switching into EC-like angiogenic cells by upregulating EC-specific cell surface markers and hypoxia-induced transcription factor signaling pathways. Our findings suggest thrombus-derived leukocytes may trans-differentiate  into EC-like angiogenic cells, which is similar to Dr. Owens’ team’s report of trans-differentiated vascular smooth muscle cell (VSMC)-derived macrophage-like cells in atherosclerotic plaques .
Our findings are based on a comprehensive database mining strategy, which allows us to understand angiogenesis in a broad perspective. However, the limitations of this study include (1) only transcriptional expression level was examined in both physiological and pathological conditions and (2) no experimental studies are available to compare with our proposed findings. To confirm these findings, further experiments with genetically modified mouse models and genetic lineage tracing method should be performed.
This work is partially supported by NIH grants to Drs. XF. Yang, H. Wang, and ET. Choi.
Availability of data and materials
The references for the information on angiogenic genes and pathways are given in the manuscript.
The databases that were used to determine the subcellular location of the proteins analyzed in the study are found at http://www.genecards.org/.
The microarray datasets that were utilized in the study were retrieved from NIH-GEO dataset database (http://www.ncbi.nlm.nih.gov/gds/) and the numbers of the datasets are as follows: GSE 77955, GSE 79973, GSE 62452, GSE 45670, GSE 45001, GSE 71963, GSE 70951, GSE 46602, GSE 36688, GSE 75037, GSE 9327, GSE 9874, GSE 27034, GSE 61144, GSE 19339, GSE 43760, GSE 55100, GSE 73034, GSE 60436, GSE 55235, GSE 57376.
HF carried out the data gathering and the data analysis and prepared the tables and figures. NV, ERX, CJ, LW, WYY, CS, JN, QC, ETC, JXM, JY, and HW aided with the analysis of the data. XFY supervised the experimental design, data analysis, and manuscript writing. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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- 27.National Center of Biotechnology Information Unigene Library. http://www.ncbi.nlm.nih.gov/unigene. Accessed March 2016.
- 28.NCBI GEO2R. http://www.ncbi.nlm.nih.gov/geo/geo2r/. Accessed May 2016.
- 29.Ingenuity Pathway Analysis. http://www.ingenuity.com/. Accessed October 2016.
- 30.Genecards. http://www.genecards.org/. Accessed December 2016.
- 31.Binder JX, Pletscher-Frankild S, Tsafou K, Stolte C, O’Donoghue SI, Schneider R, et al. COMPARTMENTS: unification and visualization of protein subcellular localization evidence. Database. 2014;2014:bau012-bau012.Google Scholar
- 33.Jeltsch M, Leppänen VM, Saharinen P, Alitalo K. Receptor tyrosine kinase-mediated angiogenesis. Cold Spring Harb Perspect Biol. 2013;5.Google Scholar
- 41.Alias S, Redwan B, Panzenböck A, Winter MP, Schubert U, Voswinckel R, et al. Defective angiogenesis delays thrombus resolution: a potential pathogenetic mechanism underlying chronic thromboembolic pulmonary hypertension. Arterioscler Thromb Vasc Biol. 2014;34:810–9.CrossRefPubMedPubMedCentralGoogle Scholar
- 46.Jakob P, Kacprowski T, Briand-Schumacher S, Heg D, Klingenberg R, Stähli BE, et al. Profiling and validation of circulating microRNAs for cardiovascular events in patients presenting with ST-segment elevation myocardial infarction. Eur Heart J. 2016:ehw563.Google Scholar
- 53.Tian H, Mcknight SL, Russell DW. Endothelial PAS domain protein 1 (EPAS1), a transcription factor selectively expressed in endothelial cells. 1997:72–82.Google Scholar
- 63.Tabas I. 2016 Russell Ross Memorial Lecture in Vascular Biology. Arterioscler Thromb Vasc Biol. 2016;:ATVBAHA.116.308036.Google Scholar
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