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(Circulation. 1999;100:583-586.)
© 1999 American Heart Association, Inc.

Brief Rapid Communication

Endothelial Growth Factor Receptors in Human Fetal Heart

Taina A. Partanen, MD; Taija Makinen, MSc; Johanna Arola, MD, PhD; Toshio Suda, MD, PhD; Herbert A. Weich, MD, PhD; Kari Alitalo, MD, PhD

From the Molecular/Cancer Biology Laboratory (T.A.P., T.M., K.A.) and Department of Pathology (J.A.), Haartman Institute, University of Helsinki, Finland; the Department of Cell Differentiation (T.S.), IMEG, Kumamoto University School of Medicine, Japan; and the Department of Gene Regulation and Differentiation (H.A.W.), Division of Molecular Biotechnology, National Research Center for Biotechnology (GBF), Germany.

Correspondence to Dr Kari Alitalo, Molecular/Cancer Biology Laboratory, Haartman Institute, POB. 21 (Haartmaninkatu 3), University of Helsinki, FIN-00014 Helsinki, Finland. E-mail kari.alitalo{at}helsinki.fi

	Abstract

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Background—Endothelial receptor tyrosine kinases include 3 members of the vascular endothelial growth factor receptor (VEGFR) family and 2 members of the angiopoietin receptor (Tie) family. In addition, the VEGF₁₆₅ isoform binds to neuropilin-1 (NP-1), a receptor for collapsins/semaphorins. The importance of these receptors for vasculogenesis and angiogenesis has been shown in gene-targeted mice, but so far, little is known about their exact expression patterns in the human vasculature.

Methods and Results—Frozen sections of human fetal heart were stained immunohistochemically with receptor-specific monoclonal (VEGFR, Tie) or polyclonal (NP-1) antibodies. The following patterns were observed: The endocardium was positive for VEGFR-1, VEGFR-2, NP-1, Tie-1, and Tie-2 but negative for VEGFR-3. The coronary vessels were positive for Tie-1, Tie-2, VEGFR-1, and NP-1 and negative for VEGFR-2 and VEGFR-3. Myocardial capillaries and epicardial blood vessels stained for VEGFR-1, VEGFR-2, NP-1, and Tie-1; myocardial capillaries and epicardial veins weakly for Tie-2; and epicardial lymphatic vessels for VEGFR-2 and VEGFR-3, weakly for Tie-1 and Tie-2, but not for VEGFR-1 or NP-1.

Conclusions—The results demonstrate differential expression of the endothelial growth factor receptors in distinct types of vessels in the human heart. This information is useful for the understanding of their roles in physiological and pathological processes and for their diagnostic and therapeutic application in cardiovascular medicine.

Key Words: angiogenesis • endothelium • endocardium • myocardium • growth substances

	Introduction

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Angiogenesis is a growth factor–driven complex multistep process by which new blood vessels are formed from the preexisting vasculature.^{1 2} The failure of angiogenesis and insufficient growth of collateral vessels is a major problem in vascular diseases, such as arteriosclerosis and restenosis. In the normal adult vessels, growth factors angiogenic in early development serve vessel maintenance functions and regulate such things as vascular permeability. An interesting question of considerable importance for therapeutic purposes is how the growth factor signaling cascade can again be recruited to an angiogenic response in adults.

Known endothelial specific receptor tyrosine kinases include the vascular endothelial growth factor receptor (VEGFR) and tyrosine kinase with immunoglobulin and epidermal growth factor homology domains (Tie) families (for reviews, see References 1 and 2^{1 2} ). Gene targeting of these receptors or their ligands usually leads to a failure to complete embryonic development because of abnormalities of vasculogenesis and/or angiogenesis. VEGF is the best-known and the most important ligand for the VEGFRs involved in angiogenic processes in physiological and pathological conditions.² Other members include the placenta growth factor, VEGF-B, VEGF-C, and VEGF-D. At least 1 VEGF isoform (VEGF₁₆₅) binds to neuropilin-1 (NP-1), a receptor for the collapsins/semaphorins.³ Interestingly, overexpression of NP-1 in chimeric mice has been shown to cause lethal anomalies of the cardiovascular system.⁴

Tie-1 and Tie-2/tunica interna endothelial cell kinase (Tek) constitute the Tie family. Angiopoietin-1 and angiopoietin-2 have been reported to be stimulatory and antagonistic ligands for Tie-2, respectively,^{5 6} whereas no ligands for Tie-1 have been identified as yet. Both of the Tie receptor tyrosine kinases are required during embryonic development for the integrity and survival of vascular endothelial cells, particularly in the regions undergoing angiogenic sprouting of capillaries, and later for endothelial cell maintenance.^{7 8}

Human VEGF is one of the most promising candidates to be used for therapeutic angiogenesis via recombinant protein or gene therapy.⁹ However, a single growth factor may be insufficient for therapeutic purposes, because the development of a functional vascular system requires a variety of factors and their receptors and signals.^{10 11} To define the molecular anatomy of the known endothelial growth factor receptors in the cardiovascular system, we studied their expression patterns in fetal heart by immunohistochemistry.

	Methods

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Fetal Tissue
The fetal hearts were obtained from one 5- to 6-week, two 13-week, two 15-week, and one 20-week legal abortions of healthy women induced with prostaglandins. One 27- to 30-week fetal heart was obtained from spontaneous abortion. The gestational age was estimated from the foot length. The study was approved by the Ethical Committee of the Helsinki University Central Hospital. Three hearts were snap-frozen, and 4 were fixed with 4% paraformaldehyde for 20 hours, dehydrated, and paraffin-embedded for sectioning.

Immunohistochemistry
Sections 4 µm thick were immunostained with mouse monoclonal antibodies against human VEGFR-3 as described earlier,¹² except that the paraffin sections were stained by use of the TSA kit (New England Nuclear Life Science Products, Inc). The other monoclonal antibodies used were against CD31 (platelet/endothelial cell adhesion molecule-1; DAKO Immunoglobulins), von Willebrand factor/factor VIII–related antigen (6.3 µg/mL; DAKO), desmoplakin 1 and 2 (5 µg/mL; Progen Biotechnik GmbH), -smooth muscle actin (0.5 mg/mL; Sigma, clone 19), an as yet molecularly undefined blood vascular endothelial antigen (PAL-E; Sanbio), VEGFR-1 (1:200 dilution of the supernatant of clone 19¹³ ), VEGFR-2 (1:800 dilution¹³ ), Tie-2 (1.32 µg/mL¹² ), and Tie-1 (8 µg/mL¹⁴ ). Immunohistochemistry for NP-1 was carried out according to Kitsukawa et al⁴ using affinity-purified rabbit IgG against mouse NP-1 (1:200, 450 µg/mL; a kind gift from Dr H. Fujisawa). After the staining procedures, all samples were examined by a trained pathologist (J.A.). Positive control slides (eg, placenta for Tie-1 and Tie-2), negative controls involving nonimmune IgG, and controls that used PBS instead of the primary antibodies were included in the stainings for each antigen.

	Results

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The localizations of the growth factor receptors in hearts of different gestational ages were similar and are summarized in Table. In the epicardial area, the coronary artery and vein as well as some capillaries were weakly positive for VEGFR-1 (Figure 1A). The coronary arteries were VEGFR-2–negative, whereas the coronary veins and adjacent capillaries were weakly positive (Figure 1B). The epicardial lymphatic vessels were negative for VEGFR-1 but strongly positive for both VEGFR-2 (Figure 1B) and VEGFR-3 (Figure 1C). These thin-walled vessels were defined as lymphatic vessels with a CD31-positive, PAL-E antigen–negative endothelium (Figure 1G and 1H), presence of desmoplakins, and lack of red cells and desmin- or smooth muscle actin–positive pericytes/smooth muscle cells.¹² The myocardial small arterioles and capillaries and the endocardium stained strongly for VEGFR-1 and VEGFR-2 (Figure 2A and 2B). Very weak VEGFR-3 signals were also observed in some myocardial capillaries (Figure 2C) but not in the endocardium (e in Figure 2C).

View this table: [in this window] [in a new window]   Table 1. Expression of Endothelial Growth Factor Receptors in the Human Fetal Heart Vasculature

View larger version (130K): [in this window] [in a new window]   Figure 1. Comparison of adjacent sections from a 15-week human fetal heart immunostained for VEGFR-1 (A), VEGFR-2 (B), VEGFR-3 (C), NP-1 (D), Tie-1 (E), Tie-2 (F), CD31 (G), PAL-E (H), and von Willebrand factor (vWF) (I). Negative control (without primary antibody) is shown in inset of (I). In epicardium, coronary artery (CA) and vein (V) were negative for VEGFR-2 (B) and VEGFR-3 (C), whereas lymphatic vessels (L) stained strongly for both of these receptors. Arrowheads identify capillaries in epicardium, which react with antibodies against VEGFR-1 (A) and VEGFR-2 (B). Staining with antigen-blocked anti–NP-1 antiserum is shown in inset in (D). Bar=68 µm.

View larger version (164K): [in this window] [in a new window]   Figure 2. Expression of VEGFR-1 (A), VEGFR-2 (B), VEGFR-3 (C), NP-1 (D), Tie-1 (E), and Tie-2 (F) in 15-week fetal myocardium and endocardium. Note that all receptors except VEGFR-3 are expressed in endocardium (e). In myocardium, some capillaries are weakly positive for VEGFR-3 (C, arrowheads). Arrows indicate myocardial (m) capillaries. C, p represents a paraffin section used to confirm weak staining obtained from frozen (f) section.

The NP-1 immunostaining pattern was similar to that of VEGFR-1 (Figure 1D). Antigen-blocking experiments indicated that the staining for NP-1 was specific (Figure 1D).

The Tie-1 and Tie-2 proteins were coexpressed in the endothelium of large and small blood vessels and in the atrial and ventricular endothelia (Figures 1E, 1F, 2E, and 2F), Tie-2 being more prominent in the venous than the arterial endothelium. The pericardial thin-walled VEGFR-3–positive vessels expressed Tie-1 and Tie-2 weakly.

	Discussion

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Our results constitute a survey of the distribution of known endothelial growth factor receptors in the developing human heart, and they are consistent with previous localization of receptor mRNAs,^{4 15 16 17 18} except that in the present study, a minor population of the myocardial capillaries also contained VEGFR-3 in 13- to 30-week fetuses. The more intense staining of VEGFR-3 in 5-week embryos (Table) suggests that the expression is downregulated in the blood vessels during the first trimester. Interestingly, a particularly strong coexpression of VEGFR-1, VEGFR-2, NP-1, Tie-1, and Tie-2 was associated with the endocardial endothelium, which is subject to a considerable degree of hemodynamic stress. In contrast, VEGFR-3 was not detected in the endocardium or other endothelia subject to shear stress. The lymphatic vessels at this stage of development seem to contain both the Tie tyrosine kinase receptors and VEGFR-2 and VEGFR-3, which they may use for signal transduction. VEGFR-2 is the only known receptor that binds both VEGF and VEGF-C. It has been speculated that the ability of VEGF-C to stimulate the migration of capillary endothelial cells and the permeability of blood vessels are mediated via VEGFR-2.¹⁹

In summary, our findings suggest that the developing vasculature of the heart requires a variety of endothelial growth factor receptors. Although the expression of VEGFR-1 and VEGFR-2 overlapped, only the former was detected in coronary arteries and myocardial capillaries and no VEGFR-3 was detected in the blood vessels or in the endocardium, where all the other receptors were coexpressed. This suggests that although VEGFR-3 is needed for early cardiovascular development,²⁰ it later serves a more specialized biological function mainly in lymphatic endothelia. In preliminary experiments, frozen sections of adult heart tissue gave qualitatively similar results, but further studies are needed to clarify the expression of endothelial growth factor receptors in ischemic adult heart.

	Acknowledgments

 
This work was supported by the Finnish Academy, the Sigfrid Juselius Foundation, the University of Helsinki Hospital (TYH 8105), the State Technology Development Center, and EU Biomed programs BMH4-CT96-0669 and 98-3380. We thank Dr Hajime Fujisawa for the anti-NP-1 antibodies.

Received March 12, 1999; revision received June 14, 1999; accepted June 18, 1999.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Korpelainen EI, Alitalo K. Signaling angiogenesis and lymphangiogenesis. Curr Opin Cell Biol. 1998;10:159–164.[Medline] [Order article via Infotrieve]

2. Ferrara N. Vascular endothelial growth factor: molecular and biological aspects. Curr Top Microbiol Immunol.. 1999;237:1–30.[Medline] [Order article via Infotrieve]

3. Soker S, Takashima S, Miao HQ, Neufeld G, Klagsbrun M. Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor. Cell. 1998;92:735–745.[Medline] [Order article via Infotrieve]

4. Kitsukawa T, Shimono A, Kawakami A, Kondoh H, Fujisawa H. Overexpression of a membrane protein, neuropilin, in chimeric mice causes anomalies in the cardiovascular system, nervous system and limbs. Development. 1995;121:4309–4318.[Abstract]

5. Maisonpierre PC, Suri C, Jones PF, Bartunkova S, Wiegand SJ, Radziejewski C, Compton D, McClain J, Aldrich TH, Papadopoulos N, Daly TJ, Davis S, Sato TN, Yancopoulos GD. Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis. Science. 1997;277:55–60.[Abstract/Free Full Text]

6. Suri C, Jones PF, Patan S, Bartunkova S, Maisonpierre PC, Yancopoulos GD. Requisite role of angiopoietin-1, a ligand for the TIE2 receptor, during embryonic angiogenesis. Cell. 1997;87:1161–1169.

7. Dumont DJ, Gradwohl G, Fong GH, Puri MC, Gertsenstein M, Auerbach A, Brietman ML. Dominant-negative and targeted null mutations in the endothelial receptor tyrosine kinase, Tek, reveal a critical role in vasculogenesis of the embryo. Genes Dev.. 1994;8:1897–1909.[Abstract/Free Full Text]

8. Puri MC, Rossant J, Alitalo K, Bernstein A, Partanen J. The receptor tyrosine kinase TIE is required for integrity and survival of vascular endothelial cells. EMBO J.. 1995;14:5884–5891.[Medline] [Order article via Infotrieve]

9. Baumgartner I, Isner JM. Stimulation of peripheral angiogenesis by vascular endothelial growth factor. Vasa. 1998;27:201–206.[Medline] [Order article via Infotrieve]

10. Asahara T, Chen D, Takahashi T, Fujikawa K, Kearney M, Magner M, Yancopoulos GD, Isner JM. Tie2 receptor ligands, angiopoietin-1 and angiopoietin-2, modulate VEGF-induced postnatal neovascularization. Circ Res. 1998;83:233–240.[Abstract/Free Full Text]

11. Peters KG. Vascular endothelial growth factor and the angiopoietins: working together to build a better blood vessel. Circ Res. 1998;83:342–343.[Free Full Text]

12. Jussila L, Valtola R, Partanen TA, Salvén P, Matikainen M-T, Renkonen R, Kaipainen A, Detmar M, Tschachler D, Alitalo R, Alitalo K. Lymphatic endothelium and Kaposi's sarcoma spindle cells detected by antibodies against the vascular endothelial growth factor receptor 3. Cancer Res. 1998;58:1599–1604.[Abstract/Free Full Text]

13. Simon M, Rockl W, Hornig C, Grone EF, Theis H, Weich HA, Fuchs E, Yayon A, Grone HJ. Receptors of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) in fetal and adult human kidney: localization and [I-125]VEGF-binding sites. J Am Soc Nephrol. 1998;9:1032–1044.[Abstract]

14. Salvén P, Joensuu H, Heikkilä P, Matikainen MT, Wasenius VM, Alanko A, Alitalo K. Endothelial Tie growth factor receptor provides antigenic marker for assessment of breast cancer angiogenesis. Br J Cancer. 1996;74:69–72.[Medline] [Order article via Infotrieve]

15. Maisonpierre PC, Goldfarb M, Yancopoulos GD, Gao G. Distinct rat genes with related profiles of expression define a TIE receptor tyrosine kinase family. Oncogene. 1993;8:1631–1637.[Medline] [Order article via Infotrieve]

16. Kaipainen A, Korhonen J, Pajusola K, Aprelikova O, Persico MG, Terman BI, Alitalo K. The related FLT4, FLT1, and KDR receptor tyrosine kinases show distinct expression pattern in human fetal endothelial cells. J Exp Med. 1993;178:2077–2088.[Abstract/Free Full Text]

17. Dumont DJ, Fong GH, Puri MC, Gradwohl G, Alitalo K, Breitman ML. Vascularization of the mouse embryo: a study of flk-1, tek, tie and vascular endothelial growth factor expression during development. Dev Dyn. 1995;203:80–92.[Medline] [Order article via Infotrieve]

18. Peters KG, De Vries C, Williams LT. Vascular endothelial growth factor receptor expression during embryogenesis. Proc Natl Acad Sci U S A. 1993;90:8915–8919.[Abstract/Free Full Text]

19. Joukov V, Kumar V, Sorsa T, Arighi E, Weich H, Saksela O, Alitalo K. A recombinant mutant vascular endothelial growth factor-C that has lost vascular endothelial growth factor receptor-2 binding, activation, and vascular permeability activities. J Biol Chem. 1998;273:6599–6602.[Abstract/Free Full Text]

20. Dumont DJ, Jussila L, Taipale J, Lymboussaki A, Mustonen T, Pajusola K, Breitman M, Alitalo K. Cardiovascular failure in mouse embryos deficient in VEGF receptor-3. Science. 1998;282:946–949.[Abstract/Free Full Text]

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Partanen, T. A. Articles by Alitalo, K. Search for Related Content PubMed PubMed Citation Articles by Partanen, T. A. Articles by Alitalo, K. Pubmed/NCBI databases Substance via MeSH