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Cardiovascular Research Advance Access originally published online on December 18, 2007
Cardiovascular Research 2008 78(2):366-375; doi:10.1093/cvr/cvm108
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2007. For permissions please email: journals.permissions@oxfordjournals.org

Developmental coronary maturation is disturbed by aberrant cardiac vascular endothelial growth factor expression and Notch signalling

Nynke M.S. van den Akker1, Vincenza Caolo2, Lambertus J. Wisse1, Patricia P.W.M. Peters2, Robert E. Poelmann1, Peter Carmeliet3,4, Daniël G.M. Molin1,2,{dagger} and Adriana C. Gittenberger-de Groot1,*,{dagger}

1 Department of Anatomy and Embryology, Leiden University Medical Center, Einthovenweg 20, PO Box 9600, 2300 RC Leiden, The Netherlands
2 Department of Physiology, CARIM, Maastricht University, Maastricht, The Netherlands
3 Center for Transgene Technology and Gene Therapy, K.U. Leuven, Leuven B-3000, Belgium
4 Department of Transgene Technology and Gene Therapy, VIB, Leuven B-3000, Belgium

* Corresponding author. Tel: +31 71 526 9301; fax: +31 71 526 8289. E-mail address: acgitten{at}lumc.nl

Received 7 September 2007; revised 21 November 2007; accepted 10 December 2007

Time for primary review: 24 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 Funding
 Supplementary material
 References
 
Aims: Currently, many potential cardiac revascularization therapies target the vascular endothelial growth factor (VEGF) pathway, with variable success. Knowledge regarding the role of the VEGF/Notch/ephrinB2 cascade in (ab)normal coronary development will provide information on the subtle balance of VEGF signalling in coronary maturation and might enhance our therapeutic possibilities.

Methods and results: The effect of VEGF isoforms on coronary development was explored in vivo using immunohistochemistry and RT–qPCR on Vegf120/120 mouse embryos solely expressing VEGF120. In vitro, human arterial coronary endothelial cells were treated with VEGF121 or VEGF165 upon which RT–qPCR was performed. In vivo, mutant coronary arterial endothelium showed a decrease in protein expression of arterial markers such as cleaved Notch1, Delta-like4, and ephrinB2 concomitant with an increase of venous markers such as chicken ovalbumin upstream promoter transcription factor II. The venous endothelium showed the opposite effect, which was confirmed on the mRNA level. In vitro, mRNA expression of arterial markers highly depended on the VEGF isoform used, with VEGF165 having the strongest effect. Also, coronary arteriogenesis was anomalous in the mouse embryos with decreased arterial and increased venous medial development as shown by staining for smooth muscle {alpha}-actin, Delta-like1, and Notch3.

Conclusion: We demonstrate that VEGF isoform-related spatiotemporal cardiac alterations in the VEGF/Notch/ephrinB2 cascade lead to disturbed coronary development. This knowledge can contribute to optimizing therapies targeting VEGF signalling by enabling balancing between angiogenesis and vascular maturation.

KEYWORDS Arteriogenesis; Cell differentiation; Coronary development; Developmental biology; Endothelial function


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 Funding
 Supplementary material
 References
 
Morphological diversity between different populations of endothelial cells (ECs) has long been recognized to relate to functional variation and as such proper physiology. More recently, differential expression profiles between arterial, microcirculatory, venous, and lymphatic endothelial populations have been described,1 further underscoring endothelial diversity. Both intrinsic and microenvironmental differences are instructive for these endothelial characteristics.2 Abnormalities can lead to endothelial dysfunction and subsequent pathogenesis, as found for maternal diabetes-related foetal endothelial dysfunction,3 increased nuchal translucency,4 and tumour angiogenesis5 but also for coronary atherosclerosis6 and adult venous bypass graft disease.7

Numerous factors associate with endothelial functioning in processes, such as vasculogenesis, angiogenesis, and arteriogenesis, with one of the key factors being vascular endothelial growth factor (VEGF).8 Therefore, it is not surprising that many potential therapies for ischaemic cardiovascular disease target VEGF, although clinical success is less than expected.8 VEGF exerts its main effects through binding to the VEGF receptor VEGFR-2.8 Its coreceptor neuropilin-1 (NP-1) can enhance this effect.9 VEGF signalling can upregulate members of the Delta-like/Jagged/Notch-family, proteins highly important in cardiovascular development.10,11 Several members of this family are specifically expressed by arterial ECs, including Notch1, Notch4, and Delta-like4 (Dll4).12 Upon stimulation of the Notch-receptor, its cytoplasmic domain becomes cleaved and translocated to the nucleus. There, it forms a complex with other nuclear proteins, which upregulates transcription of Notch signalling specific transcription factors (i.e. Hes, Hey). These can enhance arterial EC-specific ephrinB2 expression,10 suggesting a role for the VEGF/Notch/ephrinB2 cascade in arterial differentiation.11

The development of the coronary vasculature is of eminent importance for the final phases of cardiogenesis. Disbalance in these processes might lead to a dysfunctional vascular bed which will predispose for coronary diseases. Coronary development starts with the occurrence of a subepicardially located primitive endothelial network. This becomes connected to the systemic circulation downstream to the right atrium and upstream to the ascending aorta through coronary orifices at the level of the left and right sinus of the semilunar valves.13 After connection to the circulation is established, arterial differentiation of the most proximal ECs becomes obvious and, subsequently, arteriogenesis is stimulated by inducing vascular smooth muscle cell (vSMC) migration and differentiation.13,14 In the coronary system, vSMCs are mainly recruited from the population of epicardium-derived cells (EPDCs), which arises through epithelial-to-mesenchymal transformation of cells of the epicardium.15 In addition, microcirculatory and venous endothelial differentiation and remodelling of the primitive endothelial network into a fully functional coronary system will take place.

We recently described the Vegf120/120 mouse model that solely expresses the non-heparin binding isoform VEGF120. Structural cardiac abnormalities such as Tetralogy of Fallot (TOF) were found concomitant with alterations in VEGF and Notch signalling.16,17 This mouse model presents postnatally with impaired myocardial angiogenesis18 and altered retinal arterio-venous differentiation.19 In this paper, we show that changes in spatiotemporal distribution of VEGF and Notch signalling—due to altered VEGF isoform expression in Vegf120/120 embryos—lead to anomalous coronary EC differentiation and disturbed arteriogenesis.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 5. Conclusions
 Funding
 Supplementary material
 References
 
2.1 Mouse experiments
All animal experiments were approved by the Animal Ethics Committee of the Leiden University and executed according to the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85–23, revised 1996). For an extensive description of mouse experiments see.17

2.2 Immunohistochemistry, in situ hybridization, and microscopy
Immunohistochemistry was performed as described.17 In short, immunohistochemistry was carried out on sections of 5 µm of 4%PFA/0.01 Mol/L phosphate buffered fixed, paraffin embedded tissue. Antigen retrieval was achieved either by heating the sections (12 min to 98ºC) in citric acid buffer (0.01 Mol/L, pH6.0) or by incubating them for 6 min in 0.1 mg/mL Pronase E (1.07433.0001, Merck, Darmstadt, Germany) in PBS. Visualization was performed with the DAB-procedure and Mayer’s haematoxilin was used as a counterstaining. Differences between genotypes were scored per immunostaining in at least three different embryos per genotype per age-group (i.e. E10.5–E14.5 or E15.5–E19.5) that were processed within one and the same experiment. The primary antibodies were checked for false positive signals. An extensive description of the primary antibodies used is listed in Supplementary material online, Table S1. The {alpha}-Notch4 antibodies listed in Supplementary material online, Table S1 were tested to bind aspecific to the material used and therefore no characteristics were mentioned. For in situ hybridization, sense and anti-sense 35S-radiolabelled Vegf-A RNA probes were transcribed using a 451-bp clone encoding for the mouse Vegf-120 isoform (pVEGF2; kindly provided by Dr G. Breier, University of Technology, Dresden, Germany). Micrographs were made using an Olympus AX70-microscope fitted with Olympus UPlanApo-objectives (Olympus, Tokyo, Japan). The camera used was an Olympus DP12 and micrographs were fitted into a panel using Adobe Photoshop CS2 (Adobe, San Jose, USA).

2.3 3D-reconstructions
3D-reconstructions were performed as described.4 In short, micrographs were made of E18.5 wild-type and Vegf120/120 hearts and converted to 3D using ResolveRTTM-software (Template Graphics Software Inc., San Diego, USA).

2.4 Morphometry
For morphometry, VEGFR-2 stained sections of eight embryos (four Vegf+/+ and four Vegf120/120; E17.5-E19.5) were used. To calculate the total heart volume and the ratio myocardial to vascular volume, we performed volume measurements for total heart volume, myocardium, and coronary veins/microvasculature using the Cavalieri method.20 Statistical analysis was performed using a Mann–Whitney test with SPSS 11.0 (SPSS Inc., Chicago, USA) software.

2.5 Cell culture
Human coronary artery endothelial cells (HCAECs) were obtained from Cambrex (Verviers, Belgium) and cultured according to the protocol of the manufacturer. Cells at passage 9 were seeded at a density of 3500 cells/cm2 and cultured to a 70% subconfluent monolayer in EGM-2-MV BulletKit without bFGF and VEGF165 supplemented with 5% Fetal Bovine Serum (Cambrex). Subsequently, HCAECs were stimulated for 24 h with 100 ng/mL recombinant human VEGF165 or VEGF121 (RELIATech GmbH, Braunschweig, Germany). Cells were harvested according to the manufacturer’s protocol using a trypsin/EDTA and a Trypsin neutralization solution (Cambrex). Cell number was determined using trypan blue dye and the Neubauer improved chamber method. mRNA was isolated as described below. Per sample, 100 000–140 000 cells were harvested. Samples stimulated with either VEGF165 or VEGF121 provided higher cell numbers than unstimulated samples (cultured without VEGF).

2.6 RT–qPCR
Total RNA from triplicate HCAEC cultures or from cardiac tissue of E18.5 wild-type and Vegf120/120 embryos (n = 3 per group) was isolated by using the RNeasy micro-kit Qiagen with DNAse treatment (Qiagen, GmbH, Hilden, Germany). Sample with sufficient RNA quality (OD260/280 > 1.9, RIN > 7.5) and content were approved for analysis. A total of 100 ng total RNA per sample was subjected to reverse transcription (RT). qPCR was performed by using SuperscriptTMIII Platinum Two-step qRT–PCR kit with SYBR green (Invitrogen, Paisley, UK) and primer concentration of 10 µM. qPCR reactions were run on a MyiQ Single-Color Real time PCR detection System (Bio-Rad, Veenendaal, the Netherlands). Primers were designed with oligoperfectTM Designer (Invitrogen), Primer3 and Mfold (http://www.idtdna.com/scitools/Application/mfold/) and were synthesized by Eurogentec (Seraing, Belgium). All primers used met the requested qPCR efficiency (between 80 and 105%) and were found to be gene-specific. Primer sequences are found in Supplementary material online, Table S2. Data analysis was performed using a Microsoft Excel spreadsheet based on the qBase program.21 Statistic significance was determined by applying one-way ANOVA/Tukey (SigmaStat v2.03, Systat Software, San Jose, CA, USA) testing. Sample with a probability value P < 0.05 were regarded to be significant different between groups. HCAEC samples were normalized for input based on both β-ACTIN and GAPDH values. For the E18.5 embryos normalization was based on the pan-endothelial marker Tie2. No significant differences were found for Tie2 between wild type and Vegf120/120 littermates as determined after normalization for β-Actin and Gapdh expression (correction for cDNA input). Using immunohistochemistry, no altered myocardial expression of the genes investigated was found. Myocardial Notch4 expression was not determined due to aspecifc binding of the antibodies tested (see Supplementary material online, Table S1); however, Notch4 has been reported to be highly selective for ECs during development.22


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 5. Conclusions
 Funding
 Supplementary material
 References
 
3.1 Anomalous coronary morphology in Vegf120/120 mouse embryos
In line with literature on murine coronary development,23 the first appearance of coronary ECs and vessels was seen at E11.5 when using an {alpha}-VEGFR-2-staining for visualization of the ECs. We observed that at this stage, primitive coronaries already connected (in)directly to the right atrium in all embryos, suggesting normal coronary development in mutant embryos (Figures 1A, B, E, and H). However, abnormally large coronary veins were discerned in 22/33 early-stage (E11.5–E13.5) mutant cases (Figure 1A–B and F–G).


Figure 1
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  		Figure 1 Abnormal coronary and aortic arch morphology and Vegf-distribution in Vegf120/120 embryos. Wild type (+/+) and mutants (120) are compared [upper left corner, age in embryonic days (E)] with the staining indicated upper right. At E11.5, a direct (arrow) and an indirect via the left cardinal vein (LCV; arrowhead) connection between the coronary system and the right atrium (RA) are seen in +/+ and 120 embryos [schematic drawings in (A) and (B), respectively]. The indirect connection is also shown in (E) (+/+) and (H) (120) and arrows in (Ei) and (Hi). The stainings indicated in (E) and (H) do not correspond, as differentially stained sections had to be chosen due to the fact that the connection can only be seen in a very small area of the embryos. Mutant coronary arteries are smaller and reveal abnormal morphology of the main branches [arrows in (C) and (D)], especially in embryos with TOF (D). Both the aortic arch (AoA) and coronary arteries are shown in red, the pulmonary trunk (PT) in green. At E11.5, an increase in size of the coronary veins (CV) is seen in mutant embryos [compare (F) and (G)]. This is maintained throughout development (J and K). An increase in size of cardiac lymphatics (CL), as visualized using a staining for the lymphatic EC-specific marker LYVE-1, is obvious (L and M). In mutant embryos, ventriculo-coronary artery connections (VCAC), visualized using an ephrinB2-staining (N–Niii; with Nii being a consecutive section of Ni stained for {alpha}-SMA and Niii a section 35 µm caudal from Nii) and arterio-venous shunts in both the coronary system visualized using a Notch3-staining (O and arrow in Oi) and the AoA (P and dotted lines in Pi) were seen. Locally increased levels of Vegf-mRNA are observed in mutant embryos [arrows in (Q) and (R)]. Abbreviations: AAo, ascending aorta; CA, coronary artery; CM, compact myocardium; LA, left atrium; LV, left ventricle; PC, pericardial cavity; RCV, right cardinal vein; RV, right ventricle. Scale bar = 60 µm (E, H, Ni–P); 20 µm (Ei, Hi, Oi); 30 µm (F, G, J, K, L, M); 200 µm (N, Pi, Q, R).

 
In wild-type embryos, two definitive coronary orifices, originating from the left and right aortic sinus, respectively, were observed in 24/24 cases of E15.5 and older. In contrast, only 15/28 Vegf120/120 embryos of these stages presented with two coronary orifices, whereas others showed only one (2/28), three (7/28) or in one case four orifices (1/28 and Table 1). After formation of the coronary orifices, maturation of coronary arteries was obvious in normal embryos. In 22/28 mutant embryos between E15.5 and E19.5, arteriogenesis was severely impaired (Table 1; Figure 1C–D and movies online). Half of the mutant embryos showed abnormalities in anatomy of the primary coronary branches (Table1; Figure 1C–D and movies online), with the main artery supplying the interventricular septum (IVS) either having its own coronary orifice and/or originating from the left instead of the right sinus, which is normal.24 All embryos in which the IVS-branch originated (in)directly from the left sinus were previously diagnosed with TOF (5/28).17 For a more detailed description of the abnormalities in coronary branching patterns, we refer to the online supplement.


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  		Table 1 Coronary abnormalities in Vegf120/120 embryos of E15.5–E19.5

 
In 50% of E15.5–E19.5 Vegf120/120 embryos (14/28) enlarged coronary veins (Table1; Figure 1J and K), already evident at earlier stages, and enlarged cardiac lymphatics (16/28; Table1; Figure 1L and M), visualized using staining for LYVE-1 of which expression is specific for lymphatic ECs, were encountered. Also, in late stage embryos, ventriculo-coronary arterial communications (VCACs) and/or coronary arterio-venous shunts were evident (Table 1; Figure 1N and O). Arterio-venous shunts between the aortic arch and vena cava superior were found in three cases (all E19.5; Figure 1P).

3.2 Spatiotemporal distribution of Vegf in normal and mutant embryonic hearts
During early cardiac development (E10.5–E14.5), the distribution of Vegf expression has been described before17 and is likely mainly related to myocardial and to a lesser extent to coronary development. In later stages (E16.5 and older), the predominant area of Vegf expression was the borderline of compact to trabecular myocardium, overlapping with the region where prominent coronary angiogenesis and arteriogenesis take place at this stage.25 Vegf levels in this area were increased in Vegf120/120 embryos (Figure 1Q and R).

3.3 Loss of coronary venous endothelial differentiation in Vegf120/120 embryos
Besides morphological differences in mutant veins, abnormal differentiation of coronary venous ECs was clearly distinguishable from E15.5 onwards (see Supplementary material online, Table S3). High expression per EC of VEGFR-2 was still seen at later stages of development (Figure 1J and K), together with anomalous expression of arterial markers including cleaved (nuclear/activated) Notch1, Notch2, Dll4, and ephrinB2 (Figure 2A, B, E, F, I, J, M, and N), suggesting increased VEGF and Notch signalling per EC. Expression of the venous EC-markers chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) and EphB4 (Figure 2Q, R, U, and V) was clearly reduced in mutant coronary venous ECs.


Figure 2
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  		Figure 2 Abnormal EC-differentiation and microvascular morphology in Vegf120/120 embryos. Stainings performed are indicated in the upper right corner and the genotype (+/+ wild type and 120 mutant) and age in embryonic days (E) upper left. ECs of the mutant coronary veins (CV) showed an increase in staining per cell for arterial markers cleaved Notch1 (A and B), Notch2 (E and F), Dll4 (I and J) and ephrinB2 (M and N) and a decrease for the venous markers COUP-TFII (Q and R) and EphB4 (U and V). Increased capillary size is seen (Y and Z). ECs of the coronary arteries (CA) show decreased expression of cleaved Notch1 (C and D), Jagged1 (G and H), Dll4 (K and L), and ephrinB2 (O and P) concomitant with an increase in COUP-TFII (S and T), EphB4 (W and X) and VEGFR-2 expression (AA and AB). Abbreviations: Cl. Notch1, cleaved Notch1; PC, pericardial cavity. Scale bars = 20 µm.

 
RT–qPCR, normalized for endothelial-specific Tie2 expression, was performed on whole hearts of either wild-type mouse embryos or Vegf120/120 littermates of E18.5. A significant increase of Notch1, Notch4, and Hes1 mRNA-levels could be found (Figure 3A). Furthermore, a significant decrease in Ephb4 mRNA was obvious, while a positive trend was noticed for expression of Dll4 and Hey1 (Figure 3A).


Figure 3
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  		Figure 3 Quantification of alterations in mRNA expression in vivo and in vitro with regard to VEGF isoforms and abnormalities in microvascular density in Vegf120/120 embryos. A significant (*) increase in Notch1, Notch4, and Hes1, a significant decrease in Ephb4 and a trend in increase of Dll4 and Hey1 mRNA expression is seen when performing RT–qPCR normalized for Tie2 on mRNA isolated from wild type (+/+) and mutant (120) hearts of E18.5 (A). Using morphometry, a significant difference (P = 0.021) in myocardial/vascular volume is found (B). When human coronary arterial ECs (HCAECs) are cultured with VEGF165 or VEGF121, it is obvious that VEGF165 can while VEGF121 cannot significantly increase expression of arterial markers (NP-1, NOTCH1, NOTCH4, DLL4, HES1, and EPHRINB2) and decrease expression of the venous marker COUP-TFII when compared with HCAECs cultured without VEGF (of which expression is set to 1) (C).

 
3.4 Abnormalities in mutant coronary microvasculature
Not only the coronary veins, but also the capillaries were enlarged and dilated in Vegf120/120 embryos (Figure 2Y and Z). To quantify these differences, morphometry was performed on late-stage embryos. Total heart volumes did not differ significantly between groups (data not shown). The ratio myocardial to coronary vascular volume was significantly decreased in mutant mouse embryos with a P-value of 0.021 (Figure 3B). The differentiation of microvascular ECs in Vegf120/120 mouse embryos, however, was indistinguishable from normal.

3.5 Coronary arterial endothelial differentiation is impaired in Vegf120/120 embryos
In mutant embryos of E15.5 and older, obvious differences in differentiation of coronary arterial ECs were observed (see Supplementary material online, Table S3). Protein expression levels indicative for arterial maturation, including cleaved Notch1, Jagged1, Dll4, and ephrinB2 (Figure 2C, D, G, H, K, L, O, and P) were lower per EC, suggesting decreased Notch signalling. Increased expression per EC of the venous markers COUP-TFII and EphB4 (Figure 2S, T, W, and X) was observed. Due to lack of Notch4-specific antibodies, its expression profiles could not be determined (see online information). Although arterial endothelial VEGFR-2 expression normally decreased upon induction of Notch signalling,26 this decrease was much less prominent in mutant embryos (Figure 2AA and AB).

To determine whether differences in arterial marker expression were VEGF isoform related, expression of differentiation-specific markers (i.e. arterial; NP-1, NOTCH1, NOTCH4, DLL4, HES1, EPHRINB2, and venous; COUP-TFII) was analysed in HCAECs in vitro. RT–qPCR on HCAECs cultured with or without VEGF165 or VEGF121 showed that VEGF121 was much less able to affect expression than VEGF165. VEGF165 significantly induced expression levels of arterial markers and decreased expression of COUP-TFII (Figure 3C).

3.6 Abnormal arteriogenesis is evident in Vegf120/120 mouse embryos
Anomalous coronary endothelial differentiation observed in mutant mouse embryos was concomitant with abnormal arteriogenesis. Coronary arteries of mutant embryos of E15.5 and older showed a decrease in medial development with a reduction in number of medial cells and abnormal morphologic and differentiation characteristics as evident by staining for the SMC-marker {alpha}SMA (Figure 4A and B). The spatiotemporal protein expression of NP-1, Dll1, Jagged2, and Notch3 was analysed and a decrease in number of cells positive for these markers was seen in the medial layer of the coronary arteries of mutant embryos (Figures4E, F, I, J, M, N, Q, and R). In contrast, the coronary veins of mutant embryos were surrounded by a larger number of {alpha}SMA positive cells when compared with wild-type littermates (Figure 4C and D). These cells were also positive for the SMC-related markers NP-1, Dll1, and Jagged2 (Figures 4G, H, K, L, O, and P). Additionally, the pericyte-coverage of the coronary microvasculature as determined by Notch3-staining showed a marked decrease in number of positive cells in mutant mouse embryos (Figure 4S and T).


Figure 4
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  		Figure 4 Abnormal coronary arteriogenesis in Vegf120/120 embryos. The expression of several different markers (upper right) in vSMCs/pericytes was compared between wild type (+/+) and mutants [120; upper left together with embryonic age in days (E)]. A decrease in medial cells expressing {alpha}SMA (A and B), NP-1 (E and F), Dll1 (I and J), Jagged2 (M and N) and Notch3 (I and J) is obvious in the coronary arterial media (CA) of mutants while an increase of {alpha}SMA (C and D), NP-1 (G and H), Dll1 (K and L) and Jagged2-positive cells (O and P) surrounds the coronary veins (CV). A decrease in number of Notch3-positive pericytes is seen in mutant coronary microvasculature (K and L). Abbreviation: PC, pericardial cavity. Scale bars = 20 µm.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
5. Conclusions
 Funding
 Supplementary material
 References
 
The loss of the Vegf164 and Vegf188 isoforms in Vegf120/120 mutants results in spatiotemporal alterations in VEGF and Notch signalling in the heart. These alterations coincide with abnormalities in coronary endothelial differentiation and subsequent arteriogenesis. The data provide us with further insight on the role of both VEGF isoform and spatiotemporal VEGF-distribution on coronary differentiation and maturation.

4.1 Anomalous endothelial differentiation of the Vegf120/120 coronary system
VEGF signalling influences vasculogenesis and angiogenesis, but also arterial endothelial differentiation.11,27 VEGF signalling in ECs can upregulate expression of members of the Notch signalling family, which instructs ephrinB2 expression and defines arterial differentiation.11,27 In venous ECs, expression of these proteins is inhibited by the vein-specific transcription factor COUP-TFII, thereby favouring EphB4 expression and venous differentiation.28 We show in vitro that addition of VEGF165, but not of VEGF121, leads to significant decrease in COUP-TFII expression in human coronary arterial ECs, indicating a possible mechanism through which VEGF signalling might favour arterial endothelial differentiation. The exact pathway by which VEGF signalling regulates COUP-TFII expression remains to be elucidated.

The main isoform expressed during normal murine cardiac development is Vegf164.17 VEGF164 has a proximate bioactivity to its place of production due to high heparin-binding capacities29 (Figures 1Q and 5A, C, and E). We show that cardiac Vegf-production in vivo overlaps in later stages (≥ E16.5) with the region where coronary arteries develop (i.e. deep in the myocardium of the ventricular free walls in the mouse). We consider that this spatial distribution will result in relatively high levels of VEGF and subsequent Notch signalling in ECs localized in the region of the developing coronary arteries due to (i) high local levels of VEGF, combined with (ii) amplification of signalling levels by artery-specific NP-128 (Figure 5C). This is reflected in vivo by inhibition of COUP-TFII and induction of Dll4, cleaved Notch1, and ephrinB2 staining in coronary arterial ECs in late embryonic stages, suggesting increased Notch signalling, and (potentially Notch-induced26) reduced VEGFR-2 expression. In contrast, veins develop subepicardially and therefore, venous ECs are mainly subjected to the less expressed and more diffuse VEGF120 isoform, with resulting low levels of Notch signalling as indicated by low cleaved Notch1 and high COUP-TFII and EphB4-staining in wild-type embryos. In combination with the fact that venous ECs do not express NP-1,28 this leads to the assumption that coronary venous ECs experience lower levels of VEGF and Notch signalling (Figure 5E). This spatiotemporal determination of arterial and venous specification also corresponds with the situation in zebrafish. Here, VEGF expression is induced in the somites next to the notochord, which is located directly caudal from the dorsal aorta. The posterial cardinal vein is positioned more ventrally, so further away from the place of VEGF-production.27


Figure 5
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  		Figure 5 VEGF expression and signalling during embryonic coronary development in normal and Vegf120/120 hearts. (A and B) Schematic drawings of transverse sections of the left ventricle (LV) of wild type (+/+) and mutant (120/120) embryos of E16.5 are shown. In +/+ embryos, Vegf-production mainly takes place in the borderzone between compact and trabecular myocardium (A; see also Figure 1Q), where coronary arteries (red) develop. As this is mainly Vegf164, protein distribution (yellow) is expected to give high protein levels around arteries and low around veins (blue). As mutants (B) only produce Vegf120, protein levels are expected to be more dispersed due to lack of heparin-binding domains. This will lead to relative low protein levels around arteries and high levels around veins. (C–F) Schemes indicating the expected VEGF signalling levels in +/+ and 120 coronary arterial endothelial cells (AECs) and venous endothelial cells (VECs) are shown. In a +/+ AEC high VEGF protein-levels combined with neuropilin-1 (NP-1)-mediated amplification leads to high levels of VEGF signalling (C), while low VEGF-concentrations and lack of NP-1 expression in VECs leads to low levels of VEGF signalling (E). In mutant embryos, relative low VEGF-levels around the AECs in combination with lack of NP-1-mediated amplification leads to relative low levels of VEGF signalling in a 120/120 AEC (D), while relative high VEGF-levels around the VECs lead to high signalling levels (F). Due to resolution, in (A) different VEGF isoform are indicated with yellow dots of the same size.

 
In Vegf120/120 mouse embryos, only the highly soluble non-heparin binding VEGF120 isoform is produced.29 This will affect the VEGF-gradient in the myocardium giving relative low levels of VEGF in the area of the coronary arteries and higher levels around the coronary veins (Figure 5B, D, and F). Because of this, combined with the fact that VEGF120 cannot induce formation of the signalling potent NP-1/VEGFR-2 complexes,30 lower VEGF, and Notch signalling levels are expected in the ECs in the area of the coronary arteries (Figure 5D), and higher levels in the ECs of the coronary veins (Figure 5F). This could lead to the alterations in EC-differentiation observed in these embryos. Besides altered differentiation, a severe decrease in coronary arterial size coincides with an increase in venous size. As VEGF is involved in endothelial proliferation and vascular fusion,31 locally increased Vegf expression in the venous region could lead to enlarged veins as observed in the mutant mouse embryos. Therefore, we speculate that the RT–qPCR data normalized for endothelial input on embryonic hearts indicate mainly venous expression. This underscores that VEGF signalling in venous ECs might induce expression of arterial markers, such as Notch1 and Notch4. Unfortunately, we could not confirm alterations in Notch4 protein expression as the anti-Notch4-antibodies tested bind aspecific (see Supplementary material online, Table S1).

Our in vitro data show that the expression of arterial markers in HCAECs varies for the VEGF isoform applied, with VEGF165 having a more prominent effect than VEGF121. This probably relates to higher levels of VEGF signalling through VEGFR-2 with VEGF165 due to NP-1-mediated amplification.9,28,30 Unfortunately, we could not directly test the effect of VEGF signalling on coronary venous ECs in vitro, as these cells cannot be obtained for culturing. However, earlier published data on HUVEC show that treatment with VEGF induces arterial phenotype of venous ECs11,32 as does ectopic VEGF expression in zebrafish.33

Total heart volumes are unchanged in mutant embryonic hearts, while a decrease in myocardial/vascular volume-ratio is seen, together with abnormal (dilated) microvascular morphology. This pleads for an increase in vascular volume rather than a decrease in myocardial volume as the underlying cause for the altered myocardial/vascular volume-ratio. This is likely caused by a disturbed VEGF-gradient, as it has been described that loss of a VEGF-gradient leads to alterations in microvascular branching morphogenesis34 and increased availability of VEGF induces hyperfusion of vessels.31 Furthermore, VEGF is able to induce proliferation of lymphatic ECs,35 located subepicardially in the heart. Another function of VEGF as Vascular Permeability Factor36 might additionally lead to the appearance of the increased size of the cardiac lymphatic system in mutant embryos.

4.2 Alterations in Vegf120/120 coronary arteriogenesis
We show that incorrect coronary arterial EC-differentiation coincides with impaired development of the media, together with a gain of arterial phenotype in coronary venous ECs and an increased number of venous pericytes. The differential state of ECs influences recruitment or proliferation and maturation,37,38 suggesting a role for VEGF signalling on arteriogenesis via manipulation of endothelial performance. A direct role for VEGF signalling in arteriogenesis is supported by the described instructive role for NP-139 and Notch signalling38,40 in differentiation of vSMCs. Concomitantly, we show that the amount of cells expressing {alpha}SMA, NP-1, Dll1, Jagged2, and Notch3 in the coronary arterial media and of Notch3-positive microvascular pericytes was impaired in mutant embryos, while an increase in number of cells surrounding the coronary veins positive for these markers was seen. Besides a positive role for Notch signalling in arteriogenesis, inhibiting roles have been described as well.41 Our data plead for an instructive rather that an inhibiting role. We suggest that lack of larger VEGF isoforms leads to a deficient development of the vSMC-layer of the coronary arteries. This can occur either indirectly via altered endothelial differentiation, or directly through a decrease in VEGF-related Notch signalling in the mutant coronary vSMCs.

4.3 Morphological abnormalities in the coronary system of mutant mouse embryos
Congenital coronary anomalies are often correlated with primary cardiac defects. In all Vegf120/120 mutants with TOF,17 the IVS was supplied by an artery coming from the left sinus, in contrast to normal where the IVS is supplied by a branch of the right coronary artery.24 The anomalies specific for TOF (overriding and/or dextroposition of the ascending aorta) might favour the coronary IVS-branch to connect to the left sinus.42 As in humans with TOF, a single orifice was occasionally encountered in mutant mouse embryos. More often, an increased number (3 or 4) of coronary orifices was found, which is regarded to be a rare variation in both humans and mice.24,43 Nevertheless, we never saw more than two orifices in the 24 wild-type embryos of E15.5 and older.

The presence of VCACs in cardiac anomalies related to pulmonary atresia is not uncommon, even before complete atresia has developed.44 A substantial part of coronary vSMCs originates from the EPDC-population15 and EPDC-performance has been suggested to play part in the appearance of VCACs.44 Abnormal development and/or differentiation of EPDCs due to altered VEGF and/or Notch signalling might explain the occurrence of VCACs in Vegf120/120 embryos. Besides VCACs, coronary arterio-venous shunts were observed. Alterations in VEGF and Notch signalling in Vegf120/120 embryos may cause arterio-venous shunts, as the presence of such shunts has been described in Notch-mutants.45 Extracardiac arterio-venous shunts were observed in the aortic arches of perinatal Vegf120/120 mouse embryos, suggesting that alterations in Notch signalling are not heart-restricted.

We already observed at E11.5, both in wild-type and mutants, two connections of the coronary venous system (in)directly to the right atrium. This is the first report on such early coronary venous drainage in mice. We describe that the connection of the coronary system to the right atrium arises before the coronary connection to the aorta. This is feasible as it enables circulation throughout the coronary system at the onset of systemic flow and pressure, which occurs several days later, without obstruction.14


   5. Conclusions
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
Funding
 Supplementary material
 References
 
We show that loss of Vegf164 and Vegf188 in Vegf120/120 embryos leads to spatiotemporal alterations in VEGF and Notch signalling in the heart reflected by altered expression of several markers involved in and regulated by these pathways. These alterations coincide with anomalous coronary EC-differentiation and ensuing arteriogenesis. The effect of different VEGF isoforms on Notch signalling and coronary EC-differentiation could be confirmed in vitro.

From our study, it can be concluded that the spatiotemporal VEGF-distribution as well as specificity of the isoforms are important for proper coronary vascular development. These new insights on instructive roles of VEGF (isoform) distribution on angiogenesis, endothelial functioning, and arteriogenesis can be of use to improve the efficacy of current therapies targeting VEGF signalling8,46 in tissue revascularization.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 Funding
Supplementary material
 References
 
The Netherlands Heart Foundation (2001B057 to N.M.S.v.d.A. and 2005B254 to D.G.M.M.) The Marie Curie FP6 early stage researcher training (MEST-CT-20005-020706 to V.C.).


   Supplementary material
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 Funding
 Supplementary material
References
 
Supplementary Material is available at Cardiovascular Research Online.


   Acknowledgements
 
The authors would like to thank Jan Lens for photographic assistance, Ron Slagter for drawings, Saskia Maas and Conny J. van Munsteren for technical assistance (all Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands), and Sanne Verbruggen (Department of Physiology, Maastricht University, Maastricht, The Netherlands) for assistance with the in vitro experiments.

Conflict of interest: none declared.


   Notes
 
{dagger} These senior authors contributed equally to this research. Back


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 Funding
 Supplementary material
 References
 

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