Skip Navigation

Cardiovascular Research 2002 55(2):361-368; doi:10.1016/S0008-6363(02)00440-6
© 2002 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Wanstall, J. C
Right arrow Articles by Kay, G. F
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wanstall, J. C
Right arrow Articles by Kay, G. F
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2002, European Society of Cardiology

Vascular endothelial growth factor-B-deficient mice show impaired development of hypoxic pulmonary hypertension

Janet C Wanstalla,*, Agatha Gambinoa, Trina K Jefferya,1, Marian M Cahillb, Daniela Bellomob,2, Nicholas K Haywardb and Graham F Kayb

aSchool of Biomedical Sciences, Department of Physiology and Pharmacology, University of Queensland, St. Lucia, Brisbane, Qld. 4072, Australia
bQCF Transgenic Laboratory, Joint Experimental Oncology Program, The Queensland Institute of Medical Research and the University of Queensland, Brisbane, Australia

wanstall{at}mailbox.uq.edu.au

* Corresponding author. Tel.: +61-7-3365-3113; fax: +61-7-3365-1766

Received 27 November 2001; accepted 29 March 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: To test the hypothesis that Vegf-B contributes to the pulmonary vascular remodelling, and the associated pulmonary hypertension, induced by exposure of mice to chronic hypoxia. Methods: Right ventricular systolic pressure, the ratio of right ventricle/[left ventricle+septum] (RV/[LV+S]) and the thickness of the media (relative to vessel diameter) of intralobar pulmonary arteries (o.d. 50–150 and 151–420 µm) were determined in Vegfb knockout mice (Vegfb–/–; n = 17) and corresponding wild-type mice (Vegfb+/+; n = 17) exposed to chronic hypoxia (10% oxygen) or housed in room air (normoxia) for 4 weeks. Results: In Vegfb+/+ mice hypoxia caused (i) pulmonary hypertension (a 70% increase in right ventricular systolic pressure compared with normoxic Vegfb+/+ mice; P<0.001), (ii) right ventricular hypertrophy (a 66% increase in RV/[LV+S]; P<0.001) and (iii) pulmonary vascular remodelling (a 27–36% increase in pulmonary arterial medial thickness; P<0.05). In contrast, in Vegfb–/– mice hypoxia did not cause any increase in either right ventricular systolic pressure or pulmonary arterial medial thickness; also right ventricular hypertrophy (41% increase in RV/[LV+S]; P<0.001) was less pronounced (P<0.05) than in Vegfb+/+ mice. Conclusion: Vegf-B may have a role in the development of chronic hypoxic pulmonary hypertension in mice by contributing to pulmonary vascular remodelling. If so, the effect of Vegf-B appears to be different from that of Vegf-A which is reported to protect against, rather than contribute to, hypoxia-induced pulmonary vascular remodelling.

KEYWORDS Growth factors; Hypertrophy; Hypoxia; Pulmonary circulation; Remodelling


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Vascular endothelial growth factors (VEGF) are a family of closely related peptides, the mammalian members of which are VEGFs-A, -B, -C and -D and placental growth factor. VEGF-A (often referred to simply as VEGF) is the most extensively studied member of the family; it binds with high affinity to two tyrosine kinase receptors, VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1), and also to neuropilin-1 [1]. VEGF-A is an endothelial cell mitogen involved in the growth and proliferation of endothelial cells and in the maintenance of endothelial cell function [1]. It plays a critical role in the regulation of vasculogenesis and angiogenesis [2], and consequently Vegfa gene knockout mice (Vegfa–/–) do not survive to term due to impairment of blood vessel development in the embryo [3,4].

VEGF-B has 45% sequence homology with VEGF-A and its tissue distribution overlaps with that of VEGF-A [5–7]. It binds to two of the same receptors as VEGF-A, namely VEGFR-1 and neuropilin-1, but not to VEGFR-2 [6,8,9]. VEGF-B, unlike VEGF-A, is not critical for vascular development [6,10] and Vegfb gene knockout mice (Vegfb–/–) survive to term and are outwardly healthy [11]. Vegfb–/– mice have a deficiency in the maintenance of coronary vascular function following an ischaemic insult [11] or a defect in atrial conduction characterised by a prolonged PQ interval [12] but, apart from these, the functions of this member of the VEGF family remain to be established [6].

VEGF-A is induced by hypoxia and, in rats, chronic hypoxia leads to increases in VEGF-A mRNA and protein [13]. Hypoxia also up-regulates VEGFR-1 and VEGFR-2 [13–16]. Consequently it has been proposed that VEGF-A may have a role during the development of chronic hypoxic pulmonary hypertension. Initially it was considered that VEGF-A might contribute to the pathophysiology of the disease [17]. However, more recent data have shown that a reportedly selective VEGFR-2 antagonist exacerbates the pulmonary vascular remodelling and pulmonary hypertension seen in hypoxic rats [18]. A similar trend was seen with a VEGF-A neutralising antibody [19,20]. Also, adenovirus-mediated over-expression of VEGF-A in rat lungs has been shown to attenuate hypoxia-induced pulmonary vascular remodelling and pulmonary hypertension [21]. Together these data raise the possibility that VEGF-A may have a protective role against some of the adverse effects of chronic hypoxia.

However, the possibility that VEGF-B might have a role (contributory or protective) in hypoxic pulmonary hypertension has not previously been investigated. VEGF-B, unlike VEGF-A, is not induced by hypoxia, at least in in vitro studies [10,22], but it binds to VEGFR-1, one of the receptors upregulated by hypoxia, where it competes with VEGF-A for binding [6,9]. Moreover VEGF-B forms heterodimers with VEGF-A leading to the suggestion that VEGF-B may regulate the bioavailability of VEGF-A [5]. Therefore VEGF-B might conceivably influence the development of hypoxic pulmonary hypertension either directly, via a mechanism involving VEGFR-1, or indirectly, via an effect on VEGF-A availability or signalling. In the present study we have used Vegfb–/– mice and corresponding wild-type littermates (Vegfb+/+) to test the hypothesis that Vegf-B contributes to the pulmonary vascular remodelling, and the related pulmonary hypertension, induced by exposure of mice to chronic hypoxia. A recent study has shown that both of the isoforms of Vegf-B are expressed in the lungs of adult mice, with Vegf-B167 predominating [23].


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Animals
The investigation conforms to the Code of Practice for Animal Experiments issued by the National Health and Medical Research Council of Australia.

Mice (strain, 129T2/SvEms) lacking the vascular endothelial growth factor gene (Vegfb–/– mice) were bred and genotyped as described in detail previously [11]. These mice were generated by standard gene targeting techniques using a targeting construct that replaced most of the coding sequence of the Vegfb gene with a β-geo selection cassette. To definitively show that the Vegfb gene was deleted and no Vegfb transcript was produced from the locus we analysed total RNA derived from both Vegfb+/+ and Vegfb–/– animals by RT-PCR to detect both Hprt and Vegfb transcripts. The total RNA was isolated from heart tissue using Trizol reagent (Sigma) according to the manufacturer's instructions and the cDNA synthesised using Superscript II Reverse Transcriptase Kit (Gibco-BRL). The RT-PCR was performed on RNA extracts, under the following conditions: 1 µg RNA was incubated at 70 °C for 10 min with 100 ng random hexamers in a total volume of 11 µl. The samples were placed on ice and a 9-µl reverse transcription mixture (0.5 mM dNTPs, 10 mM DTT, 200 units SuperscriptTM II, 40 units Rnasin and Superscript buffer) was added to the samples which were incubated at 45 °C for 1 h, followed by 95 °C for 5 min, then 30 µl of water was added to bring the final volume to 50 µl. An aliquot of the cDNA product (1 µl) was then PCR amplified in a 50 µl reaction mixture (2.5 units AmpliTaq Gold, 1.5 mM MgCl2, 0.2 mM dNTPs. PCR buffer and 10 pmol of each of the two oligonucleotide primers necessary to amplify either Hprt or Vegfb. PCR was performed for 1 cycle of 12.5 min at 94 °C, followed by 30 cycles of: 94 °C for 1 min, 60 °C for 1 min and 72 °C for 1.5 min. The primers used were as follows: for Hprt, Hprtrnaf, 5'-cct gct gga tta cat taa agc act g-3' and Hprtrnar, 5'-gtc aag ggc ata tcc aac aac aaa c-3'; for Vegfb, AM1, 5'-gtc aaa caa cta gtg ccc ag-3' and AM2, 5'-tgt ctg ggt tga gct cta ag-3'. RT-PCR products were then run out on 2% agarose gels with ethidium bromide staining for visualisation.

Female Vegfb+/+ and Vegfb–/– mice were housed either in room air (control, normoxic mice) or in a hypoxic chamber containing 10–11% oxygen (hypoxic mice) for 4 weeks, as previously described for hypoxic rats [24]. The weights of the mice at the completion of the period of exposure to normoxia (n = 8) or hypoxia (n = 9) were (g): Vegfb+/+ normoxic 20±0.5, hypoxic 18±0.4; Vegfb–/– normoxic 19±0.8, hypoxic 18±0.8. During the 4-week exposure to normoxia, mice from both groups had gained weight (Vegfb+/+ 15±1.7%; Vegfb–/– 10±1.0%). In contrast, during the 4-week exposure to hypoxia mice had lost weight (Vegfb+/+ –6±1.4%; Vegfb–/– –6±1.3%). Mice were removed from the hypoxic chambers before anaesthetising them for measurement of right ventricular pressure, as described below. In preliminary experiments it was shown that a shorter period of hypoxic exposure (2 weeks), which is sufficient to induce pulmonary hypertension in rats [25], did not cause pulmonary hypertension in Vegfb+/+ mice, inasmuch as there was no elevation in right ventricular systolic pressure (RVSP). Elevations in RVSP parallel elevations in pulmonary artery pressure [26] (Wanstall, unpublished data).

2.2 Assessment of hypoxia-induced pulmonary hypertension
Normoxic and hypoxic Vegfb+/+ and Vegfb–/– mice were anaesthetised with pentobarbitone (60 mg/kg i.p.; Rhone Merieux, Brisbane, Australia). The mice were artificially ventilated with room air (60 strokes/min) with a Ugo Basile Rodent Ventilator, via a tracheal cannula, modified to deliver a stroke volume of 0.5 ml. The thorax was opened, a heparin-filled hypodermic needle (23 G) was inserted into the right ventricle and right ventricular pressure was rapidly (in less than 30 s after opening the thorax) recorded on a Ugo Basile Gemini recorder. Six consecutive values of RVSP were averaged. A blood sample was taken for the determination of haematocrit. The heart was removed and divided into right ventricle (RV) and left ventricle+septum (LV+S), blotted and weighed. The ratios of the weights RV/(LV+S) and RV/body weight were calculated.

2.3 Morphological measurements in histological sections of lungs
After removing the heart, the lungs and trachea were removed en bloc. The lungs were distended by filling them, via the tracheal cannula, with 10% formalin at a pressure of 25 cmH2O. The trachea was then tied off and the lungs were fixed by immersion in 10% formalin for ≥1 week. After fixation, longitudinal sections, 3 µm thick, were taken from the left lobe and stained with Miller's stain for elastin and Van Gieson's stain for smooth muscle. One section per animal was obtained, always from the same region of the left lobe, and sections from different animals were coded and analysed ‘blind’.

Images of individual pulmonary arteries (50–420 µm o.d.; a total of four to 10 arteries per section) were examined at x100 or x40 magnification, captured using a video camera (Video 7, 8 bit 765x512 pixel CCD), mounted on a light microscope (Olympus BH5), linked to a computer (Macintosh Centris 650, with scion LG3 frame grabber). Measurements of vessel diameter and medial wall thickness were obtained from these images using a computer programme (NIH image adapted for PC by Scion) and the method described previously [24]. Vessel diameter was defined as the distance between two diametrically opposed external elastic laminae; two measurements were made, (i) along the line corresponding to the longest distance and (ii) along the line perpendicular to it, and these two values were averaged to give ‘mean arterial diameter’. Medial wall thickness was defined and measured as the distance between internal and external elastic laminae; four measurements were obtained, one in each of the four quadrants defined by the perpendicular lines used to measure mean arterial diameter (see above), and averaged to give ‘mean medial thickness’. Medial wall thickness was then expressed as a percentage of mean arterial diameter according to the formula: ‘% medial wall thickness’=(2xmean medial wall thickness)x100÷mean vessel diameter. Vessels were selected on the basis that (i) they had both internal and external elastic laminae, (ii) were cut transversely (i.e., not obliquely) and (iii) had outer diameter (o.d.) ≥50 µm. The data were divided into two groups, comprising arteries 50–150 and 151–520 µm o.d., respectively

2.4 Statistical analyses
Mean values, calculated from data obtained from a number of different animals, are quoted together with their S.E. mean. The data were analysed by analysis of variance (ANOVA) followed by Student–Newman–Keuls multiple comparison post test using the statistics program, Graphpad Instat.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Confirmation that the knockout mice do not produce Vegfb gene product
RT-PCR analysis (Fig. 1) for Vegfb gene product in total heart RNA derived from Vegfb+/+ mice showed the expected products at 446 bp for Vegf-B186 and 345 bp for Vegf-B167, the two alternative splice forms of Vegfb transcript (Fig. 1, lane 5). No Vegfb gene products were amplified in the RNA derived from Vegfb–/– mice (Fig. 1, lane 6) confirming that the knockout had indeed generated a null allele of Vegfb.


Figure 1
View larger version (78K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 RT-PCR analysis of Vegfb+/+ and Vegfb–/– total heart RNA. Lanes: (1) 1-kb ladder; (2–4) RT-PCR for Hprt transcript (positive control) in Vegfb+/+, Vegfb–/– and negative water control, respectively; (5–7) RT-PCR for Vegfb transcript in Vegfb+/+, Vegfb–/– and negative water control, respectively.

 
3.2 Right ventricular systolic pressure (RVSP), right ventricular hypertrophy and haematocrit
Hypoxic Vegfb+/+ mice had elevated RVSP, when compared with the value in corresponding normoxic Vegfb+/+ mice (Figs. 2a and 3aGo). They also had right ventricular hypertrophy as evidenced by significant increases in the ratios RV/(LV+S) and RV/body wt (Fig. 3b,c). In contrast hypoxic Vegfb–/– mice did not have elevated RVSP (Figs. 2b and 3aGo) and, although there was significant right ventricular hypertrophy, the values for RV/(LV+S) and RV/body wt in hypoxic Vegfb–/– mice were significantly less than the corresponding value in hypoxic Vegfb+/+ mice (Fig. 3b,c).


Figure 2
View larger version (51K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Measurements of right ventricular pressure. Representative records obtained from (a) Vegfb+/+ mice and (b) Vegfb–/– mice exposed to room air (normoxia) or 10% oxygen (hypoxia) for 4 weeks.

 

Figure 3
View larger version (21K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 (a) Right ventricular systolic pressure (RVSP), (b) RV/(LV+S) and (c) RV/body wt in normoxic (open bars) and hypoxic (filled bars) Vegfb+/+ and Vegfb–/– mice (n = 8–9 mice in each group). *Value significantly greater than corresponding value in normoxic mice (**P<0.01, ***P<0.001). #Value significantly less than corresponding value in Vegfb+/+ mice (P<0.05).

 
Haematocrit was elevated in hypoxic Vegfb+/+ mice (normoxic, 46±1.1, n = 8; hypoxic, 53±0.8, n = 9; P<0.001) and hypoxic Vegfb–/– mice (normoxic, 48±1.3, n = 8; hypoxic, 55±1.4, n = 9; P<0.01), but in both normoxic and hypoxic mice there was no difference between the Vegfb+/+ and Vegfb–/– groups (P>0.05).

3.3 Medial thickness of intralobar pulmonary arteries
In Vegfb+/+ mice, chronic hypoxia caused significant increases in medial wall thickness of intralobar pulmonary arteries 50–150 and 151–420 µm o.d. (Fig. 4). This was not seen in hypoxic Vegfb–/– mice and, consequently, values for medial thickness in hypoxic Vegfb–/– mice were significantly smaller than the corresponding values in hypoxic Vegfb+/+ mice (Fig. 4).


Figure 4
View larger version (24K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Medial wall thickness (expressed as a percentage of vessel diameter) in intralobar pulmonary arteries, (a) o.d. 151–420 and (b) 50–150 µm, from normoxic (open bars) and hypoxic (filled bars) Vegfb+/+ and Vegfb–/– mice (n = 15–35 vessels, seven to nine mice in each group). *Value significantly greater than corresponding value in normoxic mice (*P<0.05, ***P<0.001). #Value significantly less than corresponding value in Vegfb+/+ mice (#P<0.05, ##P<0.01).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The results of this study have shown that mice lacking the Vegfb gene, unlike their wild-type littermates, did not develop sustained pulmonary hypertension in response to chronic hypoxia. For the purposes of this study an elevation in RVSP was taken as indicative of pulmonary hypertension since RVSP closely parallels pulmonary artery pressure [26] (Wanstall, unpublished). In Vegfb+/+ mice the 70% increase in RVSP caused by 4 weeks of hypoxia was within the range of increases reported in other studies in mice after 3–5 weeks of hypoxia, viz. 42–100% increase [26–30]. The different values for RVSP in hypoxic Vegfb–/– and hypoxic Vegfb+/+ mice could not be attributed to differences in blood viscosity since both groups of mice showed the same degree of polycythemia. The absence of pulmonary hypertension in the Vegfb–/– knockout mice did, however, reflect the absence of pulmonary vascular remodelling, as assessed by increases in pulmonary arterial medial thickness. An increase in pulmonary arterial medial thickness is a characteristic feature of hypoxic pulmonary hypertension, reflecting both hypertrophy of the smooth muscle and an increase in extracellular connective tissue [31,32]. Thickening of the media was seen in vessels from hypoxic Vegfb+/+ mice but not hypoxic Vegfb–/– mice. The data suggest that Vegf-B may be one of the factors responsible, directly or indirectly, for the alterations in pulmonary vascular structure caused by chronic hypoxia in vessels of the size range examined. Alternatively, the absence of Vegf-B may promote various compensatory changes, one or more of which might hinder hypoxic vascular remodelling.

Despite the absence of any elevation in RVSP, the hypoxic Vegfb–/– mice did exhibit some right ventricular hypertrophy, albeit less pronounced than in the pulmonary hypertensive wild-type mice. In pulmonary hypertensive animals, right ventricular hypertrophy is considered to reflect an increase in afterload and there is generally a good correlation between right ventricular hypertrophy and elevations in pulmonary artery pressure or RVSP [33,34]. However, there are occasions when these two changes do not occur together. For example, rats exposed to intermittent hypoxia (4 h/day, for a total of 24 exposures) had right ventricular hypertrophy with no increase in RVSP [35]. There are various possible explanations for the presence of right ventricular hypertrophy in the hypoxic Vegfb–/– mice, in the absence of a sustained elevation in RVSP. For example, there may have been a temporary elevation in pulmonary artery pressure, occurring only while the mice were in the hypoxic chamber and due to acute hypoxic pulmonary vasoconstriction rather than vascular remodelling. The resulting temporary increase in afterload could lead to right ventricular hypertrophy [35], even though there was no sustained increase in pressure once the mice were removed from the chamber and ventilated with room air. Alternatively right ventricular hypertrophy may reflect, in part, a direct effect of hypoxia on the right ventricle, i.e., part of the hypertrophy may be unrelated to an increase in pressure. Finally we cannot exclude the possibility that the RVSP values may have been under-estimated in the hypoxic Vegfb–/– mice. This would theoretically be possible if there was a greater hypotensive effect of the anaesthetic in the knockout mice, or even if there was more pronounced blood loss (although in all mice blood loss was negligible). However we think this third explanation is unlikely in view of the fact that there was not only no increase in RVSP in the hypoxic Vegfb–/– mice but also no pulmonary vascular remodelling. It should be emphasised that the right ventricular hypertrophy in these mice was significantly less than seen in hypoxic wild-type mice. The possibility that this reflects a specific role for VEGF-B in right ventricular hypertrophy cannot be excluded.

Our data suggest that VEGF-B contributes to the pathology of hypoxic pulmonary hypertension. If this conclusion is correct, then it appears that VEGF-B has a very different role from that of VEGF-A in the response to chronic hypoxia. Recent studies in rats have indicated that VEGF-A may be protective in hypoxic pulmonary hypertension. Partovian et al. showed that over-expression of VEGF-A in lungs (achieved with adenovirus-mediated gene transfer) attenuated the rise in pulmonary artery pressure and the pulmonary vascular remodelling induced by chronic hypoxia, and suggested that this beneficial effect was due in part to protection of endothelial cell function [21]. Taraseviciene-Stewart et al. showed that in hypoxic rats treated with SU5416, a reportedly selective antagonist at VEGFR-2 receptors (which bind VEGF-A but not VEGF-B), elevations in pulmonary artery pressure and thickening of the medial layer of pulmonary arteries were exaggerated when compared with data in untreated hypoxic rats [18]. Also, clusters of proliferating endothelial cells obstructing the lumen were observed in the SU5416-treated rats [18]. Pulmonary hypertension was also enhanced in hypoxic rats treated with a monoclonal antibody to VEGF-A [19,20]. Furthermore SU5416 caused the development of pulmonary hypertension even in normoxic rats [18]. Treatment with a broad-spectrum caspase inhibitor (at a concentration previously shown to inhibit caspase-3) prevented the exacerbation of hypoxic pulmonary hypertension induced by this VEGFR-2 antagonist [18]. The authors hypothesised that in the absence of VEGF-A signalling via VEGFR-2 and the presence of chronic hypoxia, pulmonary endothelial cell dysfunction and cell death occurs, leading to the emergence of an apoptosis-resistant, proliferating endothelial cell phenotype that causes obstruction of the vascular lumen. They also suggested a link between the perturbation of endothelial cell function and an increase in vascular smooth muscle in the media [36]. Collectively, the findings from these studies imply that VEGF-A, via an action on VEGFR-2, may have a protective effect against pulmonary vascular remodelling induced by hypoxia.

VEGF-B does not bind to VEGFR-2 but it does bind to VEGFR-1, and these receptors are up-regulated by hypoxia in various tissues including lungs [13,16].The functions associated with activation of VEGFR-1 are still unclear, but a recent report suggested that VEGFR-1 might negatively regulate the endothelial mitogenic effect mediated via VEGFR-2 [37]. In light of this, activation of VEGFR-1 by VEGF-B might conceivably have the same net effect as blockade of VEGFR-2 with SU5416 (see above). In other words, VEGF-B might contribute to hypoxic pulmonary vascular remodelling indirectly, by inhibiting the beneficial (protective) effect of VEGF-A.

An alternative (or additional) possibility is that activation of VEGFR-1 by VEGF-B might contribute to pulmonary vascular remodelling independently of the effects of VEGF-A. One possible mechanism could be via matrix proteinase production, which has been linked with activation of VEGFR-1 in vascular smooth muscle cells [38]. Wang and Keiser showed that VEGF-A increased matrix metalloproteinase mRNA and protein in vascular smooth muscle cells [38]. This evidently occurred via activation of VEGFR-1, because (i) there were VEGFR-1 but no VEGFR-2 receptors present in the smooth muscle cells and (ii) a VEGFR-1-selective ligand mimicked the effect of VEGF-A [38]. Extracellular matrix-degrading enzymes facilitate the growth and migration of smooth muscle cells [38,39] and permit the release of growth factors bound to extracellular matrix proteins [40,41]. Therefore one pathway whereby VEGF-B, through its ability to bind to VEGFR-1, might conceivably contribute to pulmonary vascular remodelling is through matrix proteinase production. Note that neither of the speculative explanations described above implies that VEGF-B, itself, necessarily has any direct mitogenic activity.

Placental growth factor is another member of the VEGF family that binds to VEGFR-1, and it has recently been stated (though the data were not shown) that placental growth factor-deficient mice, like the Vegfb–/– mice examined in this study, had impaired hypoxic pulmonary vascular remodelling [42]. Also, data very similar to those obtained in the present study were obtained in a study in mice partially deficient in hypoxia-inducible factor 1{alpha} [27], which regulates, among other genes, the gene encoding VEGFR-1 [43].

The possibility that the differential response to chronic hypoxia in the Vegfb–/– and Vegfb+/+ mice may be a consequence of some compensatory mechanism(s) existing in the knockout mice, rather than a direct consequence of the absence of Vegf-B, cannot be ruled out; this is a limitation of any study in knockout animals. Nevertheless, the simplest conclusion from the data obtained in this study is that, in some way, Vegf-B contributes to pulmonary vascular remodelling during the development of hypoxic pulmonary hypertension in mice. Other growth factors that have been implicated in pulmonary vascular remodelling include platelet-derived growth factor, basic fibroblast growth factor and insulin-like growth factor-1, as well as various vasoactive factors that also have mitogenic properties, viz. angiotensin II, endothelin-1 and 5-hydroxytryptamine [44]. The present data suggest that VEGF-B, too, may have a role in this disease.

Time for primary review 41 days.


   Acknowledgements
 
Financial support from the National Health and Medical Research Council of Australia and AMRAD is gratefully acknowledged. JCW and NKH are NHMRC Senior Research Fellows.


   Notes
 
1 Current address: Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge, UK. Back

2 Current address: Science Park Raf Spa, Via Olgettina 558, 20132 Milan, Italy. Back


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

  1. Ferrara N. VEGF: an update on biological and therapeutic aspects. Curr Opin Biotech (2000) 11:617–624.[CrossRef][Web of Science][Medline]
  2. Zachary I., Gliki G. Signaling transduction mechanisms mediating biological actions of the vascular endothelial growth factor family. Cardiovasc Res (2001) 49:568–581.[Abstract/Free Full Text]
  3. Ferrara N., Carver-Moore K., Chen H., et al. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature (1996) 380:439–442.[CrossRef][Medline]
  4. Carmeliet P., Ferreira V., Breier G., et al. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature (1996) 380:435–439.[CrossRef][Medline]
  5. Olofsson B., Pajusola K., Kaipainen A., et al. Vascular endothelial growth factor B. a novel growth factor for endothelial cells. Proc Natl Acad Sci USA (1996) 93:2576–2581.[Abstract/Free Full Text]
  6. Olofsson B., Jeltsch M., Eriksson U., Alitalo K. Current Biology of VEGF-B and VEGF-C. Curr Opin Biotech (1999) 10:528–535.[CrossRef][Web of Science][Medline]
  7. Lagercrantz J., Farnebo F., Larsson C., Tvrdik T., Weber G., Piehl F. A comparative study of the expression patterns for vegf, vegf-b/vrf and vegf-c in the developing and adult mouse. Biochim Biophys Acta (1998) 1398:157–163.[Medline]
  8. Ferrara N. Role of vascular endothelial growth factor in regulation of physiological angiogenesis. Am J Physiol Cell Physiol (2001) 280:C1358–C1366.[Abstract/Free Full Text]
  9. Olofsson B., Korpelainen E., Pepper M.S., et al. Vascular endothelial growth factor B (VEGF-B) binds to VEGF receptor-1 and regulates plasminogen activator activity in endothelial cells. Proc Natl Acad Sci USA (1998) 95:11709–11714.[Abstract/Free Full Text]
  10. Li X., Eriksson U. Novel VEGF family members: VEGF-B, VEGF-C and VEGF-D. Int J Biochem Cell Biol (2001) 33:421–426.[CrossRef][Web of Science][Medline]
  11. Bellomo D., Headrick J.P., Silins G.U., et al. Mice lacking the vascular endothelial growth factor-B gene (Vegfb) have smaller hearts, dysfunctional coronary vasculature and impaired recovery from cardiac ischaemia. Circ Res (2000) 86:E29–E35.[Web of Science][Medline]
  12. Aase K., von Euler G., Li X., et al. Vascular endothelial growth factor-B-deficient mice display an atrial conduction defect. Circulation (2001) 104:358–364.[Abstract/Free Full Text]
  13. Tuder R.M., Flook B.E., Voelkel N.F. Increased gene expression for VEGF and the VEGF receptors KDR/Flk and Flt in lungs exposed to acute or to chronic hypoxia. J Clin Invest (1995) 95:1798–1807.[Web of Science][Medline]
  14. Waltenberger J., Mayr U., Pentz S., Hombach V. Functional upregulation of the vascular endothelial growth factor receptor KDR by hypoxia. Circulation (1996) 94:1647–1654.[Abstract/Free Full Text]
  15. Christou H., Yoshida A., Arthur V., Morita T., Kourembanas S. Increased vascular endothelial growth factor production in the lungs of rats with hypoxia-induced pulmonary hypertension. Am J Respir Cell Mol Biol (1998) 18:768–776.[Abstract/Free Full Text]
  16. Marti H.H., Risau W. Systemic hypoxia changes the organ-specific distribution of vascular endothelial growth factor and its receptors. Proc Natl Acad Sci USA (1998) 95:15809–15814.[Abstract/Free Full Text]
  17. Voelkel N.F., Hoeper M., Maloney J., Tuder R.M. Vascular endothelial growth factor in pulmonary hypertension. Ann NY Acad Sci (1996) 796:186–193.[Medline]
  18. Taraseviciene-Stewart L., Kasahara Y., Alger L., et al. Inhibition of the VEGF receptor 2 combined with chronic hypoxia causes cell death-dependent pulmonary endothelial cell proliferation and severe pulmonary hypertension. FASEB J (2001) 15:427–438.[Abstract/Free Full Text]
  19. Tuder R.M., Allard J., Voelkel N.F. Role of vascular endothelial growth factor in hypoxia and monocrotaline induced pulmonary hypertension. Circulation (Abstract) (1996) 94:I647.
  20. Tuder R.M., Taraseviciene L. Inhibition of the VEGFR receptor causes severe pulmonary hypertension (PAH) in rats. Circulation (Abstract) (1999) 100:I241.
  21. Partovian C., Adnot S., Raffestin B., et al. Adenovirus-mediated lung vascular endothelial growth factor overexpression protects against hypoxic pulmonary hypertension in rats. Am J Respir Cell Mol Biol (2000) 23:762–771.[Abstract/Free Full Text]
  22. Enholm B., Paavonen K., Ristimäki A., et al. Comparison of VEGF, VEGF-B, VEGF-C and Ang-1 mRNA regulation by serum, growth factors, oncoproteins and hypoxia. Oncogene (1997) 14:2475–2483.[CrossRef][Web of Science][Medline]
  23. Li X., Aase K., Li H., et al. Isoform-specific expression of VEGF-B in normal tissues and tumors. Growth Factors (2001) 19:49–59.[Web of Science][Medline]
  24. Jeffery T.K., Wanstall J.C. Perindopril, an angiotensin converting enzyme inhibitor, in pulmonary hypertensive rats: comparative effects on pulmonary vascular structure and function. Br J Pharmacol (1999) 128:1407–1418.[CrossRef][Web of Science][Medline]
  25. Wanstall J.C., Hughes I.E., O'Donnell S.R. Reduced relaxant potency of nitroprusside on pulmonary artery preparations taken from rats during the development of hypoxic pulmonary hypertension. Br J Pharmacol (1992) 107:407–413.[Web of Science][Medline]
  26. Steudal W., Scherrer-Crosbie M., Bloch K.D., et al. Sustained pulmonary hypertension and right ventricular hypertrophy after chronic hypoxia in mice with congenital deficiency of nitric oxide synthase 3. J Clin Invest (1998) 101:2468–2477.[Web of Science][Medline]
  27. Yu A.Y., Shimoda L.A., Iyer N.V., et al. Impaired physiological responses to chronic hypoxia in mice partially deficient for hypoxia-inducible factor 1{alpha}. J Clin Invest (1999) 103:691–696.[Web of Science][Medline]
  28. Geraci M.W., Gao B., Shepherd D.C., et al. Pulmonary prostacyclin synthase overexpression in transgenic mice protects against development of hypoxic pulmonary hypertension. J Clin Invest (1999) 103:1509–1515.[Web of Science][Medline]
  29. Zhao L., Long L., Morrell N.W., Wilkins M.R. NPR-A-deficient mice show increased susceptibility to hypoxia-induced pulmonary hypertension. Circulation (1999) 99:605–607.[Abstract/Free Full Text]
  30. Zhao L., Mason N.A., Morrell N.W., et al. Sildenafil inhibits hypoxia-induced pulmonary hypertension. Circulation (2001) 104:424–428.[Abstract/Free Full Text]
  31. Meyrick B., Reid L. Hypoxia-induced structural changes in the media and adventitia of the rat hilar pulmonary artery and their regression. Am J Pathol (1980) 100:151–178.[Abstract]
  32. McKenzie J.C., Klein R.M. Protein synthesis in the rat pulmonary trunk during the early development of hypoxia-induced pulmonary hypertension. Blood Vessels (1983) 20:283–294.[Web of Science][Medline]
  33. Ghodsi F., Will J.A. Changes in pulmonary structure and function induced by monocrotaline intoxication. Am J Physiol (1981) 240:H149–H155.[Web of Science][Medline]
  34. Jeffery T.K., Wanstall J.C. Comparison of pulmonary vascular function and structure in early and established hypoxic pulmonary hypertension in rats. Can J Physiol Pharmacol (2001) 79:227–237.[CrossRef][Web of Science][Medline]
  35. Ostadal B., Widimsky J. Pulmonary blood vessels in lung disease. Widimsky J., Herget J., eds. (1990) Basel: Karger. 1–11. Progress in Respiratory Research.
  36. Voelkel N.F., Tuder R.M. Hypoxia-induced pulmonary vascular remodeling: a model for what human disease? J Clin Invest (2000) 106:733–738.[Web of Science][Medline]
  37. Rahim N., Dayanir V., Lashkari K. Receptor chimeras indicate that the vascular endothelial growth factor receptor-1 (VEGFR-1) modulates mitogenic activity of VEGFR-2 in endothelial cells. J Biol Chem (2000) 275:16986–16992.[Abstract/Free Full Text]
  38. Wang H., Keiser J.A. Vascular endothelial growth factor upregulates the expression of matrix metalloproteinases in vascular smooth muscle cells. Role of flt-1. Circ Res (1998) 83:832–840.[Abstract/Free Full Text]
  39. Southgate K.M., Davies M., Booth R.F., Newby A.C. Involvement of extracellular-matrix-degrading metalloproteinases in rabbit aortic smooth-muscle cell proliferation. Biochem J (1992) 288:93–99.[Web of Science][Medline]
  40. Taipale J., Keski-Oja J. Growth factors in the extracellular matrix. FASEB J (1997) 11:51–59.[Abstract]
  41. Thompson K., Rabinovitch M. Exogenous leukocyte and endogenous elastases can mediate mitogenic activity in pulmonary artery smooth muscle cells by release of extracellular matrix-bound basic fibroblast growth factor. J Cell Physiol (1996) 166:495–505.[CrossRef][Web of Science][Medline]
  42. Carmeliet P., Moons L., Luttun A., et al. Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions. Nat Med (2001) 7:575–583.[CrossRef][Web of Science][Medline]
  43. Gerber H.P., Condorelli F., Park J., Ferrara N. Differential transcriptional regulation of the two vascular endothelial growth factor receptor genes. Flt-1, but not Flk-1/KDR, is upregulated by hypoxia. J Biol Chem (1997) 272:23659–23667.[Abstract/Free Full Text]
  44. Jeffery T.K., Wanstall J.C. Pulmonary vascular remodelling: a target for therapeutic intervention in pulmonary hypertension. Pharmacol Ther (2001) 92:1–20.[CrossRef][Web of Science][Medline]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Proc. Natl. Acad. Sci. USAHome page
F. Zhang, Z. Tang, X. Hou, J. Lennartsson, Y. Li, A. W. Koch, P. Scotney, C. Lee, P. Arjunan, L. Dong, et al.
VEGF-B is dispensable for blood vessel growth but critical for their survival, and VEGF-B targeting inhibits pathological angiogenesis
PNAS, April 14, 2009; 106(15): 6152 - 6157.
[Abstract] [Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
X. Li, M. Tjwa, I. Van Hove, B. Enholm, E. Neven, K. Paavonen, M. Jeltsch, T. D. Juan, R. E. Sievers, E. Chorianopoulos, et al.
Reevaluation of the Role of VEGF-B Suggests a Restricted Role in the Revascularization of the Ischemic Myocardium
Arterioscler. Thromb. Vasc. Biol., September 1, 2008; 28(9): 1614 - 1620.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Wanstall, J. C
Right arrow Articles by Kay, G. F
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wanstall, J. C
Right arrow Articles by Kay, G. F
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?