© 2000 American Heart Association, Inc.
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Mice Lacking the Vascular Endothelial Growth Factor-B Gene (Vegfb) Have Smaller Hearts, Dysfunctional Coronary Vasculature, and Impaired Recovery From Cardiac Ischemia
From the QCF Transgenic Laboratory and Human Genetics Laboratory, Joint Experimental Oncology Program, the Queensland Institute of Medical Research (D.B., G.U.S., C.A.P., M.G., A.M., M.M.C., I.D.T., S.M.G., S.T., N.K.H., G.F.K.), and the Department of Pathology, University of Queensland (M.C.C.), Brisbane, Australia; Griffith University (J.P.H.), Gold Coast Campus, Southport, Australia; Department of Cardiothoracic Surgery (P.S.T.), Imperial College School of Medicine, National Heart and Lung Institute, London, UK; and Centre for Molecular and Cellular Biology (C.W., M.L.), University of Queensland, Brisbane, Australia.
Correspondence to Graham Kay, Queensland Institute of Medical Research, Post Office Royal Brisbane Hospital, Queensland 4029, Australia. E-mail grahamK{at}qimr.edu.au
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Abstract |
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Abstract—Vascular endothelial growth factor-B (VEGF-B) is closely related to VEGF-A, an effector of blood vessel growth during development and disease and a strong candidate for angiogenic therapies. To further study the in vivo function of VEGF-B, we have generated Vegfb knockout mice (Vegfb-/-). Unlike Vegfa knockout mice, which die during embryogenesis, Vegfb-/- mice are healthy and fertile. Despite appearing overtly normal, Vegfb-/- hearts are reduced in size and display vascular dysfunction after coronary occlusion and impaired recovery from experimentally induced myocardial ischemia. These findings reveal a role for VEGF-B in the development or function of coronary vasculature and suggest potential clinical use in therapeutic angiogenesis. The full text of this article is available at http://www.circresaha.org.
Key Words: angiogenesis • cardiac ischemia • coronary vasculature
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Vascular endothelial growth factor-B (VEGF-B)1 2 3 is a secreted growth factor that has strong sequence homology with VEGF-A, a primary regulator of angiogenesis in development, corpus luteum formation, wound healing, and cancer.4 VEGF-B can form stable heterodimers with VEGF-A3 and is generally coexpressed with VEGF-A.5 6 VEGF-B can bind to two of the VEGF-A receptors, VEGFR-17 and neuropilin-1,8 suggesting that it may regulate the bioavailability and/or action of VEGF-A.5 Although VEGF-B has been reported to behave as an endothelial cell mitogen,2 part of the mitogenic activity reported may be due to VEGF-B/VEGF-A heterodimers.5
Several mouse models have been generated by gene knockout technology where the genes encoding Vegf-A or its receptors have been mutated. Both Vegfa-/- and Vegfa+/- mice are unable to survive to term due to a general impairment of blood vessel formation in the early embryo.9 10 Vegfa120/120 mice, where only two of the three major Vegf-A isoforms have been knocked out, die postnatally after cardiac failure due to widespread myocardial ischemia.11 Vegfr1-/- mice die as embryos due to defects in angiogenesis,12 but partial knockout mice, where only the tyrosine kinase–encoding portion of the Vegfr1 gene is deleted, develop normal vasculature.13
To study the in vivo role of Vegf-B, we have generated a knockout mouse line and found that, unlike the Vegf-A–related knockouts, Vegfb-/- mice appear outwardly normal and fertile. Because Vegfb transcripts are expressed predominantly in the heart during murine embryogenesis and adult life,1 14 15 16 suggesting a specific role for Vegf-B during cardiac development, we have concentrated on studying the cardiac phenotype in these mice. Vegfb-/- hearts are reduced in size compared with hearts of Vegfb+/+ littermates and display clinical symptoms of impaired recovery from experimentally induced ischemia. The results suggest an essential role for Vegf-B in establishment of a fully functional coronary vasculature and highlight the potential of this cytokine for application in the emerging field of therapeutic angiogenesis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Generation of Vegfb+/- Mice
All mice used for the present study were supplied by the Animal Resources Centre (Western Australia), and their treatment was in accordance with the National Health and Medical Research Council (NH&MRC) guidelines for the care of experimental animals.
Targeted inactivation of the Vegfb gene was achieved
by replacing exons 3 to 7 (Figure 1a
) with a promoter-less
ß-geo cassette. The ß-geo gene was preceded
by an internal ribosomal entry site to give cap-independent translation
of ß-geo.17 Targeted 129/SvJ ES cells (C1368) were
injected into C57BL/6J blastocysts to produce chimeras. Progeny of
germline-transmitting chimeras were genotyped by polymerase
chain reaction (PCR) amplification of tail-tip DNA using PCR1, 5'-TTT
GAT GGC CCC AGC CAC-3'; PCR2, 5'-CCC CCA GCT GAC TGC TCG-3'; and PCR3,
5'-CTA GTG GAT CCC CCG GGC-3' (Figure 1b).
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ß-Gal Staining and Immunohistochemistry
Frozen sections and whole embryos were stained for ß-Gal as
described.18 For quantitation of capillary density,
transverse sections of the left ventricle (LV) were cut at comparable
levels in Vegfb+/+ and
Vegfb-/- P30 hearts (30 days
postpartum) (4 hearts each), immunostained with
anti–PECAM-1 (clone M13, Pharmingen), and the capillaries counted
using ImagePlus software on 7 randomly chosen fields (x40
magnification, 
0.06 mm2 per field) in the
epicardial, endocardial, and midmyocardial portion of the LV. Because
no difference between genotypes was found within each portion,
capillary density data were averaged for each heart. Coronary
vessels were counted as anti–smooth muscle 
-actin (FITC conjugated,
clone 1A4, Sigma)–stained vessels in whole sections.
Heart Weight and LV Thickness
Vegfb+/+,
Vegfb+/-, and
Vegfb-/- mice of either 129/SvJ or
C57BL/6Jx129/SvJ background were weighed. After dissection, the hearts
were trimmed of surrounding tissue and weighed. A subset of the P25
hearts was fixed in formalin and microdissected to obtain a similar
angle of section. LV thickness was measured on sections with a stage
micrometer (n=10 Vegfb+/+
hearts; n=16 Vegfb+/- hearts; n=14
Vegfb-/- hearts).
Langendorff Perfusion
Hearts were isolated from mice anesthetized with 60
mg/kg sodium pentobarbital. Vegfb+/+
(161±7 mg wet heart weight [WHW], n=15),
Vegfb+/- (152±6 mg WHW, n=14), and
Vegfb-/- mice (155±7 mg WHW, n=16)
hearts were perfused in the Langendorff mode as
described.19
For ischemia, baseline measurements were recorded from
Vegfb+/+ (n=8),
Vegfb+/- (n=8), and
Vegfb-/- hearts (n=8) after 30
minutes of stabilization. Global normothermic
ischemia was initiated for 20 minutes before 30 minutes of
aerobic reperfusion. To examine reactive hyperemia, a subset of
hearts (n=7 for Vegfb+/+, n=6 for
Vegfb+/-, and n=8 for
Vegfb-/-) was perfused as described
above and after stabilization was subjected to a single 20-second
period of zero flow followed by reperfusion at 90 mm Hg perfusion
pressure. The coronary flow response was recorded, peak
hyperemic flows were measured in individual experiments, and
percentage of flow-debt repayment over the initial 60 seconds of
reperfusion was calculated as follows:
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Statistical Analyses
Body/heart weight, LV thickness, and capillary density data were
analyzed using unpaired Student’s t tests.
Body/heart weight data were also analyzed using the generalized
estimation equation.20 Hyperemia data were
analyzed via one-way ANOVA and functional
parameters by two-way ANOVA for repeated measures. Where
significant effects were detected, the Tukey’s HSD post hoc test was
used. In all tests, significance was accepted at
P<0.05.
An expanded Materials and Methods section is available online at http://www.circresaha.org.
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Results |
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Generation of the Vegfb-/- Mouse
The Vegfb knockout mice generated with the modified Vegfb locus shown in Figure 1a
were produced in normal mendelian ratios, were healthy and fertile, and
did not display any overt phenotype. The genotype of
mice was determined by PCR amplification of tail-tip DNA from P10 pups
(Figure 1b). Rather than producing Vegf-B, this modified locus
results in ß-Gal expression under the control of the
endogenous Vegfb promoter (herein referred to as
Vegf-B/ß-Gal). Because the Vegfb+/-
mice generated in this manner had no obvious developmental defects, we
assumed that Vegf-B/ß-Gal expression in these mice accurately
reflects the endogenous Vegfb expression
pattern.
Cardiac Vegf-B/ß-Gal Expression Pattern in the
Vegfb+/- Mouse
Using ß-Gal staining, Vegf-B/ß-Gal expression was first
detected in the heart at E10.5 (embryonic day 10.5), it became
prominent at E12.5 (Figure 2a) and
further increased thereafter (Figure 2b). Throughout
development, Vegf-B/ß-Gal expression appeared to be restricted to the
myocardium (Figures 2c through 2g) and subepicardium
(Figures 2g and 2h) and remained undetectable in
endothelial cells, including those of the endocardium
and coronary endothelium. Endocardial
derivatives, such as the valve leaflets were always devoid of
Vegf-B/ß-Gal expression. During development, the highest
concentration of Vegf-B/ß-Gal–expressing cells was seen in the right
ventricular myocardium and right aspect of the
interventricular septum (Figures 2a through 2c).
Lower Vegf-B/ß-Gal expression was detectable in the LV (Figures 2a and 2c) and the right atrial appendage (Figure 2d).
The lowest expression was found in the atrial wall (Figures 2c, 2d, and 2f), where coronary angiogenesis is less conspicuous
(Figures 2e and 2f). Within the right ventricle (RV),
Vegf-B/ß-Gal staining was prevalent in the trabeculations
(Figure 2d). The intensity of Vegf-B/ß-Gal expression
increased further in the neonate heart (Figures 2i and 2j) in
correlation with the massive early postnatal coronary capillary
and vessel growth.11 The prevalence of Vegf-B/ß-Gal
expression switches from the RV to the LV in the early neonatal period
(Figures 2i and 2j) reflects the predominant early postnatal
capillarization of this chamber.21 In the juvenile heart,
the ventricular prevalence of expression is lost, and the
density of Vegf-B/ß-Gal–expressing cells is similar in the
ventricles and the atria (data not shown).
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The great arteries in the heart expressed low levels of Vegf-B/ß-Gal
at all stages of development (eg, Figure 2b) and in juvenile
mice (data not shown). Vegf-B/ß-Gal was undetectable in the tunica
intima and media of coronary vessels (Figures 2j, arrowheads, and 2k and 2l), although we found Vegf-B/ß-Gal expression
in other vessels in the body (eg, the intralobar component of the
pulmonary arteries) (data not shown).
Postnatal Heart Growth in
Vegfb-/- Mice
Although histological examination of all organs
revealed no differences between genotypes,
Vegfb-/- hearts frequently appeared
marginally smaller than their Vegfb+/+
and Vegfb+/- littermates (Figure 3a). We recorded the total body and
heart weight of 122 animals including male and female
Vegfb+/+,
Vegfb+/-, and
Vegfb-/- mice at several ages
between P3 and P91. These mice were grouped as P3 to P9 mice
(Vegfb+/+, n=15;
Vegfb+/-, n=20; and
Vegfb-/-, n=13) and P25 or older
(Vegfb+/+, n=20;
Vegfb+/-, n=28; and
Vegfb-/-, n=27). We found no
consistent genotype-dependent decrease in body weigh;
however, heart weight was always reduced in
Vegfb-/- mice. To account for the
inherent interlitter and intralitter variability in body weight, due to
sex, age, and genetic background, we used heart/body ratios to display
the results. Although we found no significant difference in heart/body
ratio in relation to sex or genetic background, statistical
analysis revealed a dramatic increase in heart/body ratio from
P3-9 to P25 (or older) in all animals regardless of genotype
(Figure 3b). There were no differences in percentage of
heart/body weight ratios among genotypes in P3-9 mice
(Vegfb+/+, 0.64±0.02;
Vegfb+/-, 0.59±0.03; and
Vegfb-/-, 0.66±0.03), but we found
a significant (P<0.05) decrease in percentage of heart/body
weight ratio in P25 (or older)
Vegfb-/- (0.78±0.02) mice compared
with Vegfb+/+ (0.87±0.04) and
Vegfb+/- (0.89±0.02) mice (Figure 3b). When familial (litter/parents) correlation among mice was
taken into account, this significant difference remained (data not
shown). No significant difference was found between
Vegfb+/+ and
Vegfb+/- mice at any stage.
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The dramatic increase in heart weight during the first few weeks after birth is well documented and appears to be mainly due to massive growth of the coronary capillaries and vessels, but cardiomyocyte proliferation and hypertrophy also contribute.22 No difference in the size of myocardial cells was found in histological sections in Vegfb+/+, Vegfb+/-, or Vegfb-/- hearts (data not shown). LV thickness was significantly decreased in P25 Vegfb-/- (0.80±0.03 mm,* n=14) compared with Vegfb+/+ (0.89±0.03 mm, n=10) and Vegfb+/- (0.91±0.02 mm, n=16) hearts (*P=0.059 versus Vegfb+/+ and P<0.05 versus Vegfb+/-). Analysis of capillary density using standard morphometric measures found no significant differences between P30 Vegfb+/+ (2321±255 capillaries/mm2) and Vegfb-/- (2334±253 capillaries/mm2) hearts. Vessel density measures in adjacent heart sections also showed no differences between Vegfb+/+ (270±10 vessels/section) and Vegfb-/- (275±14 vessels/section) hearts.
Reactive Hyperemic Responses in
Vegfb+/+,
Vegfb+/-, and
Vegfb-/- Hearts
Baseline contractile function and coronary flow were
equivalent in Langendorff-perfused hearts from all three groups under
normoxic conditions (see Table online, http://www.circresaha.org). To
test whether alterations in vascular function would be more evident
during active responses to modified myocardial O2
delivery, we exposed hearts to transient (20 seconds) coronary
occlusion and studied the hyperemic response on reperfusion.
The reactive hyperemic responses differed subtly between groups
(Figure 4). Although peak
hyperemic flow was comparable in all three groups of hearts (32
to 36 mL · min-1 ·
g-1) (Figure 4a), overall flow-debt
repayment during the initial 60 seconds of reperfusion (during which
flow recovered to preocclusion levels) was significantly lower in
Vegfb-/- mice (60%) versus the
other two groups (100%) (Figure 4b). There were no
differences in repayment between
Vegfb+/+ and
Vegfb+/- hearts. These findings
indicate that the functional status of the coronary vasculature
is impaired in Vegfb-/- mice.
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Responses to Ischemia-Reperfusion in
Vegfb+/+,
Vegfb+/-, and
Vegfb-/- Hearts
As noted, baseline functional parameters were
comparable in hearts from Vegfb+/+,
Vegfb+/-, and
Vegfb-/- mice (see Table online,
http://www.circresaha.org). Global normothermic
ischemia completely abolished contractile function in all
hearts within 2 to 3 minutes and caused a rapid rise in
diastolic pressure. Time to onset of contracture and
peak-developed contracture are indicators of the severity of
ischemic injury. Although no difference was found in the rate
of contracture development, peak contracture during ischemia
was greater in Vegfb-/- compared
with Vegfb+/+ and
Vegfb+/- hearts (Figure 5a). Diastolic pressure was
significantly elevated in Vegfb-/-
hearts compared with Vegfb+/+ and
Vegfb+/- hearts during reperfusion
and recovered minimally (73 mm Hg) relative to the other two
groups (35 mm Hg) (Figure 5a). Recovery of contractile
function was slightly depressed throughout reperfusion in
Vegfb-/- hearts, with the
rate-pressure product being significantly lower at 30 minutes
compared with Vegfb+/+ and
Vegfb+/- hearts (Figure 5b).
Coronary flow responses did not differ between the three groups
at any time point (Figure 5c).
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Discussion |
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In the present study, we have used promoter trap LacZ expression in the Vegfb+/- mouse to correlate Vegf-B/ß-Gal expression to processes of vascularization in the heart, the major site of Vegf-B expression. The general developmental pattern of Vegf-B expression has previously been interpreted to reflect a paracrine action of Vegf-B on the developing vasculature, although no attempts were made to correlate expression patterns with developmental processes of vascularization in any individual organ.16 Although Vegfb transcripts are produced in the heart from E8.5, Vegf-B protein expression is not detected until E10.5.16 We can first detect Vegf-B/ß-Gal at E10.5 in the interventricular septum and propose that Vegf-B/ß-Gal (and therefore Vegf-B) production increases substantially at this time point, in spatial and temporal correlation with the commencement of coronary endothelial growth in the heart.23 Indeed, Vegf-B/ß-Gal levels increase both throughout development and after birth, closely correlating with the progression of cardiac angiogenesis. Vegf-B/ß-Gal is conspicuous in the subepicardium where heart angiogenesis has been shown to commence.24 It is more densely expressed in the ventricles than the atria and correlates with the degree of coronary angiogenesis that is more advanced in the ventricles than in the atria at fetal stages. After birth, Vegf-B/ß-Gal expression increases further in the LV, at a time when substantial capillary growth occurs predominantly in this chamber.21 22 The disparity between LV and RV capillary growth rates decreases several days after birth.21 Accordingly, by P25, Vegf-B/ß-Gal expression becomes even throughout the heart with levels of expression similar in RV, LV, and the atria. Despite the correlation between capillarization and Vegf-B/ß-Gal expression, such expression was not found in the smooth muscle cells of the differentiated coronary vessels. It is therefore likely that Vegf-B may exert its paracrine action on the microvasculature surrounding the expressing myocardium, rather than on the endothelium of the coronary vessels.
We find that Vegfb-/- hearts appear morphologically and functionally normal in the unstressed animal but do not undergo the same extent of postnatal growth as those of Vegfb+/+ and Vegfb+/- animals. Postnatal heart growth appears to be mainly due to the substantial increase in the coronary microvasculature and vessels.11 22 This increase has been attributed to the action of Vegf-A164 and Vegf-A188,11 because mice lacking these Vegf-A isoforms die as a consequence of severe heart ischemia due to an almost total absence of postnatal capillary and coronary vessel growth. Postnatal ablation of Vegf-A (and possibly Vegf-B) function by administering a soluble Flt-1 receptor (mFlt(1–3)-IgG)25 is also lethal. In the heart, this treatment leads to cardiomyocyte necrosis and massive capillary and vessel density reduction.25 Because Vegf-B is coexpressed with Vegf-A in the myocardium of the ventricles,6 can form biologically active heterodimers with Vegf-A, and also binds Flt-1,7 it is likely that the abnormal coronary angiogenesis described above is a result of interference with the normal function of both Vegf-A and Vegf-B. We tested whether the observed Vegfb-/- reduction in heart weight was a consequence of impaired growth of the vascular network by measures of coronary capillary and vessel density. We found no significant differences between Vegfb-/- and Vegfb+/+ hearts, although additional studies measuring lumen size, patency, and permeability of capillaries and vessels in the heart will reveal whether any structural abnormalities in the vascular network of Vegfb-/- hearts may be responsible for reduced volume of this organ. Alternatively, the observed microcardia could be attributed to an effect of Vegf-B on cardiomyocyte growth. Vegf-B effect on heart muscle could be mediated by the Vegf-B167 (and Vegf-A165) receptor, neuropilin-1, which is expressed in the developing cardiac muscle. However, this is unlikely, because cardiomyocytes do not appear affected in size or function in the Vegfb-/- heart, and we cannot rule out a direct effect of Vegf-B ablation on myocytes. It is worth noting, nevertheless, that cardiomyocytes are normal in the Vegfa120/120 mouse, where neuropilin-1 ligand Vegf-A165 has been ablated.11 A slight decrease in left ventricular thickness in the Vegfb-/- heart may indicate that some developmental hypoplasia, resulting from suboptimal vascularization, could be responsible for the observed microcardia.
Ablating Vegfb expression reduced the ability to repay coronary flow after a transient coronary occlusion. This occurred despite baseline coronary flow in the Vegfb-/- heart appearing normal, which was not unexpected given that moderate impairment of vascularization or vascular function that might result from deletion of the Vegfb gene could be compensated by enhanced intrinsic vasodilatation. Impairment of flow-debt repayment, despite similar peak flows, suggests inhibition of flow-mediated dilatation, which occurs subsequent to the immediate hyperemic response, indicating that the functional status of the coronary vasculature is impaired in some way in Vegfb-/- hearts. Reactive hyperemia is thought to be mediated by the combined actions of nitric oxide (NO) and adenosine,26 with potential involvement of KATP channels.27 The prolongation of the hyperemic response is thought to be at least partially NO dependent.28 Thus, one possible mechanism contributing to this change is an impaired NO production. However, deletion of the endothelial NO synthase gene fails to alter peak hyperemic flow, flow repayment, and adenosine responses in murine hearts.29
Heart rate was almost identical in hearts of all genotypes before and after ischemia, and no significant differences existed for heart rate between any groups at any time. Interestingly, deletion of Vegfb reduced functional recovery from ischemia-reperfusion and appeared to worsen contracture during ischemia. The mechanism of contracture is not well understood but may involve rigor-bond formation as a result of impaired glycolytic ATP formation.30 During reperfusion, diastolic dysfunction was significantly greater in knockout mice; the difference was wholly due to a change in contractile force and not rate. Recovery of the rate-pressure product was slightly reduced whereas coronary flow was similar in all three groups. Although a reduced reflow or perfusion could have explained the dysfunction, this was not supported by the measures of total myocardial perfusion. However, this does not exclude a more subtle change in flow distribution that is not reflected in the total flow response. The postischemic elevation in diastolic pressure is likely to reflect altered Ca2+ handling in reperfused tissue, resulting in enhanced diastolic Ca2+ levels.31 Ca2+ handling is energy dependent, particularly at the level of the sarcoplasmic reticulum. Knockout of the Vegfb gene could conceivably lead to impaired postischemic recovery of energy metabolism, owing to maldistribution of coronary flow, such that myocardial handling of Ca2+ is impaired. Further experiments addressing patency, permeability, and responses to vasodilatory stimuli in the ventricular microvasculature of Vegfb-/- hearts will reveal whether this is indeed the case. The increased diastolic dysfunction during ischemia is largely independent of the coronary vasculature and may reflect a developmental effect of Vegfb deletion on heart growth or function, as suggested by the smaller hearts and reduced left ventricular thickness in Vegfb-/- mice.
In the present study, we have shown that, despite heart morphology and function being normal in Vegfb-/- mice, the response to coronary occlusion and myocardial recovery from ischemia are compromised. Thus, although Vegf-B may play a redundant role in establishing the coronary vasculature, our results define a unique role in the development and maintenance of function in response to ischemic insult.
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Acknowledgments |
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This work was supported by the Queensland Cancer Fund and AMRAD Corporation Ltd. Nicholas K. Hayward is a recipient of a NH&MRC Senior Research Fellowship, and Sean M. Grimmond holds a NH&MRC C.J. Martin Traveling Fellowship. Graham F. Kay and Melissa Little are Fellows of the Sylvia and Charles Viertel Charitable Foundation. We would like to thank Michael Walsh for histological services and Dr Patrick Ward for advanced statistical analysis.
Received December 29, 1999; accepted December 29, 1999.
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