Blood Flow Remodels Growing Vasculature During Vascular Endothelial Growth Factor Gene Therapy and Determines Between Capillary Arterialization and Sprouting Angiogenesis
Background— For clinically relevant proangiogenic therapy, it would be essential that the growth of the whole vascular tree is promoted. Vascular endothelial growth factor (VEGF) is well known to induce angiogenesis, but its capability to promote growth of larger vessels is controversial. We hypothesized that blood flow remodels vascular growth during VEGF gene therapy and may contribute to the growth of large vessels.
Methods and Results— Adenoviral (Ad) VEGF or LacZ control gene transfer was performed in rabbit hindlimb semimembranous muscles with or without ligation of the profound femoral artery (PFA). Contrast-enhanced ultrasound and dynamic susceptibility contrast MRI demonstrated dramatic 23- to 27-fold increases in perfusion index and a strong decrease in peripheral resistance 6 days after AdVEGF gene transfer in normal muscles. Enlargement by 20-fold, increased pericyte coverage, and decreased alkaline phosphatase and dipeptidyl peptidase IV activities suggested the transformation of capillaries toward an arterial phenotype. Increase in muscle perfusion was attenuated, and blood vessel growth was more variable, showing more sprouting angiogenesis and formation of blood lacunae after AdVEGF gene transfer in muscles with ligated PFA than in normal muscles. Three-dimensional ultrasound reconstructions and histology showed that the whole vascular tree, including large arteries and veins, was enlarged manifold by AdVEGF. Blood flow was normalized and enlarged collaterals persisted in operated limbs 14 days after AdVEGF treatment.
Conclusions— This study shows that (1) blood flow modulates vessel growth during VEGF gene therapy and (2) VEGF overexpression promotes growth of arteries and veins and induces capillary arterialization leading to supraphysiological blood flow in target muscles.
Received February 15, 2005; revision received October 4, 2005; accepted October 11, 2005.
Vascular endothelial growth factor (VEGF) is an absolute requirement for embryonic blood vessel formation, and its overexpression results in strong angiogenesis in various tissues in adults, suggesting that it could be used for the treatment of myocardial and peripheral ischemia.1–4 Recent studies from our laboratory and elsewhere have shown that the overexpressions of VEGF and the mature form of VEGF-D, another VEGFR-2 ligand, induce efficient proliferation of both vascular endothelial cells and pericytes via VEGFR-2 and NO-mediated mechanisms, leading to strong capillary enlargement in skeletal muscle and myocardium.1–3,5–7 This kind of angiogenesis is clearly different from the Clinical Perspective p 3946 traditional concept that VEGF mainly induces sprouting angiogenesis in adults, resulting in new daughter vessels. There is also substantial controversy regarding whether VEGF or other VEGFR-2 ligands can, directly or indirectly, promote the growth of larger blood vessels than capillaries. Previously, VEGF has not been recognized as a molecule that promotes arteriogenesis, ie, collateral artery growth. However, for clinically relevant therapeutic blood vessel growth, it would be essential that the candidate growth factor is able to promote arteriogenesis in addition to angiogenesis.
We hypothesized that local blood flow influences vessel growth during VEGF overexpression. To test this hypothesis, we injected adenoviral (Ad) VEGF (1011 viral particles/mL) intramuscularly into semimembranous muscles of rabbits with or without the ligation of the major arterial branch supplying blood to this muscle, the profound femoral artery (PFA). It was found that blood inflow remodels growing capillaries and that AdVEGF can promote the growth of large arteries and veins, leading to a 23- to 27-fold increase in muscle perfusion, a value that has not been described with any treatment before.
Please see the online-only Data Supplement for Methods. The video files and legends can also be found in the online-only Data Supplement.
Ligation of PFA Leads to a Selective Vascular Defect in the Semimembranous Muscle
Digital subtraction angiography obtained 6 days after AdLacZ gene transfer (GT) shows normal vascularity in the rabbit hindlimb without any extravasation of x-ray contrast agent (Figure 1a). The ligation of PFA results in a selective vascular defect in the semimembranous muscle (Figure 1b). AdVEGF overexpression increases vascular permeability in the transduced muscle, resulting in the leakage of the contrast agent (Figure 1c and 1d). After PFA ligation, collaterals grow from the distal arterial branches in the knee area (Figure 1d). Quantitatively, AdVEGF increased plasma protein extravasation 42- and 54-fold (AdLacZ 2.0-fold) in semimembranous muscles of normal and operated limbs, respectively, as assessed by modified Miles assay (P=NS, AdVEGF normal versus operated; P<0.05 versus AdLacZ).
VEGF-Induced Blood Vessel Growth Is Modulated by Blood Flow
In normal and operated limbs, native power Doppler signal is very low or absent 6 days after AdLacZ GT, respectively (Figure 2a and 2b; for Videos 1 and 2, see the online-only Data Supplement). AdVEGF treatment increases perfusion strongly in normal muscle, whereas in operated limbs the response is attenuated (Figure 2c and 2d; Videos 3 and 4).
Transversal CD31-immunostained histological sections of the whole semimembranous muscles reveal highly increased vascularity 6 days after AdVEGF treatment in comparison to AdLacZ controls (Figure 2e to 2h). Higher magnifications demonstrate a more variable microvascular growth response to AdVEGF in muscles with ligated PFA than in normal muscles (Figure 2i to 2l). Importantly, no significant infiltrations of leukocytes or necrotic/regenerating myofibers were observed outside the needle track region despite rapid vascular growth and edema (Figure 2i to 2q).
In CD31+α-smooth muscle actin (α-SMA) double immunostainings at high magnification, capillaries are small and pericytes can hardly be observed in AdLacZ control (Figure 2m). Round, enlarged capillaries with thickened but loose pericyte coverage predominate in normal muscles after AdVEGF treatment (Figure 2n). In contrast, sprouting angiogenesis and formation of blood lacunae were observed after AdVEGF injections in muscles with ligated PFA (Figure 2o to 2q). Figure 2s to 2v demonstrates that normal and enlarged capillaries as well as blood lacunae are perfused, as shown by intra-arterial lectin injection resulting in red fluorescence on endothelium and intravascular red blood cells. The intramuscular injections of adenoviruses led to widespread transduction of skeletal muscle (Figure 2w), leading to abundant amounts of transduced VEGF165 in muscles (Figure 2y).
Videos 5 to 8 in the online-only Data Supplement show both low-power overviews and high magnifications of the whole CD31-immunostained semimembranous muscles of all groups, demonstrating distinct forms of blood vessel growth with AdVEGF.
Capillary Arterialization and Growth of Arteries and Veins by AdVEGF
Figure 3a to 3c shows normal appearance of capillaries, arteries, and veins in AdLacZ control muscle. Strong remodeling and enlargement of capillaries, arteries, and veins occur with AdVEGF (Figure 3d to 3f). To study the phenotype of enlarged microvessels, we used a histochemical staining for alkaline phosphatase (AP), which is known to be expressed in arterial capillaries but not in larger arteries or veins.8 AP was expressed in normal capillaries of AdLacZ-transduced muscles, as shown by CD31+AP double stainings (Figure 3g to 3i). Interestingly, AP staining was completely negative in enlarged capillaries by AdVEGF (Figure 3j to 3l). To exclude the possibility that the enlarged microvessels are venules, a histochemical staining for venous capillaries and venules, dipeptidyl peptidase IV (DPP), was used.8 As shown by triple AP+DPP+CD31 stainings, enlarged microvessels were also negative for DPP (Figure 3m and 3n).
Contrast-Enhanced Ultrasound Imaging and 3D Reconstructions of the Vasculature After Angiogenic Therapy
Figure 4a to 4d and Videos 9 to 12 in the online-only Data Supplement illustrate the difference in blood flow between AdLacZ- and AdVEGF-treated normal limbs and limbs with ligated PFA as assessed by power Doppler imaging and intravenous bolus administration of the second-generation contrast agent (SonoVue). In limbs with PFA ligation, AdVEGF increases blood flow primarily in the distal region, while a perfusion deficit is still present in the proximal part of the muscle because of PFA litigation (Figure 4d). 3D reconstructions of the vasculature were performed to visualize the effect of AdVEGF on perfusion and collateral growth within the whole muscles (Figure 4e to 4h and Videos 13 to 16).
Relative Blood Volume Measurement With Contrast-Enhanced MRI
To confirm the strong perfusion increase by AdVEGF measured with contrast-enhanced ultrasound (CEU) imaging, contrast-enhanced and dynamic susceptibility contrast (DSC) MRI protocols were performed for relative blood volume and perfusion measurements, respectively, exploiting intravenous contrast agent SHU-555A (carboxydextran-coated superparamagnetic iron oxide particles [Resovist]).9,10 As shown by contrast-enhanced MRI (Figure 5), the relative blood volume was found to be strongly increased by AdVEGF in normal muscle. In postcontrast images taken at the steady state of contrast agent distribution (Figure 5b and 5e), regions with high blood volume appear black in the AdVEGF-treated limb because superparamagnetic contrast agents decrease the MRI signal by enhancing T2* relaxation. The effect of AdVEGF versus AdLacZ on regional blood volume is best visualized by ΔR2* maps that show the difference between T2* relaxation rates before and after contrast administration, ie, the contrast agent concentration and thus blood volume in muscles (Figure 5c and 5f).
CEU Perfusion Measurement Correlates Significantly With DSC-MRI and Provides Information on the Structure and Function of the Vascular Bed
The CEU signal intensity–time curves illustrate the kinetics of intravenous bolus–injected SonoVue contrast agent in semimembranous muscles 6 days after AdVEGF or AdLacZ GT (Figure 6a). CEU perfusion index between the transduced and contralateral intact muscle was calculated by using the ratio of the peak intensity values. Furthermore, the time to the arrival of the contrast agent was derived from the curves, which gives information on peripheral vascular resistance.11 The shape of the curve reflects different vessel types in the vascular bed: The presence of large arteries and shunting without a normal capillary bed produces a steep curve with a high peak intensity (such as AdVEGF in normal muscle), whereas a normal vascular network with normal-sized capillaries yields a flat curve (AdLacZ).12 Furthermore, after AdVEGF GT in limbs with ligated PFA, the peak intensity is lower and the shape of the curve is flatter, reflecting the absence of this large conducting artery (Figure 6a).
DSC-MRI data were acquired and quantified analogously to the CEU data with the use of intravenous bolus injection of Resovist and a FLASH pulse sequence. The calculated ΔR2* is directly proportional to concentration of the contrast agent, and therefore the resulting ΔR2*-time curve (Figure 6b) was similar to that obtained by CEU imaging. The slight differences in the curve shape between CEU and MRI probably indicate some differences in the contrast agent behavior or detection between these 2 different imaging modalities.
As shown in Figure 6c, a comparison of 4 different methods to calculate a perfusion index in normal muscles transduced with AdVEGF or AdLacZ was performed. In all methods, the peak tracer signal intensities between the transduced and contralateral limbs were used. Importantly, the microsphere method likely underestimates perfusion in cases in which potent angiogenic growth factors such as VEGF significantly enlarge capillary size (diameter >15 μm) so that microspheres (15 μm) cannot be retained in them.2,5 The perfusion indices calculated with the use of native power Doppler ultrasound, CEU, or DSC-MRI were very similar after AdVEGF GT (23- to 27-fold). Furthermore, it was found that the CEU perfusion index correlated significantly with the corresponding index measured with DSC-MRI (r=0.89, P<0.01), showing that muscle perfusion assessment with the novel CEU technique is comparable to the previously established method.
Growth of the Whole Vascular Tree by AdVEGF Results in Supraphysiological Perfusion and a Decrease in Peripheral Resistance
The CEU perfusion index was significantly enhanced (8.3-fold) 3 days after AdVEGF injections in normal muscles (Figure 7a). After reaching the maximal 27-fold increase at 6 days, perfusion decreased rapidly, reaching baseline at 2 weeks (1.4-fold). After PFA ligation, the maximal perfusion with AdVEGF was clearly attenuated but still significantly elevated 2 weeks after GT (2.7-fold, 0.4-fold in AdLacZ operated).
Total areas (percentage) of arteries, veins, and microvessels were measured quantitatively in the transduced muscles (Figure 7b). AdVEGF induced manifold increase in the total arterial, venous, and capillary areas in both groups. However, vascular growth was even more pronounced in muscles with ligated PFA because of the effect of blood lacunae and enlarged collateral arteries. Collaterals persisted in operated AdVEGF animals, but abundantly enlarged capillaries and large blood lacunae disappeared completely only 2 weeks after GT (Figure 7b and 7c).
In CEU imaging, the contrast agent arrived &2-fold faster in normal muscles treated with AdVEGF than AdLacZ, reflecting a significant reduction in peripheral vascular resistance as the result of microvascular enlargement (Figure 7d). Contrast agent arrival was also quicker in operated AdVEGF animals at 6 days than in normal AdLacZ controls, probably because of shunting via large blood lacunae.
VEGF has been generally considered as an angiogenic but not very arteriogenic growth factor. This study demonstrates that VEGF, likely via the effects of increased blood flow, is a strong modulator of the whole vascular tree, including large arteries and veins, in both normal and hypoperfused muscles. In normal muscles, capillaries are enlarged and transformed toward an arterial phenotype by VEGF in a process that we call capillary arterialization. Unlike in arteriogenesis (collateral artery growth from preexisting small arterial anastomoses),13 in capillary arterialization it is the capillary vessels that start to enlarge and strengthen their wall. This kind of capillary arterialization by AdVEGF results in a significant reduction in peripheral resistance and a very high perfusion increase (23- to 27-fold). On the other hand, in muscles with compromised perfusion, AdVEGF generates variable blood vessel growth also composed of sprouting angiogenesis and formation of large blood lacunae. The excess vasculature regresses in normal limbs, but collateral arteries persist in ischemic limbs after VEGF withdrawal.
Imaging of growing vasculature and quantitative measurement of perfusion are very important in the assessment of clinical efficacy of angiogenic therapies. Strong vascular growth by AdVEGF involving manifold capillary enlargement and increased vascular permeability interferes with many standard vascular imaging and perfusion measurement techniques such as x-ray angiography, the microsphere method, and, as shown previously, contrast-enhanced MRI with the use of low-molecular-weight extracellular contrast agents such as gadolinium.2,5 In these circumstances, the contrast agent must not extravasate and the method must not assume physiological capillary size. CEU imaging is very attractive for quantitative perfusion measurement after angiogenic therapies because it meets these requirements, is noninvasive, is approved for humans, provides quantitative data on blood flow kinetics, and enables 3D reconstruction of vasculature without nephrotoxic contrast agents or ionizing radiation. DSC-MRI with the use of relatively large contrast agent particles is feasible as well but is more time consuming and technically challenging. CEU is increasingly used for perfusion measurement in various tissues including human skeletal muscle.11,14 Here, the perfusion indices measured with the 2 independent methods, CEU and DSC-MRI, were in good agreement.
AdVEGF at 1011 viral particles/mL increased perfusion 23- to 27-fold in normal rabbit skeletal muscle 6 days after intramuscular injections. To the best of our knowledge, a perfusion increase of this magnitude has not been described previously with any medical treatment. In fact, the perfusion achieved with AdVEGF in resting muscles is about the same order of magnitude as the increase observed during maximal skeletal muscle exercise.15 Thus, VEGF overexpression seems to bypass the physiological control of muscle perfusion, which is normally tightly adapted to metabolic needs, leading to supraphysiological perfusion. The enormous blood flow enhancement is easier to understand when the theoretical effect of Poiseuille’s law (blood flow in cylindrical vessel is proportional to the fourth power of the vessel radius) is combined with the fact that the capillary mean size was increased 20- to 28-fold by AdVEGF.
AP, an enzyme capable of hydrolyzing organic phosphate esters, is expressed on the arterial side of the capillaries and arterioles but not in large arteries,8 and thus it has been widely used to demonstrate increases in capillary density after various angiogenic therapies. However, we found that both AP and DPP, a venous capillary and venule marker,8 activities were absent in AdVEGF-induced enlarged capillaries. Similar AP downregulation has been found to occur in tumor vasculature and in hypoxic skeletal muscle,16,17 both of which involve increased vascular growth and VEGF. The downregulation of AP and DPP activity in enlarged microvessels together with the enhanced coverage with α-SMA–positive pericytes suggests that capillaries were transformed toward an arterial phenotype in muscles treated with AdVEGF. Furthermore, the downregulation of AP by VEGF overexpression corroborates the previously known fact that AP is not an optimal staining method for capillaries after angiogenic therapies.
The short arrival times (decreased peripheral resistance) and the steep shape of CEU and DSC-MRI signal intensity–time curves indicate the presence of large arteries and enlarged capillaries and the lack of normal small-sized capillary bed12 after AdVEGF treatment. The enlargement and decreased peripheral vascular resistance suggest that the transformed capillaries conduct large amounts of blood with a high velocity and therefore function like shunts. Our findings are similar to those in some tumors that have high blood velocity likely because of shunting.11 Although excess blood flow may not be very useful to the local environment, decreased peripheral resistance increases blood flow in upstream collaterals, which probably contributes to arteriogenesis via enhanced shear stress and wall strain. Thus, preoperative or perioperative AdVEGF treatment could be useful, eg, in combination with coronary artery or peripheral bypass surgery to avoid “poor runoff” due to high peripheral resistance and thereby improve the patency of the grafts.
VEGF likely promotes capillary arterialization and arteriogenesis indirectly because pericyte and smooth muscle cell proliferation has not been demonstrated to occur directly on VEGF stimulation in vitro. Previously, blood flow has been shown to be a critical determinant of vessel maintenance and growth.18,19 Elevated circumferential wall strain may force vessels exposed to increased blood pressure to strengthen their wall by hypertrophy of their pericyte and smooth muscle cell coverage.20 Furthermore, a very high degree of arteriogenesis was recently achieved in a model with the use of an arteriovenous fistula, which strongly increased blood flow in collaterals after the ligation of the main artery.21 Thus, mechanical factors derived from increased blood flow and pressure, occurring subsequently to microvascular effects by VEGF, appear necessary for capillary arterialization and growth of arteries and veins (Figure in the online-only Data Supplement).
Our results showing that VEGF may be arteriogenic agree with previous studies in which mice deficient in VEGF120 and VEGF164 show impaired development of retinal arteries but not veins, and transgenic cardiac overexpression of VEGF164 leads to increased arteriolar but decreased venular capillary formation.22,23 In 2003, 3 independent research groups reported transformation of capillaries toward an arterial phenotype by VEGF or VEGF-DΔNΔC overexpression in skeletal muscle.2,6,7 Furthermore, a decade ago VEGF administration was reported to enhance collateral artery growth,24 and more recently VEGFR-2 receptor antagonism was shown to completely inhibit25 collateral growth. These results suggest an important role for VEGF in the growth of muscular vessels. Although VEGF may also directly contribute to the arterialization of blood vessels because VEGF appears to promote the arterial fate even before the onset of circulation,26 the results of the present study with the use of the arterial ligation model suggest that blood flow is crucial for capillary arterialization and collateral growth by VEGF in adults.
Capillary arterialization played the predominant role in normal muscles after AdVEGF injections, whereas in operated limbs with a perfusion deficit a different response was observed, with other forms of blood vessel growth being more prevalent. Classic sprouting angiogenesis, which is not the most common form of capillary growth in normal muscles with AdVEGF, took place in muscles with the ligation of PFA. Furthermore, massive blood lacunae were formed from preexisting capillaries in other areas of the same muscles. The lacunae were perfused as shown by lectin injections and intravascular erythrocytes and formed in between the myofibers, in contrast to a study using AAV-VEGF overexpression in which the lacunae grew inside myocytes.7 In addition to the microenvironmental VEGF concentration,27 local blood flow appears crucial in molding the growing capillaries into ones that best serve the needs of current circumstances: Into daughter vessels via sprouting angiogenesis if newly formed vessels are needed or into large shunts to direct excess blood flow elsewhere. In future studies, it is necessary that the significance of the different forms of VEGF-induced neovascularization on local muscle metabolism and exercise tolerance will be carefully addressed. In addition, the intramuscular AdVEGF165 dose and injection volumes must be optimal in clinical trials to obtain a clinically relevant effect on muscle perfusion but avoid excess tissue edema and formation of blood lacunae.
In nonischemic skeletal muscle, all the effects by AdVEGF were transient, lasting no longer than adenoviral gene expression (<2 weeks). Thus, in normal tissues increased blood flow and immature pericyte coverage alone are insufficient to protect the enlarged vessels from regression without the presence of VEGF. Mathematical models also suggest that shear stress alone does not lead to stabilization of vessels, but additional factor(s) must be present.28 However, it appears that long-term VEGF overexpression may stabilize the vessels and produce persistent increases in perfusion in most normal tissues.6,7,27,29 Collateral arteries in the operated limbs resisted regression, probably because they provide crucial blood flow to muscles with compromised perfusion and they are formed from structurally mature preexisting arteriolar anastomoses. The persistence of collateral blood flow resulted in normal or even slightly elevated (2.7-fold) perfusion values in the operated AdVEGF-treated limbs at 2 weeks.
In conclusion, the results of this study indicate that adenoviral VEGF overexpression alone can orchestrate the growth of the whole vascular tree and induce supraphysiological perfusion increases in the target muscle. Thus, VEGF gene therapy is very promising for the induction of clinically relevant therapeutic vascular growth in patients with ischemic disease, but the correct dosage and duration of expression are the key issues for clinical success.
This study was supported by grants from Finnish Academy, Sigrid Juselius Foundation, EU grant to European Vascular Genomics Network (EVGN LSHM-CT-2003-503254), Finnish Medical Foundation, Finnish Cultural Foundation of Northern Savo, and Paavo Nurmi Foundation. Technicians in the group of Molecular Medicine and at the National Experimental Animal Center at Kuopio University are acknowledged for their expert technical help. We thank Bracco for providing SonoVue for these experiments.
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Vascular endothelial growth factor (VEGF) is the master regulator of blood vessel growth that has been previously thought to induce effects exclusively at the capillary level. However, the growth of collaterals bypassing the stenosis or occlusion in the conducting artery would be highly desirable in patients with myocardial or peripheral ischemia. This study shows that adenoviral VEGF overexpression promotes the growth of the whole vasculature in both normal and ischemic rabbit hindlimbs. This occurs secondarily to the great enlargement of capillaries that results in very high capillary perfusion and reduced peripheral resistance, leading to compensatory growth of upstream arteries and downstream veins. In normal muscles, excess blood flow resulting from VEGF overexpression transformed capillaries toward arterioles that functioned as shuntlike vessels. Muscle perfusion increased 23- to 27-fold under these conditions. In ischemic muscles, VEGF induced capillary sprouting in addition to the formation of large vessel structures. These results show the enormous potential of VEGF to reshape the existing vasculature. Clinical success in future trials will require optimization of the indications of therapy as well as the dose and duration of VEGF overexpression. VEGF gene therapy could potentially be used in combination with conventional revascularization procedures as adjuvant therapy to enhance capillary perfusion, reduce peripheral resistance, and improve patency of the grafts via better blood flow.
The online-only Data Supplement can be found with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.105.543124/DC1.