Published online before print February 2, 2009, doi: 10.1161/CIRCULATIONAHA.108.816454
(Circulation. 2009;119:845-856.)
© 2009 American Heart Association, Inc.
Molecular Cardiology |
Vascular Endothelial Growth Factor-B Induces Myocardium-Specific Angiogenesis and Arteriogenesis via Vascular Endothelial Growth Factor Receptor-1– and Neuropilin Receptor-1–Dependent Mechanisms
From the Department of Biotechnology and Molecular Medicine (J.E.L., M.T.L., A.K., T.H., T.T.R., E.V., P.K., S.Y.-H.), A.I. Virtanen Institute, University of Kuopio, Kuopio, Finland; Ludwig Institute for Cancer Research Ltd, Karolinska Institutet (C.R., A.F., J.K., U.E.), Stockholm, Sweden; Molecular and Cancer Biology Laboratory (B.E., K.A.), Haartman Institute, University of Helsinki, Helsinki, Finland; and Flanders Interuniversity Institute for Biotechnology (P.C.), Katholieke Universiteit Leuven, Leuven, Belgium.
Reprint requests to Seppo Ylä-Herttuala, MD, PhD, FESC, Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute, University of Kuopio, PO Box 1627, FIN-70211 Kuopio, Finland. E-mail Seppo.Ylaherttuala{at}uku.fi
Received December 5, 2007; accepted December 9, 2008.
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Abstract |
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Background— New revascularization therapies are urgently needed for patients with severe coronary heart disease who lack conventional treatment options.
Methods and Results— We describe a new proangiogenic approach for these no-option patients using adenoviral (Ad) intramyocardial vascular endothelial growth factor (VEGF)-B186 gene transfer, which induces myocardium-specific angiogenesis and arteriogenesis in pigs and rabbits. After acute infarction, AdVEGF-B186 increased blood vessel area, perfusion, ejection fraction, and collateral artery formation and induced changes toward an ischemia-resistant myocardial phenotype. Soluble VEGF receptor-1 and soluble neuropilin receptor-1 reduced the effects of AdVEGF-B186, whereas neither soluble VEGF receptor-2 nor inhibition of nitric oxide production had this result. The effects of AdVEGF-B186 involved activation of neuropilin receptor-1, which is highly expressed in the myocardium, via recruitment of G-protein-
interacting protein, terminus C (GIPC) and upregulation of G-protein- interacting protein. AdVEGF-B186 also induced an antiapoptotic gene expression profile in cardiomyocytes and had metabolic effects by inducing expression of fatty acid transport protein-4 and lipid and glycogen accumulation in the myocardium.
Conclusions— VEGF-B186 displayed strikingly distinct effects compared with other VEGFs. These effects may be mediated at least in part via a G-protein signaling pathway. Tissue-specificity, high efficiency in ischemic myocardium, and induction of arteriogenesis and antiapoptotic and metabolic effects make AdVEGF-B186 a promising candidate for the treatment of myocardial ischemia.
Key Words: angiogenesis • gene therapy • metabolism • myocardial infarction
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Introduction |
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Severe coronary heart disease is still a leading cause of death in developed countries in spite of improved management of risk factors and more effective treatments. It is estimated that approximately 5 million people in the United States and the European Union have ischemic heart disease; however, a steadily increasing number of patients fall into a category in which currently available revascularization techniques cannot be applied. This is especially true of elderly patients who have had multiple bypass and stenting operations.1 It is estimated that these patients represent up to 3% to 5% of all patients in specialty cardiology clinics. Thus, there is a clear need to develop efficient, minimally invasive procedures for the treatment of these no-option patients.
Clinical Perspective p 856
Therapeutic vascular growth (ie, angiogenesis and arteriogenesis) with genes or proteins has been suggested as an alternative approach for the treatment of these patients.2 Vascular endothelial growth factors (VEGFs) are potent inducers of vascular growth via binding to 3 tyrosine kinase receptors (VEGFRs). VEGFR-2 is the main regulator of angiogenesis, exerting its function via nitric oxide production, whereas the role of VEGFR-1 is far less defined.3 VEGF-B4 and placental growth factor (PlGF)3 share structural and functional characteristics and bind to VEGFR-1, whereas VEGF-A5 binds to both VEGFR-1 and VEGFR-2.3 VEGF-B has 2 isoforms, VEGF-B167 and VEGF-B186.4 Both isoforms of VEGF-B, PlGF, and VEGF-A165 have been shown to bind neuropilin receptor-1 (Nrp-1),4,6 but their binding is not identical. VEGF-A165 binds to 2 different domains of Nrp-1, whereas PlGF only binds to one.7 Although both isoforms of VEGF-B bind to Nrp-1, the binding of VEGF-B167 is mediated via a heparin-binding domain, whereas the binding of VEGF-B186 requires proteolytic processing.4 Nrp-1 interactions with VEGFRs may alter their signaling properties,8 and recently, Nrp-1 has also been shown to convey intracellular signaling.9 In addition, diffusion properties of VEGFs regulate their biological effects. VEGF-A165 and VEGF-B167 bind to heparin sulfates, whereas VEGF-B186 and PlGF are more freely soluble.
In addition to enhancing blood flow, the altering of myocardial metabolism and inhibition of apoptosis may also help to protect hibernating myocardium after myocardial infarction. Metabolism is altered in energy-depleted myocardium, and, for example, glycogen accumulation has been shown to protect ischemic myocardium and limit structural damage.10 Although several angiogenic and metabolic factors have been identified, none has shown relative specificity for the heart. Here, we describe a new cardiac-specific proangiogenic gene therapy approach that could be applicable for the treatment of patients with severe ischemic heart disease.
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Methods |
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Animal Models
Acute myocardial infarction in pigs was induced in the anterolateral wall of the left ventricle by occluding the distal part of the left anterior descending coronary artery with a VortX-18 occlusion coil (Boston Scientific, Natick, Mass). After the appearance of ischemic ECG changes and detection of a wall-motion defect by intracardiac ultrasound (Acunav, Acuson Sequoia, Siemens, Erlangen, Germany), total occlusion of the coronary artery was confirmed by angiography. Hindlimb ischemia was induced in New Zealand White rabbits as described previously.11
Gene Transfer
Domestic pigs (n=121) and New Zealand White rabbits (n=68) received an intramuscular or intramyocardial gene transfer of adenoviruses (Ad) encoding human VEGF-A165, PlGF-2, VEGF-B167, VEGF-B186, or a marker gene, LacZ. Experiments were performed in 2 large-animal models to reduce the possibility of species-specific effects. In addition, a subgroup of animals received intramuscular or intramyocardial injections of adenoviruses encoding soluble (s) VEGFR-1, sVEGFR-2, or sNrp-1. In the myocardium, 10 injections with 1012 viral particles (vp)/mL were given with a custom-made injection catheter in rabbits (injection volume 50 µL) and an 8F NOGA catheter system (Biosense Webster, Diamond Bar, Calif), with an injection volume of 200 µL in pigs, as described previously.12 In ischemic pigs, gene transfer was performed 30 minutes after acute myocardial infarction. A total of ten 200-µL injections were given around the infarct area, the border zone of the movement defect, and the normally contracting myocardium proximal to the infarction area where collateral vessel formation was expected (referred to as the maximal transduction area). In the hindlimbs, 5 injections with virus concentration of 1011 vp/mL were given into the semimembranosus muscles (25-gauge needle, volume 0.1 mL in rabbits; 20-gauge needle, injection volume 1 mL in pigs). In ischemic hindlimbs, rectus femoris and tibialis anterior muscles were also transduced. Human clinical GMP-grade (first-generation, serotype 5) replication-deficient adenoviruses analyzed to be free from contaminants were used. The nitric oxide synthase inhibitor L-NAME (NG-nitro-L-arginine methyl ester; 50 mg · kg–1 · d–1; Sigma-Aldrich, St. Louis, Mo) was given to a subgroup of animals twice a day after the gene transfer. Propofol and fentanyl were used for pig anesthesia and medetomidin and ketamine for rabbit anesthesia. All animal experiments were approved by the Experimental Animal Committee, University of Kuopio.
Echocardiography and Contrast-Enhanced Ultrasound Imaging of Perfusion
Echocardiography was performed at baseline, after acute myocardial infarction, and before animals were euthanized on day 6 after gene transfer with an Acunav catheter inserted into the right atrium. Ejection fraction was quantified with ultrasound. Contrast-enhanced ultrasound perfusion measurement was done in skeletal muscles as described previously13 after a bolus injection of a contrast agent (SonoVue, Bracco, Milan, Italy) via the ear vein.
Microsphere Perfusion and Angiography
Myocardial perfusion was measured with red fluorescent microsphere particles (Molecular Probes/Invitrogen, Carlsbad, Calif; 15 µm, 5x106 particles) as described previously.12 Angiography of the left anterior descending coronary artery to visualize collateral vessels was performed at the time of euthanasia (Siremobil 2000, Siemens). The last images before venous filling are shown.
Blood Vessel Measurements
Mean blood vessel area (µm2) was measured from CD31-immunostained sections of rabbit muscles13 and platelet and endothelial cell adhesion molecule-1–stained pig muscle sections at 200x magnification. Vessels from the infarction edge were analyzed at 100x magnification. All measurements were performed with AnalySIS software (Soft Imaging System) in a blinded manner from 5 to 10 different randomly selected fields from each section. Means of the measurements are reported.
Immunoprecipitation and Western Blot
Snap-frozen tissue samples from normoxic and ischemic transduced myocardium and skeletal muscles were homogenized in protein extraction buffer (T-Per, Pierce, Rockford, Ill). Immunoprecipitations were done with Nrp-1 antibody (C-19, Santa Cruz Biotechnology, Santa Cruz, Calif) and protein A Sepharose (Amersham Biosciences, Uppsala, Sweden). Immunoblotting from native samples was performed with antibodies for Nrp-1 (C-19, Santa Cruz Biotechnology) and G-protein- interacting protein (GAIP, N-17, Santa Cruz Biotechnology) and from Nrp-1–immunoprecipitated samples with antibodies for VEGF-B (MAB 3373, R&D Systems, Minneapolis, Minn) and GAIP-interacting protein (GIPC; N-19, Santa Cruz Biotechnology).
Statistical Analyses
Results are expressed as mean±SEM. Statistical significance was evaluated with linear mixed models (SPSS version 14.0, SPSS, Inc, Chicago, Ill), which are based on generalized estimating equations, or by ANOVA followed by Student’s t test. P<0.05 was considered statistically significant.
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
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AdVEGF-B186 Induces Myocardium-Specific Angiogenesis
It has remained unknown which growth factor(s) might be best suited for the treatment of myocardial ischemia.1,2,12,13 In the present study, we tested several VEGFR-1 ligands side by side in large-animal models using a new pig infarction model based on a percutaneous coil-mediated occlusion of the left anterior descending coronary artery. The infarction area was confirmed with a wall-motion defect and ECG changes. In contrast to previous infarction models based on thoracotomy and surgical coronary occlusion, this model preserves an intact pericardium, avoids excess fibrous scar formation in the thorax and around the heart, and allows precise imaging of the myocardium with ultrasound. Gene delivery was done percutaneously into the infarction border zone and adjacent myocardium with a NOGA mapping-injection catheter.12
Of the VEGFR-1 ligands (VEGF-B167, VEGF-B186, PlGF, and VEGF-A165), both VEGF-B isoforms showed angiogenic effects only in the myocardium (Figure 1A through 1E). Both in normal and in ischemic skeletal muscles, the 2 AdVEGF-B isoforms were ineffective (Figure 1D, d4–5, d9–10, and d14–15), whereas AdVEGF-A and AdPlGF had significant angiogenic effects that increased tissue perfusion (Figure 1D, d2, d7, and d12 and d3, d8, and d13, respectively; Data Supplement Figure I). AdVEGF-B186 was particularly efficient in myocardium after acute myocardial infarction (Figure 1A, a6 through a10). Mean vessel size in the infarction border zone was increased almost 3-fold compared with the AdLacZ controls (Figure 1C). AdPlGF also increased the mean vessel number in the infarction edge (Figure 1A, a8 and data not shown), but the increase in mean vessel area was not significant (Figure 1C). The angiogenic effects of AdVEGF-B186 and AdVEGF-A persisted in the maximal transduction area in the periinfarct zone 3 weeks after gene therapy (Data Supplement Figure II). Transient production of angiogenic factors by adenoviral overexpression is therefore sufficient to induce angiogenic neovessels that are further regulated by blood flow and the metabolic needs of the surrounding tissue.
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) 6 days after infarction and gene transfer in AdLacZ-transduced animals (a6). In AdVEGF-A–transduced animals, vessels were significantly larger in the infarction edge (a7). Lacunae and large glomerular structures were also observed (data not shown). Modest angiogenic response was observed with AdPlGF- and AdVEGF-B167–transduced animals (a8 and a9, respectively). AdVEGF-B186 induced an efficient angiogenic response in the border zone, with thick-walled vessels with an arteriolar phenotype (a10). B and C, AdVEGF-B186 induced a significant increase in mean capillary area in healthy (B) and ischemic (C) myocardium. B, In the maximal transduction areas of AdVEGF-A–transduced myocardium, mean capillary area was increased by 5.1-fold compared with AdLacZ controls (PECAM-1 staining, magnification 200x). AdPlGF induced a similar but slightly weaker angiogenic response (increase in mean capillary size 3.2-fold compared with AdLacZ controls). Both AdVEGF-B167 and AdVEGF-B186 induced angiogenesis in the myocardium, although the effect was greater in AdVEGF-B186–transduced animals (2.8-fold compared with AdLacZ controls). C, In the infarction edge, AdVEGF-A gene transfer led to a 2.2-fold increase in vessel area, AdPlGF and AdVEGF-B167 gene transfer led to a 1.4- and 1.6-fold increase, respectively; AdVEGF-B186 gene transfer induced the largest (2.9-fold) increase in mean vessel area among the VEGFs compared with the AdLacZ control. D and E, AdVEGF-B186 did not induce angiogenesis in either normoxic or ischemic skeletal muscle, whereas VEGF-A and PlGF were angiogenic (D, d1–d10. and E). AdLacZ did not change capillary size or morphology 6 days after gene transfer (d1 and d6). AdVEGF-A (d2 and d7) and AdPlGF (d3 and d8) induced a robust angiogenic response in normoxic skeletal muscle (20- and 43-fold increase in capillary size [e] compared with AdLacZ, respectively) and ischemic skeletal muscle. AdVEGF-B167 and AdVEGF-B186 were ineffective in both settings (d4, d5, d9, and d10). AdVEGF-B186 did not increase perfusion in skeletal muscle (d11–d15). Perfusion measured by contrast-enhanced ultrasound imaging (CEU) 6 days after gene transfer showed significant increases in perfusion in AdVEGF-A–transduced (d12) and AdPlGF-transduced (d13) animals (29- and 44-fold compared with AdLacZ, respectively). Both AdVEGF-B167 and AdVEGF-B186 were ineffective (d14 and d15). Quantification of CEU is found in supplementary Figure I. F and G, VEGFR-1 ligands differentially stimulated cell proliferation in the pig myocardium (f and g1–g5). Double staining for PECAM-1 (blue) and proliferating cell nuclear antigen (PCNA; brown), showed very few proliferating cells in AdLacZ-transduced hearts (g1; magnification 400x). AdVEGF-A induced a strong proliferative response (g2), with a 15-fold increase in proliferative cells/field compared with AdLacZ in the maximally transduced area, and most of these were endothelial cells (F). A similar but slightly weaker effect was observed in AdPlGF-transduced hearts (g3 and F). AdVEGF-B167 had only a weak effect on cell proliferation (g4). In contrast, in AdVEGF-B186–transduced myocardium, the total number of proliferating cells was comparable to AdVEGF-A, but the distribution between the different cell types was strikingly different, with the majority of the PCNA-positive nuclei being nonendothelial cells in the perivascular space (g5, F). g6–g10, VEGFR-1 ligands displayed differences in pericyte recruitment in the pig myocardium. Double staining for PECAM-1 and
The angiogenic phenotypes induced by the different VEGFs were clearly distinct. AdVEGF-B186 predominantly stimulated proliferation of abundant -smooth muscle actin–positive cells (Figure 1F and 1G, g5 and 1g10), whereas AdPlGF and AdVEGF-A mainly induced proliferation of endothelial cells (Figure 1F and 1G, g2 and 1g3) in normal myocardium. AdVEGF-B167–induced vessels had low pericyte coverage, and only a few proliferating endothelial cells were seen (Figure 1F and 1G, g4 and g9). Thus, the effects of AdVEGF-B186 in the myocardium were clearly different from the other VEGFs.
AdVEGF-B186–induced angiogenesis also improved the functional outcome in the myocardial ischemia model. AdVEGF-B186 induced the formation of angiographically visible collateral arteries around the infarction area (Figure 2A). Ejection fraction improved by 18% and 21% after AdVEGF-B186 and AdVEGF-A treatments, respectively (Figure 2B), as well as blood flow in the myocardium as measured by microsphere trapping (Figure 2C). Thus, AdVEGFB186 treatment is a promising new candidate for acute myocardial infarction treatment because of its unique myocardium-specific properties.
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AdVEGF-B Induces Angiogenesis Through VEGFR-1 and Nrp-1 but Not VEGFR-2
Even though both VEGF-B186 and PlGF bind to VEGFR-1, the angiogenic effects induced by these 2 ligands were clearly distinct. The effect of AdVEGF-B186 was dependent on VEGFR-1, but unlike AdPlGF, it was not dependent on VEGFR-2 or upregulation of endogenous VEGF-A, as determined by simultaneous transductions with AdsVEGFR-1 or AdsVEGFR-2 (Figure 3A through 3D). Moreover, angiogenesis induced by AdVEGF-B186 was not blocked by inhibition of nitric oxide production by L-NAME, which completely inhibits the angiogenic effects of AdPlGF and AdVEGF-A (Data Supplement Figure III).
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The angiogenic effects of AdVEGF-B186 in normal myocardium were instead dependent on membrane-bound Nrp-1, because AdsNrp-1 significantly reduced AdVEGF-B186–induced capillary growth (Figure 3E, e4, and 3F). Interestingly, AdsNrp-1 enhanced the angiogenic effects of AdVEGF-A (Figure 3Em, e3, and 3F), which may be due to enhanced VEGFR-2 activity,14 further suggesting different angiogenic mechanisms for VEGFR-1 and VEGFR-2.
Mechanistic studies were also performed in ischemic myocardium. AdsNrp-1 increased mean vessel area by 2.3-fold compared with AdLacZ (Figure 3G, g3, and 3H), which was likely due to an enhancement of the vessel growth induced by endogenous VEGF-A produced in the ischemic tissue (data not shown). In contrast, AdVEGF-B186–induced angiogenesis was reduced both by AdsNrp-1 (by 69%; Figure 3G, g4) and by AdsVEGFR-1 (by 47%; Figure 3G, g6).
In line with the cardiac-specific effects of VEGF-B186, Nrp-1 was abundantly present in both normal (Figure 4A and 4B) and ischemic myocardium (data not shown) but not in skeletal muscle (Figure 4A and 4B). In AdLacZ-, AdVEGF-A–, and AdPlGF-transduced hearts, Nrp-1 was observed mainly in the capillary endothelium in the angiogenic area and in the endothelial layer of arteries and small veins (Figure 4A, a5 through a7), whereas in AdVEGF-B186–transduced myocardium, expression was also seen in cardiomyocytes (Figure 4A, a8 and a12). Western blot analysis confirmed strong expression of Nrp-1 in both AdLacZ- and AdVEGF-B186–transduced myocardium but not in skeletal muscle (Figure 4B).
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The role of Nrp-1 in VEGF-B186–induced cardiac angiogenesis warranted analysis of the intracellular signaling events downstream of the VEGF-B186 binding to Nrp-1. Immunostainings, Western blotting, and pull-down assays of GIPC, an intracellular Nrp-1 adaptor protein,9 and GAIP, a GIPC interacting protein and a regulator of G-protein signaling (also known as RGS19), were performed. GIPC expression was mainly localized to endothelial cells in AdLacZ-, AdVEGF-A–, and AdPlGF-transduced myocardium (Figure 4C, c5 through c7), whereas no expression was detected in skeletal muscle (Figure 4C, c1 through c4); however, in AdVEGF-B186–transduced hearts, polarized GIPC expression was also found in the cardiomyocytes (Figure 4C, c12).
VEGF-B186 bound to Nrp-1 in AdVEGF-B186–transduced myocardial samples, as shown by pull-down assays of tissue lysates with Nrp-1 antibodies followed by Western blotting with antibodies to VEGF-B (Figure 4D). Pull-down assays with Nrp-1 antibodies also showed GIPC binding to Nrp-1 in AdVEGF-B186–transduced myocardium but not in skeletal muscle (Figure 4E). Only small amounts of Nrp-1–associated GIPC were seen in AdLacZ-transduced myocardium (Figure 4E), which suggests a VEGF-B–dependent recruitment of GIPC to Nrp-1. Similar activation of Nrp-1 in ischemic myocardium was also confirmed by Western blot (data not shown).
GAIP was expressed in endothelial cells in AdLacZ-, AdVEGF-A–, AdPlGF-, and AdVEGF-B186–transduced myocardium (Figure 4F, f5 through f8). Cardiomyocytes in AdLacZ-, AdVEGF-A–, and AdPlGF-transduced hearts were positive for GAIP (Figure 4F, f9 through f11); however, in AdVEGF-B186–transduced myocardium, a strong polarized, granular GAIP staining was observed in cardiomyocytes in an overlapping staining pattern with Nrp-1 and GIPC (Figure 4F, f12). Upregulation of GAIP was confirmed by Western blot analyses that showed strong GAIP expression in AdVEGF-B186–transduced myocardium but not in AdLacZ-transduced myocardium or skeletal muscle (Figure 4G). These findings suggest that Nrp-1 signaling via G proteins at least partially mediates the cardiac effects of VEGF-B186 and that this effect is specific for VEGF-B compared with the other VEGFs.
AdVEGF-B186 Induces Secondary Changes in Metabolism and Apoptotic Signaling in Cardiomyocytes
Because proliferation was largely seen in nonendothelial, -smooth muscle actin–positive cells in vivo (Figure 1F and 1G, g5, and g10), possible effects of VEGF-B on cardiomyocyte growth and metabolism were also addressed. Recently, both VEGF-B and Nrp-1 have been implicated in antiapoptotic signaling.15,16 We therefore studied apoptosis in the infarction edge of ischemic myocardium using terminal dUTP nick end-labeling staining. The number of apoptotic cells was significantly decreased after AdVEGF-B186 gene transfer compared with AdLacZ-, AdPlGF-, and AdVEGF-A–transduced hearts (Data Supplement Figure IV).
To study whether VEGF-B had a direct antiapoptotic effect on cardiomyocytes or whether it mediated its effects through VEGFR1- and Nrp-1–expressing endothelial cells, bEnd3 endothelial cells and HL-1 cardiomyocytes were studied in vitro. Whereas bEnd3 cells express VEGFR-1, VEGFR-2, and Nrp-1, HL-1 cardiomyocytes express Nrp-1 but do not express VEGFR-1 (data not shown), similar to the situation in vivo. Treatment of HL-1 cardiomyocytes with VEGF-B186 did not alter levels of the fatty acid transporters Fatp1 and Fatp4 or of the apoptotic factors Bik1, Bmf1, or Bad1 (Figure 5A), which suggests that VEGFR-1 indeed is essential for VEGF-B–mediated signaling. In contrast, VEGF-B186 treatment of bEnd3 endothelial cells increased Fatp4 expression by 1.7-fold, whereas it had no direct effect on the apoptotic genes (Figure 5B) (C. Rosenlew and U. Eriksson, unpublished data, 2007). Most importantly, we found that although VEGF-B186 did not have direct effects on cultured cardiomyocytes, conditioned media from both VEGF-B167– and VEGF-B186–treated endothelial cells significantly decreased the expression of Bik1, Bmf1, and Bad in cardiomyocytes (Figure 5C). These results thus indicate an indirect antiapoptotic effect of both VEGF-B isoforms both in vivo and in vitro, mediated through endothelial cells by unknown soluble factors.
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We furthermore assayed for AdVEGF-B186–mediated metabolic changes in the myocardium. Fatp4 upregulation was confirmed from the transduced pig myocardium by reverse-transcription polymerase chain reaction (Figure 5D) (C. Rosenlew and U. Eriksson, unpublished data, 2007). Higher accumulation of lipids (4-fold compared with AdLacZ) was also observed in cardiomyocytes after AdVEGF-B186 gene transfer (Figure 5E, e1 through 5e4, and 5F), whereas fewer lipid droplets were seen in AdVEGF-A– and AdPlGF-transduced hearts (Figure 5E, e2 and e3). Glycogen has been shown to be important for heart metabolism, especially under ischemic conditions, and its accumulation is cardioprotective in the hibernating myocardium.10 Periodic acid-Schiff staining showed a marked glycogen accumulation in cardiomyocytes after AdVEGF-B186 gene transfer (Figure 5F, f8), whereas smaller amounts of glycogen were observed after AdLacZ, AdVEGF-A, and AdPlGF gene transfers (Figure 5F, f5 through f7). Thus, AdVEGF-B186, but not transduction of the other VEGFs, led to changes in cardiac metabolism that likely further protected the myocardium after infarction.
On the basis of the findings in both normoxic and ischemic myocardium, we propose a model for VEGF-B function in the heart (Figure 6). Because membrane-bound Nrp-1 appears to be essential for the cardiac effects of AdVEGF-B186 (Figure 3E, 3F, 3G, and 3H), and Nrp-1 is activated in AdVEGF-B186–transduced tissues (Figure 4E), VEGF-B186 functions may be mediated at least in part via recruitment of GAIP to the cell membrane (Figure 4F, f12) and G-protein–mediated signaling.
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VEGF-B186 induced a myocardium-specific angiogenic effect that was qualitatively and mechanistically different from that induced by other VEGFR-1 ligands. In previous studies, we have shown that AdVEGF-B167 and AdVEGF-B186 do not have any angiogenic effects in periadventitial tissue around carotid arteries.17 In contrast to our results, VEGF-B has been shown to induce angiogenesis in mouse hindlimb ischemia.18 This discrepancy may be explained by the use of a small-animal model and differences in capillary growth mechanisms between small- and large-animal species. Interestingly, a recent publication by Li et al19 also suggests a restricted function for VEGF-B in mouse myocardium. It is interesting to note that VEGF-B–deficient mice appear healthy and fertile and display only minor heart phenotypes.20,21 PlGF-deficient mice are also healthy and fertile.22 Although neither growth factor is regulated by hypoxia, both VEGF-B and PlGF knockout mice are reported to have an impaired angiogenic response in ischemic tissues.20,22 However, the specificity of VEGF-B in the myocardium has not been reported previously.
In addition to angiogenesis, AdVEGF-B186 also induced collateral artery growth in the ischemic heart (Figure 2). Collateral artery growth may have been induced by either direct effects on smooth muscle cells (Figure 1G) or increased shear stress in collateral arteries induced by increased blood flow in the infarction edge.
Nrp-1 expression was strongly positive in the capillary endothelium in angiogenic areas and in the endothelium of arterioles and venules in the myocardium, but it was below the detection limit in the skeletal muscles. It is possible that Nrp-1 localizes the downstream effects of VEGF-B186 to the myocardium because of the differences in Nrp-1 expression in the myocardium and skeletal muscle (Figure 4A). Nrp-1 has been shown to be involved in embryonic angiogenesis.23 In contrast to its functions in the developing nervous system, intracellular signaling events have recently been shown to play a role in angiogenic functions of Nrp-1.9,24 Interestingly, AdVEGF-B186 also induced Nrp-1 expression in cardiomyocytes in the gene transfer area (Figure 4A).
In the present study, we showed an activation of the Nrp-1 signaling pathway after AdVEGF-B186 gene transfer in vivo. In contrast, the effects of AdPlGF were mediated primarily via upregulation of endogenous VEGF-A, which could be blocked by simultaneous transduction with AdsVEGFR-2 (Figure 3A). The angiogenic effects of AdVEGF-A and AdPlGF were also blocked by the nitric oxide synthase inhibitor L-NAME, whereas this treatment had no effect on the angiogenic effects of AdVEGF-B186. It is therefore likely that AdVEGF-B186 and AdPlGF induce different signaling events, and AdVEGF-B186–dependent recruitment of the Nrp-1 downstream target molecules GAIP and GIPC to myocardial cell membranes was detected only after AdVEGF-B186 administration (Figure 4a12, 4c12, and 4f12). On the basis of cell culture studies, the effects of AdVEGF-B186 were mediated via endothelial cells, and no direct effect on cultured cardiomyocytes could be detected. Interestingly, VEGF-B186 also has indirect metabolic effects on cardiomyocytes, increasing Fatp4 expression and lipid and glycogen accumulation in AdVEGF-B186 hearts, which may make cardiomyocytes more resistant to ischemia. Karpanen at al25 also recently published a report on the metabolic effects of VEGF-B. However, we cannot exclude other possibilities, such as the presence of a new VEGF receptor, a coreceptor, or another type of structure capable of preferentially binding VEGF-B186 in cardiac cells, nor can we rule out the possibility of secretion of an unidentified molecule from endothelial cells that mediates the effects of VEGF-B186 on cardiomyocytes. In addition to Nrp-1 activation, different diffusion kinetics of the transduced growth factors contribute to the different angiogenic profiles, because VEGF-B186 is known to be freely soluble and capable of diffusing from the site of transduction, whereas VEGF-B167 and VEGF-A bind tightly to heparan sulfate proteoglycans.
The exact mechanisms for the myocardial specificity of VEGF-B186 and the collateral artery formation in the AdVEGF-B186-treated hearts need to be clarified further; however, the increased proliferation and appearance of apoptosis-resistant cardiomyocytes around the infarction border zone in the AdVEGF-B186–treated hearts, together with increases in Fatp4 expression and lipid and glycogen accumulation, clearly suggest that VEGF-B186 may have favorable cardioprotective properties in addition to its unique heart specificity. We conclude that AdVEGF-B186 is a promising new therapeutic candidate for the treatment of patients with severe coronary heart disease.
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Acknowledgments |
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The authors would like to thank Seija Sahrio, Johanna Kilpijoki, Teemu Takalo, Tessa Heino, Atte Räty, Anssi Uutela, Maarit Kosonen, Essi Kattainen, Mikko Taina, and Valtteri Reinman for technical assistance; Tiina Koponen and Sari Järveläinen for virus production; and Heikki Pekonen and Heikki Karhunen from National Laboratory Animal Center for assistance in animal work. The authors also thank Dr William C. Claycomb for the generous gift of the HL-1 cells.
Sources of Funding
This study was supported by grants from the Dr Peter Wallenberg Foundation for Economy and Technology, the Finnish Academy, the Ludwig Institute for Cancer Research, the Sigrid Juselius Foundation, the Maud Kuistila Foundation, the Finnish Medical Foundation, the Ida Montini Foundation, the Swedish Research Council, the Novo Nordisk Foundation, the Swedish Cancer Foundation, the Karolinska Institute, and Zenyth Operations Pty Ltd.
Disclosures
None.
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The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.108.816454/DC1.
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