HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) Erratum All Versions of this Article: 19/10/1365 05-3720fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Pajusola, K. Articles by Alitalo, K. Search for Related Content PubMed PubMed Citation Articles by Pajusola, K. Articles by Alitalo, K.

Stabilized HIF-1 is superior to VEGF for angiogenesis in skeletal muscle via adeno-associated virus gene transfer

Katri Pajusola^*, Jaana Künnapuu^*,1, Sanna Vuorikoski^*,1, Jarkko Soronen^*,1, Helder André, Teresa Pereira, Petra Korpisalo, Seppo Ylä-Herttuala, Lorenz Poellinger and Kari Alitalo^*,2

^* Molecular/Cancer Biology Laboratory and Ludwig Institute for Cancer Research, Biomedicum Helsinki, University of Helsinki, Finland;
A.I. Virtanen Institute and Department of Medicine, University of Kuopio, Kuopio, Finland; and
Karolinska Institutet, Department of Cell and Molecular Biology, Stockholm, Sweden

²Correspondence: Molecular/Cancer Biology Laboratory, Biomedicum Helsinki, P.O.B. 63 (Haartmaninkatu 8), 00014 University of Helsinki, Finland. E-mail: kari.alitalo{at}helsinki.fi

SPECIFIC AIMS

Cardiovascular diseases comprise the largest health problem in the western world. Angiogenic gene therapy aims at solving the problems arising from reduced oxygen supply in the affected tissues by introducing new vasculature via overexpression of angiogenic growth factors. Vascular endothelial growth factor (VEGF) is one of the most potent angiogenic growth factors and has been the prime target for development of proangiogenic therapy. In this study, we wanted to compare the vascular phenotypes resulting from either direct overexpression of VEGF or overexpression of a stabilized form of hypoxia-inducible factor 1- (mHIF-1). HIF-1 is known to induce expression of several growth factors required for the angiogenic response, including VEGF. As the gene transfer vehicle, we used adeno-associated virus (AAV), because of its optimal transduction profile for muscle-directed gene therapy and lack of inflammatory responses.

In addition to growth factors for endothelial cells, synthesis of functional neovasculature requires factors that influence the cells of the outer vascular wall. These arteriogenic growth factors include angiopoietin-1 (Ang-1) and platelet-derived growth factor-B (PDGF-B), which have been shown to be important for vessel maturation and stability. Therefore, we tested Ang-1 and PDGF-B for their contribution to the observed angiogenic responses.

Finally, the different growth factor combinations were assayed for their ability to functionally improve blood circulation in mouse skeletal muscle. For the functional performance, we measured two parameters, vascular permeability and general perfusion in the AAV transduced muscles.

PRINCIPAL FINDINGS

1. mHIF-1 induced significant capillary proliferation and sprouting, whereas overexpression of VEGF led to increased numbers of endothelial cells without their proper arrangement into new capillary structures (Fig. 1 ). Furthermore, our experiments demonstrated species-specific effects of VEGF as expression of human VEGF in mice resulted in a significantly milder endothelial cell phenotype than its murine counterpart.

View larger version (65K): [in this window] [in a new window]   Figure 1. Angiogenic response analyzed by immunohistochemistry of mouse tibialis anterior TA muscles transduced with rAAVs expressing growth factor or control protein. A–D) Coexpression of PECAM-1 (green) and NG2 (red). Insets: Immunohistochemistry of the common leukocyte antigen CD45-positive leukocytes recruited to the site of expression. 20x original magnification. E) Intensity of the combined PECAM-1 and NG2 signals was analyzed with the IMAGE J program. The fold increase in the signal intensities of the test situation in B-D was compared with the HSA control and presented as black bars. F–H) Analysis of the endothelial cell sprouting and proliferation status in the transduced TA muscles. Staining for PECAM-1 (green) and PHH3 (red). Note that mHIF-1 overexpression is associated with capillary sprouting (arrows, G), while VEGF120 stimulates just proliferation of endothelial cells (arrowheads, H). HSA-rAAV control; note that the PHH3-positive cells are not stained for PECAM-1; they are likely proliferating leukocytes (F). 63x original magnification. Scale bars 100 µm (A–D), 20 µm (F–H).

2. The function of the synthesized de novo capillaries was tested for permeability and vascular perfusion. Evans Blue permeability assays showed that, as expected, vessel permeability was strongly increased in the VEGF overexpressing muscles. In contrast, the neo-vessels in mHIF-1 expressing muscles were not leaky and demonstrated permeability corresponding to that of the control muscles (Fig. 2 ). Moreover, vascular perfusion, when measured with Doppler ultrasound imaging and compared with the controls, was significantly improved in the mHIF-1-expressing muscles. Both assays demonstrated, however, that the tested arteriogenic growth factors Ang-1 and PDGF-B did not further improve the performance of mHIF-1 in the corresponding coexpression experiments.

View larger version (31K): [in this window] [in a new window]   Figure 2. The integrity of the synthesized neo-capillaries upon rAAV transduction was studied by using the Evans Blue permeability assay. Columns represent the Evans Blue content measured at A620 nm after formamide extraction of the transduced mouse TA muscles indicated. Error bars represent the standard deviation of several tested samples. Insets: examples of TA muscles after Evans Blue injection. HSA (A), VEGF-A120+Ang-1 (B), VEGF-A164 (C), mHIF-1 (D), mHIF-1+Ang-1 (E), mHIF-1+PDGF-B (F).

CONCLUSIONS AND SIGNIFICANCE

VEGF has, in numerous studies over 15 years, been shown to be one of the most important growth factors for angiogenesis. Yet in many of the VEGF gene therapy trials, an unsatisfactory outcome has been encountered. Proangiogenic gene therapy involves direct application of therapeutic genes into the target tissue. This results in a high local concentration of the gene products, which may lead to inappropriate function of the overexpressed proteins. In our experimental setup, we employed viral gene transfer of mHIF-1 using recombinant AAV transduction. This approach allowed us to circumvent the aforementioned problem because HIF-1 is known to induce the required angiogenic growth factors endogenously. Indeed, direct VEGF gene transfer led to unwanted and disorganized overproliferation of endothelial cells. In conclusion, our study provides important new results on VEGF-induced angiogenesis, which are highly relevant for development of improved gene therapy applications for cardiovascular diseases. The top priorities for our future studies will therefore include a closer characterization of the effects of HIF-1 on ischemic muscles in mice and larger animals.

View larger version (33K): [in this window] [in a new window]   Figure 3. Schematic diagram that depicts the main angiogenic responses induced by direct VEGF overexpression or overexpression of mHIF-1. A) Top: recombinant AAV-VEGF is injected into mouse TA muscle. Middle: cross section of the muscle. Arrows indicate the capillaries in a resting TA. AAV-VEGF transduction induces synthesis of VEGF gradients. Bottom: endothelial cells (arrowhead) of the muscle vessels proliferate strongly in response to rAAV-transduced VEGF. B) Top: AAV-mHIF-1 is injected into mouse TA muscle. Middle: AAV-mHIF-1 transduction is likely to induce endogenous expression of several angiogenic growth factors, including VEGF, PDGF-B and angiopoietin (Ang). Bottom: the resting endothelial cells start to sprout and proliferate, which leads to increased numbers of new capillaries (arrowheads). EC: endothelial cell.

FOOTNOTES

¹ These authors contributed equally to this work.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-3720fje;

This article has been cited by other articles:

				P. Korpisalo, T. T. Rissanen, T. Bengtsson, T. Liimatainen, S. Laidinen, H. Karvinen, J. E. Markkanen, O. H. Grohn, and S. Yla-Herttuala Therapeutic angiogenesis with placental growth factor improves exercise tolerance of ischaemic rabbit hindlimbs Cardiovasc Res, August 5, 2008; (2008) cvn195v2. [Abstract] [Full Text] [PDF]

				M. Bosch-Marce, H. Okuyama, J. B. Wesley, K. Sarkar, H. Kimura, Y. V. Liu, H. Zhang, M. Strazza, S. Rey, L. Savino, et al. Effects of Aging and Hypoxia-Inducible Factor-1 Activity on Angiogenic Cell Mobilization and Recovery of Perfusion After Limb Ischemia Circ. Res., December 7, 2007; 101(12): 1310 - 1318. [Abstract] [Full Text] [PDF]

				G. L. Semenza Regulation of tissue perfusion in mammals by hypoxia-inducible factor 1 Exp Physiol, November 1, 2007; 92(6): 988 - 991. [Abstract] [Full Text] [PDF]

				N. Dehne, U. Kerkweg, T. Otto, and J. Fandrey The HIF-1 response to simulated ischemia in mouse skeletal muscle cells neither enhances glycolysis nor prevents myotube cell death Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2007; 293(4): R1693 - R1701. [Abstract] [Full Text] [PDF]

				K. Takeda, A. Cowan, and G.-H. Fong Essential Role for Prolyl Hydroxylase Domain Protein 2 in Oxygen Homeostasis of the Adult Vascular System Circulation, August 14, 2007; 116(7): 774 - 781. [Abstract] [Full Text] [PDF]

				S. Rajagopalan, J. Olin, S. Deitcher, A. Pieczek, J. Laird, P. M. Grossman, C. K. Goldman, K. McEllin, R. Kelly, and N. Chronos Use of a Constitutively Active Hypoxia-Inducible Factor-1{alpha} Transgene as a Therapeutic Strategy in No-Option Critical Limb Ischemia Patients: Phase I Dose-Escalation Experience Circulation, March 13, 2007; 115(10): 1234 - 1243. [Abstract] [Full Text] [PDF]

				Y. Luo, C. Jiang, A. J. Belanger, G. Y. Akita, S. C. Wadsworth, R. J. Gregory, and K. A. Vincent A Constitutively Active Hypoxia-Inducible Factor-1{alpha}/VP16 Hybrid Factor Activates Expression of the Human B-Type Natriuretic Peptide Gene Mol. Pharmacol., June 1, 2006; 69(6): 1953 - 1962. [Abstract] [Full Text] [PDF]

				M. E. Wilhide and W. K. Jones Potential Therapeutic Gene for the Treatment of Ischemic Disease: Ad2/Hypoxia-Inducible Factor-1{alpha} (HIF-1)/VP16 Enhances B-Type Natriuretic Peptide Gene Expression via a HIF-1-Responsive Element Mol. Pharmacol., June 1, 2006; 69(6): 1773 - 1778. [Abstract] [Full Text] [PDF]

				D. Trentin, H. Hall, S. Wechsler, and J. A. Hubbell Tissue Engineering Special Feature: Peptide-matrix-mediated gene transfer of an oxygen-insensitive hypoxia-inducible factor-1{alpha} variant for local induction of angiogenesis PNAS, February 21, 2006; 103(8): 2506 - 2511. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) Erratum All Versions of this Article: 19/10/1365 05-3720fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Pajusola, K. Articles by Alitalo, K. Search for Related Content PubMed PubMed Citation Articles by Pajusola, K. Articles by Alitalo, K.