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Fibroblast growth factor 4 induces vascular permeability, angiogenesis and arteriogenesis in a rabbit hindlimb ischemia model ¹

TUOMAS T. RISSANEN^*, JOHANNA E. MARKKANEN^*, KATJA ARVE^*, JUHA RUTANEN^*, MIKKO I. KETTUNEN, ISMO VAJANTO^*,, SUVI JAUHIAINEN^*, LINDA CASHION, MARCIN GRUCHALA^*, OUTI NÄRVÄNEN^*, PEKKA TAIPALE^||, RISTO A. KAUPPINEN, GABOR M. RUBANYI and SEPPO YLÄ-HERTTUALA^*,,2

^* Department of Molecular Medicine,
Department of Biomedical NMR and National Bio-NMR Facility, A.I. Virtanen Institute, and
Department of Medicine, Kuopio University, Finland;
Department of Thoracic and Cardiovascular Surgery, Kuopio University Hospital, Kuopio, Finland;
Department of Gene Therapy, Berlex Biosciences, Richmond, California, USA; and
^|| Department of Gynecology and Obstetrics, and
Gene Therapy Unit, Kuopio University Hospital, Finland

²Correspondence: Department of Molecular Medicine, A. I. Virtanen Institute, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland. E-mail: Seppo.Ylaherttuala{at}uku.fi

SPECIFIC AIMS

Previous studies have shown that fibroblast growth factor-1 (FGF-1), FGF-2 and FGF-5 induce therapeutic angiogenesis. Here we investigated the potential of FGF-4 on therapeutic angiogenesis and arteriogenesis compared to vascular endothelial growth factor (VEGF) using adenoviral (Ad) gene transfer (GT) in a novel rabbit hind limb ischemia model with ischemia restricted to the calf.

PRINCIPAL FINDINGS

1. FGF-4 induces endothelial cell (EC) proliferation but not tube formation in vitro
Supernatant from AdFGF-4 transduced RaaSMCs failed to promote EC tube formation in Matrigel assay in contrast to the positive control AdVEGF supernatant, which efficiently induced EC tube formation. However, rhFGF-4 and AdFGF-4 supernatant caused a similar dose-dependent stimulation of EC proliferation.

2. Calf but not thigh ischemia in the modified hind limb ischemia model
Five weeks after resection of the superficial femoral artery and ligation of two reentry branches for collaterals, ischemic changes were found in calf muscles in 48% of animals (n=60), but in none of the thigh muscles. Contrast-enhanced MRI (Fig. 1 a, b) and similar muscle perfusion in operated and intact semimembranosus muscles of the control groups further support the findings that the thigh is nonischemic in this new model. However, the phosphocreatine/ATP ratio by ³¹P-MRS at rest from the gastrocnemius muscle of the calf was 2.78 ± 0.09 in intact muscle, 2.08 ± 0.15 7 days after the total resection of the whole femoral artery (P<0.001 vs. intact muscle) and 2.35 ± 0.12 7 days after the current modified model (P<0.05 vs. intact muscle and P=NS vs. total resection). Together, these findings demonstrate that in the modified rabbit hind limb ischemia model, the calf but not the thigh musculature is ischemic at rest.

View larger version (92K): [in this window] [in a new window]   Figure 1. Dose-dependent edema is detected in rabbit midthighs 5 days after AdVEGF and AdFGF-4 i.m. GT by GdDTPA-BMA-enhanced T2* weighted MRI. Transversal sections of treated limbs (left), and intact limbs (right). No vascular leakage or edema but only the scar of hind limb operation (arrow) is visible as bright GdDTPA-BMA contrast in a) PBS control, b) AdLacZ control, c) AdVEGF 1010 vp i.a., and d) AdVEGF 1011 vp i.a.-treated thighs. In contrast, in limbs transduced i.m. with e) AdVEGF 1010 vp, f) AdVEGF 1011 vp, g) AdFGF-4 1010 vp, and h) AdFGF-4 1011 vp GdDTPA-BMA has extravasated due to increased vascular permeability (arrowheads).

3. AdFGF-4 and AdVEGF cause macroscopic hind limb edema
MRI 5 days after GT showed that hind limbs administered intramuscular (i.m.) AdLacZ, PBS i.m., or AdVEGF intraarterial (i.a.) did not induce vascular permeability or edema (Fig. 1a-d ). In contrast, a dose-dependent vascular permeability effect and subsequent edema was detected in AdFGF-4 and AdVEGF i.m. transduced muscles (Fig. 1e-h ). Edema reached its maximum 5 to 6 days after GT and diminished thereafter in a couple of days. Transudates yielding bright GdDTPA-BMA contrast are seen under the skin, in semimembranosus muscle and muscle fascias, and in fat tissue between the medial and lateral muscle compartments. These edemic regions contained high amounts of VEGF both in AdVEGF- and AdFGF-4-treated limbs as shown by immunohistochemistry (see below).

4. AdFGF-4 and AdVEGF induce angiogenesis and collateral remodeling in vivo
Control gene transfer with AdLacZ (10¹¹vp i.m.) caused only a moderate inflammatory reaction in transduced muscles but not angiogenesis (Fig. 2 a, d). On the other hand, AdFGF-4 and AdVEGF given i.m. induced remarkable angiogenic effects as shown by histological sections 6 days after GT (Fig. 2b, c, e, f ). Although intense, these effects on capillaries were local and occurred only in the injection spread area. Capillary morphology was different in AdVEGF and AdFGF-4 transduced muscles. Consistent with in vitro findings, in vivo AdVEGF induced stronger tube formation and capillary enlargement effects than AdFGF-4 (Fig. 2b, c, e, f ).

View larger version (144K): [in this window] [in a new window]   Figure 2. Angiogenesis, capillary enlargement and collateral artery remodeling 6 days after AdVEGF and AdFGF-4 GT (1011 vp i.m.). a–f) CD31 immunostainings (DAB) of the semimembranosus muscles transduced with the indicated viruses. a, d) AdLacZ control. b, e) Strong capillary enlargement in the injection spread area after AdVEGF GT. c, f) Capillaries are not strongly enlarged but show significant growth after AdFGF-4 transduction. d–f) Inserts demonstrate EC proliferation in the enlarged capillaries [BrdU (DAB) + CD31 (Vector blue) double immunostaining]. g–i) BrdU (DAB) + CD31 (Vector blue) double immunostainings. g) Only a few proliferating cells in a collateral artery (arrowhead) in AdLacZ control. A high cell proliferation rate is detected around and in the wall of collateral arteries (arrowheads) in h) AdVEGF- and i) AdFGF-4-treated muscles. j–l) VEGF (DAB) + CD31 (Vector blue) double immunostainings. j) AdLacZ control. k) Abundant VEGF in the edemic muscle fascia after AdVEGF treatment. Enlarged capillaries and glomeruloid bodies (insert) are present adjacent to VEGF-rich areas. l) Up-regulation of endogenous VEGF in fascia after AdFGF-4 GT. m) Recombinant VEGF164 but not FGF-4 protein induces vascular permeability in the Miles assay in rabbit skin using the doses indicated and PBS as a control. Arrowheads denote the sites of injected protein. Bar = 1000 µm (a–c); 50 µm in the inserts of panels d–f; and 25 µm elsewhere.

BrdU labeling showed that capillary vessel growth 6 days after AdVEGF and AdFGF-4 GT involved strong EC proliferation (inserts in Fig. 2e, f ). Furthermore, cell proliferation rate was high in the wall of remodeling collateral arteries in AdVEGF- and AdFGF-4-treated muscles and involved non-ECs such as smooth muscle cells (SMCs) (Fig. 2h, i ). Glomeruloid bodies were found only in AdVEGF injected muscles in the vicinity of abundant amounts of transduced VEGF (Fig. 4k). AdFGF-4 did not cause this kind of extreme capillary growth although endogenous rabbit VEGF was up-regulated in AdFGF-4 transduced muscles (Fig. 4l). Although AdFGF-4 transduced limbs were edemic in MRI 6 days after GT, recombinant FGF-4 protein did not directly induce acute vascular permeability in the Miles assay in rabbit skin (Fig. 4m).

5. Capillary vessel enlargement, muscle perfusion, and vascular permeability effects of AdFGF-4 and AdVEGF
AdFGF-4 and AdVEGF increased capillary/myocyte ratio 6 days after GT. At the same time, a much more significant induction was observed in the capillary size as the mean capillary area increased 3.9- and 6.5-fold by AdFGF-4 and AdVEGF, respectively. There was also a strong increase in regional perfusion (4-fold) in both treatment groups compared with AdLacZ and PBS controls at 6 days. By 4 wk, the effects of AdFGF-4 and AdVEGF on angiogenesis and perfusion had returned to baseline. With the modified Miles assay, it was found that AdFGF-4 and AdVEGF caused 10- and 19-fold increases in extravasation of plasma proteins in transduced muscles 6 days after GT, respectively. AdLacZ did not increase vascular permeability over PBS control. Mouse VEGF₁₆₄ and human FGF-4 proteins were detected by ELISA in transduced muscles 6 days after i.m. GT but not in plasma at any time after i.m. or i.a. GT.

6. AdFGF-4 and AdVEGF induce arteriogenesis and increase collateral-dependent blood flow
In AdFGF-4 and AdVEGF i.m. transduced hind limbs collateral formation was more pronounced than in controls or AdVEGF i.a.-treated animals. Quantitatively, 10¹⁰ and 10¹¹ vp of AdVEGF i.m. caused 2.1- and 2.4-fold increases in collateral growth compared with PBS control, respectively. With AdFGF-4 the respective increases were 1.8- and 1.7-fold. Consistent with the angiography data, Doppler ultrasound measurements showed a significant increase in popliteal blood flow 7 and 21 days after AdVEGF i.m. GT and 14 days after AdFGF-4 i.m. GT.

CONCLUSIONS AND SIGNIFICANCE

We report here for the first time that AdFGF-4 i.m. GT induces vascular permeability and therapeutic angiogenesis and collateral artery growth comparable to AdVEGF. Our data suggest that some effects (such as vascular permeability) promoted by AdFGF-4 may be mediated by endogenous VEGF, which was up-regulated by FGF-4 overexpression. We further demonstrate that the i.a. route with adenoviral vector is ineffective for therapeutic vascular growth in rabbit skeletal muscle. Finally, contrast-enhanced MRI could be used as a noninvasive tool to detect changes in vascular permeability after GT with angiogenesis growth factors. The schematic illustration summarizes the vascular effects of FGF-4 overexpression in skeletal muscle (Fig. 3 ).

View larger version (13K): [in this window] [in a new window]   Figure 3. Schematic illustration of the proposed effects induced by FGF-4 gene transfer in skeletal muscle.

In the current modified rabbit hind limb ischemia model, the angiogenic effects of transduced genes are distinguished more reliably from those caused by endogenous growth factors up-regulated in response to ischemia. Our in vitro and in vivo experiments showed that compared with VEGF, FGF-4 potently induced EC proliferation but not tube formation. In contrast to previous studies using naked DNA-based GT methods, in vivo AdVEGF mainly enlarged the preexisting capillaries leading to the formation of "mother" vessels whereas the capillary density was only moderately increased. Vessels generated by AdVEGF and AdFGF-4 were leaky, indicating a role for VEGF, the most potent vascular permeability factor, in AdFGF-4 induced angiogenesis. Despite the regression of excess capillaries and perfusion by 4 wk, significant increases in collateral growth persisted after AdFGF-4 and AdVEGF GT. Collateral arteries supply the ischemic regions of the leg and thus are necessary and prevail. VEGF and FGF-4 probably induced growth of muscular vessels through production of nitric oxide and by creating a stimulating extracellular matrix environment via an increase in plasma protein extravasation, up-regulation of proteases, and additional growth factors. Thus, in normoperfused thigh muscles, transient adenoviral expression of FGF-4 and VEGF seems to be beneficial for the stimulation of a persistent arteriogenesis effect but not an angiogenesis effect.

We conclude that adenoviral FGF-4 GT is a potential new treatment for lower limb ischemia with beneficial effects on angiogenesis and arteriogenesis.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0377fje; to cite this article, use FASEB J. (November 15, 2002) 10.1096/fj.02-0377fje

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