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Enhanced collateral growth by double transplantation of gene-nucleofected fibroblasts in ischemic hindlimb of rats.

ABSTRACT: BACKGROUND: Induction of neovascularization by releasing therapeutic growth factors is a promising application of cell-based gene therapy to treat ischemia-related problems. In the present study, we have developed a new strategy based on nucleofection with alternative solution and cuvette to promote collateral growth and re-establishment of circulation in ischemic limbs using double transplantation of gene nucleofected primary cultures of fibroblasts, which were isolated from rat receiving such therapy. METHODS AND RESULTS: Rat dermal fibroblasts were nucleofected ex vivo to release bFGF or VEGF165 in a hindlimb ischemia model in vivo. After femoral artery ligation, gene-modified cells were injected intramuscularly. One week post injection, local confined plasmid expression and transient distributions of the plasmids in other organs were detected by quantitative PCR. Quantitative micro-CT analyses showed improvements of vascularization in the ischemic zone (No. of collateral vessels via micro CT: 6.8±2.3 vs. 10.1±2.6; p<0.05). Moreover, improved collateral proliferation (BrdU incorporation: 0.48±0.05 vs. 0.57±0.05; p<0.05) and increase in blood perfusion (microspheres ratio: gastrocnemius: 0.41±0.10 vs. 0.50±0.11; p<0.05; soleus ratio: soleus: 0.42±0.08 vs. 0.60±0.08; p<0.01) in the lower hindlimb were also observed. CONCLUSIONS: These results demonstrate the feasibility and effectiveness of double transplantation of gene nucleofected primary fibroblasts in producing growth factors and promoting the formation of collateral circulation in ischemic hindlimb, suggesting that isolation and preparation of gene nucleofected cells from individual accepting gene therapy may be an alternative strategy for treating limb ischemia related diseases.

SUBMITTER: Zhang Z

PROVIDER: S-EPMC3081850 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Enhanced collateral growth by double transplantation of gene-nucleofected fibroblasts in ischemic hindlimb of rats.

Zhang Ziyang Z Slobodianski Alex A Ito Wulf D WD Arnold Astrid A Nehlsen Jessica J Weng Shaoxiang S Lund Natalie N Liu Jihong J Egaña José-Tomás JT Lohmeyer Jörn A JA Müller Daniel F DF Machens Hans-Günther HG

PloS one 20110425 4

<h4>Background</h4>Induction of neovascularization by releasing therapeutic growth factors is a promising application of cell-based gene therapy to treat ischemia-related problems. In the present study, we have developed a new strategy based on nucleofection with alternative solution and cuvette to promote collateral growth and re-establishment of circulation in ischemic limbs using double transplantation of gene nucleofected primary cultures of fibroblasts, which were isolated from rat receivin ...[more]

PMID: 21547081

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Project description:While tissue perfusion and angiogenesis subsequent to acute femoral artery occlusion are suppressed in NADPH oxidase 2 (Nox2)-null (Nox2(-/-)) mice, studies have not established the role of Nox2 in collateral artery enlargement. Rac2 is a small GTPase that binds Nox2 and activates Nox2-based NAD(P)H oxidase but, unlike Nox2, is primarily restricted to bone marrow-derived cells. In this study, we used Rac2-null (Rac2(-/-)) and Nox2(-/-) mice with a novel method of identifying primary hindlimb collaterals to investigate the hypothesis that collateral growth requires these molecules. When initial experiments performed with femoral ligation demonstrated similar perfusion and collateral growth in Rac2(-/-) and wild-type C57BL/6J (BL6) mice, subsequent experiments were performed with a more severe ischemia model, femoral artery excision. After femoral excision, tissue perfusion was suppressed in Rac2(-/-) mice relative to BL6 mice. Histological assessment of ischemic injury including necrotic and regenerated muscle fibers and lipid and collagen deposition demonstrated greater injury in Rac2(-/-) mice. The diameters of primary collaterals identified during Microfil injection with intravital microscopy were enlarged to a similar extent in BL6 and Rac2(-/-) mice. Intimal cells in collateral cross sections were increased in number in both strains and were CD31 positive and CD45 negative. Circulating leukocytes and CD11b(+) cells were increased more in Rac2(-/-) than BL6 animals. Experiments performed in Nox2(-/-) mice to verify that the unexpected results related to collateral growth were not unique to Rac2(-/-) mice gave equivalent results. The data demonstrate that, subsequent to acute femoral artery excision, perfusion recovery is impaired in Rac2(-/-) and Nox2(-/-) mice but that collateral luminal expansion and intimal cell recruitment/proliferation are normal. These novel results indicate that collateral luminal expansion and intimal cell recruitment/proliferation are not mediated by Rac2 and Nox2.

Dataset Information

Enhanced collateral growth by double transplantation of gene-nucleofected fibroblasts in ischemic hindlimb of rats.

Publications

Enhanced collateral growth by double transplantation of gene-nucleofected fibroblasts in ischemic hindlimb of rats.

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