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

TY - JOUR

T1 - Enhanced collateral growth by double transplantation of gene-nucleofected fibroblasts in ischemic hindlimb of rats

AU - Zhang, Ziyang

AU - Slobodianski, Alex

AU - Ito, Wulf D

AU - Arnold, Astrid

AU - Nehlsen, Jessica

AU - Weng, Shaoxiang

AU - Lund, Natalie

AU - Liu, Jihong

AU - Egaña, José-Tomás

AU - Lohmeyer, Jörn A

AU - Müller, Daniel F

AU - Machens, Hans-Günther

PY - 2011/4/25

Y1 - 2011/4/25

N2 - 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.

AB - 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.

KW - Animals

KW - Cells, Cultured

KW - Collateral Circulation/genetics

KW - Fibroblast Growth Factor 2/genetics

KW - Fibroblasts/metabolism

KW - Hindlimb/pathology

KW - Ischemia/therapy

KW - Male

KW - Neovascularization, Physiologic/genetics

KW - Plasmids/genetics

KW - Rats

KW - Vascular Endothelial Growth Factor A/genetics

U2 - 10.1371/journal.pone.0019192

DO - 10.1371/journal.pone.0019192

M3 - SCORING: Journal article

C2 - 21547081

VL - 6

SP - e19192

JO - PLOS ONE

JF - PLOS ONE

SN - 1932-6203

IS - 4

ER -