Skip to main content
NCBI home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1996 Dec 15;98(12):2894–2902. doi: 10.1172/JCI119118

Particle-mediated gene transfer with transforming growth factor-beta1 cDNAs enhances wound repair in rat skin.

S I Benn 1, J S Whitsitt 1, K N Broadley 1, L B Nanney 1, D Perkins 1, L He 1, M Patel 1, J R Morgan 1, W F Swain 1, J M Davidson 1
PMCID: PMC507757  PMID: 8981938

Abstract

Based on preliminary but variable results with direct DNA transfer into wounds, we evaluated in vivo gene transfer by particle-mediated DNA delivery to rat skin to determine whether overexpression of TGF-beta1 at the site of skin incisions would result in a significant improvement in repair. Optimization of the method with viral promoter-luciferase reporter constructs indicated that expression of luciferase activity persisted up to 5 d and was promoter, pressure, and site dependent (ventral > dorsal). Using cytomegalovirus (CMV)-driven human alpha1-antitrypsin, transgene expression was immunolocalized within keratinocytes of the stratum granulosum at 24 h. We measured tensile strength of skin incisions at 11-21 d in both normal and diabetic rats transfected with TGF-beta1 expression vectors at surgery. Native murine TGF-beta1 under an SV40 promoter produced positive effects, while wound strengthening was more pronounced in diabetic animals using a CMV-driven construct. Transfection of rat skin with constitutively active, mutant porcine TGF-beta1 under the control of the CMV and Moloney murine leukemia virus promoters significantly increased tensile strength up to 80% for 14-21 d after surgery. Transfection 24 h before surgery was more effective. Particle-mediated gene delivery can be used to deliver viral promoter-cytokine expression constructs into rat skin in a safe, efficient, and reproducible fashion. The extent of wound repair, as evidenced by enhanced tensile strength, can be markedly improved in tissues transfected with TGF-beta1 expression constructs.

Full Text

The Full Text of this article is available as a PDF (315.1 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Andree C., Swain W. F., Page C. P., Macklin M. D., Slama J., Hatzis D., Eriksson E. In vivo transfer and expression of a human epidermal growth factor gene accelerates wound repair. Proc Natl Acad Sci U S A. 1994 Dec 6;91(25):12188–12192. doi: 10.1073/pnas.91.25.12188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Attisano L., Wrana J. L., López-Casillas F., Massagué J. TGF-beta receptors and actions. Biochim Biophys Acta. 1994 May 26;1222(1):71–80. doi: 10.1016/0167-4889(94)90026-4. [DOI] [PubMed] [Google Scholar]
  3. Bandara G., Mueller G. M., Galea-Lauri J., Tindal M. H., Georgescu H. I., Suchanek M. K., Hung G. L., Glorioso J. C., Robbins P. D., Evans C. H. Intraarticular expression of biologically active interleukin 1-receptor-antagonist protein by ex vivo gene transfer. Proc Natl Acad Sci U S A. 1993 Nov 15;90(22):10764–10768. doi: 10.1073/pnas.90.22.10764. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bernad A., Varas F., Gallego J. M., Almendral J. M., Bueren J. A. Ex vivo expansion and selection of retrovirally transduced bone marrow: an efficient methodology for gene-transfer to murine lympho-haemopoietic stem cells. Br J Haematol. 1994 May;87(1):6–17. doi: 10.1111/j.1365-2141.1994.tb04863.x. [DOI] [PubMed] [Google Scholar]
  5. Border W. A., Noble N. A. Transforming growth factor beta in tissue fibrosis. N Engl J Med. 1994 Nov 10;331(19):1286–1292. doi: 10.1056/NEJM199411103311907. [DOI] [PubMed] [Google Scholar]
  6. Bradham D. M., Igarashi A., Potter R. L., Grotendorst G. R. Connective tissue growth factor: a cysteine-rich mitogen secreted by human vascular endothelial cells is related to the SRC-induced immediate early gene product CEF-10. J Cell Biol. 1991 Sep;114(6):1285–1294. doi: 10.1083/jcb.114.6.1285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Branchereau S., Calise D., Ferry N. Factors influencing retroviral-mediated gene transfer into hepatocytes in vivo. Hum Gene Ther. 1994 Jul;5(7):803–808. doi: 10.1089/hum.1994.5.7-803. [DOI] [PubMed] [Google Scholar]
  8. Broadley K. N., Aquino A. M., Hicks B., Ditesheim J. A., McGee G. S., Demetriou A. A., Woodward S. C., Davidson J. M. The diabetic rat as an impaired wound healing model: stimulatory effects of transforming growth factor-beta and basic fibroblast growth factor. Biotechnol Ther. 1989 1990;1(1):55–68. [PubMed] [Google Scholar]
  9. Broadley K. N., Aquino A. M., Hicks B., Ditesheim J. A., McGee G. S., Demetriou A. A., Woodward S. C., Davidson J. M. The diabetic rat as an impaired wound healing model: stimulatory effects of transforming growth factor-beta and basic fibroblast growth factor. Biotechnol Ther. 1989 1990;1(1):55–68. [PubMed] [Google Scholar]
  10. Buckley A., Davidson J. M., Kamerath C. D., Wolt T. B., Woodward S. C. Sustained release of epidermal growth factor accelerates wound repair. Proc Natl Acad Sci U S A. 1985 Nov;82(21):7340–7344. doi: 10.1073/pnas.82.21.7340. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Buckley A., Davidson J. M., Kamerath C. D., Woodward S. C. Epidermal growth factor increases granulation tissue formation dose dependently. J Surg Res. 1987 Oct;43(4):322–328. doi: 10.1016/0022-4804(87)90088-6. [DOI] [PubMed] [Google Scholar]
  12. Chapman G. D., Lim C. S., Gammon R. S., Culp S. C., Desper J. S., Bauman R. P., Swain J. L., Stack R. S. Gene transfer into coronary arteries of intact animals with a percutaneous balloon catheter. Circ Res. 1992 Jul;71(1):27–33. doi: 10.1161/01.res.71.1.27. [DOI] [PubMed] [Google Scholar]
  13. Cheng L., Ziegelhoffer P. R., Yang N. S. In vivo promoter activity and transgene expression in mammalian somatic tissues evaluated by using particle bombardment. Proc Natl Acad Sci U S A. 1993 May 15;90(10):4455–4459. doi: 10.1073/pnas.90.10.4455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  15. Davis H. L., Demeneix B. A., Quantin B., Coulombe J., Whalen R. G. Plasmid DNA is superior to viral vectors for direct gene transfer into adult mouse skeletal muscle. Hum Gene Ther. 1993 Dec;4(6):733–740. doi: 10.1089/hum.1993.4.6-733. [DOI] [PubMed] [Google Scholar]
  16. Davis H. L., Whalen R. G., Demeneix B. A. Direct gene transfer into skeletal muscle in vivo: factors affecting efficiency of transfer and stability of expression. Hum Gene Ther. 1993 Apr;4(2):151–159. doi: 10.1089/hum.1993.4.2-151. [DOI] [PubMed] [Google Scholar]
  17. Fisher L. J., Ray J. In vivo and ex vivo gene transfer to the brain. Curr Opin Neurobiol. 1994 Oct;4(5):735–741. doi: 10.1016/0959-4388(94)90017-5. [DOI] [PubMed] [Google Scholar]
  18. Flaumenhaft R., Kojima S., Abe M., Rifkin D. B. Activation of latent transforming growth factor beta. Adv Pharmacol. 1993;24:51–76. doi: 10.1016/s1054-3589(08)60933-3. [DOI] [PubMed] [Google Scholar]
  19. Flotte T. R., Afione S. A., Conrad C., McGrath S. A., Solow R., Oka H., Zeitlin P. L., Guggino W. B., Carter B. J. Stable in vivo expression of the cystic fibrosis transmembrane conductance regulator with an adeno-associated virus vector. Proc Natl Acad Sci U S A. 1993 Nov 15;90(22):10613–10617. doi: 10.1073/pnas.90.22.10613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Geary R. L., Clowes A. W., Lau S., Vergel S., Dale D. C., Osborne W. R. Gene transfer in baboons using prosthetic vascular grafts seeded with retrovirally transduced smooth muscle cells: a model for local and systemic gene therapy. Hum Gene Ther. 1994 Oct;5(10):1211–1216. doi: 10.1089/hum.1994.5.10-1211. [DOI] [PubMed] [Google Scholar]
  21. Giro M. G., Davidson J. M. Elastin. Methods Enzymol. 1988;163:656–673. [PubMed] [Google Scholar]
  22. Giro M. G., Hill K. E., Sandberg L. B., Davidson J. M. Quantitation of elastin production in cultured vascular smooth muscle cells by a sensitive and specific enzyme-linked immunoassay. Coll Relat Res. 1984 Jan;4(1):21–34. doi: 10.1016/s0174-173x(84)80026-6. [DOI] [PubMed] [Google Scholar]
  23. Hinegardner R. T. An improved fluorometric assay for DNA. Anal Biochem. 1971 Jan;39(1):197–201. doi: 10.1016/0003-2697(71)90476-3. [DOI] [PubMed] [Google Scholar]
  24. Kanzaki T., Olofsson A., Morén A., Wernstedt C., Hellman U., Miyazono K., Claesson-Welsh L., Heldin C. H. TGF-beta 1 binding protein: a component of the large latent complex of TGF-beta 1 with multiple repeat sequences. Cell. 1990 Jun 15;61(6):1051–1061. doi: 10.1016/0092-8674(90)90069-q. [DOI] [PubMed] [Google Scholar]
  25. Kim S. J., Jeang K. T., Glick A. B., Sporn M. B., Roberts A. B. Promoter sequences of the human transforming growth factor-beta 1 gene responsive to transforming growth factor-beta 1 autoinduction. J Biol Chem. 1989 Apr 25;264(12):7041–7045. [PubMed] [Google Scholar]
  26. Kondaiah P., Van Obberghen-Schilling E., Ludwig R. L., Dhar R., Sporn M. B., Roberts A. B. cDNA cloning of porcine transforming growth factor-beta 1 mRNAs. Evidence for alternate splicing and polyadenylation. J Biol Chem. 1988 Dec 5;263(34):18313–18317. [PubMed] [Google Scholar]
  27. Kulkarni A. B., Huh C. G., Becker D., Geiser A., Lyght M., Flanders K. C., Roberts A. B., Sporn M. B., Ward J. M., Karlsson S. Transforming growth factor beta 1 null mutation in mice causes excessive inflammatory response and early death. Proc Natl Acad Sci U S A. 1993 Jan 15;90(2):770–774. doi: 10.1073/pnas.90.2.770. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Le Gal La Salle G., Robert J. J., Berrard S., Ridoux V., Stratford-Perricaudet L. D., Perricaudet M., Mallet J. An adenovirus vector for gene transfer into neurons and glia in the brain. Science. 1993 Feb 12;259(5097):988–990. doi: 10.1126/science.8382374. [DOI] [PubMed] [Google Scholar]
  29. Levine J. H., Moses H. L., Gold L. I., Nanney L. B. Spatial and temporal patterns of immunoreactive transforming growth factor beta 1, beta 2, and beta 3 during excisional wound repair. Am J Pathol. 1993 Aug;143(2):368–380. [PMC free article] [PubMed] [Google Scholar]
  30. Mansford K. R., Opie L. Comparison of metabolic abnormalities in diabetes mellitus induced by streptozotocin or by alloxan. Lancet. 1968 Mar 30;1(7544):670–671. doi: 10.1016/s0140-6736(68)92103-x. [DOI] [PubMed] [Google Scholar]
  31. Massagué J., Andres J., Attisano L., Cheifetz S., López-Casillas F., Ohtsuki M., Wrana J. L. TGF-beta receptors. Mol Reprod Dev. 1992 Jun;32(2):99–104. doi: 10.1002/mrd.1080320204. [DOI] [PubMed] [Google Scholar]
  32. McGee G. S., Broadley K. N., Buckley A., Aquino A., Woodward S. C., Demetriou A. A., Davidson J. M. Recombinant transforming growth factor beta accelerates incisional wound healing. Curr Surg. 1989 Mar-Apr;46(2):103–106. [PubMed] [Google Scholar]
  33. Morén A., Olofsson A., Stenman G., Sahlin P., Kanzaki T., Claesson-Welsh L., ten Dijke P., Miyazono K., Heldin C. H. Identification and characterization of LTBP-2, a novel latent transforming growth factor-beta-binding protein. J Biol Chem. 1994 Dec 23;269(51):32469–32478. [PubMed] [Google Scholar]
  34. Mustoe T. A., Pierce G. F., Thomason A., Gramates P., Sporn M. B., Deuel T. F. Accelerated healing of incisional wounds in rats induced by transforming growth factor-beta. Science. 1987 Sep 11;237(4820):1333–1336. doi: 10.1126/science.2442813. [DOI] [PubMed] [Google Scholar]
  35. Nabel E. G., Plautz G., Nabel G. J. Site-specific gene expression in vivo by direct gene transfer into the arterial wall. Science. 1990 Sep 14;249(4974):1285–1288. doi: 10.1126/science.2119055. [DOI] [PubMed] [Google Scholar]
  36. Nabel E. G., Shum L., Pompili V. J., Yang Z. Y., San H., Shu H. B., Liptay S., Gold L., Gordon D., Derynck R. Direct transfer of transforming growth factor beta 1 gene into arteries stimulates fibrocellular hyperplasia. Proc Natl Acad Sci U S A. 1993 Nov 15;90(22):10759–10763. doi: 10.1073/pnas.90.22.10759. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. O'Malley B. W., Jr, Adams R. M., Sikes M. L., Ledley F. D. Gene transfer into human thyroid follicular cells. Laryngoscope. 1994 Nov;104(11 Pt 1):1330–1336. doi: 10.1288/00005537-199411000-00004. [DOI] [PubMed] [Google Scholar]
  38. Parker B. A., Stark G. R. Regulation of simian virus 40 transcription: sensitive analysis of the RNA species present early in infections by virus or viral DNA. J Virol. 1979 Aug;31(2):360–369. doi: 10.1128/jvi.31.2.360-369.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Quaglino D., Jr, Nanney L. B., Kennedy R., Davidson J. M. Transforming growth factor-beta stimulates wound healing and modulates extracellular matrix gene expression in pig skin. I. Excisional wound model. Lab Invest. 1990 Sep;63(3):307–319. [PubMed] [Google Scholar]
  40. Roberts A. B., Sporn M. B., Assoian R. K., Smith J. M., Roche N. S., Wakefield L. M., Heine U. I., Liotta L. A., Falanga V., Kehrl J. H. Transforming growth factor type beta: rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4167–4171. doi: 10.1073/pnas.83.12.4167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Samuel S. K., Hurta R. A., Kondaiah P., Khalil N., Turley E. A., Wright J. A., Greenberg A. H. Autocrine induction of tumor protease production and invasion by a metallothionein-regulated TGF-beta 1 (Ser223, 225). EMBO J. 1992 Apr;11(4):1599–1605. doi: 10.1002/j.1460-2075.1992.tb05205.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Sun W. H., Burkholder J. K., Sun J., Culp J., Turner J., Lu X. G., Pugh T. D., Ershler W. B., Yang N. S. In vivo cytokine gene transfer by gene gun reduces tumor growth in mice. Proc Natl Acad Sci U S A. 1995 Mar 28;92(7):2889–2893. doi: 10.1073/pnas.92.7.2889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Taipale J., Miyazono K., Heldin C. H., Keski-Oja J. Latent transforming growth factor-beta 1 associates to fibroblast extracellular matrix via latent TGF-beta binding protein. J Cell Biol. 1994 Jan;124(1-2):171–181. doi: 10.1083/jcb.124.1.171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Torre-Amione G., Beauchamp R. D., Koeppen H., Park B. H., Schreiber H., Moses H. L., Rowley D. A. A highly immunogenic tumor transfected with a murine transforming growth factor type beta 1 cDNA escapes immune surveillance. Proc Natl Acad Sci U S A. 1990 Feb;87(4):1486–1490. doi: 10.1073/pnas.87.4.1486. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Ulmer J. B., Donnelly J. J., Parker S. E., Rhodes G. H., Felgner P. L., Dwarki V. J., Gromkowski S. H., Deck R. R., DeWitt C. M., Friedman A. Heterologous protection against influenza by injection of DNA encoding a viral protein. Science. 1993 Mar 19;259(5102):1745–1749. doi: 10.1126/science.8456302. [DOI] [PubMed] [Google Scholar]
  46. Vogt P. M., Thompson S., Andree C., Liu P., Breuing K., Hatzis D., Brown H., Mulligan R. C., Eriksson E. Genetically modified keratinocytes transplanted to wounds reconstitute the epidermis. Proc Natl Acad Sci U S A. 1994 Sep 27;91(20):9307–9311. doi: 10.1073/pnas.91.20.9307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Walsh C. E., Nienhuis A. W., Samulski R. J., Brown M. G., Miller J. L., Young N. S., Liu J. M. Phenotypic correction of Fanconi anemia in human hematopoietic cells with a recombinant adeno-associated virus vector. J Clin Invest. 1994 Oct;94(4):1440–1448. doi: 10.1172/JCI117481. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Wolff J. A., Malone R. W., Williams P., Chong W., Acsadi G., Jani A., Felgner P. L. Direct gene transfer into mouse muscle in vivo. Science. 1990 Mar 23;247(4949 Pt 1):1465–1468. doi: 10.1126/science.1690918. [DOI] [PubMed] [Google Scholar]
  49. Wolff J. A., Williams P., Acsadi G., Jiao S., Jani A., Chong W. Conditions affecting direct gene transfer into rodent muscle in vivo. Biotechniques. 1991 Oct;11(4):474–485. [PubMed] [Google Scholar]
  50. Yang N. S., Burkholder J., Roberts B., Martinell B., McCabe D. In vivo and in vitro gene transfer to mammalian somatic cells by particle bombardment. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9568–9572. doi: 10.1073/pnas.87.24.9568. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES