Transforming Growth Factor-β1 Fails to Stimulate Wound Healing and Impairs Its Signal Transduction in an Aged Ischemic Ulcer Model
1999; Elsevier BV; Volume: 154; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)65276-5
ISSN1525-2191
AutoresLiancun Wu, Yuping Xia, Sanford I. Roth, Elliott Gruskin, Thomas A. Mustoe,
Tópico(s)Planarian Biology and Electrostimulation
ResumoClinical trials of exogenous growth factors in treating chronic wounds have been less successful than expected. One possible explanation is that most studies used animal models of acute wounds in young animals, whereas most chronic wounds occur in elderly patients with tissue ischemia. We described an animal model of age- and ischemia-impaired wound healing and analyzed the wound-healing response as well as the transforming growth factor (TGF)-β1 effect in this model. Rabbits of increasing ages were made ischemic in the ear where dermal ulcers were created. Histological analysis showed that epithelium ingrowth and granulation tissue deposition were significantly impaired with increased age under ischemia. TGF-β1 stimulated wound repair under both ischemic and non-ischemic conditions in young animals, although it showed no statistical difference in aged animals. Procollagen mRNA expression decreased under ischemic conditions and with aging. Neither TGF-β1 nor procollagen α1(I) mRNA expression increased in response to TGF-β1 treatment under ischemia in aged animals. Therefore, the wound-healing process is impaired additively by aging and ischemia. The lack of a wound-healing response to TGF-β1 in aged ischemic wounds may play a role in the chronic wounds. Clinical trials of exogenous growth factors in treating chronic wounds have been less successful than expected. One possible explanation is that most studies used animal models of acute wounds in young animals, whereas most chronic wounds occur in elderly patients with tissue ischemia. We described an animal model of age- and ischemia-impaired wound healing and analyzed the wound-healing response as well as the transforming growth factor (TGF)-β1 effect in this model. Rabbits of increasing ages were made ischemic in the ear where dermal ulcers were created. Histological analysis showed that epithelium ingrowth and granulation tissue deposition were significantly impaired with increased age under ischemia. TGF-β1 stimulated wound repair under both ischemic and non-ischemic conditions in young animals, although it showed no statistical difference in aged animals. Procollagen mRNA expression decreased under ischemic conditions and with aging. Neither TGF-β1 nor procollagen α1(I) mRNA expression increased in response to TGF-β1 treatment under ischemia in aged animals. Therefore, the wound-healing process is impaired additively by aging and ischemia. The lack of a wound-healing response to TGF-β1 in aged ischemic wounds may play a role in the chronic wounds. Many growth factors tested in animal models appear to be promising therapeutic agents promoting wound healing.1Pierce GF Mustoe TA Pharmacologic enhancement of wound healing.Annu Rev Med. 1995; 46: 467-481Crossref PubMed Scopus (186) Google Scholar However, clinical trials of growth factors on treating chronic wounds have been less encouraging.2Mustoe TA Cutler NR Allman RM Goode PS Deuel TF Prause J Bear M Serdar CM Pierce GF A phase II study to evaluate recombinant platelet-derived growth factor-BB in the treatment of stage 3 and 4 pressure ulcers.Arch Surg. 1994; 129: 213-219Crossref PubMed Scopus (160) Google Scholar, 3Richard JL Parer-Richard C Daures JP Clouet S Vannereau D Bringer J Rodier M Jacob C Comte-Bardonnet M Effect of topical basic fibroblast growth factor on the healing of chronic diabetic neuropathic ulcer of the foot: a pilot, randomized, double-blind, placebo-controlled study.Diabetes Care. 1995; 18: 64-69Crossref PubMed Scopus (187) Google Scholar We reasoned that preclinical studies using young animals may not be suitable for predicting growth factor effect on human chronic wounds, which are primarily a problem of aged patients who have local tissue hypoxia. Previously, most preclinical studies have been done on acute wounds in healthy young animals.1Pierce GF Mustoe TA Pharmacologic enhancement of wound healing.Annu Rev Med. 1995; 46: 467-481Crossref PubMed Scopus (186) Google Scholar, 4Pierce GF Tarpley JE Yanagihara D Mustoe TA Fox GM Thomason A Platelet-derived growth factor (BB homodimer), transforming growth factor-β1, and basic fibroblast growth factor in dermal wound healing.Am J Pathol. 1992; 140: 1375-1388PubMed Google Scholar Impaired wound-healing models that have been used in many previous studies include young animals with the impaired conditions produced by injecting glucocorticoids, treating with radiation, or decreasing the blood supply to the wound.5Beck LS DeGuzman L Lee WP Xu Y Siegel MW Amento EP One systemic administration of transforming growth factor-β1 reverses age or glucocorticoid-impaired wound healing.J Clin Invest. 1993; 92: 2841-2849Crossref PubMed Scopus (224) Google Scholar, 6Pierce GF Mustoe TA Lingelbach J Masakowski VR Gramates P Deuel TF Transforming growth factor β reverses the glucocorticoid induced wound healing deficit in rats and is regulated by platelet derived growth factor in macrophages.Proc Natl Acad Sci USA. 1989; 86: 2229-2233Crossref PubMed Scopus (287) Google Scholar, 7Mustoe TA Purdy J Gramates P Deuel TF Thomason A Pierce GF Reversal of impaired wound healing in irradiated rats by platelet-derived growth factor-BB.Am J Surg. 1989; 158: 345-350Abstract Full Text PDF PubMed Scopus (145) Google Scholar, 8Albertson S Hummel RP Breeden M Greenhalgh DG PDGF and FGF reverse the healing impairment in protein-malnourished diabetic mice.Surgery. 1993; 114: 368-374PubMed Google Scholar, 9Wu L Mustoe TA Effect of ischemia upon growth factor enhancement of incisional wound healing.Surgery. 1995; 117: 570-576Abstract Full Text PDF PubMed Scopus (39) Google Scholar Some preclinical studies have examined age effects on wound healing, but truly aged animals (defined as those at the age when one-half of the studied population has died due to natural causes) under the condition of tissue ischemia have not been studied.5Beck LS DeGuzman L Lee WP Xu Y Siegel MW Amento EP One systemic administration of transforming growth factor-β1 reverses age or glucocorticoid-impaired wound healing.J Clin Invest. 1993; 92: 2841-2849Crossref PubMed Scopus (224) Google Scholar, 10Cohen BJ Danon D Roth GS Wound repair in mice as influenced by age and antimacrophage serum.J Gerontol. 1987; 42: 295-301Crossref PubMed Scopus (56) Google Scholar, 11Cox DA, Kunz S, Cerletti N, McMaster GK, Burk RR: Wound healing in aged animals: effects of locally applied transforming growth factor beta 2 in different model systems. 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Angiogenesis: Key Principles-Science-Technology-Medicine.Birkhauser Verlag, Basel, Switzerland, pp 287-295Google Scholar, 12Danon D Kowatch MA Roth GS Promotion of wound repair in old mice by local injection of macrophages.Proc Natl Acad Sci USA. 1989; 86: 2018Crossref PubMed Scopus (203) Google Scholar However, most chronic wounds occur in aged patients with varying degrees of local tissue ischemia secondary to scarring, fibrin cuffing, edema and increased venous pressure in venous stasis, pressure in pressure ulcers, small artery disease, and edema in diabetic ulcers.2Mustoe TA Cutler NR Allman RM Goode PS Deuel TF Prause J Bear M Serdar CM Pierce GF A phase II study to evaluate recombinant platelet-derived growth factor-BB in the treatment of stage 3 and 4 pressure ulcers.Arch Surg. 1994; 129: 213-219Crossref PubMed Scopus (160) Google Scholar, 13Phillips TJ Chronic cutaneous ulcers: etiology and epidemiology.J Invest Dermatol. 1994; 102: 38S-41SCrossref PubMed Scopus (104) Google Scholar, 14Falanga V Chronic wounds: pathophysiologic and experimental considerations.J Invest Dermatol. 1993; 100: 721-725Abstract Full Text PDF PubMed Google Scholar, 15Falanga V Growth factors and chronic wounds: the need to understand the microenvironment.J Invest Dermatol. 1992; 19: 667-672Google Scholar Therefore, the questions are whether some of the growth factors are less effective in promoting wound healing of aged ischemic animals and whether those altered effects are due to altered gene expression and signal transduction resulting from aging and ischemia. In general, wounds heal more slowly in healthy elderly human and animals.16Ashcroft GS Horan MA Ferguson MW The effects of aging on cutaneous wound healing in mammals.J Anat. 1995; 187: 1-26PubMed Google Scholar, 17Herrick SE Ireland GW Simon D McCollum CN Ferguson MW Venous ulcer fibroblasts compared with normal fibroblasts show differences in collagen but not fibronectin production under both normal and hypoxic conditions.J Invest Dermatol. 1996; 106: 187-193Crossref PubMed Scopus (83) Google Scholar, 18Holt DR Kirk SJ Effect of age on wound healing in healthy human beings.Surgery. 1992; 112: 293-298PubMed Google Scholar, 19Sussman MD Aging of connective tissue: physical properties of healing wounds in young and old rats.Am J Physiol. 1973; 224: 1167-1171PubMed Google Scholar Studies suggest that aging is accompanied by altered inflammatory response,20Forcher BK Cecil HC Some affects of age on the biochemistry of acute inflammation.Gerontologia. 1958; 2: 174-182Crossref PubMed Scopus (10) Google Scholar, 21Ashcroft GS Horan MA Ferguson MW Aging alters the inflammatory and endothelial cell adhesion molecule profiles during human cutaneous wound healing.Lab Invest. 1998; 78: 47-58PubMed Google Scholar decreased fibroblast proliferation,22Bruce SA Deamond SF Longitudinal study of in vivo wound repair and in vitro cellular senescence of dermal fibroblasts.Exp Gerontol. 1991; 26: 17-27Crossref PubMed Scopus (49) Google Scholar delayed angiogenesis,23Yamura H Matsuzawa T Decrease in capillary growth during aging.Exp Gerontol. 1980; 15: 145-150Crossref PubMed Scopus (48) Google Scholar reduced deposition of specific extracellular matrix components,24Ashcroft GS Horan MA Ferguson MW Aging is associated with reduced deposition of specific extracellular matrix components, an upregulation of angiogenesis, and an altered inflammatory response in a murine incisional wound healing model.J Invest Dermatol. 1997; 108: 430-437Crossref PubMed Scopus (201) Google Scholar, 25Platt D Ruhl W An age dependent determination of lysosomal enzyme activities, as well as the measurements of the incorporation of 14-c-proline and 14-c-glucosamine in subcutaneously implanted polyether sponge.Gerontologia. 1972; 18: 96-112Crossref PubMed Google Scholar and slower re-epithelialization.18Holt DR Kirk SJ Effect of age on wound healing in healthy human beings.Surgery. 1992; 112: 293-298PubMed Google Scholar However, the effect of tissue ischemia, a common denominator of a number of other disease processes, such as stroke, myocardial infarction and ischemia reperfusion injuries that have a high incidence in the elderly population, has not been studied in aged systems. As the aged population grows, wound healing under impaired conditions secondary to ischemia will increase patient morbidity and mortality after surgery or tissue injury. Clinicians have observed that aged healthy patients can have surgery and heal with few complications.26Chick LR Walton RL Reus W Colen L Sasmor M Free flaps in the elderly.Plast Reconstr Surg. 1992; 90: 87-94Crossref PubMed Scopus (92) Google Scholar However, aged patients with the additive conditions that contribute to local wound tissue ischemia tend to have a higher incidence of surgical wound dehiscence and are at higher risk for developing chronic wounds. Thus, we hypothesize that aging and ischemia have an additive effect on wound healing, and growth factor effects in promoting wound healing may be minimized under ischemic conditions. Transforming growth factor (TGF)-β1, one of the strongest stimulators of wound healing as shown in preclinical studies,1Pierce GF Mustoe TA Pharmacologic enhancement of wound healing.Annu Rev Med. 1995; 46: 467-481Crossref PubMed Scopus (186) Google Scholar, 5Beck LS DeGuzman L Lee WP Xu Y Siegel MW Amento EP One systemic administration of transforming growth factor-β1 reverses age or glucocorticoid-impaired wound healing.J Clin Invest. 1993; 92: 2841-2849Crossref PubMed Scopus (224) Google Scholar, 27Mustoe TA Pierce GF Thomason A Gramates P Sporn MB Deuel TF Accelerated healing of incisional wounds in rats induced by transforming growth factor-β.Science. 1987; 237: 1333-1335Crossref PubMed Scopus (851) Google Scholar, 28Kane CJM Hebda PA Mansbridge JN Hanawalt PC Direct evidence for spatial and temporal regulation of transforming growth factor beta 1 expression during cutaneous wound healing.J Cell Physiol. 1991; 148: 157-173Crossref PubMed Scopus (200) Google Scholar, 29Mustoe TA Pierce GF Morishima C Deuel TF Growth factor-induced acceleration of tissue repair through direct and inductive activities in a rabbit dermal ulcer model.J Clin Invest. 1991; 87: 694-703Crossref PubMed Scopus (290) Google Scholar has been associated with various stages of tissue repair.27Mustoe TA Pierce GF Thomason A Gramates P Sporn MB Deuel TF Accelerated healing of incisional wounds in rats induced by transforming growth factor-β.Science. 1987; 237: 1333-1335Crossref PubMed Scopus (851) Google Scholar, 29Mustoe TA Pierce GF Morishima C Deuel TF Growth factor-induced acceleration of tissue repair through direct and inductive activities in a rabbit dermal ulcer model.J Clin Invest. 1991; 87: 694-703Crossref PubMed Scopus (290) Google Scholar TGF-β1 has been shown to stimulate wound healing in young, normal, and ischemic animal models29Mustoe TA Pierce GF Morishima C Deuel TF Growth factor-induced acceleration of tissue repair through direct and inductive activities in a rabbit dermal ulcer model.J Clin Invest. 1991; 87: 694-703Crossref PubMed Scopus (290) Google Scholar when applied locally. Systemic administration of TGF-β1 stimulated wound healing in a middle-aged rat incisional model.5Beck LS DeGuzman L Lee WP Xu Y Siegel MW Amento EP One systemic administration of transforming growth factor-β1 reverses age or glucocorticoid-impaired wound healing.J Clin Invest. 1993; 92: 2841-2849Crossref PubMed Scopus (224) Google Scholar A study of the reduced healing rate seen in aged human females suggested a possible role of reproductive hormones in wound healing, and analysis showed a significant difference in re-epithelialization with aging.30Ashcroft GS Dodsworth J van Boxtel E Tarnuzzer RW Horan MA Schultz GS Ferguson MW Estrogen accelerates cutaneous wound healing associated with an increase in TGF-beta1 levels.Nature Med. 1997; 3: 1209-1215Crossref PubMed Scopus (494) Google Scholar In profound wound healing, deficits of age and ischemia are unknown. Aged mouse dermal cells express less TGF-β1 mRNA in vivo.31Schmid P Kunz S Cerletti N McMaster G Cox D Injury-induced expression of TGF-beta 1 mRNA is enhanced by exogenously applied TGF-beta.Biochem Biophys Res Commun. 1993; 194: 399-406Crossref PubMed Scopus (31) Google Scholar It has also been shown in vitro that TGF-β1 binding affinity is impaired under hypoxic conditions in young dermal fibroblasts.32Falanga V Takagi H Ceballos PI Pardes JB Low oxygen tension decreases receptor binding of peptide growth factors in dermal fibroblast cultures.Exp Cell Res. 1994; 213: 80-84Crossref PubMed Scopus (23) Google Scholar Our experimental data suggest that aging and ischemia may have additive effects on TGF-β1 mRNA expression. Collagen synthesis and deposition into the wound is essential during wound healing, and TGF-β1 is the strongest stimulator of collagen synthesis in wound healing.27Mustoe TA Pierce GF Thomason A Gramates P Sporn MB Deuel TF Accelerated healing of incisional wounds in rats induced by transforming growth factor-β.Science. 1987; 237: 1333-1335Crossref PubMed Scopus (851) Google Scholar Thus, it is very important to study collagen gene expression under different conditions during wound healing and after TGF-β1 treatment in both young and aged animals. This research will help evaluate the effects of aging and ischemia and the potential benefit of TGF-β1 treatment in wound healing. It will also help us learn about the signal transduction of growth factors and the potential effect of aging and ischemia on signal transduction at the molecular level. New Zealand White male rabbits (Hazelton, Norwalk, CT), young adult (6 months, ranging from 5 to 7 months), retired breeder rabbits, middle-aged (30 months, ranging from 29 to 31 months), upper middle-aged (36 months, ranging from 35 to 37 months), and aged (60 months, ranging from 58 to 62.5 months) were acclimated and kept under standard conditions in the Northwestern University Animal Care Center. As the lifespan of retired breeder laboratory rabbits is ∼5 years, which is shorter than that of normal laboratory rabbits, this age is comparable to that of humans in their 7th to 8th decades. This study and its surgical procedures have been approved by the Northwestern University Animal Care and Use Committee. The surgical procedures were performed as previously described9Wu L Mustoe TA Effect of ischemia upon growth factor enhancement of incisional wound healing.Surgery. 1995; 117: 570-576Abstract Full Text PDF PubMed Scopus (39) Google Scholar, 33Ahn B Mustoe TA Effects of ischemia on ulcer wound healing: a new model in the rabbit ear.Ann Plast Surg. 1990; 24: 17-23Crossref PubMed Scopus (131) Google Scholar after anesthetizing the rabbits with Ketamine (60 mg/kg) and Xylazine (5 mg/kg). Briefly, one of the rabbit ears was made ischemic by dissecting the rostral and central arteries and interrupting the entire dermal circulation, preserving only the major three veins and the smallest caudal artery. Three full-thickness, 6-mm-diameter circular wounds were created extending down to bare cartilage. The contralateral ear vessels were left undisturbed and served as matched non-ischemic controls. All of the wounds were covered with an occlusive polyurethane dressing (Tegaderm, 3M, Minneapolis, MN) for 12 days. The wounds were bisected and analyzed histologically based on a previous study that showed these wounds were minimally contracted.29Mustoe TA Pierce GF Morishima C Deuel TF Growth factor-induced acceleration of tissue repair through direct and inductive activities in a rabbit dermal ulcer model.J Clin Invest. 1991; 87: 694-703Crossref PubMed Scopus (290) Google Scholar Re-epithelialization rate, percentage of full re-epithelialization, and new granulation tissue formation in all matched wounds were measured as previously described.29Mustoe TA Pierce GF Morishima C Deuel TF Growth factor-induced acceleration of tissue repair through direct and inductive activities in a rabbit dermal ulcer model.J Clin Invest. 1991; 87: 694-703Crossref PubMed Scopus (290) Google Scholar, 33Ahn B Mustoe TA Effects of ischemia on ulcer wound healing: a new model in the rabbit ear.Ann Plast Surg. 1990; 24: 17-23Crossref PubMed Scopus (131) Google Scholar Recombinant human (rh)TGF-β1 (1 μg/wound; Amgen, Thousand Oaks, CA) was topically applied once to the wounds immediately after wounding. TGF-β1 was also topically applied to the ischemic wounds made in young, middle-aged, and aged rabbit ears. The dose of TGF-β1 (1 μg/wound) was chosen based on previous studies that demonstrated it was the optimal dose for ischemic wound healing.34Zhao LL Davidson JD Roth SI Mustoe TA Effect of hyperbaric oxygen and growth factors on rabbit ear ischemic ulcers.Arch Surg. 1994; 129: 1043-1049Crossref PubMed Scopus (147) Google Scholar In all rabbits, whether young or aged, the wounds on contralateral ears served as a paired control and were treated with vehicle alone (PBS). The growth factors were purified to homogeneity by conventional techniques and assayed for endotoxin before use. This growth factor has been tested in vitro and in vivo, and no difference in biological effects was found between recombinant and natural growth factor derived from macrophage.35Bourdrel L Lin C-H Lauren SL Elmore RH Sugarman BJ Hu S Westcott KR Recombinant human transforming growth factor-β1: expression by Chinese hamster ovary cells, isolation, and characterization.Protein Expres Purif. 1993; 4: 130-140Crossref PubMed Scopus (19) Google Scholar The rabbit ear ulcers were harvested and evaluated histologically at day 12 after wounding as previously described.5Beck LS DeGuzman L Lee WP Xu Y Siegel MW Amento EP One systemic administration of transforming growth factor-β1 reverses age or glucocorticoid-impaired wound healing.J Clin Invest. 1993; 92: 2841-2849Crossref PubMed Scopus (224) Google Scholar, 29Mustoe TA Pierce GF Morishima C Deuel TF Growth factor-induced acceleration of tissue repair through direct and inductive activities in a rabbit dermal ulcer model.J Clin Invest. 1991; 87: 694-703Crossref PubMed Scopus (290) Google Scholar, 34Zhao LL Davidson JD Roth SI Mustoe TA Effect of hyperbaric oxygen and growth factors on rabbit ear ischemic ulcers.Arch Surg. 1994; 129: 1043-1049Crossref PubMed Scopus (147) Google Scholar All of the wounds were created and harvested in a matched fashion, and the data were collected in the same manner allowing paired analyses with each animal serving as its own control. A paired two-tailed Student's t-test (Epistat program, Epistat Service, Richardson, TX) was used to detect differences between non-ischemic and ischemic wounds in each age group and between TGF-β1-treated wounds and matched control wounds. Analysis of one-way variance was used to analyze the differences among different age groups in model development and TGF-β1-treated wounds. The χ2Mustoe TA Cutler NR Allman RM Goode PS Deuel TF Prause J Bear M Serdar CM Pierce GF A phase II study to evaluate recombinant platelet-derived growth factor-BB in the treatment of stage 3 and 4 pressure ulcers.Arch Surg. 1994; 129: 213-219Crossref PubMed Scopus (160) Google Scholar test was used to analyze the differences in the percentage of full re-epithelialization. Multivariate analysis (Epistat program) was used to analyze the relative responsiveness of adult versus young or aged animals. The wound granulation tissue in each rabbit was harvested as previously described.36Brucker MJ Gruskin E Farrell CL Siddiqui A Mustoe TA Differential expression of platelet-derived growth factor receptor-beta in an aging model of wound repair.Wound Rep Regul. 1996; 4: 219-223Crossref PubMed Scopus (15) Google Scholar, 37Wu L Brucker M Gruskin E Roth SI Mustoe TA Differential effects of PDGF-BB in accelerating wound healing on aged versus young animals: the impact of tissue hypoxia.Plast Reconstruct Surg. 1997; 99: 815-822Crossref PubMed Scopus (34) Google Scholar All three wounds from each ear were processed, and the total cellular RNA was extracted with guanidine thiocyanate/phenol-based reagent according to the manufacturer's instructions (TRI Reagent, Cincinnati, OH).38Chomczynski PA Reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples.BioTechniques. 1993; 15: 532-537PubMed Google Scholar All reverse transcription (RT) reactions were performed simultaneously using a master mix to eliminate variability of RT efficiency. A total of 5.0 μg of each RNA sample, with acceptable purity (A260/A280 ratio ≥1.8) was converted to cDNA using Moloney murine leukemia virus reverse transcriptase and random primers (Gibco BRL, Grand Island, NY). Specific polymerase chain reaction (PCR) primers for rabbit glyceraldehyde-3-phosphate dehydrogenase (GAPDH), TGF-β1, and procollagen 1(I) were designed using conserved sequences from published Genbank complete and partial mRNA sequences of various species. Sequence information of all PCR primers used and the reaction conditions are listed in Table 1. The PCR products were confirmed by subcloning each into TA cloning vectors (TA cloning kit, Invitrogen, San Diego, CA) and sequencing analysis.39Byravan S Foster LM Phan T Verity AN Campagnoni AT Murine oligodendroglial cells express nerve growth factor.Proc Natl Acad Sci USA. 1994; 91: 8812-8816Crossref PubMed Scopus (75) Google Scholar, 40Taglialatela M Wible BA Caporaso R Brown AM Specification of pore properties by the carboxyl terminus of inwardly rectifying K+ channels.Science. 1994; 264: 844-847Crossref PubMed Scopus (146) Google Scholar Nonhomologous competitive PCR fragments (mimic) were used as an internal standard to measure the desired product.41Siebert PD Larrick JW PCR MIMICS: competitive DNA fragments for use as internal standards in quantitative PCR.BioTechniques. 1993; 14: 244-249PubMed Google Scholar, 42Siebert PD Larrick JW Competitive PCR.Nature. 1992; 359: 557-558Crossref PubMed Scopus (673) Google Scholar Mimic was created using a commercial kit (Clontech, Palo Alto, CA). Serial dilution of a known quantity of mimic was co-amplified with a constant amount of the cDNA sample, which resulted in varying band intensities depending on the ratio between the mimic and the cDNA of interest. The reaction products were electrophoresed on 2% agarose gels. Gel photographs were quantified by densitometry imaging (Imaging Densitometer GS-670, Bio Rad, Richmond, CA), and the ratio of gene product to mimic was plotted against the known quantity of the mimic. At a ratio of one, each curve gives the corresponding concentration of gene product cDNA. Competitive PCR reactions designed to compare the experimental condition with the control were run simultaneously to allow relative comparison of the extrapolated ratios. Results were confirmed by repeating experiments within the same RNA extraction and with multiple rabbits.Table 1Primer Sequences and PCR Cycle InformationGAPDHTGF-β1Procollagen 1(I)Upper Primer5′-CCA TGT TCG TCA TGG GTG TGA ACC A-3′5′-CTT CAG CTC CAC AGA GAA GAA CTG C-3′5′-TTC AGC TTT GTG GAC CTC CGG CTCLower Primer5′-CAT GAG TCC TTC CAC GAT ACC AAA G-3′5′-CAC GAT CAT GTT GGA CAA CTG CTC C-3′5′-CTG AGG GAT GCC ATC TCG GCC-3′Cycling (25 cycles each)94 °C for 45 seconds, 55 °C for 45 seconds, 72 °C for 90 seconds94 °C for 45 seconds, 55 °C for 45 seconds, 72 °C for 90 seconds94 °C for 45 seconds, 50 °C for 45 seconds, 72 °C for 90 seconds Open table in a new tab Histology studies showed depressed wound healing by age and ischemia. Young non-ischemic wounds had the most healing (Figure 1A), and young ischemic and aged non-ischemic wounds healed to a lesser degree (Figure 1, B and C). Aged ischemic wounds had severely impaired healing (Figure 1D), showing essentially no healing on day 12. Quantitative analysis of new granulation tissue deposition showed a moderate and progressive decrease with aging under non-ischemic conditions and a sharp decrease under ischemic conditions in both middle-aged and aged animals (Figure 2). Multivariate analysis showed that under non-ischemic condition, significant decrease of wound granulation tissue formation was found between age 30- and 6-month-old rabbits (41.7% decrease, P < 0.01) and 60- and 30-month-old rabbits (70.0% decrease, P < 0.05). Under ischemic condition, the decrease was 92.9% (P < 0.01) between 6- and 30-month-old rabbits, and no significant change was detected between 60- and 30-month-old rabbits. Young ischemic wounds showed a 65% decrease in new granulation tissue formation compared with their age-matched non-ischemic controls. Middle-aged and aged rabbits each had a more than 95% decrease compared with their age-matched control wounds (Figure 2). Histological analysis of hematoxylin and eosin (H&E)-stained wound tissue sections revealed a significant reduction of re-epithelialization by age and ischemia (P < 0.01) (Figure 3) as well. Multivariate analysis showed significant difference in re-epithelialization with aging: in non-ischemic wounds, 57.1% (P < 0.01) decrease between 30- and 6-month-old rabbits and 76.7% (P < 0.01) decrease between 30- and 60-month-old rabbits. In ischemic wounds, an 80% decrease (P < 0.01) was found between 30- and 6-month-old rabbits, and a 100% (P < 0.05) decrease was found between 30- and 60-month-old rabbits.Figure 2Effect of aging and ischemia on new granulation tissue formation. Day 12 wounds were harvested from both ischemic and non-ischemic animals of increasing age and stained with H&E, followed by quantification of granulation tissue formation using a calibrated lens reticule under a light microscope (×10). Reduction of granulation tissue formation was observed with increasing donor age and ischemia. The paired Student's t-test was used for statistical analysis.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Effect of aging and ischemia on re-epithelialization. Wounds were harvested on day 12 after wounding from both ischemic and non-ischemic rabbit ears. Re-epithelialization was quantified histologically on H&E-stained tissue sections. Significant decreases in the percentage of complete re-epithelialization among different age groups and under ischemia versus non-ischemia were observed. The paired Student's t-test was used for statistical analysis.View Large Image Figure ViewerDownload Hi-res image Download (PPT) TGF-β1 has been found to stimulate wound healing in several models of young animal, including ischemic, diabetic, and radiation-impaired healing.1Pierce GF Mustoe TA Pharmacologic enhancement of wound healing.Annu Rev Med. 1995; 46: 467-481Crossref PubMed Scopus (186) Google Scholar In this study, we found that TGF-β1 stimulated partial healing of non-ischemic wounds of aged animals at day 12 (Figure 4C), at which time wounds of young animals showed complete epithelialization (Figure 4A). Under ischemic conditions, TGF-β1 stimulated wound healing of young rabbits (Figure 4B), but it failed to promote the
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