Comparison of the effectiveness of gene therapy with vascular endothelial growth factor or shock wave therapy to reduce ischaemic necrosis in an epigastric skin flap model in rats.

The effect of gene therapy with adenovirus-mediated (Ad) vascular endothelial growth factor (VEGF) was compared to that of shock wave (SW) therapy on skin flap survival in a rat model, using the epigastric skin flap, based solely on the right inferior epigastric vessels. Thirty male Sprague-Dawley rats were randomly divided into three groups (SW-group, Ad-VEGF-group, and Control-group) of 10 rats each. Immediately after surgery, the SW-group was administered 2500 impulses at 0.15mJ/mm(2), in the Ad-VEGF-group injections were made to the subdermal space whereas the Control-group received no treatment. Flap viability was evaluated on day 7 after the operation. Standardised digital pictures of the flaps were taken and transferred to the computer, and necrotic zones relative to total flap surface area were measured and expressed as percentages. Overall, significantly smaller areas of necrotic zones were noted in the SW-group and the Ad-VEGF-group compared with the Control-group (SW-group: median 2.23% (range: 0-5.1) versus Control-group: median 17.4% (range: 11.8-22.8) (p<0.05); Ad-VEGF-group: median 9.25% (range: 7.6-11.9) versus Control-group: median 17.4% (range: 11.8-22.8) (p<0.05)). Furthermore, in the SW-group, areas of necrotic zones were significantly smaller than in Ad-VEGF-group (SW-group: median 2.23% (range: 0-5.1) versus Ad-VEGF-group: median 9.25% (range: 7.6-11.9) (p<0.05)). We conclude that treatment with SW enhances epigastric skin flap survival significantly more than Ad-VEGF treatment and also represents a feasible and cost effective technique to improve blood supply in ischaemic tissue.     

Adenovirus-mediated transforming growth factor-beta ameliorates ischemic necrosis of epigastric skin flaps in a rat model

BACKGROUND: Gene therapy has been recently introduced as a novel approach to treat ischemic tissues by using the angiogenic potential of certain growth factors. We investigated the effect of adenovirus-mediated gene therapy with transforming growth factor-beta (TGF-beta) delivered into the subdermal space to treat ischemically challenged epigastric skin flaps in a rat model. MATERIAL AND METHODS: A pilot study was conducted in a group of 5 animals pretreated with Ad-GFP and expression of green fluorescent protein in the skin flap sections was demonstrated under fluorescence microscopy at 2, 4, and 7 days after the treatment, indicating a successful transfection of the skin flaps following subdermal gene therapy. Next, 30 male Sprague Dawley rats were divided into 3 groups of 10 rats each. An epigastric skin flap model, based solely on the right inferior epigastric vessels, was used as the model in this study. Rats received subdermal injections of adenovirus encoding TGF-beta (Ad-TGF-beta) or green fluorescent protein (Ad-GFP) as treatment control. The third group (n = 10) received saline and served as a control group. A flap measuring 8 x 8 cm was outlined on the abdominal skin extending from the xiphoid process proximally and the pubic region distally, to the anterior axillary lines bilaterally. Just prior to flap elevation, the injections were given subdermally in the left upper corner of the flap. The flap was then sutured back to its bed. Flap viability was evaluated seven days after the initial operation. Digital images of the epigastric flaps were taken and areas of necrotic zones relative to total flap surface area were measured and expressed as percentages by using a software program. RESULTS: There was a significant increase in mean percent surviving area between the Ad-TGF-beta group and the two other control groups (P < 0.05). (Ad-TGF-beta: 90.3 +/- 4.0% versus Ad-GFP: 82.2 +/- 8.7% and saline group: 82.6 +/- 4.3%.) CONCLUSIONS: In this study, the authors were able to demonstrate that adenovirus-mediated gene therapy using TGF-beta ameliorated ischemic necrosis in an epigastric skin flap model, as confirmed by significant reduction in the necrotic zones of the flap. The results of this study raise the possibility of using adenovirus-mediated TGF-beta gene therapy to promote perfusion in random portion of skin flaps, especially in high-risk patients.     

Tissue expression of transforming growth factor-β1 and transforming growth factor-α during wound healing in human skin explants

In the dynamic and complex process of wound healing, locally produced growth factors are important mediators, although their actual roles have not been fully established. In the present study, the presence of transforming growth factor-beta1 and -alpha during the re-epithelialization of full-thickness wounds was investigated in an in vitro model of wound healing in human skin. The amounts of transforming growth factor-beta1 and -alpha secreted from the wound area were measured with enzyme immunoassays, and immunohistochemistry was used to study the localization of these two growth factors in the healing wound. The wounds were followed until they were completely re-epithelialized. The results showed a continuous increase in secreted transforming growth factor-beta1 throughout the re-epithelialization phase of healing followed by a decrease after its completion. The keratinocytes migrating out from the wound edges showed intense staining for transforming growth factor-beta1 which declined to the level of the surrounding epidermis after the wound was covered by a new epidermis. After the skin was wounded, a decrease both in secreted transforming growth factor-alpha and in immunostaining for this growth factor was apparent. Even though a minor increase in the immunoreactivity for transforming growth factor-alpha occurred after the completion of re-epithelialization, no increase in secreted transforming growth factor-alpha could be detected by enzyme immunoassay. These data suggest that keratinocytes modulate their expression of transforming growth factor-beta1 and -alpha during the wound healing process in human skin and that these changes may be controlled in part by autocrine pathways.     

Differential proliferative response of fetal and adult human skin fibroblasts to transforming growth factor-β

Since pronounced differences exist between the fetal and adult repair processes, we studied the proliferative response of skin fibroblasts from these two stages to transforming growth factor-beta (TGF-beta), a cytokine with a broad range of activities in tissue repair. Here, we present evidence that TGF-beta inhibits fetal human skin fibroblasts, while it is stimulatory for adult ones. This proliferative effect of TGF-beta was found to be concentration- dependent, but isoform-independent. Furthermore, even a transient exposure of the cells to this growth factor was sufficient to exert its stimulatory or inhibitory action. Accordingly, we have studied the immediate responses provoked by TGF-beta in major signaling pathways, and we have found that it induces a rapid activation of the SMAD pathway, i.e., phosphorylation and nuclear translocation of SMAD2, followed by dephosphorylation, most probably due to degradation by the proteasome. However, similar intensity and kinetics of this activation have been observed in both fetal and adult fibroblasts. On the other hand, curcumin, a natural product with wound healing properties that inhibits several intracellular signaling pathways, was found to completely abrogate the inhibitory effect of TGF-beta1 on human fetal skin fibroblasts, without affecting the stimulatory action on fibroblasts from adult donors. In conclusion, there is a major radical in the proliferative response of fetal and adult human skin fibroblasts to TGF-beta, possibly reflecting the different repair strategies followed in these two stages of development.     

Increased transforming growth factor-β1 and tubulointerstitial fibrosis in rats with congenital hydronephrosis

OBJECTIVES: Most of our knowledge concerning renal obstruction has been derived from experimental animal models, and it is not yet well defined in spontaneous hydronephrosis. The aim of our study is to evaluate the roles of transforming growth factor-beta1 (TGF-beta1) and apoptosis in congenital hydronephrotic kidneys in comparison with experimental models. METHODS: We made histological studies on kidneys from 6-week-old Wistar-Imamichi rats with congenital unilateral hydronephrosis as well as surgical models of complete or partial unilateral ureteral obstruction. The severity of hydronephrotic kidneys was evaluated on routine hematoxylin and eosin (H&E) stained sections, and the tubulointerstitial fibrosis analyzed morphometrically on Masson's trichrome stained sections. Renal tubular atrophy was assessed on periodic acid Schiff (PAS) stained sections, and tubular cell apoptosis assessed with TUNEL technique. The renal TGF-beta1 level was determined by a sandwich enzyme-linked immunosorbent assay (ELISA). RESULTS: We observed a significant loss of kidney weight with profound compensatory growth of the contralateral kidney in rats with congenital hydronephrosis. Most of the hydronephrotic kidneys were markedly enlarged with dilatation of the collecting system, renal parenchymal thinning, tubular atrophy, interstitial infiltration and fibrosis. The renal TGF-beta1 level was markedly elevated in hydronephrotic kidneys as compared with normal controls (326.01 +/- 30.64 pg/mg protein vs 227.81 +/- 11.07 pg/mg protein, P < 0.01). The tubular apoptotic score in hydronephrotic kidneys was also significantly higher than normal controls (2.17 +/- 0.50/HPF [high power field]vs 0.14 +/- 0.04/HPF, P < 0.01). The increased TGF-beta1 and apoptotic status paralleled the histological changes of tubulointerstitial fibrosis and tubular atrophy. Similar findings were also obtained in experimental obstructive models. CONCLUSION: In comparison with surgical models of partial and complete ureteral obstruction, our data provide solid morphological and molecular evidences of renal obstruction in rats with congenital hydronephrosis.     

Shock wave therapy reduces necrotic flap zones and induces VEGF expression in animal epigastric skin flap model

The effect of extracorporeal shock wave (ESW) therapy on skin flap survival and growth factor expression was investigated in a rat model using epigastric skin flap. Treatment and control groups each contained 20 animals. ESW effectively enhanced epigastric skin flap survival by significant reduction of areas of necrotic zones. At day 7 after the operation, necrotic zones of 4.2% were found in the ESW-treated group compared with 18.3% in the control group ( P < 0.01). Concomitantly, in tissue samples adjacent to the necrosis areas, increased vascular endothelial growth factor expression was observed in the ESW-treated animals (median 84.5%, range 57.4 to 94.5%) compared with the control group (median 46.7%, range 29.1 to 93.1%; P < 0.1). However, for expression of basic fibroblast growth factor, no difference was found between the two groups. The authors conclude that the success of the shock wave treatment may partly be due to modulation of growth factor expression.     

Reversal of the wound healing deficit in diabetic rats by combined basic fibroblast growth factor and transforming growth factor-β1 therapy

Wound healing is impaired in the diabetic state because of, at least in part, low expression of growth factors. Individual growth factors can partially activate healing, yet the actual wound environment presents a dynamic continuum of multiple cellular signals. Complex interactions among growth factors and target cells can have synergistic effects, and several examples of combinatorial, in vivo activity are evident in the literature. In this study, the implantation of a combination of basic fibroblast growth factor and transforming growth factor-beta in rats induced fivefold to sevenfold increases in granulation tissue formation in comparison with implantation of each growth factor alone. Diabetes was induced in adult, male Sprague-Dawley rats with streptozotocin. Incisional wounds and sponge granulation tissue were produced in separate groups and then treated with an injection of 2 microg transforming growth factor-beta1 combined with 10 microg basic fibroblast growth factor on day 3. DNA, collagen, and protein were analyzed in granulation tissue on days 7 and 9, and biomechanical properties of incisions were tested on days 7, 14, and 21. The combination of transforming growth factor-beta1 and basic fibroblast growth factor had marked, positive effects on biochemical parameters of wound healing and reversed the tensile strength deficit of diabetic wounds. Nonradioactive in situ hybridization showed increased expression of messenger RNA for type I and III procollagen and transforming growth factor-beta1 in normal and diabetic wounds, whereas ultrastructural examination showed a marked reorganization of collagen fibrils. These findings reinforce the concept that appropriate mixtures of cytokines rather than individual cytokines may more adequately stimulate tissues in cases of impaired wound healing.     

Urinary transforming growth factor-β1 is an indicator of histological chronicity in IgA nephropathy

SUMMARY: This study examined urinary excretion of transforming growth factor-β1 (TGF-β1) in adult IgA nephropathy and compared this with clinical and histological parameters. TGF-β1 was measured by enzyme-linked immunosorbent assay in 24-h urine specimens from 25 patients with IgA nephropathy (17 men, eight women). Urine from eight age-matched control subjects served as the control. Serum TGF-β1 was also measured in 16 out of the 25 patients and six age-matched control subjects. TGF-β1 was detected in the urine in 72% of IgA nephropathy patients(18/25), but was not detected in any of the control subjects. Patients with decreased renal function (creatinine clearance (Ccr) < 80 mL/min) had higher urine levels of TGF-β1 than those with normal renal function (Ccr ≥ 80 mL/min) (141.8 ± 60.0 ng/day vs 39.7 ± 33.2 ng/day, P < 0.01). The level of TGF-β1 gave a negative correlation with Ccr ( r = −0.62, P = 0.001), but not with proteinuria ( r = 0.11, P = 0.58). Twenty-two of the patients were evaluated histologically. The urinary TGF-β1 levels correlated with both global sclerosis and interstitial volume ( r = 0.52, P < 0.05, and r = 0.51, P < 0.05, respectively). However, there was no correlation between TGF-β1 levels and glomerular intracapillary inflammatory cell score, mesangial proliferation score or the matrix score. No correlation was found between TGF-β1 levels and the number of glomerular or interstitial CD68⁺ cells. No significant differences in serum TGF-β1 levels were observed between patients with normal Ccr and those with decreased Ccr. Also, its levels were found to be independent of the urine levels. In conclusion, 24-h urinary TGF-β1 excretion was found to correlate with Ccr, global sclerosis and interstitial volume, demonstrating that urinary TGF-β1 is an indicator of chronicity in adult IgA nephropathy.     

Topical application of transforming growth factor-β1 in acute traumatic tympanic membrane perforations: an experimental study in rats

High transforming growth factor-beta1 (TGF-beta1) expression in combination with fibrotic scar was observed in human tympanic membranes containing a chronic perforation. The purpose of this study was to investigate whether application of exogenous TGF-beta1 to experimentally created tympanic membrane perforations might induce a nonhealing tympanic membrane perforation. Bilateral myringotomies were performed in 20 rats. In 10 animals, a single dose of TGF-beta1 (0.1 microg) was topically applied to the left tympanic membrane after perforation. In the second part of the study with 10 animals, repeated applications of TGF-beta1 at a higher concentration were tested. In both groups, the opposite ears received vehicle alone. Tympanic membranes were observed for a total of 5 weeks. The effect of TGF-beta1 on the healing of the tympanic membranes was evaluated by closure rates and histology. In the single application group, the healing process was not delayed. Repeated applications of TGF-beta1 did cause a faster perforation closure and a thicker tympanic membrane. Topical TGF-beta1 applied to a traumatic tympanic membrane perforation does not create a chronic perforation at the concentrations and modes of application used in this study.     

Histopathological changes and tumour necrosis factor-α, transforming growth factor-β and tenascin expression in patients with primary type I membranoproliferative glomerulonephritis in remission

Aim:   Primary type I membranoproliferative glomerulonephritis (MPGN) is a rare cause of glomerular disease with a high relapse rate and poor prognosis. The aim of this study was: (i) to evaluate the histopathological findings associated with remission; and (ii) to document the possible clinical and histopathological factors predicting relapses.
Methods:   Eleven type I MPGN patients (five men, six women; mean age, 38.8 ± 13.5 years) who were in remission for at least 1 year after the cessation of immunosuppressive drugs were re-biopsied. The intensity of immunostaining for tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β1, and tenascin was graded from 0 (no staining) to 3+ (maximum staining).
Results:   Mean baseline mesangial cellularity score and tubulointerstitial infiltration score were reduced and mesangial matrix expansion score was increased at protocol re-biopsies compared to baseline. The glomerular and tubulointerstitial staining scores for TGF-β1 and tenascin were higher than that of baseline. Reduced tubulointerstitial TNF-α expression was found in re-biopsy specimens compared to baseline. Patients have been followed for a mean time of 51.5 ± 22.2 months after the protocol biopsy. Eight patients had a relapse. Mesangial cellularity score and glomerular tenascin expression at re-biopsy specimens were higher in relapsed patients compared to those without a relapse.
Conclusion:   Our study shows that mesangial cellularity and tubulointerstitial cell infiltration are reducing whereas mesangial matrix expansion, glomerular and tubulointerstitial TGF-β1 and tenascin expression are increasing with remission. The higher mesangial cell proliferation and glomerular tenascin scores in remission are associated with the development of relapse.     

Extracorporal shock wave may enhance skin flap survival in an animal model.

Several methods have been used in an attempt to increase blood supply and tissue perfusion in ischemic tissues. The authors investigated the effect of extracorporal shock wave (ESW) treatment on compromised skin flaps. For this purpose, the epigastric skin flap model in rats, based solely on the right inferior epigastric vessels was used. Twenty male Sprague-Dawley rats were divided into two groups (ESW-group, Control group) of 10 rats each. The ESW-group was administered 2500 impulses at 0.15 mJ/mm(2) immediately after surgery, whereas, the control group received no treatment. Flap viability was evaluated on day 7 after the operation. Standardised digital pictures of the flaps were taken and transferred to the computer, and necrotic zones relative to total flap surface area were measured and expressed as percentages. Overall, there was a significant reduction in the surface area of the necrotic zones of the flaps in the ESW group compared to the control group (ESW group: 2.2+/-1.9% versus control: 17.4+/-4.4% (p < 0.01). In this study, the authors demonstrated that treatment with ESW enhanced epigastric skin flap survival, as confirmed by the significant reduction of necrotic flap zones. ESW treatment seems to represent a feasible and cost effective method to improve blood supply in ischemic tissue.     

Differential expression of platelet-derived growth factor and transforming growth factor-β in relation to progression of IgA nephropathy

SUMMARY: The role of platelet-derived growth factor (PDGF) and transforming growth factor-β1 (TGF-β1) in relation to mesangial matrix expansion and progressive glomerulosclerosis in IgA nephropathy (IgAN) is not clearly defined. Expression of PDGF B, TGF-β1, and extracellular matrix proteins in glomeruli was assessed by immunohistochemistry in 42 biopsies with IgAN and six renal biopsies with no detectable abnormalities. the mRNA expression of PDGF B, TGF-β1, α1(IV) collagen, laminin B1 and fibronectin genes was further evaluated by in situ hybridization in 25 biopsies with IgAN and six controls. In IgAN, the intensity of immunostaining for PDGF B, type IV collagen, laminin and fibronectin, but not for TGF-β1, was increased in the mesangium compared with controls. the immunoreactivity of PDGF B was closely correlated with that of type IV collagen and laminin. the number of PDGF B mRNA-, α1(IV) collagen mRNA-, laminin B1 mRNA-, and TGF-β1 mRNA-expressing cells/glomerular section, but not the number of fibronectin mRNA-expressing cells, was increased in IgAN compared with controls. the number of PDGF B mRNA-expressing cells correlated significantly with the percentage of glomerulosclerosis. In the cellular lesions of focal segmental glomerulosclerosis (FSGS), expression of TGF-β1 protein and mRNA was markedly increased in visceral glomerular epithelial cells (GEC). These results suggest that PDGF B mainly overproduced by mesangial cells may cause mesangial matrix expansion, whereas TGF-β1 produced by GEC may be related to the formation of FSGS in IgAN. Thus, PDGF B and TGF-β1 may play differential roles in the pathogenesis of renal fibrosis and the progression of IgAN.     

Abrogation of the fibrotic effect of transforming growth factor-β in dermal wound healing

The growth factor, transforming growth factor-beta1, which under normal circumstances promotes wound healing by stimulating local fibroblasts to produce collagen and other extracellular matrix proteins, has also been implicated as the primary causative agent of fibrosis. Because transforming growth factor-beta1 is capable of stimulating its own production by fibroblasts, its normally beneficial effects may become amplified to the point where excess extracellular matrix accumulation occurs, thereby causing abnormal scarring. Therefore, strategies that block or counter the effects of transforming growth factor-beta1 may be useful in preventing or decreasing fibrosis. One such strategy is the use of glucocorticoid steroids such as dexamethasone, which normally have the opposite effect of transforming growth factor-beta1, namely the impairment of wound healing. When used in conjunction with transforming growth factor-beta1, glucocorticoid steroids may normalize the effect of transforming growth factor-beta1 on collagen synthesis, thereby reducing excessive collagen deposition and fibrosis.     

pcD2/hVEGF对缺血皮瓣存活的影响

目的 观察并探讨pcD2/hVEGF对缺血皮瓣血循环重建和皮瓣存活率的影响.方法 SD大鼠24只,分2组,每组12只.在鼠背部制作蒂位于尾侧的缺血皮瓣(7cm×1cm).实验组皮下注射pcD2/hVEGF,对照组皮下注射生理盐水,术后7天观察皮瓣存活面积比、单位面积微血管计数和及VEGF基因表达水平.结果 2组中pcD2VEGF组皮瓣存活面积比、单位面积微血管密度与对照组有显著性差异.结论 pcD2VEGF能促进缺血皮瓣存活.     

Inhibition of wound closure by transforming growth factor-β and dexamethasone in a fetal mouse limb organ culture model

Tissue repair comprises several physiologic processes including the deposition of a newly synthesized connective tissue matrix and the regeneration of the epidermis by reepithelialization. A fetal mouse limb organ culture system facilitates the investigation of both reepithelialization and connective tissue deposition in repair within a controlled environment. A sutured closed wound in an intact 18.5-day old fetal mouse limb completely heals by 7 days. Including dexamethasone in the media inhibited both reepithelialization and connective tissue deposition. Concurrent administration of transforming growth factor-beta with dexamethasone restored the deposition of connective tissue but did not restore reepithelialization. When transforming growth factor-beta was given alone, connective tissue deposition was enhanced at both the wound site and in contiguous dermis, but reepithelialization did not proceed. Transforming growth factor-beta inhibited wound closure by blocking the migration of epidermal cells. The organ-cultured, wounded fetal mouse limb system is sufficiently sensitive to show both mesenchymal cell-enhancing activity as well as epithelial migration inhibiting activity by transforming growth factor-beta. The in vitro repair of fetal mouse limbs may serve as an in vitro system to test the influence of soluble agents on the repair process.     

Modeling the effects of transforming growth factor-β on extracellular matrix alignment in dermal wound repair

We present a novel mathematical model for collagen deposition and alignment during dermal wound healing, focusing on the regulatory effects of transforming growth factor-beta (TGFbeta.) Our work extends a previously developed model which considers the interactions between fibroblasts and an extracellular matrix composed of collagen and a fibrin based blood clot, by allowing fibroblasts to orient the collagen matrix, and produce and degrade the extracellular matrix, while the matrix directs the fibroblasts and control their speed. Here we extend the model by allowing a time varying concentration of TGFbeta to alter the properties of the fibroblasts. Thus we are able to simulate experiments which alter the TGFbeta profile. Within this model framework we find that most of the known effects of TGFbeta, i.e., changes in cell motility, cell proliferation and collagen production, are of minor importance to matrix alignment and cannot explain the anti-scarring properties of TGFbeta. However, we find that by changing fibroblast reorientation rates, consistent with experimental evidence, the alignment of the regenerated tissue can be significantly altered. These data provide an explanation for the experimentally observed influence of TGFbeta on scarring.