Enhanced vascularization of cultured skin substitutes genetically modified to overexpress vascular endothelial growth factor

Cultured skin substitutes have been used as adjunctive therapies in the treatment of burns and chronic wounds, but they are limited by lack of a vascular plexus. This deficiency leads to greater time for vascularization compared with native skin autografts and contributes to graft failure. Genetic modification of cultured skin substitutes to enhance vascularization could hypothetically lead to improved wound healing. To address this hypothesis, human keratinocytes were genetically modified by transduction with a replication incompetent retrovirus to overexpress vascular endothelial growth factor, a specific and potent mitogen for endothelial cells. Cultured skin substitutes consisting of collagen-glycosaminoglycan substrates inoculated with human fibroblasts and either vascular endothelial growth factor-modified or control keratinocytes were prepared, and were cultured in vitro for 21 d. Northern blot analysis demonstrated enhanced expression of vascular endothelial growth factor mRNA in genetically modified keratinocytes and in cultured skin substitutes prepared with modified cells. Furthermore, the vascular endothelial growth factor-modified cultured skin substitutes secreted greatly elevated levels of vascular endothelial growth factor protein throughout the entire culture period. The bioactivity of vascular endothelial growth factor protein secreted by the genetically modified cultured skin substitutes was demonstrated using a microvascular endothelial cell growth assay. Vascular endothelial growth factor-modified and control cultured skin substitutes were grafted to full-thickness wounds on athymic mice, and elevated vascular endothelial growth factor mRNA expression was detected in the modified grafts for at least 2 wk after surgery. Vascular endothelial growth factor-modified grafts exhibited increased numbers of dermal blood vessels and decreased time to vascularization compared with controls. These results indicate that genetic modification of keratinocytes in cultured skin substitutes can lead to increased vascular endothelial growth factor expression, which could prospectively improve vascularization of cultured skin substitutes for wound healing applications.     

Keratinocytes suppress transforming growth factor-beta1 expression by fibroblasts in cultured skin substitutes

Transforming growth factor (TGF)-beta1 is a multipotent growth factor with an important role in tissue homeostasis. This growth factor regulates cell proliferation, adhesion, migration and differentiation, as well as extracellular matrix deposition. The temporal secretion and activation of latent TGF-beta1 is thus of major importance to physiological and pathological processes and in wound healing and tumour formation. Cultured skin substitutes, as used to treat extensive acute or chronic skin wounds, offer an attractive model to investigate cellular interactions in cytokine and growth factor expression and response in vitro. In the present investigation, expression of TGF-beta1 was analysed in keratinocyte, fibroblast and melanocyte monolayer cultures, as well as in the dermal vs. epidermal components of reconstituted human skin. Immunohistology, enzyme-linked immunosorbent assay (ELISA) and Northern blotting were used to demonstrate expression at the RNA and protein level. In the monolayer cultures, levels of TGF-beta1 synthesized by melanocytes were observed to be considerably elevated when compared with keratinocytes. Most TGF-beta1, however, was secreted by fibroblasts. The relative contribution of the epidermal and dermal components of the skin substitutes to overall TGF-beta1 levels was determined by comparing results obtained for either component in the presence and absence of fibroblasts and keratinocytes. From results obtained by ELISA it was apparent that TGF-beta1 levels generated predominantly by fibroblasts within the skin substitutes were greatly reduced over time in the presence of keratinocytes. Suppression of fibroblast TGF-beta1 expression in the presence of keratinocytes was also demonstrable at the RNA level by Northern blotting. Results obtained by immunohistochemistry suggest that most, if not all, of the growth factor was present in the latent form. It is therefore most likely that the observed effect results from a factor secreted by keratinocytes, which is capable of suppressing TGF-beta1 synthesis by fibroblasts. These results suggest that expression of TGF-beta1 by fibroblasts is downregulated by paracrine actions of keratinocytes in healing skin.     

Transiently increased insulin-like growth factor. I. Immunoreactivity in UVB-irradiated mouse skin

UVB-irradiation during 3 d for 90, 180, and 180 sec, respectively, at a daily dose of 0.1 and 0.2 joule/cm2, respectively, induced slight inflammatory reactions in the mouse ear. The insulin-like growth factor I (IGF-I) immunoreactivity, normally demonstrable only in scattered basal epidermal cells, rapidly increased in intensity and frequency in the epidermis. After 3 d of UVB irradiation almost all epidermal cells were outlined by IGF-I immunoreactivity in their plasma membrane. The Langerhans cells expressed intense IGF-I immunoreactivity throughout their cytoplasm. The elevated IGF-I immunoreactivity ceased after 5-7 d and was normalized in 3 weeks. The number of Ia positive epithelial Langerhans cells did not seem to be affected by UVB irradiation. It is concluded that the increased IGF-I immunoreactivity is likely to reflect formation of the trophic peptide IGF-I, most evidently by Langerhans cells, in early events of the inflammatory, reactive response of the skin to UVB irradiation.     

Keloid fibroblasts resist ceramide-induced apoptosis by overexpression of insulin-like growth factor I receptor

Keloids are benign dermal tumors, characterized by overgrowth of lesions, invasiveness beyond the original boundary of the insult, and recurrence of lesions. The exact etiology is unknown, however. Our hypothesis is that keloids are acquired as a result of an abnormal or prolonged wound healing process, with persistent proliferation and extracellular matrix production of fibroblasts that should otherwise discontinue in normal wound healing. In this study, we examined the response of keloid fibroblasts to proapoptotic signaling. Cell-permeable ceramide, N-acetyl-D-sphingosine, induced apoptosis of dermal fibroblasts in a dose- and time-dependent manner, which was detected by phase contrast microscopy, fluorescent microscopy, the TUNEL method, flow cytometric analysis, and WST-1 assay. In contrast, keloid fibroblasts resisted apoptosis induced by N-acetyl-D-sphingosine (percent survival with 40 mM ceramide treatment for 12 h, normal versus keloid: 9.6% +/- 6.6% vs 66.8% +/- 5.5%). Western blotting analysis showed insulin-like growth factor I receptor overexpression in keloid fibroblasts, but not in normal fibroblasts. Exogenously added insulin-like growth factor I enhanced the resistance of keloid fibroblasts to ceramide-induced apoptosis. Wort- mannin, a phosphatidylinositol 3 kinase inhibitor, suppressed the antiapoptotic action of insulin-like growth factor I in keloid fibroblasts. Our results suggest that keloid fibroblasts overexpressing insulin-like growth factor I receptor are resistant to apoptosis, thus allowing persistent proliferation and production of excessive extracellular matrix. J Invest Dermatol 115:1065-1071 2000     

Induction of connective tissue growth factor expression by sphingosylphosphorylcholine in cultured human skin fibroblasts

Sphingosylphosphorylcholine (SPC) is a bioactive sphingolipid metabolite that can enhance wound healing. In an effort to find downstream effectors of SPC, we performed microarray analysis and found that the expression of the gene for connective tissue growth factor (CTGF) was significantly affected in human skin fibroblasts cultured in vitro. Northern blot analysis showed that SPC markedly induced CTGF mRNA expression in a dose- and time-dependent manner. Consistent with this result, Western blot analysis also showed that SPC significantly induced the CTGF production. Pretreatment with cycloheximide did not prevent the CTGF induction by SPC, indicating that SPC stimulates CTGF mRNA expression without the increased synthesis of a regulatory protein. Inhibition by pretreatment with Y27632, but not by PD98059 (a mitogen-activated protein kinase 1/2 inhibitor) and LY294002 (a phosphatidylinositol 3-kinase inhibitor), indicated that rho-kinase pathway was involved in SPC-induced CTGF expression. Together, these results reveal the potential importance of CTGF induction as a downstream event in SPC-induced cellular responses.     

Expression of insulin-like growth factor-I in lesional and non-lesional skin of patients with morphoea

Background  Morphoea (scleroderma) is a chronic disorder characterized by circumscribed sclerotic plaques with the hallmark of increased fibroblast activation and fibrosis. Through its effect on connective tissue cells and immune cells, insulin-like growth factor (IGF)-I has been found to play a role in some autoimmune connective tissue diseases and has been implicated in the pathogenesis of several fibrotic disorders.
Objectives  To evaluate the role of IGF-I in the pathogenesis of morphoea.
Methods  The study was carried out on 15 patients with morphoea and nine healthy controls. Two 5-mm punch skin biopsies were taken from every patient (one from lesional and one from non-lesional skin) and a single biopsy was taken from the normal skin of each control. A 10-mL blood sample was also taken from each patient and control. Quantitative detection of tissue and serum levels of IGF-I was done using an enzyme-linked immunosorbent assay technique.
Results  IGF-I in lesional skin was significantly higher than in non-lesional and control skin ( P  =   0·001 and P  =   0·021, respectively). Moreover, a significantly higher level of IGF-I was detected in patient serum when compared with control serum ( P  <   0·001). A direct significant correlation existed between lesional and non-lesional skin level ( r  =   0·618, P  =   0·014), and between lesional skin level and Rodnan score ( r  =   0·538, P  =   0·039).
Conclusions  Despite the small sample size, this study suggests that IGF-I plays an important role in the pathogenesis of fibrosis, characteristic of morphoea. Studies on a larger number of patients with morphoea as well as on patients with systemic sclerosis are recommended. Furthermore, therapeutic trials using IGF-I antagonist (octreotide) are highly recommended in patients with morphoea.     

Up-regulation of putative hyaluronan synthase mRNA by basic fibroblast growth factor and insulin-like growth factor-1 in human skin fibroblasts

Recently, cDNAs for the three putative human hyaluronan synthase (HAS) genes, HAS1, HAS2 and HAS3, have been cloned. In this study we investigated the effects of basic fibroblast growth factor (bFGF) and insulin-like growth factor-1 (IGF-1) on the expression of HAS genes in cultured skin fibroblasts. Northern blot analyses showed that treatment of fibroblasts with bFGF enhanced the mRNA levels of all three genes. HAS2 gene expression showed the strongest up-regulation with a more than 10-fold increase at 50 ng/ml of bFGF. bFGF also increased hyaluronan production. Incubation of fibroblasts with IGF-1 increased HAS1, HAS2, and HAS3 mRNA levels, as well as hyaluronan production. Our results suggest that up-regulation of the HAS genes by bFGF and IGF-1 is closely associated with the stimulation of hyaluronan synthesis, and that effects of growth factors on HAS gene expression may have important implications for tissue remodeling, such as in development and wound healing.     

Maintenance of human skin in organ culture: role for insulin-like growth factor-1 receptor and epidermal growth factor receptor

Recent studies have shown that adult skin incubated in low-Ca2+ (0.15 mM) medium rapidly degenerates but that normal architecture is maintained when the tissue is incubated in high-Ca2+ medium (1.4 mM Ca2+). To investigate whether the skin cell-produced growth factors insulin-like growth factor-1 (IGF-1) and epidermal growth factor (EGF) play a role in these events, 2-mm skin punch biopsies were obtained and maintained for 8 to 10 days in a basal medium containing 0.15 mM Ca2+ with and without growth factors, or containing 1.4 mM Ca2+ with and without antibodies to the same growth factors. In parallel experiments, cultured human keratinocytes were incubated for 2 days in the same basal medium in the presence or absence of the same growth factors and antibodies. Consistent with previous reports, organ cultures incubated in the low-Ca2+ (0.15 mM) medium rapidly degenerated. Neither IGF-1 nor EGF prevented the complete degeneration of epidermis and dermis in these organ cultures. Interestingly, the addition of an anti-IGF-1 receptor (IGF-1R) antibody to the organ cultures maintained in high-Ca2+ medium induced changes reminiscent of those seen when the organ cultures were maintained in low-Ca2+ medium, i.e. tissue degeneration. In contrast, antibodies to EGF receptor, used for comparison, only produced focal areas of epidermal necrosis. In vitro, IGF-1 is a known mitogen for keratinocytes. In cultured human keratinocytes, anti-IGF-1R antibody partially inhibited the IGF-1-mediated stimulation of human keratinocyte proliferation without affecting normal spontaneous growth. Additionally, IGF-1R immunolocalized to basal keratinocytes in vivo, exhibited specific binding to IGF-1 in vitro. This indicated a critical role for IGF-1R in both organ cultures ex vivo and cultured cells in vitro. Messenger RNA encoding both IGF-1 and IGF-1R were readily detected by RT-PCR in organ cultures incubated in both low- and high-Ca2+ medium. There were no detectable differences in IGF-1 mRNA in organ cultures growing in the low- or high-Ca2+ medium, but lower levels of IGF-1R mRNA were observed in the organ cultures maintained in low-Ca2+ medium than in those in high Ca2+ medium. These findings are consistent with homeostatic changes in the tissue grown under different calcium concentrations. IGF-1 mRNA was detected in several skin cell populations in vitro, even though it was undetectable in cultured keratinocytes. Taken together these findings indicate that (1) the IGF-1/ IGF-1R loop is critically involved in maintenance of human skin organ cultures ex vivo, and (2) IGF-1, locally produced by skin cells other than keratinocytes, interacts with its receptor, predominantly expressed in basal keratinocytes, to maintain tissue homeostasis.     

Differential roles of insulin receptor and insulin-like growth factor-1 receptor in differentiation of murine skin keratinocytes

The insulin receptor and the insulin-like growth factor-1 receptor are widely expressed tyrosine kinases that mediate insulin and insulin-like growth factor-1 signaling. Both receptors are expressed in many cells in which insulin stimulation does not result in an increase in glucose transport, and the distinct role of the insulin receptor in these tissues, is not known. We have studied the regulation of insulin receptor and insulin-like growth factor-1 receptor in the differentiation of cultured murine keratinocytes. Both receptors are expressed in skin keratinocytes and their expression was unchanged in all stages of calcium-induced differentiation. Insulin binding to skin keratinocytes, however, increased during calcium-induced differentiation, whereas insulin-like growth factor-1 binding decreased. Ligand-induced autophosphorylation was also changed during differentiation. In proliferating keratinocytes both receptors became phosphorylated upon ligand binding, insulin-like growth factor-1 receptor to a greater extent. Terminal differentiation resulted in a decrease in insulin receptor autophosphorylation, whereas insulin-like growth factor-1 receptor autophosphorylation was abolished. There was no change in the cellular localization of the proteins, their intrinsic activity, or their internal structure. Finally, due to the change in the receptor's activity during keratinocyte differentiation, the role of insulin and insulin-like growth factor-1 in the differentiation process was examined. The expected increase in the expression of keratins 1 and 10 during calcium-induced differentiation was facilitated in the presence of insulin, whereas this induction was inhibited in the presence of insulin-like growth factor-1. In conclusion, these results demonstrate that insulin and insulin-like growth factor-1 signaling pathways are differentially involved in skin differentiation, suggesting that abnormal insulin signaling, as occurs in diabetes, may lead to skin pathology.     

人肝细胞生长因子对体外培养的黑素细胞生物学活性的影响

目的 探讨人肝细胞生长因子（rhHGF）对体外培养正常人黑素细胞生物学活性的影响。方法 分别用不同浓度的rhHGF（0.1，1，10，20，60，80，100 μg/L）作用于体外培养的正常人黑素细胞，采用噻唑蓝（MTT）法检测细胞增殖及酪氨酸酶活性，用倒置显微镜对比观察不同时期rhHGF（60 μg/L）处理组黑素细胞形态的改变，并用流式细胞仪分析黑素细胞周期及凋亡情况。结果 0.1 ~ 100 μg/L rhHGF有促进黑素细胞增殖作用，在20，60，80 μg/L浓度时促增殖比较显著（P ＜ 0.01），rhHGF各浓度组均增加黑素细胞酪氨酸酶活性（P ＜ 0.01或P ＜ 0.05）；浓度为60 μg/L的rhHGF能使黑素细胞数目增多、胞体增大、树突增多，并有促进黑素细胞由G0/G1期进入S期及抑制细胞凋亡的作用。结论 rhHGF可增强体外培养的人黑素细胞的生物学活性。     

胰岛素样生长因子结合蛋白-3在原代培养的阴茎海绵体平滑肌细胞中的表达及定位

目的:观察胰岛素样生长因子结合蛋白-3(insulin-like growth factor binding protein 3,IGFBP-3)在阴茎海绵体平滑肌细胞(corpus cavernosum endothelial cells,CCSMCs)中的表达及定位情况。方法:原代培养大鼠CCSMCs并鉴定,RT-PCR检测IGFBP-3基因mRNA表达,用荧光免疫细胞化学法检测IGFBP-3在CCSMCs中的定位及蛋白表达。结果:RT-PCR能够检测到IGFBP-3 802bp特异性条带的表达,荧光免疫细胞化学法可见IGFBP-3蛋白表达并同时定位于CCSMCs胞质和胞核中。结论:CCSMCs可表达IGFBP-3基因,其蛋白定位于胞质和胞核,可能是ED治疗的新靶点。     

阿维A对HaCaT细胞体外凋亡和胰岛素样生长因子结合蛋白7及血管内皮生长因子表达的影响

目的 探讨阿维A对HaCaT细胞体外凋亡和胰岛素样生长因子结合蛋白7（IGFBP7）及血管内皮生长因子（VEGF）表达的影响。 方法 将10-5、10-6、10-7、10-8 mol/L的阿维A分别作用于HaCaT细胞24、48、72 h后,采用CCK8法检测细胞增殖情况;流式细胞仪检测阿维A对HaCaT细胞凋亡率的影响;Western印迹和RT-PCR法检测阿维A对HaCaT细胞IGFBP7、VEGF蛋白及其mRNA表达的影响。 结果 10-8 mol/L阿维A处理HaCaT细胞48 h时表现出对细胞增殖的抑制作用,随着时间延长和药物浓度升高,抗增殖作用亦增强,当药物浓度增加到10-5 mol/L时,48 h和72 h的抑制率分别为39.94% ± 2.27%和49.77% ± 1.87%。与对照组相比,10-5 mol/L阿维A作用48 h后,凋亡率由1.803% ± 0.313%（对照组）上升至7.617% ± 0.767%（阿维A组）（P ＜ 0.05）,IGFBP7的表达由0.436 ± 0.013上升至0.939 ± 0.040（P ＜ 0.05）,IGFBP7 mRNA的表达由0.190 ± 0.056上升至0.872 ± 0.079（P ＜ 0.05）,VEGF的表达由0.798 ± 0.036下降至0.213 ± 0.032（P ＜ 0.05）,VEGF mRNA的表达由0.933 ± 0.054下降至0.274 ± 0.041（P ＜ 0.05）。 结论 阿维A可促进HaCaT细胞的体外凋亡,并可在蛋白及mRNA水平上调IGFBP7及下调VEGF的表达。     

血管内皮生长因子在某些增生性皮肤病皮损表皮的表达

血管内皮生长因子(vascular endothelial growth factor,VEGF)在离体实验中可引起血管内皮细胞的增殖,诱导血管新生和血管通透性增加^[1],在大疱性类天疱疮、结节性痒疹等皮损中VEGF有高表达^[2].     

Infrared radiation does not enhance the frequency of ultraviolet radiation-induced skin tumors, but their growth behaviour in mice

There is increasing concern about the interaction between infrared radiation (IR) and ultraviolet radiation (UVR) with regard to carcinogenesis because prolonged solar exposure is associated with an increased cumulative load not only of UVR but also of IR. We recently demonstrated that IR-pretreatment reduces UVR-induced apoptosis. As this might support the survival of UVR-damaged cells and thus carcinogenesis, we performed an in vivo photocarcinogenesis study. One group of mice were treated with IR prior to each UVR exposure; additional groups were treated with IR or UVR alone. IR alone did not induce skin cancer. UVR-induced tumor formation was not enhanced in IR-pretreated mice, but, in contrast, seemed to occur with delay. This correlated with a reduction of p53 mutated clones in the skin. However, once developed, tumors in IR-pretreated mice grew faster which was confirmed by their enhanced Ki-67 expression. The enhanced aggressiveness of tumors derived from IR-pretreated mice was associated with a higher prevalence of sarcomas than epithelial tumors. Hence, the impact of IR on UVR-induced carcinogenesis has to be interpreted with caution. Although IR may delay the onset of UVR-induced tumors, it might contribute to a worse outcome by shifting these tumors into a more aggressive phenotype.     

Expression of vascular endothelial growth factor in normal epidermis, epithelial tumors and cultured keratinocytes

Vascular endothelial growth factor (VEGF), an endothelium-specific growth factor and microvessel hypermeability factor, is expressed and secreted by several kinds of cells and is implicated in angiogenesis of tumors. The present study was performed to determine the relationship between the expression of VEGF in normal skin, benign and malignant epithelial lesions and cultured keratinocytes and the proliferative activity and degree of differentiation of keratinocytes. Skin lesions were studied immunohistochemically by staining with two anti-VEGF antibodies and secretion and production of VEGF by keratinocyte cultures were evaluated using an enzyme-linked immunosorbent assay. Low to moderate VEGF expression was observed in normal epidermis. In epithelial tumors, different reactivity patterns were observed and different areas of the same tumor expressed different amounts of VEGF. A more prominent labelling occurred in proliferative layers and/or more differentiated cells of virus-induced lesions, squamous cell carcinomas and Bowen’s disease, whereas basal cell carcinomas always stained weakly for VEGF. In cultured keratinocytes, the amount of cell-associated and secreted VEGF increased with time, and the constitutively produced VEGF was mostly released extracellularly. High calcium concentrations upregulated the intracellular content of VEGF but downregulated its release. Taken together, these results showed a modulated expression and release of VEGF in relation to the stage of cell differentiation and in rapidly growing or activated keratinocytes. Received: 22 April 1996     

Further characterization of the keratinocyte somatomedin-C/insulin-like growth factor I (SM-C/IGF-I) receptor and the biological responsiveness of cultured keratinocytes to SM-C/IGF-I

Somatomedin-C/insulin-like growth factor I (SM-C/IGF-I) binds to both cultured keratinocytes trypsinized to single-cell suspensions and fresh epidermal sheets derived from 1 M NaBr-treated 6-mm punch biopsies. A gradual increase of 1-4% in specific binding of SM-C/IGF-I to fresh epidermal sheets was observed during a 5-hour incubation period at 15 degrees C. The human keratinocyte SM-C/IGF-I receptor was isolated by polyacrylamide gel electrophoresis after cross-linking with iodinated SM-C/IGF-I and migrated at 135,000 daltons under reducing conditions. Both a partially purified preparation of SM-C/IGF-I, as well as recombinant DNA-derived thr-59-SM-C/IGF-I (25-100 ng/ml), stimulated keratinocyte replication in a serum-free system, with an approximately equivalent effect as insulin (10 micrograms/ml). Since fibroblasts produce SM-C/IGF-I, our data suggest that some dermal-epidermal interactions in diseases such as psoriasis may be mediated, in part, by SM-C/IGF-I.