Upregulation of transforming growth factor-β1 and vascular endothelial growth factor in cultured keloid fibroblasts: relevance to angiogenic activity

Keloids are tumor-like lesions that result from excessive scar formation during healing of wounds. Histologically, keloids show an increased blood vessel density compared with normal dermis or normal scars. However, the angiogenic activity of keloid fibroblasts remains unknown. In this study, we investigated angiogenic activity of keloid fibroblasts. Transforming growth factor-β1 (TGF-β1) and vascular endothelial growth factor (VEGF) were investigated as elements of the angiogenic factors. Expressions of TGF-β1 and VEGF in conditioned medium were measured with enzyme-linked immunosorbent assay (EIA) and Northern blot analysis. Participation of TGF-β1 in the production of VEGF was also investigated with addition of TGF-β1 and a neutralizing anti-TGF-β1 antibody. A modified Boyden chamber assay was performed to assess the chemotactic activity of vascular endothelial cells. Angiogenic activity in vivo was evaluated by neovascularization of nodules formed by implantation of fibroblasts into severe combined immunodeficiency (SCID) mice. EIA showed that the concentrations of TGF-β1 and VEGF in conditioned medium were increased 2.5- and 6-fold, respectively, after the culture of keloid fibroblasts compared with normal fibroblasts. Northern blot analysis revealed that the expression of TGF-β1 and VEGF mRNA was upregulated 3.6- and 6-fold, respectively, in keloid fibroblasts compared with normal fibroblasts. Addition of TGF-β1 to keloid fibroblast cultures increased VEGF production by 3.5-fold, while there was a 6-fold in culture of normal fibroblasts. A neutralizing anti-TGF-β1 antibody reduced VEGF secretion to control levels, suggesting that TGF-β1 mediated the upregulation of VEGF expression. A modified Boyden chamber assay demonstrated that the chemotactic activity of vascular endothelial cells was more strongly (sevenfold) induced by keloid fibroblast-conditioned medium than by normal fibroblast-conditioned medium. Anti-VEGF antibody inhibited chemotaxis to basal levels. When SCID mice underwent implantation of fibroblasts into the back, the nodules formed by keloid fibroblasts were three times larger than those formed by normal fibroblasts. Although abundant neovascularization was observed in keloid fibroblast nodules, neovascularization was scarce in normal fibroblast nodules. Both in vitro and in vivo studies confirmed the significantly higher angiogenic activity of keloid fibroblasts compared with normal fibroblasts, and TGF-β1 and VEGF were clearly shown to be involved. These results suggest that angiogenesis in keloids is promoted by endogenous TGF-β1 and VEGF.     

The role of heat shock protein 60, vascular endothelial growth factor and antiphospholipid antibodies in Behçet disease

Background Behçet disease is a systemic inflammatory disease of unknown aetiology. T cells in this disease proliferate vigorously in response to a specific peptide of heat shock protein (HSP) 60 in an antigen‐specific fashion. Vascular endothelial cell growth factor (VEGF) is a cytokine participating in the inflammatory process. One of the prominent features of Behçet disease is vasculitis as a result of endothelial dysfunction. Antiphospholipid antibodies (APA) may play a role in the development of thrombosis by inhibiting production of prostacyclin by endothelial cells. Objectives To investigate the role of HSP60, VEGF and APA in Behçet disease and their relation to clinical manifestations and disease activity. Methods Thirty patients with Behçet disease were included; 17 were in the active stage and 13 were in the inactive. Fifteen age‐ and sex‐matched healthy subjects served as controls. Complete clinical examination and Doppler examination were done. Serum levels of HSP60, VEGF and APA were performed. Results Serum levels of HSP60, VEGF and APA were significantly higher in patients than in controls; however, their level did not correlate with disease activity. The serum level of VEGF correlated significantly with the presence of vascular manifestations and ocular involvement. The serum level of APA was greater in patients with thrombosis. HSP60 has an important role in aetiopathogenesis of Behçet disease, which sheds new light on its autoimmune nature. Conclusions An elevated serum level of VEGF may be a risk factor for the development of ocular disease contributing to poor visual outcome.     

Successful surgical management of giant condyloma acuminatum (Buschke-Löwenstein tumor) in the genitoanal region: a case report and evaluation of current therapies

Giant condyloma acuminatum (GCA; Buschke-Lowenstein tumor) is a human-papillomavirus-induced cauliflower-like tumor of the genitoanal region. It is characterized by its size, capability of local infiltration and high recurrence rate. We report on a 50-year-old patient presenting with a maximum finding of GCA with deep infiltration into the adductor and perineal musculature, the scrotum, the penis and the para-rectum. After performing a temporary loop colostomy, the tumor was removed by wide radical excision following plastic reconstruction with a myocutaneous gracilis flap. During a follow-up period of more than 5 years, no recurrence developed. Many treatment strategies (e.g. chemotherapy, radiation) have been published in the literature. Most authors recommend the radical surgical excision, allowing a complete histological examination and assessment of tumor-free resection margins. Despite the benign histological pattern of GCA in most cases, transformations into verrucous carcinoma and squamous-cell carcinoma have been described. In our case, the GCA seems to represent a continuum between normal condyloma acuminatum and an initial verrucous carcinoma.     

Specific inhibition of human skin fibroblast chemotaxis to platelet-derived growth factor A-chain homodimer by transforming growth factor-β1

Platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta) have been suggested to play important roles in wound healing. We investigated the effect of TGF-beta1 on the mitogenic and chemotactic activities of PDGF A-chain homodimer (PDGF-AA) and B-chain homodimer (PDGF-BB) in primary cultures of human skin fibroblasts. TGF-beta1 inhibited the growth-promoting activity of both PDGFs. Proliferative responses to basic fibroblast growth factor and epidermal growth factor were also restricted by TGF-beta1. A Boyden chamber chemotaxis assay revealed that the chemotactic migration to PDGF-AA was inhibited by TGF-beta1 pretreatment, but in contrast, the response to PDGF-BB was not affected by the same treatment. Western blot analysis showed that TGF-beta1 downregulated PDGF alpha-receptors, but not beta-receptors, indicating that the isoform-specific inhibition of chemotaxis is related to differential effects of TGF-beta1 on PDGF receptor expression. The present findings suggest that TGF-beta1 may act antagonistically towards PDGFs in humans under certain conditions, and this antagonistic nature of TGF-beta1 must be considered when it is applied to human wounds as a therapeutic agent.     

Effects of tumor necrosis factor-α on connective tissue metabolism in normal and scleroderma fibroblast cultures

Recent studies have demonstrated that tumor necrosis factor-(TNF-) selectively decreases production of collagens I and III, the major types of collagen in the dermis, and increases production of collagenase in cultured dermal fibroblasts. The effects of TNF- on collagens I, III and VI, fibronectin and collagenase gene expression by fibroblasts derived from normal individuals and patients with systemic sclerosis (SSc) were studied. SSc is characterized by excessive accumulation of collagen in the skin and in certain organs. TNF- inhibited collagen production and mRNA levels of collagens I and III and of fibronectin, and stimulated collagenase activity and collagenase mRNA levels in SSs fibroblasts. Levels of mRNA for 1(VI) and 3(VI) collagen and for -actin were unaltered in SSc fibroblasts incubated with TNF-. Similar results were observed for mRNA levels in normal fibroblasts incubated with TNF-. These results suggest that TNF- could be expected to be beneficial in the treatment of SSc. In addition, our results indicated that collagen-VI expression is regulated independently from expression of collagens I and III, and expression of fibronectin and collagens I and III are regulated in parallel in fibroblasts treated with TNF-.     

Absence of tumor necrosis factor-α in suction blister fluids and stratum corneum from patients with psoriasis

Summary Tumor necrosis factor (TNF)- is a cytokine with multiple biological properties, particularly proinflammatory, apart from the induction of tumor necrosis. In order to elucidate the role of TNF- in the pathogenesis of psoriasis, we have carried out bioassay and enzyme-linked immunosorbent assay for TNF- in suction blister fluids and horny tissue extracts from psoriatic skin. Although bioassay showed some activities in the suction blister fluids and horny tissue extracts, there were no significant differences between the levels of activities from normal and psoriatic skin. They were at the background level and pretreatment of the samples with anti-TNF- antiserum failed to abrogate the activities. ELISA confirmed the absence of TNF-. Therefore, the present study could not verify that TNF- plays a major role in the pathogenesis of psoriasis.     

Increased nerve growth factor- and tyrosine kinase A-like immunoreactivities in prurigo nodularis skin – an exploration of the cause of neurohyperplasia

Neurotrophins and their receptors play an important role in cutaneous nerve development and reconstruction after injury. Recent developments indicate that this group of molecules not only exert a neurotrophic action, but are also involved in immune responses and inflammation. Prurigo nodularis is a skin disease characterized by neurohyperplasia and intense itch. In the present study, the localization and distribution of nerve growth factor (NGF) and its receptors were explored by immunohistochemical methods, with the aim of detecting the cause of the neurohyperplasia in the disease. In normal healthy volunteers and in uninvolved skin, NGF immunoreactivity was seldom seen in the basal layer of the epidermis or in the dermis. In prurigo nodularis skin, there was also very little NGF immunoreactivity in the epidermis. However, in the dermis, a huge number of cells showed an NGF-like immunoreactivity. In normal skin of healthy volunteers, only a weak staining for tyrosine kinase A (trkA) was seen in the epidermis, whereas in the dermis, there was no trkA staining seen at all. However, in the prurigo nodularis tissue, the hyperplastic nerves clearly showed trkA immunoreactivity, and it seemed that the staining was only present in the axons. By NGF and p75 NGF receptor double-labelling, both immunoreactivities showed weak staining in the epidermis and dermis of normal skin. However, in the dermis of prurigo nodularis, strong staining for both NGF and NGF receptor antibodies was seen. NGF receptor-immunoreactive nerves were more dense in areas where there were more NGF-immunoreactive cells. The results indicate that in prurigo nodularis skin, NGF is overexpressed, locally infiltrated inflammatory cells may be the source of this NGF, and NGF and its receptors may contribute to the neurohyperplasia of the disease.     

Decisive role of tumor necrosis factor-α for spongiosis formation in acute eczematous dermatitis

Apoptosis of single keratinocytes (KC) is a characteristic feature of spongiosis formation, the histopathologic hallmark of acute eczematous dermatitis. In acute eczema, activated dermis-infiltrating T cells secrete several proinflammatory cytokines which might be decisive for KC apoptosis or survival. We analyzed the role of tumor necrosis factor alpha (TNF-α) in the determination of KC fate during spongiosis formation in acute eczematous dermatitis. Supernatants of activated human CD4⁺ T cells induced apoptosis in primary KC, which could be fully inhibited by individual blockade of interferon-γ (IFN-γ) and CD95 but not by neutralization of TNF-α activity. As compared to CD95-triggering alone, synchronous CD95 and TNF receptor cross-linking in the presence of IFN-γ only marginally enhanced KC apoptosis. Importantly, pre-treatment of KC with TNF-α followed by CD95 stimulation, but not vice versa, significantly amplified KC apoptosis as compared to CD95 stimulation alone. This TNF-α-mediated sensitization to CD95-induced KC cell death could be abrogated by blocking TNF receptor 1 (TNF-R1) but not TNF-R2 mAb. In eczematous dermatitis, the CD95 receptor was expressed throughout the epidermis, whereas immunohistological detection of TNF-R1 was rather restricted to KC around spongiotic vesicle formation. Thus, TNF-α primes KC for CD95-mediated signals which results in an increased susceptibility to apoptosis. TNF-R1 expression and spatial action of TNF-α restricted to spongiotic vesicles promote both CD95-induced KC apoptosis and limitation of spreading KC damage.     

The effects of prostaglandins E1 and F2α on epidermal growth

Summary The effects of prostaglandins E₁ and F₂ (PGE₁ and PGF_2a) on the growth of guinea pig epidermis have been studied in vivo. The prostaglandins were injected intradermally into guinea pigs, and tritiated thymidine was injected intraperitoneally prior to sacrifice. The autoradiographic labelling indices (L.I.) were assessed and a significant increase was found 4 b after injection of PGE₁-an effect which lasted for up to 72 h. PGF_2a injections had no significant effect on the L.I.

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Zusammenfassung Der Effekt der Prostaglandine E₁ und F₂ auf das Wachstum der Epidermis von Meerschweinchen wurde in vivo untersucht. Die Prostaglandine wurden intradermal injiziert; mit Tritium markiertes Thymidin wurde intraperidonial vor dem Töten der Tiere injiziert. Die autoradiographischen Daten (labelling indices) wurden bestimmt. Ein signifikanter Anstieg wurde 4 h nach der Injektion von PGE₁ ermittelt, ein Effekt der mindestens 72 h anhielt. PGF_2a-Injektionen hatten keinen Einfluß auf den L.I.

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Based on a paper read at meeting of the Investigative Group of the British Association of Dermatologists, January 1975     

Serum levels of interleukin-8, tumor necrosis factor-α and γ-interferon in Egyptian psoriatic patients and correlation with disease severity

Psoriatic plaques have been shown to contain increased levels of pro-inflammatory cytokines. Also, serum levels of several cytokines have been reported elevated in psoriatic patients. It is postulated that changes in cytokine production both locally and systemically could be useful in monitoring disease activity. The aim of this study was to evaluate serum cytokine profile of interleukin (IL)-8, γ-interferon (IFN-γ) and tumor necrosis factor-α (TNF-α) in Egyptian psoriatic patients by enzyme-linked immunosorbent assay (ELISA) technique and to correlate these levels with disease severity. We analyzed serum samples from 60 Egyptian patients (31 females and 29 males) with a mean age of 40.2 ± 17.4 years with active psoriasis, and 21 healthy volunteers for major T-helper type 1 cytokines using the ELISA technique. The disease severity, including erythema, induration and scales, was assessed by Psoriasis Area and Severity Index (PASI) score. TNF-α and IFN-γ were markedly elevated in all sera from psoriatic patients. TNF-α was found a more efficient predictor for disease severity than IL-8 and IFN-γ using three receiver-operator curves with accuracy. IL-8 was also moderately elevated and correlated with the age of patients (r = 0.28). We have obtained evidence that TNF-α in our study was found to be more useful than the other two tested cytokines, IL-8 and IFN-γ as a follow-up marker for monitoring disease severity in Egyptian psoriatic patients. A positive correlation between lL-8 and the age of the patients was also noted.     

Effects of high power-pulsed Nd:YAG laser irradiation on the release of transforming growth factor-beta (TGF-β) and vascular endothelial growth factor (VEGF) from human gingival fibroblasts

The purpose of this study was to investigate the effect of different high-power energy settings of a neodymium:yttrium-aluminum-garnet (Nd:YAG) laser (1064 nm) on cell viability of human gingival fibroblasts (GFs) and release of transforming growth factor-beta (TGF-β) and vascular endothelial growth factor (VEGF) on these cells. GFs were isolated from human gingival connective tissues during the crown lengthening procedure. GFs were irradiated with different laser parameters as follows: group 1: 1 W (100 mJ, 10 Hz) 10 seconds; group 2: 1.5 W (150 mJ, 10 Hz) 10 seconds; group 3: 2 W (200 mJ, 10 Hz) 10 seconds; group 4: 1 W (100 mJ, 10 Hz) 20 seconds; group 5: 1.5 W (150 mJ, 10 Hz) 20 seconds; and group 6: 2 W (200 mJ, 10 Hz) 20 seconds. Cell viability/cell proliferation was analyzed with XTT (tetrazolium salt, cell proliferation kit) staining. The release levels of TGF-β and VEGF were analyzed by the enzyme-linked immunosorbent assay. No significant differences were observed in the different laser irradiation groups compared to the control group in terms of cell viability (p > 0.05). The release of TGF-β was not affected by different laser irradiation settings (p > 0.05). Only group 6 promoted significantly higher VEGF release from GFs in 24 hours compared to the control group (p ? 0.05). These findings suggest that high-power Nd:YAG laser is probably safe but has a very limited effect for wound healing.     

Receptor activator of nuclear factor κB ligand/osteoprotegerin axis and vascular calcifications in patients with chronic kidney disease

Vascular calcifications are commonly observed in patients with chronic kidney disease (CKD) and contribute to the excessive cardiovascular morbidity and mortality rates observed in these patients populations. Although the pathogenetic mechanisms are not yet fully elucidated, recent evidence suggests a link between bone metabolism and the development and progression of vascular calcifications. Moreover, accumulating data indicate that receptor activator of nuclear factor κB ligand/osteoprotegerin axis which plays essential roles in the regulation of bone metabolism is also involved in extra-osseous bone formation. Further studies are required to establish the prognostic significance of the above biomarkers as predictors of the presence and severity of vascular calcifications in CKD patients and of cardiovascular morbidity and mortality. Moreover, randomized clinical trials are needed to clarify whether inhibition of osteoclast activity will protect from vascular calcifications.