Extracorporal Shock Waves Activate Migration, Proliferation and Inflammatory Pathways in Fibroblasts and Keratinocytes, and Improve Wound Healing in an Open-Label, Single-Arm Study in Patients with Therapy-Refractory Chronic Leg Ulcers.

BACKGROUND/AIMS: Chronic leg ulcers (CLUs) are globally a major cause of morbidity and mortality with increasing prevalence. Their treatment is highly challenging, and many conservative, surgical or advanced therapies have been suggested, but with little overall efficacy. Since the 1980s extracorporal shock wave therapy (ESWT) has gained interest as treatment for specific indications. Here, we report that patients with CLU showed wound healing after ESWT and investigated the underlying molecular mechanisms. METHODS: We performed cell proliferation and migration assays, FACS- and Western blot analyses, RT-PCR, and Affymetrix gene expression analyses on human keratinocytes and fibroblasts, and a tube formation assay on human microvascular endothelial cells to assess the impact of shock waves in vitro. In vivo, chronic therapy-refractory leg ulcers were treated with ESWT, and wound healing was assessed. RESULTS: Upon ESWT, we observed morphological changes and increased cell migration of keratinocytes. Cell-cycle regulatory genes were upregulated, and proliferation induced in fibroblasts. This was accompanied by secretion of pro-inflammatory cytokines from keratinocytes, which are known to drive wound healing, and a pro-angiogenic activity of endothelial cells. These observations were transferred "from bench to bedside", and 60 consecutive patients with 75 CLUs with different pathophysiologies (e.g. venous, mixed arterial-venous, arterial) were treated with ESWT. In this setting, 41% of ESWT-treated CLUs showed complete healing, 16% significant improvement, 35% improvement, and 8% of the ulcers did not respond to ESWT. The induction of healing was independent of patient age, duration or size of the ulcer, and the underlying pathophysiology. CONCLUSIONS: The efficacy of ESWT needs to be confirmed in controlled trials to implement ESWT as an adjunct to standard therapy or as a stand-alone treatment. Our results suggest that EWST may advance the treatment of chronic, therapy-refractory ulcers.

Green tea extract reduces induction of p53 and apoptosis in UVB-irradiated human skin independent of transcriptional controls.

Ultraviolet (UV) irradiation plays a pivotal role in human skin carcinongenesis. Preclinically, systemically and topically applied green tea extract (GTE) has shown reduction of UV-induced (i) erythema, (ii) DNA damage, (iii) formation of radical oxygen species and (iv) downregulation of numerous factors related to apoptosis, inflammation, differentiation and carcinogenesis. In humans, topical GTE has so far only been tested in limited studies, with usually very high GTE concentrations and over short periods of time. Both chemical stability of GTE and staining properties of highly concentrated green tea polyphenols limit the usability of highly concentrated green tea extracts in cosmetic products. The present study tested the utility of stabilized low-dose GTE as photochemopreventive agents under everyday conditions. We irradiated with up to 100 mJ/cm(2) of UVB light skin patches which were pretreated with either OM24-containing lotion or a placebo lotion. Biopsies were taken from both irradiated and un-irradiated skin for both immunohistochemistry and DNA microarray analysis. We found that while OM24 treatment did not significantly affect UV-induced erythema and thymidine dimer formation, OM24 treatment significantly reduced UV-induced p53 expression in keratinocytes. We also found that OM24 treatment significantly reduced the number of apoptotic keratinocytes (sunburn cells and TUNEL-positive cells). Carefully controlled DNA microarray analyses showed that OM24 treatment does not induce off-target changes in gene expression, reducing the likelihood of unwanted side-effects. Topical GTE (OM24) reduces UVB-mediated epithelial damage already at low, cosmetically usable concentrations, without tachyphylaxis over 5 weeks, suggesting GTE as suitable everyday photochemopreventive agents.

Reduced pSmad2 immunodetection correlates with increased primary melanoma thickness.

Cutaneous melanoma is the most aggressive of cutaneous neoplasms. Identifying patients with an increased risk for the development of metastases is critical. This study investigates phospho-Smad2, a central factor of the transforming growth factor beta pathway, on formalin-fixed, paraffin-embedded tissues from 60 primary cutaneous melanomas (Breslow >1 mm), for its candidacy for being a prognostic marker in primary cutaneous melanoma. Phospho-Smad2 positivity was assessed for correlation with clinical parameters including Breslow index, melanoma type, survival, development of metastases, sentinel lymph node status and age. Phospho-Smad2 positivity was not associated with survival or development of metastases, suggesting that it would not be a useful prognostic marker. Despite this, we found phospho-Smad2 positivity to be correlated with low tumour thickness, indicating that as the primary tumour grows there is an increased inhibition of transforming growth factor beta signalling resulting in suppressed Smad2 phosphorylation. Additionally, phosphorylation of Smad2 in neighbouring melanoma cells and keratinocytes was interrelated, which is a further indication that Smad2 phosphorylation in primary melanoma is affected by local area microenvironmental factors. We hypothesize that the observed decrease in transforming growth factor beta signalling in thicker primary melanomas is due to the increased production of signalling inhibitors.

p16 expression in primary malignant melanoma is associated with prognosis and lymph node status.

Lymph node (LN) status is an important prognostic factor in melanoma patients. p16 expression and proliferation rate (MIB-1) of primary melanomas have been suggested as a marker of metastatic potential. In this study, the correlation of p16 expression and the proliferation rate (MIB-1) with LN status and tumor-specific survival was investigated in primary melanomas. MIB-1 and p16 expression were analyzed by immunohistochemistry in 64 patients with primary cutaneous melanoma. Thirty four nevi were used as control. All patients underwent sentinel lymph node staging. Three different p16 staining patterns were observed: a combination of nuclear and cytoplasmic staining, only cytoplasmic staining and absence of p16 expression. All 34 nevi displayed a nuclear and cytoplasmic p16 staining, whereas p16 was negative in 14 of 64 (22%) melanomas. The level of p16 expression gradually decreased from benign nevi to melanoma without metastasis to melanoma with metastasis. There was a significant correlation between cytoplasmic p16 expression and absence of metastasis (p < 0.05). Death of disease correlated with absence of p16 immunostaining (p = 0.01). MIB-1 expression was not associated with survival. These results confirm the relevance of p16 expression as a prognostic marker in melanoma patients. In addition, it was shown that cytoplasmic immunostaining for p16 in primary melanoma might serve as a predictor of the LN status. Therefore, immunohistochemical evaluation for p16 expression is of potential value for treatment planning in melanoma surgery.