Targeting leiomyomas with all-trans-retinoic acid at phosphoinositide 3-kinase pathway suppression: Effective roles of ß-catenin and of signaling interactions.

AIM: Leiomyomas, monoclonal tumors developed by the transformation of myometrium somatic stem cells, are a major health concern that can severely impair quality of life. Pathological alterations of signaling pathways have been recognized as a key feature in a variety of human diseases. Our objective was to analyze treatment with all-trans-retinoic acid (ATRA) by suppression of the phosphoinositide 3-kinase (PI3K) pathway on growth, signaling pattern and interactions among PI3K/B-cell lymphoma 2 (Bcl2)/retinol leiomyoma proteins. METHODS: Cultures of paired myometrium and leiomyoma cells from premenopausal women undergoing hysterectomy were collected. Western blot and analysis of variance were used for analysis. RESULTS: Significant differences were detected between treatment with ATRA alone or with LY294002 (a PI3K growth suppressor) in response to treatment and among cell samples and cell numbers. Leiomyoma cells were less affected. Immunochemical analysis of signaling patterns demonstrated that treatments affected most of the examined protein levels differently. Significant differences between the cell type responses to treatment in pyruvate phosphate dikinase 1 (pPDK1), Bad and pß-catenin levels were identified. The pß-catenin level showed highly significant interaction between response to treatment and cell type. CONCLUSIONS: ATRA treatment on PI3K pathway suppression significantly affected growth, signaling pattern and interactions among PI3K/Bcl2/retinol proteins involved in the growth, survival and apoptosis of leiomyomas. Interpretation of our results suggests that increasing knowledge of the role of signaling interplay in the pathogenesis of leiomyomas may present an opportunity to use specific signal transduction inhibitors for treating and preventing this disorder.

Survival-apoptosis associated signaling in GNE myopathy-cultured myoblasts.

GNE Myopathy (GNEM) is a neuromuscular disorder caused by mutations in the GNE gene. It is a slowly progressive distal and proximal muscle weakness sparing the quadriceps. In this study, we applied our model of mutated M743T GNE enzyme skeletal muscle-cultured myoblasts and paired healthy controls to depict the pattern of signaling proteins controlling survival and/or apoptosis of the PI3K/AKT, BCL2, ARTS/XIAP pathways, examined the effects of metabolic changes/stimuli on their expression and activation, and their potential role in GNEM. Immunoblot analysis of the GNEM myoblasts indicated a notable increased level of activated PTEN and PDK1 and a trend of relative differences in the expression and activation of the examined signaling molecules with variability among the cultures. ANOVA analysis showed a highly significant interaction between the level of PTEN and the patients groups. In parallel, the interaction between the level of BCL2, BAX and PTEN with the specific PI3K/AKT inhibitor-LY294002 was highly significant for BCL2 and nearly significant for PTEN and BAX. The pattern of the ARTS/XIAP signaling proteins of GNEM and the paired controls was variable, with no significant differences between the two cell types. The response of the GNEM cells to the metabolic changes/stimuli: serum depletion and insulin challenge, as indicated by expression of selected signaling proteins, was variable and similar to the control cells. Taken together, our observations provide a clearer insight into specific signaling molecules influencing growth and survival of GNEM muscle cells.

Changes of PI3K/AKT/BCL2 signaling proteins in congenital Giant Nevi: melanocytes contribute to their increased survival and integrity.

Congenital Giant Nevi (CGN) are rare melanocytic lesions with the potential to regress into malignant melanoma. Simultaneous up-regulation and cooperative interactions of signaling pathways are crucial events in the pathogenesis of melanocytes. Our study aimed to identify changes in the expression and activation of proteins controlling survival and/or apoptosis of the key signaling pathways PI3K/AKT/BCL2 and Wnt/ß-catenin of CGN melanocytes. We applied a model of cultured melanocytes from paired CGN and normal appearing skin, and Western blot (WB) analyzed the expression and activation profile of survival and anti-apoptotic proteins of these signaling pathways, growth pattern, cell cycle and apoptosis. WB analysis demonstrated a significant higher expression level of activated AKT and of BCL2 proteins in the CGN melanocytes compared with paired melanocytes from normal appearing skin. A relative increase in the level of GSK3 and FOXO1 proteins, down stream targets of AKT, as well as of pß-catenin was also detected in the CGN melanocytes compared with the controls. These changes were not affected by growth of the CGN melanocytes in reduced serum (starvation). Both cell populations shared a similar growth pattern, with no significant differences in the proportion of apoptotic cells and in cell cycle fractions. These data demonstrate for the first time, changes in signaling proteins of cultured CGN melanocytes. Further, suggesting that the changes in AKT/BCL2 signaling molecules might mediate growth and anti-apoptosis processes at least in part, thus increasing the survival potential of CGN melanocytes and maintaining their integrity.

Characterization of factors that determine lentiviral vector tropism in skin tissue using an ex vivo model.

BACKGROUND: Lentiviral tropism to a solid tissue may be determined by receptor availability, the differentiation state of cells and the three-dimensional architecture of the tissue. METHODS: Using skin organ cultures, lentiviral vector tropism was compared with that of keratinocytes in cell culture. Furthermore, the tropism of lentiviral vector to mouse and human tissues was compared ex vivo, in attempt to validate the mouse skin as an experimental system for human gene therapy of skin diseases. RESULTS: The results obtained indicated that although early progenitor keratinocytes (keratin 15+ and p63+), when grown in culture are permissive to lentiviral vector, they are resistant to transduction in their native 'niche' in the skin tissue. Transiently amplifying keratinocytes (keratin 14+) on the other hand, are permissive to lentiviral vector transduction, in cell culture and in the skin, after separation of the epidermis from the dermis layer. Keratinocytes (keratin 14+) in the hair follicle of human skin are resistant to lentiviral transduction, even after partial digestion of the extracellular matrix collagen. By contrast, collagenase pretreatment of mouse tissue facilitated transduction of keratinocytes within the hair follicle. Because lentivirus pseudotyped by two envelopes (amphotropic murine leukemia virus and vesicular stomatitis virus G glycoprotein) display the same tropism, we suggest that receptor availability is not the critical factor in the pattern of skin tissue transduction. CONCLUSIONS: Taken together, the results obtained in the present study indicate that lentiviral vector tropism in the three-dimensional skin tissue is distinct from the tropism to keratinocytes in culture and is dependent on a complex interplay of extracellular restrictions.

Efficient transgene expression from naked DNA delivered into an arterio-venous fistula model for kidney dialysis.

BACKGROUND: Patients with kidney failure frequently require the formation of an arterio-venous fistula (AVF) in which a vein is connected to an artery resulting in arterialization of the vein to allow adequate blood flow into an external 'artificial kidney'. In most patients, neo-intimal hyperplasia (NIH) ensues, causing narrowing and subsequent occlusion of the vein, leading to significant morbidity. The cellular events causing venous NIH may serve as ideal targets for molecular-based therapies. However, therapeutic gene delivery into the vascular system is seriously impeded by problems related to the low efficacy and toxicity of targeted viral vector delivery. MATERIALS AND METHODS: To explore the feasibility of a plasmid-based vascular gene delivery system, we established a rat AVF model that develops NIH. Plasmids encoding for reporter or therapeutic genes were delivered into the blood vessels at the time or after AVF formation. RESULTS: Intra-luminal injection of plasmid into the AVF resulted in extensive and long-term reporter gene expression at the venous limb mainly at the site of NIH formation. Transgene expression was confined to endothelial cells and myofibroblasts that migrate inwards from the adventitia and form the NIH lesion. There was no detrimental tissue reaction to plasmid delivery, contrasting with the severe inflammatory response observed after adenovirus infection. Intra-vascular delivery of a plasmid carrying the endothelial nitric oxide synthase gene resulted in sustained production of nitric oxide, previously shown to mitigate NIH formation. CONCLUSIONS: These findings open the possibility of vascular transduction with naked DNA bearing therapeutic genes in areas prone to NIH to ameliorate vein graft pathologies using simple and clinically applicable vector delivery methods.

Flame burn protection: assessment of a new, air-cooled fireproof garment.

A new, air-cooled fireproof garment for tank crewmen was assessed regarding its efficacy for burn protection. A pig model was developed with a flame infliction instrument specially designed for this experiment. This pneumatic tool can initiate eight simultaneous flame injuries where the distance of skin from burn source and exposure time are adjustable. In the study, 1,000 degrees C, 5-second exposure flame burns were inflicted upon anesthetized pigs. Full-thickness injuries were caused to exposed animals or to animals that were protected by the single layer of old type Nomex protective garments. On day 21, the original burn size diminished to 42.3% +/- 6.3% and 41.2% +/- 7.9%, respectively. When the animals were dressed with the new type of air-cooled Nomex, only small and superficial burns could be detected when the air compressor was operating, and moderate burns were demonstrated when the compressor was not working. On day 21, postburn original burn size was diminished to 1.9% +/- 1.9% and to 17% +/- 6.5%, respectively. Quantitative burn wound histology followed the same trends with almost normal skin architecture after 7 days in the air-inflated new garments, moderate pathology, and an advanced wound healing process in the affected area when the compressor was not working and severe damage with only initial wound healing in the exposed skin or the areas that were protected by old type, single-layered fireproof garments. This new type of air-cooled fireproof garment was significantly better than the old garment under the experiment condition and seems to be very promising in burn prevention among tank crewmen.

Human papillomavirus types detected in skin warts and cancer differ in their transforming properties but commonly counteract UVB induced protective responses in human keratinocytes.

In the present study, E6E7 and E6 proteins of human papillomaviruses (HPVs) associated with skin warts and cancer were compared for their transforming and carcinogenic abilities in primary human keratinocytes (PHKs). We show that E6E7 of cancer associated beta HPV types, notably 49 and 24, were able to extend the life span and enhance the clonogenic efficiency of PHKs when maintained in serum free/low calcium medium. Activities of the beta HPV E6E7 were lower than those of HPV16 E6E7. In contrast, E6 proteins from HPV types detected in skin warts or cancer, notably 10, 49 and 38, attenuated UVB induced protective responses in PHKs including cell death, proliferation arrest and accumulation of the proapoptotic proteins, p53, bax or bak. Together, this investigation revealed functional differences and commonalities between HPVs associated with skin warts and cancer, and allowed the identification of specific properties of beta HPVs supporting their involvement in skin carcinogenesis.

Comparison of topical iodine and silver sulfadiazine as therapies against sulfur mustard burns in a pig model.

Sulfur mustard (SM) is a powerful vesicant used as an agent of chemical warfare. The severity of lesions incurred after exposure to SM reiterated the need for an efficient and rapid neutralizing agent against SM. Previous studies have shown that postexposure treatment with iodine is effective against SM lesions in rodents. In the current study we used the pig model to emulate SM-induced burn lesions, and observed the immediate effect of a single dose of iodine formulation treatment on these burns. SSD, a common agent recommended for use in both chemical and thermal burns was used as control. Results indicated that 1.27 mg of SM caused deep lesions and histopathological changes in the pig skin as scored in the biopsies obtained. A single application of an iodine formulation 20 minutes from exposure to SM exhibited no protective action on the skin as evident in the biopsies obtained 1, 3, 5, 10, and 21 days after treatment. SSD treatment induced the least protective action. The SSD-treated wounds also took the longest to heal. Attempts to neutralize the SM action with iodine compounds were not successful in the pig model. Currently, other compounds are being investigated. Attention must be drawn to the adverse effect of SSD on SM-induced wounds. Further studies must be initiated to elucidate this phenomenon.