bluuebunny/crossref_metadata_2025_split huggingface dataset:
080 Synergistic Effect of TGF Beta 1 and HIF‐1A on the Expression of a Hypoxia‐Responsive Element‐Containing Promoter
Authors: Roy Nakshatra K. , Sull Alan C. , Mustoe Thomas A. 
Published: March 2007

Accumulating data strongly suggests that under hypoxic conditions the adaptive physiological response of the cells involves co‐operation between oxygen sensing and growth factor signals that cause HIF‐1 mediated gene expression. The HIF‐1 has been identified as a central and critical molecule in oxygen sensing and possibly a master switch. Of the many cytokines that have been shown to modulate collagen production, TGFβ1 appears to be crucial as it can sustain stimulation of collagen production as well as autoinduction of its own synthesis. Several genes that are known to be hypoxia inducible are also up‐regulated by TGFβs, and the promoter of TGFβ3, a member of the family, has a hypoxia‐response element (HRE). Recent reports indicate that HIF‐1α physically interacts with Smad3 and that the TGFβ and hypoxia signaling pathways synergize at the transcriptional level to regulate gene expression. To determine if this interaction upregulates gene expression through the HRE element, we have co‐transfected cultured human dermal fibroblasts with the mammalian expression plasmid for HIF‐1α(pCEP1‐HIF‐1α) with a reporter construct 5HRE‐luc, containing a concatemer of five copies of HRE derived from human vascular endothelial growth factor (VEGF), a minimal cytomegalovirus (CMV) promoter and a reporter gene,luciferase.). When treated with 2 ng/ml of TGFβ1 protein, co‐ transfected hypoxic aged and young human dermal fibroblasts showed significant synergistic upregulation of the reporter gene expression. To further confirm the TGFβ‐HIF‐1α interaction we have created a TGFβ1 ‐RNAi construct by subcloning a 19‐nucleotide sequence derived from rabbit in a mammalian expression vector (p‐SUPER) that directs the synthesis of small interfering RNAs (siRNAs). Hypoxic rabbit cultured dermal fibroblasts co‐transfected with TGFβ1‐RNAi and 5HRE‐luc showed down‐regulation of reporter gene expression.As a prerequisite to understanding the biology of physiological and pathological ischemic tissue repair process it is important to delineate the molecular basis of regulation of collagen gene expression in fibroblasts. Each step in the TGFβ signaling cascade is a potential target for highly specific therapy and information about how hypoxic low‐oxygen microenvironments affect the TGFβ cascade could be useful to develop novel therapeutic strategies that will involve agonist and antagonist agents that directly interfere with these steps.
002 Australian Taipan Snake Venom to Synthetic Oxynor for Wound Healing
Authors: Lipps Binie V. 
Published: March 2007

At Ophidia Products, Inc. we have discovered Oxynor a synthetic therapeutic from snake venom for wound healing. The venom of Australian taipan snake (Oxyuranus s. scutellatus) is extremely potent due to the presence of taipoxin. The intact complex molecule of taipoxin having molecular weight 45.6 kDa is composed of three subunits α, β and γ. Fractionation of crude venom by High pressure chromatography (HPLC) using ion exchange column permits separation of the subunits. The mitogenic activity was revealed in non toxic β taipoxin. Non toxic β taipoxin showed mitogenic activity on variety of eukaryotic cells. Its activity as a mitogen extended to wound healing in experimental animals. We identified the active domain for wound healing in β taipoxin. after trypsin digestion were separated on HPLC, resolved into 11 different fragments. Each fragment was tested on PC12 cells to test mitogenic/neurotrophic activity. The fragment which showed the highest activity consisted of 21 amino acids. A synthetic peptide consisting of ten amino acids, from the N‐terminal –– was named Oxynor.Oxynor mimics the biological properties of natural β taipoxin when tested in vitro and in vivo systems. The biological results showed that Oxynor was not toxic at the concentration of 100 μg/ml for PC12 cells and 500 μg/adult mouse. Oxynor was mitogenic to various eukaryotic cells. Oxynor is neurotrophic showing neurite outgrowth on PC12 cells. Growth of human skin fibroblast cells indicates that Oxynor has keratinous activity. Experimentally produced 4 mm punched wounds on the back of mice were treated with β taipoxin 100 μg/mouse or Oxynor at 500 μg/mouse for seven consecutive days. The results showed complete closure of the wounds after six days while the wounds of control treated with PBS were open. Histology examination of the skin around the wounds revealed that treated mice showed re‐epithelization of the epidermis, which looked close to the normal mouse skin biopsy. Whereas the controls showed distortion of epithelium. β taipoxin and Oxynor were tested for in vivo at the concentration of 100 μg/ml in hydrogel vehicle applied once daily to help heal 6 mm ischemic skin wounds in rats. At days 7 and 14 wound treated with β taipoxin were about 15% smaller than the wounds treated with Oxynor. This shows that synthetic Oxynor is as efficacious as natural β taipoxin under the conditions of the test. On increasing the concentration of Oxynor to 500 μg/ml should give equivalent wound closure to 100 μg/ml of β taipoxin. In 1997 the US FDA approved for sale the first recombinant human platelet‐derived growth factor (rhPDGF) for treatment of human non‐healing wounds. Oxynor will be next therapeutic for wound healing Currently, Oxynor is in clinical trials, it has passed toxicity tests.
083 Transforming Growth Factor‐Beta Ligand, Receptor, and SMAD Expression Patterns in Fetal and Adult Keratinocytes
Authors: Lorenz HP , Faudoa R , Longaker MT , Krummel TM 
Published: March 2007

Introduction: The transforming growth factor‐beta (TGF‐ß) cytokine family regulates cellular proliferation, differentiation, and migration with important function during both development and repair. Moreover, the TGF‐ßs are growth inhibitory for keratinocytes, proliferative for fibroblasts, and generally profibrotic during repair. In order to better define the influence of keratinocyte TGF‐ß during these processes, we examined the TGF‐ß isoform, receptor, and signal messenger Smad expression in fetal and postnatal keratinocytes. Methods: Sprague‐Dawley rat keratinocytes were isolated in primary culture from fetal at E15 and E17 (term = E22), newborn, and 6 week old adults. Open fetal rat wounds heal without scar at E17 gestation. Quantitative‐polymerase chain reaction was performed for TGF‐ß1, ‐ß2, and ‐ß3 ligand; TGF‐beta receptor1 and ‐receptor2; Smad3, Smad4, and Smad7 expression. All cells were passage 2 or 3. Results: TGF‐ß1 expression did not change appreciably from E15 to adult age in keratinocytes. TGF‐ß2 and ‐ß3 expression increased (8 fold and 2 fold respectively) from E17 to newborn ages. Overall, the expression of TGF‐ß1 was 5–8 fold greater compared to ‐ß2 and ‐ß3 in both fetal and adult keratinocytes. TGF‐ß receptor1 expression increased 2 fold whereas ‐receptor2 expression showed little change from E17 to adult age. Smad3 expression increased over 50 fold, with Smads 4 and 7 showing a smaller increase from E17 to adult age. Conclusions: The TGF‐beta system has differential expression in fetal compared to postnatal keratinocytes, which suggests function during skin differentiation. TGF‐ß1 expression is relatively greater than ‐ß2 and ‐ß3 in both fetal and adult keratinocytes. However, increases in isoforms ‐ß2 and ‐ß3, receptor1 and the Smads occurs at ages associated with scarring, implying increased function of the pro‐fibrotic TGF‐beta response during ages associated with scarring.
088 Topical Substance P Modulates Inflammatory Responses in Healing Wounds in Nitric Oxide Synthase Knockout Mice
Authors: Muangman P , Tamura RN , Muffley LA , Isik F Frank , Gibran NS 
Published: March 2007

Introduction: Substance P (SP), a proinflammatory neuropeptide released by sensory nerves in response to cutaneous injury, increases nitric oxide (NO) production by target cells. SP upregulates three nitric oxide synthase (NOS) isoforms: neuronal (nNOS), inducible (iNOS) and endothelial (eNOS). Topical SP application improves wound healing kinetics in NOS KO mice. Since nitric oxide may be a key intermediary mediator for SP induced response to injury, this study was designed to determine whether SP promotes inflammatory cell density in early wounds of NOS KO mice. Methods: Mice of each NOS null strain and appropriate background controls (C57BL/6J – i/eNOS control;B6129SF2/J – nNOS control) were anesthetized with intraperitoneal ketamine and xylazine. After shaving, full‐thickness 1.5 × 1.5 sq. cm. dorsal excisional wounds were covered with a semi‐occlusive dressing. Mice were randomly assigned to daily topical infusion of 10−7 M SP or NS onto wound beds. Wounds were harvested at day 3 and 7 (3 mice per group). Cells were isolated by sequential digestion in dispase and hyaluronidase. Cell counts were performed to determine total cell density. Flow cytometry was performed after staining cells with FITC – labeled antibodies against F4/80 (macrophages), CD45 (pan leukocytes), CD31 (endothelial cells) or CD11c (dendritic cells). Results: Total cell number was higher at day 7 post‐wounding than at day 3 (P < 0.05) in both control and NOS null mice. Macrophage, pan‐leukocyte and dendritic cell density was greater in the SP‐treated wounds than in NS‐treated wounds at both day 3 and 7 post‐wounding (P < 0.05) in all NOS KO mice. Whereas SP increased endothelial cell number in nNOS and iNOS mice, SP had no effect on endothelial cell number in the eNOS KO mice (P > 0.05). SP did not affect cell density in background mice at either time point (P > 0.05). Conclusion: SP upregulated inflammatory cell numbers in all NOS null mice suggesting that either SP activates NOS isoforms that are not reduced by targeted deletion or that SP induces inflammatory cell migration by NO‐independent pathways. The lack of SP effect on endothelial cell number in eNOS KO wounds suggests that eNOS may mediate SP‐induced wound angiogenesis.
089 Tensile Strength Deficiency in Skin and Wounds Of Substance P Receptor (NK1R) Knockout Mice
Authors: Carlisle C D , DasGupta J , Ansel J , Davidson J M 
Published: March 2007

Diabetic neuropathy leads to reduced innervation that may in turn diminish the availability of tachykinins such as Substance P (SP). In diabetic mice, exogenous SP has positive effects on wound closure. The role of SP was tested by inactivating the murine SP receptor gene (neurokinin‐1 receptor, NK1R). Age‐matched, NK1R‐/‐ mice and C57/BL6 control mice were each given 2 full thickness 6 mm excisional wounds on their dorsal surface and creation of two dead space wounds by the insertion of two 1 × 3 mm PVA sponge disks under the skin through a 1.5 cm long ventral midline incision. Digital images for wound closure analysis were collected until day 10 when tissues were harvested for biochemical, histological and tensiometric analysis.There was a striking reduction (2.5 fold, p < 0.05) in the tensile strength of intact, unwounded skin of the NK1R‐/‐ mice, and tensile strength of 10 day incisions was likewise reduced (1.8 fold, p < 0.0126). Wound closure rates were also reduced in the knockout mice, but not to a significant level. Sponge granulation tissue had equivalent protein and DNA content at 10 day post‐implantation while collagen content was diminished. Histological examination of excisions, normal skin and sponges showed no readily observable difference in thickness or organization however NK1R‐/‐ mice exhibited visibly less collagen in excisional wounds. The principal dermal effect seen in this gene deletion appears to be the accumulation and organization of connective tissue. Our preliminary results with a non‐diabetic NK1R‐/‐ mouse do not indicate a marked difference in reëpithelization, but instead a significant decrease in biomechanics. The parallel diminution of intact skin and wound strength suggests an intrinsic defect in collagen fiber maturation that may be reflected in ultrastructural organization or covalent crosslinking.
097 Comparative Analysis of HLA‐G Expression by Keratinocytes Derived from Pemphigus Vulgaris Patients vs. Normal Individuals
Authors: Yari F. , Hosseini A. Zavaran , Nemat‐Gorgani M. , Mansouri Parvin , Khorramizadeh M.R. 
Published: March 2007

Background: Human leukocyte antigen‐G (HLA‐G), is a non‐classical HLA class I molecule. Over the past few years, HLA‐G has been considered as a tolerogenic antigen and a tissue protective molecule in inflammatory diseases. Objectives: This study was performed to analyze HLA‐G expression in pemphigus vulgaris (PV) patients and to assess its possible anti‐inflammatory role in skin disorders. Methods: With observing ethical issues, skin biopsies from 4 normal subjects and 3 PV patients were prepared. The epidermal layer of these biopsies was separated using Dispase enzyme treatment. After trypsinization, single cell suspensions of keratinocytes were cultured in MCDB‐153 serum‐free medium. Three weeks supernatants were collected and applied for a sandwich ELISA to detect total soluble isoforms of HLA‐G antigen. Two species of antibodies used for ELISA were made in our laboratory, i.e. anti‐HLA‐G polyclonal and 5E6H7 monoclonal. A reverse transcriptase polymerase chain reaction (RT‐PCR) was developed for pan HLA‐G transcript to assess the regulation level of HLA‐G expression. Results: The data on ELISA experiments showed significant HLA‐G expression by PV kertainocytes (p < 0.001). No traces of HLA‐G production were seen in normal cells. RT‐PCR revealed, however, variation in HLA‐G mRNA expression. These findings indicate that regulation of HLA‐G gene might occur both at transcriptional and translational levels. Conclusions: Collectively, our data show the presence, and hence, advocate the possible protective contribution of HLA‐G isoforms in skin inflammatory processes.
098 Smoking, Atherosclerosis, and MCP‐1 Expression
Authors: Wong Lina , Yuan Hongwei , Martins‐Green Manuela 
Published: March 2007

The inflammatory events of atherosclerosis involve monocytes, endothelial cells, smooth muscle cells, and several cytokines, including chemokines. Atherosclerosis begins with recruitment of circulating monocytes to sites of blood vessel injury. In the vessel wall, these cells are activated to become macrophages that scavenge toxic material such as oxidized LDL and transition into “foam cells” which will eventually lead to plaque formation. Tobacco smoke is known to exacerbate cardiovascular disease but the mechanisms by which this occurs are not known. The chemokine MCP‐1 is involved in recruiting monocytes to the injured blood vessel wall and is commonly found in atherosclerotic plaques. Our data show that both “first‐hand” and “second‐hand” cigarette smoke stimulate MCP‐1 production in cultured primary human aortic endothelial cells (EC), suggesting that cigarette smoke may contribute to plaque formation by stimulating aortic EC to produce MCP‐1 in blood vessels after smoke injury. To test whether MCP‐1 links smoking and cardiovascular disease, we are studying the expression of this molecule in mice that are either transgenic for human ApoB or are null for MCP‐1 or its receptor, CCR2, by exposing the mice to smoke in a way that closely resembles human smoking. Blood samples from the mice were tested for carboxyhemoglobin, triglycerides, LDL and HDL, and MCP‐1 at successive weeks after initiation of smoking and the aortas isolated to analyze for plaque size and MCP‐1 expression in the tissue. The carboxyhemoglobin test shows that the mice are exposed to smoke. Plasma MCP‐1 and HDL levels are reduced in the smoking groups, whereas LDL, triglyceride, and total cholesterol remain unchanged. On one hand, the decrease in circulating HDL levels suggests that the reason smokers develop atherosclerosis may be because the rate of cholesterol clearance is slowed down. On the other hand, a decrease in circulating MCP‐1 level may reflect that this chemokine accumulates in blood vessel walls, conferring stronger ability for attraction of monocytes.This work is supported by TRDRP and Phillip Morris.
100 An Open, Randomised, Explorative, Phase II, Investigation Comparing Enamel Matrix Derivative Proteins (EMD) and Control In Patients With Pressure Ulcer Stage II
Authors: Dierick AM. , Van De Looverbosch Dirk , Heyman Hilde , Van De Looverbosch Dieter 
Published: March 2007

Background: Enamel Matrix Derivative proteins (EMD) have been used as local adjunct to periodontal surgery in more than 600.000 patients. EMD is approved in Europe, USA and Japan as a medical device class III. There have been no negative reports concerning the safety of the EMD. This is the first reported controlled investigation in a series of ongoing investigations on EMD application in chronic wounds and burns. Aim of the study: The investigation was designed as an open randomised investigation with EMD (30 mg/ml) or vehicle control that consists of propylene glycol alginate (PGA) only. Ten patients were planned to be included. Male or female patients were >18 years of age and had had pressure ulcer present for more than 1 month. Patients with controlled diabetes were also included in the study. Patients with sacral pressure ulcer were to be excluded. EMD or vehicle control was applied weekly for up to 8 weeks. All patients received a secondary dressing (Mepilex® Border). Ulcer status and area were measured at week 0, 2, 4, 6, and 8. The primary objective was to measure the wound area and the percentage of viable/non‐viable tissue. The secondary objectives were to evaluate ulcer tissue in biopsies taken before application (baseline) and at last visit. Tissue morphology was evaluated on haematoxylin‐eosin‐stained sections and the presence of EMD, selected growth factors, proliferating cells and myofibroblasts in tissue sections stained with monoclonal antibodies.Regulatory authority and Ethical committee in Belgium approved the investigation. Results: At the time of the abstract submission, results are not final. The histology and immunohistochemical evaluations will be performed November/December 2003.
104 Effect of Connective Tissue Growth Factor on Protein Kinase Expression and Activity in Human Corneal Fibroblasts
Authors: Radhakrishnan S.S. , Blalock T.D. , Schultz G.S. 
Published: March 2007

Purpose: To evaluate expression patterns of protein kinases and protein kinase activities in human corneal fibroblasts treated with connective tissue growth factor (CTGF). Methods: Human corneal fibroblast cultures were grown to confluence and treated with CTGF for 0, 5, and 15 minutes. Cytoplasmic protein extracts were obtained; protein kinase expression and activity arrays were performed using Kinetworks™ analysis (screening for expression of 75 different protein kinases and 31 different phosphoproteins). Further studies using extended time courses of CTGF exposure in corneal fibroblasts (0, 1, 2, 3, 4, 5, 10, 15, 30, and 60 minutes) were performed using immunoblot analysis to detect expression of protein kinase A catalytic subunit (PKA‐cat), and focal adhesion kinase (FAK). All results and were normalized by comparison to beta‐actin. Results: After 5 minutes of exposure to CTGF, levels of active proteins increased for 21 of the 75 kinases analyzed. Some notable protein kinases that were induced include: death‐associated kinase 1, focal adhesion kinase (FAK), G‐protein coupled receptor kinase 2, protein kinase A catalytic subunit (PKA‐cat), protein kinase B alpha, and protein kinase C (ε, μ, and ζ, subunits). Extended time course analysis of PKA‐cat and FAK showed statistically significant increases in expression following CTGF stimulation within 15 minutes. Conclusion: CTGF increased the levels of active protein kinases in human corneal fibroblast cultures, including PKA‐cat and FAK after 5 minutes of exposure. These results further our understanding of the signal transduction mechanism activated by CTGF in corneal fibroblasts. This suggests that CTGF mediates the effects of transforming growth factor beta (TGF‐β) on protein kinase expression and phosphorylation of second messengers in processes such as cell proliferation and collagen synthesis. Acknowledgments: Funding source: NEI 05587
117 Care of the Thermally Injured Breast
Authors: Ortega R.E. , Riina L.H. , Davenport T.A. , Glickman L.T. , Simpson R.L. , Burger K.M. , Hangan D. 
Published: March 2007

Introduction: Thermal injury to the female breast is one of the most challenging aspects of aesthetic burn surgery today. As the ability to provide coverage for large body surface injuries has progressed greatly in recent years; attention can now be directed towards achieving aesthetically pleasing results. Breast reconstruction in the burn patient can be accomplished in several ways. In planning the reconstruction, one must account for gender, age, and stage of breast development at the time of injury. The following is the treatment protocol utilized at our institution. After determining the level of injury the injured area is cleansed and treated with topical anti‐microbial agents such as Silvadene. If the decision is made to excise and graft the injured area one of two algorithms is followed. The first choice involves excising the burn and placing a split thickness skin graft to the area involved. This is done by placing a sheet graft and using aerosolized fibrin sealant to affix it to the wound bed. If the burn involves deeper elements of tissue then a second approach is taken which includes excision of the burn down to the level of fascia with preservation of the breast mounds and the nipple areola complex (NAR). The (NAR) is spared excision and allowed to heal. Reconstruction of the (NAR) can be deferred for a secondary procedure depending upon the response to primary healing. A split thickness skin graft is then applied to the area of injury. Again a sheet graft is preferred and fibrin sealant is utilized to improve graft fixation and contour. We attribute our excellent results to the sheet grafts and fibrin sealant used. It should be noted that the increased vascularity of the breast fat when compared to fat located elsewhere in the body allows the grafts to adhere and survive on this generally difficult to graft surface. Methods: We identified five female patients at our institution over the last 18 months with thermal injuries to the breasts. Each patient was placed into one of the two treatment algorithms. Results: The five patients had excellent outcomes. Breast mounds and symmetry were preserved. Further development of the breast was allowed in each patient. One patient even underwent a breast augmentation after surviving a 50% TBSA injury. Proper use of fibrin sealant and sheet grafts account for the excellent results seen at this institution. Conclusion: Following careful evaluation of the burned female breast cosmetically and functionally acceptable results can be attained when following our institution’s protocol for breast reconstruction in the female burn victim.