bluuebunny/crossref_metadata_2025 huggingface dataset:
035 Leptin Plays a Key Role in the Modulation of Wound Angiogenesis
Authors: Nath A.K. , Murad A. , Cha S.T. , Sierra‐Honigmann M.R.
Published: March 2007

Leptin is a pleiotropic cytokine constitutively expressed in adipose tissue and upregulated by hypoxia in sites of tissue injury and in the placenta. Leptin has been demonstrated to play a role in normal and pathological wound healing and it is gene is acutely expressed in experimental wounds. Angiogenesis is among the most salient and well‐documented biological actions of leptin. Therefore it was hypothesized that leptin may play in the modulation of wound neovascularization. Using a murine model of experimental wounds the modulation of wound angiogenesis was investigated by treatment of the wounds to either recombinant leptin or a neutralizing anti‐leptin antibody. The parameters measured were: a)woundblood flow using laser Doppler imaging; b)vesseldensity by counting the number of CD‐31 positive vessels in wound sections; c)angiogenic transcriptionalprofiling using quantitative real‐time RT‐PCR; and d)validation of transcriptionalprofiling by immunohistochemistry and immunoblotting. The treatment of wounds with leptin had a marked effect in wound blood flow that was most significant at 24hours post‐wounding with average increase of 55% with respect to the controls. The effect diminished significantly by 72 hours (24%) and became negligible thereafter. Leptin also accelerate wound neovascularization by 24 hours where the number of small caliber vessels averaged 30/mm2 in leptin treated vessels and 13/mm2 in the controls (n = 10; P < 0.005). However, the number of vessels measured by 72 hours not significantly different between controls and leptin‐treated wounds. In addition transcriptional profiling reveled changes in gene expression of molecules related to angiogenesis such as VEGF, PlGF, Tie‐1, Tie‐2, Flt‐1, Flk‐1, FGF‐2 and endothelial markers like CD‐31, endoglin and eNOS. Overall, time course analysis throughout the healing process showed that leptin treatment induced an early shift in the expression of many of the analyzed transcripts. The most salient changes could be observed as early as 6 hours post‐wounding and included: TSP‐1 (+8.7); FLK‐1(+9); Ang‐2 (+18); CD31(+5.3); endoglin (+7.6); eNOS (+4.2); FGF‐2 (+4.3); etc. Leptin mRNA was also significantly increased (+10) as well as the long‐form of its receptor ObR‐b (+9). Conversely, when wounds were exposed to anti‐leptin antibodies, healing progression was impaired and changes in the transcriptional profile showed diminished expression of several genes related to wound neovascularization. Some examples by 24 hours: Endoglin(−2.5); Flk‐1(−2.8); eNOS (−5.1); Ang‐2 (−5.1); CD31(−3); VEGF‐A (−3.6). In conclusion, leptin had and early and significant effect in modulating wound neovascularization. The effect of treatment of wounds with exogenous leptin did not elicit hypervascularization of the wound and appeared to only accelerate what appeared to be a self‐limited process of wound angiogenesis.

Funded by a Cedars‐Sinai grant from the Skirball‐Kenis Center for Plastic and Reconstructive Surgery.
038 Adenoviral overexpression of MCP‐2 induces macrophages and improves wound healing in diabetic rats
Authors: Shi Y. , Liu J. , Bittner M. , Goppelt A. , Davidson J.M.
Published: March 2007

The recruitment of leukocyte subsets is mainly driven by chemokines. Monocyte chemotactic protein‐2 (MCP‐2) belongs to the CC subfamily of chemokines, and it is especially a chemoattractant for monocytes. Of all CC subfamily members only MCP‐2 interacts with multiple receptors, and its N‐terminal truncated isoform is a natural chemokine inhibitor. Gene expression profiling in excisional wounds showed MCP‐2 was down‐regulated 40% in wild type mice and 69.2% in diabetic mice at 24 h after wounding. To determine the biological response to MCP‐2 during wound healing, we constructed an adenoviral vector (Ad‐MCP‐2). Ad‐MCP‐2 infected cell conditioned medium contained 460 ng/ml secreted MCP‐2 (ELISA) and had chemotactic activity similar to recombinant full‐length MCP‐2 but not to MCP‐2 N‐terminal 8–13aa peptides in a THP‐1cell migration assay. In an incisional wound model in STZ induced diabetic rats, the breaking strength of Ad‐MCP‐2 infected skin wounds increased by 43%(108 PFU, p < 0.05) and 30%(107 PFU, p < 0.05) at 7d after Ad‐MCP‐2 injection compared with Ad‐LacZ control. The wound closure strength of 107 PFU Ad‐MCP‐2 infected wounds still increased by 21%(p < 0.05) at 10d after injection. In normal rats, the breaking strength of Ad‐MCP‐2 and Ad‐Lacz infected wounds was not significantly different. In rats, we injected either Ad‐MCP‐2 or Ad‐LacZ (108 PFU) into each PVA sponge at 3d after implantation. Histological analyses revealed numerous ED‐1 positive macrophages infiltrating in the experimental granulation tissue at 7d after Ad‐MCP‐2 injection. Our data provide evidence that MCP‐2 improved wound healing in diabetic rats, and the recruitment of macrophages into the wound granulation tissues was the one of the mechanisms.
041 Aged Rats Display Increased Ischemia‐Reperfusion Skin Injury in the in vivo Magnet Model
Authors: Rosenberg David S. , Lu Leonard , Gurjala Anandev , Mustoe Thomas A.
Published: March 2007

Introduction: Ischemia‐reperfusion (IR) injury is commonly associated with numerous pathologies including pressure sores, venous stasis ulcers, and lower extremity diabetic ulcers. The purpose of this study is to further investigate the relationship between age and ischemia‐reperfusion skin injury in a rat model utilizing magnets for the purpose of injury creation. Methods: Magnets were designed for subcutaneous placement and calibrated such that a second magnet placed externally over them would cause compression that exceeds capillary perfusion pressure (ischemia). Removing the external magnet results in reperfusion of the skin. After placing subcutaneous magnets in aged and young Fisher 344 rats, repeated cycles of external magnet placement and removal were performed.  Results: Visual analysis of the skin revealed statistically significant greater areas of injury in the aged rats relative to their younger counterparts (37.4 ± 13.3% vs. 24.1 ± 14.8%, P < .02) Conclusions: Aged rats demonstrate an increased degree of injury relative to their younger counterparts in response to ischemia‐reperfusion injury. Future studies will attempt to delineate differences in the markers of IR injury (such as myeloperoxidase and vitamin E levels) in aged versus young rats, giving insight to the mechanisms responsible for the impaired wound healing seen in the elderly.
045 Adenoviral HTERT Transfection Results in Altered Phospho‐erk Activity in Human Dermal Fibroblasts
Authors: Lu Leonard , Rosenberg David S. , Mogford Jon E. , Mustoe Thomas A.
Published: March 2007

Introduction: Telomeres are nucleoprotein structures at the ends of each chromosome. Due to the inability of DNA polymerase to replicate the full length of the chromosome, up to 50–200 base pairs of the telomere are lost during each successive round of cell division. In adult human somatic cells, telomerase is not active resulting in progressive loss of telomere length and entry into replicative senescence as observed in cell culture. hTERT is the catalytic subunit of telomerase, an enzyme which maintains telomere length. Transfection of human dermal fibroblasts (HDFs) by hTERT has been shown to reverse the senescent phenotype seen in aging HDFs in vitro. ERK (p44/42) is a MAP kinase which functions as a critical intermediary in the determination of cell growth and differentiation. Activation of ERK occurs through phosphorylation of threonine and tyrosine residues.  Methods: In order to delineate some of the cellular mechanisms by which hTERT functions, we treated adenoviral hTERT (Ad‐hTERT) transfected HDFs with TGFB1, and assayed phosphorylated ERK activity by Western blotting. Results: Ad‐hTERT treated HDFs demonstrated a 2–3 fold increase in phospho‐ERK activity. In addition, our preliminary findings show that Ad‐hTERT transfected HDFs have increased TGFB1, TGFB1‐Receptor I and II, and COL1A1 gene expression by real‐time rtPCR.  Conclusions: Increased phosphor‐ERK activity as well as increased TGFB1, TGFB1‐Receptor I and II, and COL1A1 gene expression is seen in hTERT transfected HDF’s. Further studies will focus on defining other intermediary changes resulting from Ad‐hTERT tranfection.

Funding source: Geron Corporation
048 Retroviral Delivery of A Dominant Negative TGF‐beta Receptor II Mitigates Scar in a Rabbit Model of Scar Hypertrophy
Authors: Reid Russell R. , Roy Nakshatra , Mogford Jon E. , Zimmerman Hannah , Lee Chung , Mustoe Thomas A.
Published: March 2007

Effective blockade of the pluripotent cytokine TGF‐beta as a means of cutaneous scar reduction is a strategy with great potential. This desired effect may be achieved through the overexpression of mutant TGF beta receptors within the wound milieu. Our goal was to examine the effects of dominant negative mutant TGF‐beta receptor II (dnTGFRII) protein expression in a well‐established rabbit ear model of hypertrophic scarring. Serial injections of a retroviral construct encoding a truncated TGFβRII and the marker green fusion protein (pMSCV‐rIIdn‐GFP) were performed in 7mm punch wounds at day 10 and day 14 (two‐day injection group) or day 8, 10, 12 (three‐day injection group) post wounding. Delivery of a null vector (pMSCV‐GFP) at the same time points served as a negative control. Histomorphometric analysis of wounds harvested at day 28 revealed a statistically significant reduction (33%) in the scar elevation index in 2‐day treated and a more modest reduction in SEI (17.5%) in the 3‐day treated arm compared to null‐treated controls. Confocal microscopy confirmed stable transfection of the construct in both peri‐wound tissue as well as rabbit dermal fibroblasts transfected in vitro. Optimization of this novel application in retroviral gene therapy could lead to effective anti‐scarring strategies.
053 Psychological Stress Impairs Healing and Oxygenation in Cutaneous Wounds
Authors: Gajendrareddy PK , Ilangovan G , Kuppusamy P , Horan MP , Eijkelkamp N , Marucha PT
Published: March 2007

Human and animal studies have shown that psychological stress impairs wound healing. Catecholamines released in response to stress could cause vascular changes that would restrict oxygen delivery to the healing wound. Hence, we hypothesized that stress impairs healing by disrupting oxygen balance in the wounds. 6–8 week, female SKH‐1 mice were subjected to stress by confinement in well‐ventilated 50‐ml conical tubes. Stressing was done three days prior to wounding and five days post wounding, during the active cycle of the mice. Wounds were placed using a standard, 3.5 mm‐diameter biopsy punch. Each mouse received two wounds on their dorsum, behind their shoulder blades. Wound oxygenation was measured by EPR oximetry. Gene expression was measured by real time PCR. Phentolamine, an alpha‐adrenergic antagonist was used in conjunction with EPR oximetry to determine catecholamine‐mediated modulation of wound oxygenation during healing. Our results on the effects of stress on wound healing were consistent with previous results in the model, and showed 19%– 30% larger wounds in the stressed animals when compared to controls. Oxygen levels in the wounds showed a 17.49 to 34.38%(p < 0.001) decrease in the stressed animals on days 1 through 5 post wounding. Furthermore, gene expression studies of a hypoxia driven gene, iNOS, showed an increase in expression on days 1 (205%; p < 0.0003), 3(96%; p < 0.04) and 5 (249%; p < 0.0003). eNOS expression increased by 66%(p < 0.02) on day 1 post‐wounding, with no significant difference in nNOS expression. Phentolamine ameliorated the effect of stress on wound oxygenation on day 1 post wounding. These findings show that stress‐impaired healing is associated with reduced tissue oxygen and altered expression of a hypoxia‐induced gene. Furthermoe, the data suggests a role for catecholamines in stress‐induced impairment of wound oxygenation during early healing.
001 A Novel Model for the Assessment of Formulated Enzymatic Debriding Agents
Authors: Shi Lei , Keen Justin , Shroot Braham
Published: March 2007

In order to closely mimic the actual mechanism of wound debridement by an enzymatic debriding agent, a synthetic wound eschar substrate (SWES) has been developed. The SWES is prepared by the enzymatic conversion of fibrinogen to fibrin in the presence of fibrous bovine collagen and elastin, to form an insoluble planar matrix composed of the three major proteins present in wound eschar. Each of the proteinaceous components is uniquely labeled prior to formation of SWES with different dye, and the debridement test is conducted using a Franz Diffusion System. The debriding agent is applied to the SWES mounted on the donor compartment of the cell, and the digested hydrolysate containing dye‐labeled peptide fragments is sampled from the receptor compartment. Accordingly, the progress of digestion is obtained in real time. Since the three proteins are labeled with spectrally distinct dyes, data on the hydrolysis of the substrates can be collected simultaneously. This method facilitates the testing of formulated debriding products in a manner that closely mimics application to a wound and provides a powerful tool for the evaluation of formulated enzymatic debriding agents.
055 The Effect of an Oxygen Emulsion on Collagen Deposition in Second‐Degree Burn Wound Repair
Authors: Li J. , Zhang Y.P. , Zhu L. , Mertz P.M. , Cazzaniga A.L. , Ricotti C. , Zalesky P.J. , Hsu L.C. , Creech J. , Eaglstein W.H. , Davis S.C.
Published: March 2007

A porcine model of second‐degree burn wound was used to evaluate the effect of a newly developed topical oxygen emulsion (TOE) on collagen deposition during wound repair. A total of 6 pigs were used for the study. The burn wounds were treated with air exposure (no treatment), vehicle control or TOE, which contains super‐saturated oxygen and releases oxygen in sustained high level when applied topically. Skin wound samples were collected at days 0, 1, 4, 7, 10, 14 and 21 after wounding. Semi‐quantitative Reverse Transcription and Polymerase Chain Reactions (RT‐PCR) were used to examine the mRNA expressions for type I and type III collagens and matrix metalloprotease‐1 (MMP‐1). RT‐PCR products were run on ethidium bromide gel and analyzed under UV light with Bio‐Rad Gel Document 2000 system. The expression intensity was recorded as the mean gray value. One‐way analysis of variance was used for statistical analysis. The results showed: 1). Higher mRNA expressions of collagen III and MMP‐1 were observed in TOE treatment group compared with air exposure and vehicle control groups. 2). MMP‐1 expression increased shortly after wounding with peak at days 4 and 7. 3). Collagen III expression increased earlier with peak at day 10 and day 14. 4). Collagen I expression level increased later than that of collagen III. Significant increase was seen after day 10 with the highest at day 21. However, there was no significant difference between vehicle control and TOE treatment groups. The data suggests that sustained high level of oxygen release by TOE may promote wound repair through the mechanism of increased expressions of type I and type III collagens. The TOE also increased the expression of MMP‐1, which might accelerate clearing damaged collagens in the earlier phase of wound healing and promote collagen remodeling in the later phase.

This study was supported by grants from DOD/DARPA and the Dermatology Foundation of South Florida.
059 Effects of Two Topical Antimicrobial Agents on Staphylococcus Aureus Biofilms in a Porcine Model
Authors: Davis SC , Cazzaniga AL , Ricotti C , Mertz PM
Published: March 2007

Biofilms are a combination of microorganisms and extrapolysaccharide matrices. Our laboratory has previously demonstrated that biofilms are present in both acute and chronic wounds. Once biofilms are established, phagocytosis and diffusion of antibiotics are impaired thus contributing to antimicrobial resistance due to increased bacterial virulence. The purpose of this study was to determine the efficacy of two topical antimicrobial agents on partial thickness wounds containing Staphylococcus aureus biofilms. All wounds were inoculated with 106 CFU/ml and covered for 48 hrs under a polyurthene film to promote biofilm formation. Wounds were divided into three treatment groups; triple antibiotic ointment (Polymyxin B sulfate, bacitracin zinc, neomycin), mupirocin cream and untreated control. Wounds were treated twice daily. Wounds were cultured for bacterial quantitation at 24, 48, 72, 96, and 120 hrs. Significant reduction in CFU/ml was observed only after several days of treatments. This finding supports the antimicrobial resistance that occurs when bacteria live within biofilms. Our previous studies demonstrated that both of these agents were able to completely eliminate planktonic S. aureus(106) at the early time points. This study demonstrates that when bacterial biofilms are established in wounds there is a longer response time for topical antimicrobial activity suggesting bacterial resistance.
063 The Human Forearm Biopsy Model for Acute Wound Healing
Authors: Serena T. , Li V. W. , Li W. W.
Published: March 2007

Background: The study of wound biology and effects of advanced modalities would benefit from a reliable human model for normal healing. Eaglstein et al. previously suggested that the forearm biopsy may be useful in acute wound healing studies (J Am Acad Dermatol 45:857, 2001). The Wound Healing Cooperative Group (WHCG) here validates this model in healthy individuals treated with topical growth factor therapy.

Methods: In our randomized, double‐blinded study design, 20 normal healthy volunteers underwent four 6 mm biopsies of the flexor surface of both forearms. Biopsy sites were randomly assigned to a control arm (daily bacitracin) or to one of three treatment arms: i) rhPDGF‐BB 0.01% gel (Q.D.), ii) rhPDGF‐BB (Q.O.D.), iii) rhPDGF‐BB (Q.D. × 7 days followed by bacitracin alone daily). The wounds were examined, measured and photographed daily until complete healing was achieved. Adverse events were monitored. Rates of healing and time‐to‐complete closure were measured. Repeat biopsies of a subset of healing wounds were performed for gene microarray analysis.

Results: The forearm biopsy model allowed direct quantitative and qualitative comparisons of acute wound healing outcomes achieved by rhPDGF‐BB regimens versus standard care alone. There were no infectious complications. Subject compliance was excellent with < 3% of visits missed.

Conclusion: The forearm biopsy model has several advantages in the study of acute wounds: it allows for comparison of topical agents in a controlled fashion; studies can be conducted easily with good patient compliance; and the forearm allows for repeat imaging and tissue harvesting for gene profiling during healing.

Acknowledgements: Funding from The Angiogenesis Foundation.
067 Reversal of ε(γ‐Glutamyl) Lysine Cross‐Linking and Down‐Regulation of Fibronectin and Tissue Transglutaminase (tTGase) Activity in Hypertrophic Scars Following Treatment with 0.8% 1,4 DAB 2HCL
Authors: Hardas B. , Dolynchuk K. , Jacob C. , Griffin M. , Panchal C.
Published: March 2007

Hypertrophic scar formation is an unfavorable condition which is difficult to predict, prevent or treat. Although much research has been done on understanding hypertrophic scar formation, the exact underlying molecular mechanism has not been fully elucidated.

Hypertrophic scars younger than 6 months are known to over‐express tTGase. It has been shown that treatment of hypertrophic scars with topical 1, 4 DAB 2HCl inhibited ε(γ‐glutamyl) lysine cross‐linking4.

In the current study, 12 paired scar biopsies, either treated or untreated with 1, 4 DAB 2HCl were examined for the presence of ε(γ‐glutamyl) lysine cross‐linking by fluorescence immunohistochemistry. In situ tTGase enzyme activity, expression of latent tissue TGF‐β binding protein‐1 (LTBP‐1) and fibronectin were also examined. Scars showed a marked reduction of ε(γ‐glutamyl) lysine cross‐linking following treatment with 1, 4 DAB 2HCl. Although the treated samples did not show any change in expression of tTGase, its in situ activity was noticeably reduced. Treated samples also demonstrated down‐regulation of fibronectin and LTBP‐1. Results suggest that topical treatment of hypertrophic scars with 1,4 DAB 2HCl not only reduced ε(γ‐glutamyl) lysine cross‐linking but also reduced tTGase activity, expression of fibronectin and LTBP‐15 which are known to play a role in extracellular matrix storage of transforming grown factor‐β(TGF‐β), responsible for wound healing and scar formation.

4Dolynchuk KN. Wound Rep Reg 1996; 4(1): 16–20.

5Verderio E, Gaudry C et al. J. Histochem 1999; 47(11): 1417–1432.
073 Expression of Caspases 3, 9, and 14 in the Epidermis of Early Wounds
Authors: Kuechle M.K. , Usui M.L. , Underwood R.A.
Published: March 2007

Caspases are a family of at least 14 highly conserved aspartate proteases that are the major effector arm of apoptosis or programmed cell death. Previous immunohistochemical and biochemical studies have implicated two of the family members, caspase 3 and caspase 14, as having a role in the process of epidermal terminal differentiation. Reepithelialization of epidermal defects after wounding requires coordination between signals that initiate growth and migration as well as signals that initiate differentiation. We therefore examined the expression of caspase 3, caspase 9, and caspase 14 in the epidermis of early (up to 48 hour) human wounds. Two excisional wounds were created on the inner arm using a 3 mm biopsy punch and allowed to heal via secondary intention. Wounds were excised at 24 and 48 hours using a 6 mm biopsy punch, placed in OCT, frozen, cryosectioned and immunohistochemically labeled using affinity purified antisera for caspases 3, 9 and 14. At 24 hours, the distribution of caspase 3 and caspase 9 expression was unchanged from non‐wounded skin. Caspase 3 was found in the upper differentiating cells of the granular layer, while caspase 9 was expressed in scattered dendritic cells in the epidermis. At 48 hours, caspase 3 expression was lost in the epithelial tongue, while caspase 9 expression was found in keratinocytes at the tip of the migrating tongue. Caspase 14 expression at 24 hours was noted at the tip of the migrating epithelial tongue, while at 48 hours, the normal expression of caspase 14 in the granular layer was lost. These results suggest that in 48 hour wounds, loss of caspase 3 and 14 expression in the migrating epithelium reflects the activated state of this tissue. The onset of caspase 9 expression at 48 hours in the leading edge of the wound suggests a role for caspase 9 in wound remodeling, as caspase 9 is known to participate in tissue remodeling during development. Characterization of the caspases involved during wound healing may lead to future therapies utilizng caspases or caspase inhibitors in situations of poor or aberrant wound healing.
075 Wound Healing is Delayed in Women and in the Aged: A Potential Role for the HPA Axis
Authors: Marucha PT , Engeland CG , Cacioppo JT
Published: March 2007

Both senescence and psychological stress have been shown to alter numerous immune components including inflammatory processes. However, whether such effects can be dissociated by gender is largely unknown. To determine the effects of gender and age on wound healing, a 3.5 mm round wound was placed under local anesthesia on the hard palates of 88 younger (18–35 years) and 56 older (50+ years) male and female volunteers. Immediately prior to wounding and 15, 30 and 60 min afterwards, blood was drawn from which cortisol and ACTH levels were determined, as both are immunosuppressive. Wounds were photographed and measured daily until healed. Preliminary analyses revealed that older individuals healed 25.6% slower than younger individuals (P < .001), and women healed 14.0% slower than men (P < .05). These results may have involved alterations in HPA activity, as blood work revealed significant differences in baseline hormone levels: cortisol levels were lower in the older group (P < .05), and ACTH levels were lower in females (P < .01). Furthermore, in women only, faster healing rates were related to both increased anxiety at the time of wounding (P < .05), and higher cortisol levels at all time points (P < .01). Moreover, preliminary results indicated that, in women, higher anxiety was also related to lowered release of IL‐1β in the first 24 h. In men these relationships were not evident, although higher ACTH levels were related to faster healing times (P < .01). Taken together, these findings indicate that aging delays oral wound healing and provide evidence for a previously unreported gender difference in wound healing rates in favor of males. Furthermore, the effects of anxiety on wound healing seem to be sexually differentiated and transient activation of the HPA axis, especially in women, appears to be associated with healing rates.
077 Human Mesenchymal Stem Cells Interact with Keratinocytes and Succesfully Accelerate Wound Healing
Authors: Akita Sadanori , Nakagawa Hiroshi , Fukui Masashi , Akino Kozo
Published: March 2007

Human mesenchymal stem cells (hMSCs) obtained from a single donor from an iliac crest, were investigated with cutaneous wound healing models using nude rat, eliminating T cell‐mediated immune reaction to the grafted stem cell of human origin. Co‐culture of the human keratinocytes and the hMSCs revealed tight basal membranous interaction by electron microscopy.

1.5 × 1.5 cm2 dorsal full‐thickness defects including panniculus carnosus of F344/NJCl‐rnu nude rats were covered by bi‐layered porcine‐derived collagen sponge artificial skin substitutes, Pelnac®, Johnson & Johnson, Tokyo, Japan, impregnated with 5 × 106 hMSCs grated to along with 0, 1, 10, or 100 μg of recombinant human basic fibroblast growth factor (bFGF). The tissues were harvested at 3, 7 42 days after grafting. The defects were remarkably epithelialized by 7 days after coverage with artificial skin substitutes and 10 μg of bFGF, while artificial substitutes with no hMSCs or artificial skin substitutes with hMSCs alone did not demonstrate such healing. The architectures of the artificial skin substitutes were integrated by day 42 in hMSCs‐treated groups. The artificial skin substitute at least plays a role in as a template or scaffold of the grafted hMSCs. Up to day 3, the mesenchymal stem cell surface markers such as CD 29 and CD 44 were remained immunopositive in the groups with hMSCs‐treated groups over the reconstructed dermis‐like tissue. By 42 days after grafting, artificial skin substitutes with hMSCs and 10 μg of bFGF demonstrated the total epithelization and the keratinocytes by this treatment exhibited the pan‐cytokeratin of human origin by immunohistochemical expressions. Thus, the hMSCs with bFGF treatment using an artificial skin substitute as a successfully heal. These results suggest that the human mesenchymal stem cells may be utilized for wound coverage together with bFGF and artificial skin substitutes.
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.
094 Fibroblast Transmigration from Collagen into Fibrin/Fibronectin Gels requires Syndecan‐4 Proteoglycan
Authors: Lin Fubao , An Jianqiang , Ren Xiangdong , Doris Greiling , Clark Richard A.F.
Published: March 2007

Fibroblast migration from the peri‐wound collagenous stroma into the fibrin‐laden wound is critical for granulation tissue formation and subsequent healing. Previously we found that fibroblast transmigration from a collagen matrix into a fibrin matrix required the presence of fibronectin (FN). Several cell surface receptors, namely integrins α4β1, α5β1 and αvβ3 were also required for this invasive migration. Here we demonstrated that syndecan‐4, a transmembrane heparan sulfate proteoglycan, known to bind FN at the highly cationic HepII domain is also required for fibroblast invasive migration of a fibrin/FN gel. This conclusion was based on fibroblast migration using two independent means of disrupting syndecan‐4: heparinase degradation of heparan sulfate glycosaminoglycans or suppression of syndecan‐4 core protein with antisense oligodeoxynucleotides. Isolated syndecan‐4 from these fibroblasts bound the Hep II recombinant constructs FN III12–15v > FN III12–15 > FN III12–14 but did not bind the alternatively spliced IIICs (V) domain. Furthermore, we found that platelet‐derived growth factor (PDGF), which is required to stimulate fibroblast migration, markedly increased cell levels of syndecan‐4 core protein in a time and dose dependent fashion. PDGF also induced up‐regulation of syndecan‐4 at transcriptional level as determined by RT‐PCR. These results demonstrate that syndecan‐4 is essential for fibroblast invasive migration into fibrin clot and that PDGF, the stimulus for migration, induces increased syndecan‐4 core protein expression.
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.