Nakshatra K. Roy

Antisense inhibition of connective tissue growth factor (CTGF/CCN2) mRNA limits hypertrophic scarring without affecting wound healing in vivo

Augmented expression of connective tissue growth factor (CTGF/CCN2) is observed in healing wounds and in a variety of fibrotic disorders. It appears to enhance many of the effects of transforming growth factor‐β and has been shown to have independent fibrogenic functions. Despite these observations, its importance to dermal wound healing and the transition from wound to scar remains poorly defined. In this study, we use established rabbit models to evaluate the roles of CTGF in dermal wound healing and hypertrophic scarring. We show that CTGF mRNA demonstrates persistent up‐regulation in hypertrophic scars. Treatment of wounds with antisense oligonucleotides to CTGF has no measurable effect on early wound closure. However, antisense therapy significantly limits subsequent hypertrophic scarring. Inhibition of CTGF is associated with a marked reduction in the number of myofibroblasts in scars and decreased transcription of TIMP‐1 and types I and III collagen. These findings confirm CTGF to be a key mediator of hypertrophic scarring in this model. Its effect on myofibroblasts in this setting suggests a mechanism whereby it plays this role. Its limited participation in early healing implies that it may be a useful and specific target for modulating hypertrophic scarring following injury.

Homeostasis of the epidermal barrier layer: A theory of how occlusion reduces hypertrophic scarring

The mechanism of hypertrophic scar reduction using silicone gel sheeting remains elusive. We hypothesize that the decrease in scar formation is due to occlusion and homeostasis of the barrier layer. Using an established model of hypertrophic scarring, rabbits were divided into four groups and scars were tape‐stripped or occluded with Kelocote, Cavilon, or Indermil, with each rabbit serving as its own internal control. All wounds were harvested on day 28 and examined histologically to measure the scar elevation index (SEI), epithelial thickness, and cellularity. Immunohistochemistry fluorescence was used to quantify inflammation in the dermis. Transepidermal water loss (TEWL) was measured for each occlusive agent and tape stripping. Ultrastructural analysis was performed by electron microscopy. Kelocote, Cavilon, and Indermil all significantly decreased SEI when compared with controls. Each of the occlusive treatments was shown to decrease TEWL while tape stripping increased TEWL. Tape stripping significantly increased the SEI, epithelial thickness, and cellularity. Immunostaining for macrophages showed increased density of inflammatory cells in the tape‐stripped scars. Under electron microscopy, the tape‐stripped wounds displayed extensive inflammation and keratinocyte damage. Both unwounded skin and occlusion‐treated scars did not display these characteristics. In conclusion, hypertrophic scarring was reduced regardless of occlusive method used. Furthermore, repeated disruption of the permeability barrier by tape stripping led to an increase in scarring. Ultrastructural analysis suggests that occluded wounds may be in an advanced state of wound repair. Occlusion may mediate its effects through establishing homeostasis of the epidermal barrier layer.

A novel murine model of cyclical cutaneous ischemia-reperfusion injury

BACKGROUND Increasing evidence points to a principal role of ischemia-reperfusion in the pathogenesis of chronic skin ulceration, including pressure sores, diabetic ulcers, and venous ulcers. An incomplete understanding of this process and the limitations of current animal models of chronic wounds mandate a reproducible model in mice, in which transgenic and knockout technology are continually evolving. MATERIALS AND METHODS A murine model of chronic skin ulceration based on cyclical magnetic compression is presented. Forty-three C57BL/6J mice underwent varying degrees of cyclical compression with defined periods of reperfusion. Injury was measured grossly as regional necrosis, and tissue was harvested for histology, DNA electrophoresis, and reverse transcription polymerase chain reaction. RESULTS Skin necrosis became apparent only 12 h post cycling, and was cycle-responsive and quantitative in cycled subjects. Histopathologic analysis revealed a statistically significant doubling of the leukocyte count in sections from compressed skin versus sham controls. Moreover, apoptotic DNA laddering was evident in post ischemic skin and absent in controls. Real-time PCR analysis revealed a 300-fold higher expression in iNOS mRNA from cyclically compressed skin compared with normal skin: such expression was temporal in nature. CONCLUSIONS A murine model of pressure necrosis, which bears all of the gross, histological, and molecular features of ischemia-reperfusion injury, has been established. Application of this model to the vast number of transgenic mice available will further our understanding of the mechanism of pressure sore development.

High-dose ultraviolet light exposure reduces scar hypertrophy in a rabbit ear model

Background: The effects of ultraviolet light exposure on scar pigmentation are well documented. There is a commonly held belief among physicians that sun exposure may also worsen the appearance of fresh scars and result in excess collagen deposition. However, few studies have documented a relationship between ultraviolet light exposure and hypertrophic scarring. This study sought to evaluate the effect of ultraviolet light exposure on scar hypertrophy in an established rabbit model of cutaneous scarring. Methods: Four 7-mm ulcers were created on the ventral ears of eight rabbits. Starting on postoperative day 15, half of the wounds were exposed to ultraviolet-B radiation daily for either 7 or 14 days. Ultraviolet-B–exposed (n = 16) and control (n = 16) scars were harvested on postoperative day 32 for histologic and reverse-transcriptase polymerase chain reaction analysis. Results: Exposure to ultraviolet-B radiation for 7 or 14 days was associated with a 52 percent (p < 0.01) or 74 percent (p < 0.05) reduction in scar volume, respectively, compared with controls. In wounds subjected to ultraviolet-B radiation for 14 days, collagen type I-α2 mRNA expression was 29 percent lower than in controls (p < 0.05). There was no difference in the mRNA expression of transforming growth factor-β1. Conclusions: These short-term observations demonstrate that ultraviolet-B radiation exposure reduces scar hypertrophy in this clinically relevant animal model. A reduction in collagen production or increase in collagen breakdown may account for this result. However, sunscreen should still be used as primary protection when skin is exposed to direct sunlight.

Alteration of Smad3 signaling in ischemic rabbit dermal ulcer wounds

Impaired reepithelialization is a hallmark of chronic, ischemic wounds; however, the pathogenesis of the delayed reepithelialization in these wounds remains poorly understood. Transforming growth factor β is involved in both the normal and hypoxic wound‐healing response and exogenous overexpression of Smad3, which has been known to accelerate reepithelialization. Recently, it was shown in the rabbit ear dermal ulcer model that Ad‐Smad3 injection enhanced reepithelialization and granulation tissue formation suggesting a positive effect of Smad3 on wound healing. However, little is known about the role of Smad3 in the ischemic wound healing process. In this study, we examined the effect of Smad3 in an ischemic wound model. Ad‐Smad3 or Ad‐LacZ (108 pfu/wound) was injected into either ear of white New Zealand rabbits. Twenty‐four hours later, these ears were rendered ischemic using an established model, and four 7 mm full‐thickness punch wounds were made on each ear. Histological evaluation showed a significant increase in reepithelialization parameters in Ad‐Smad3–transfected wounds (p<0.01). In contrast, granulation tissue parameters were not affected by Smad3 in ischemia. Smad4 and Smad7 mRNA‐expression was not affected by Smad3 overexpression. Connective tissue growth factor protein was up‐regulated under ischemic conditions but was unaffected by Smad3 transfection in both ischemic and nonischemic wounds. Our results suggest an enhancing effect of Smad3 on reepithelialization in an ischemic wound model that, in turn, might provide novel therapeutic options. Furthermore, the lack of alteration of Smad‐dependent intermediates by Smad3 overexpression suggests the activation of Smad‐independent pathways in ischemia.

Use of hypoxia-inducible factor signal transduction pathway to measure O2 levels and modulate growth factor pathways

Tissue PO2 levels are known to directly modulate numerous processes involved in the reparative response to cutaneous tissue injury, including cell differentiation and migration, extracellular matrix synthesis and maturation, and effectiveness of endogenous and exogenous growth factors. Oxygen is therefore likely the critical variable determining the healing capabilities of any tissue. Significant advances in the understanding of cutaneous wound healing progressed with advances in the measurement of tissue PO2, which has advanced over the past several decades from implantable probes to now include molecular tools such as the transcription factor hypoxia inducible factor‐1 (HIF‐1). HIF‐1 modulates the expression of genes that drive the cellular adaptive response to hypoxia and possess the HIF‐1 binding sequence named hypoxia response element within their promoter sequence. Molecular biology techniques are now allowing exploitation of the HIF‐1/hypoxia response element pathway to drive the expression of potential vulnerary ectopic genes. Here we show the utility of the hypoxia response element for hypoxia‐driven expression of the transforming growth factor‐β–signaling component Smad3 in vitro and the in vivo detection of ischemic hypoxia using luciferase. Smad3 is a positive effector of transforming growth factor‐β superfamily signal transduction. Such approaches are the latest evolution of work championed by Hunt and colleagues over the past 4 decades. (WOUND REP REG 2003;11:496–503)

Quantifying tissue level ischemia: Hypoxia response element-luciferase transfection in a rabbit ear model

Ischemia is a common underlying factor in a number of pathologic conditions ranging from cardiac dysfunction to delayed wound healing. Previous efforts have shown the resulting hypoxia activates the hypoxia inducible factor, a transcription factor with signaling effects through an intranuclear hypoxia response element (HRE). We hypothesized that ischemic conditions should activate these hypoxic signaling pathways in a measurable manner. We tested our hypothesis using variations of an established rabbit ear ischemic wound model and an HRE‐luciferase‐reporter gene construct. This plasmid construct was transfected into the ears of young, female New Zealand White rabbits, harvested at day 7 and processed to yield a reactive solution. Luminometry was used to quantify luciferase expression in each solution as a marker for HRE activation in each wound. Quantitative readings of hypoxic signaling as measured by luminescence yielded profound and statistically significant differences between the various ischemic models. Our results suggest that the biologic systems for hypoxic signaling can be used to detect local ischemia. HRE‐luciferase transfection is an effective tool for quantifying the degree of tissue hypoxia. The caudal ischemic rabbit ear model showed significantly higher levels of hypoxia. Use of a validated model that produces sufficient tissue levels of hypoxia is recommended for meaningful study of ischemic wound healing.