Dose-dependent reduction of somatic expansions but not Htt aggregates by di-valent siRNA-mediated silencing of MSH3 in HdhQ111 mice.

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by CAG trinucleotide repeat expansions in exon 1 of the HTT gene. In addition to germline CAG expansions, somatic repeat expansions in neurons also contribute to HD pathogenesis. The DNA mismatch repair gene, MSH3, identified as a genetic modifier of HD onset and progression, promotes somatic CAG expansions, and thus presents a potential therapeutic target. However, what extent of MSH3 protein reduction is needed to attenuate somatic CAG expansions and elicit therapeutic benefits in HD disease models is less clear. In our study, we employed potent di-siRNAs to silence mouse Msh3 mRNA expression in a dose-dependent manner in HdhQ111/+ mice and correlated somatic Htt CAG instability with MSH3 protein levels from simultaneously isolated DNA and protein after siRNA treatment. Our results reveal a linear correlation with a proportionality constant of ~ 1 between the prevention of somatic Htt CAG expansions and MSH3 protein expression in vivo, supporting MSH3 as a rate-limiting step in somatic expansions. Intriguingly, despite a 75% reduction in MSH3 protein levels, striatal nuclear HTT aggregates remained unchanged. We also note that evidence for nuclear Msh3 mRNA that is inaccessible to RNA interference was found, and that MSH6 protein in the striatum was upregulated following MSH3 knockdown in HdhQ111/+ mice. These results provide important clues to address critical questions for the development of therapeutic molecules targeting MSH3 as a potential therapeutic target for HD.

A MicroRNA-29 Mimic (Remlarsen) Represses Extracellular Matrix Expression and Fibroplasia in the Skin.

MicroRNA-29 (miR-29) negatively regulates fibrosis and is downregulated in multiple fibrotic organs and tissues, including in the skin. miR-29 mimics prevent pulmonary fibrosis in mouse models but have not previously been tested in the skin. This study aimed to identify pharmacodynamic biomarkers of miR-29 in mouse skin, to translate those biomarkers across multiple species, and to assess the pharmacodynamic activity of a miR-29b mimic (remlarsen) in a clinical trial. miR-29 biomarkers were selected based on gene function and mRNA expression using quantitative reverse transcriptase polymerase chain reaction. Those biomarkers comprised multiple collagens and other miR-29 direct and indirect targets and were conserved across species; remlarsen regulated their expression in mouse, rat, and rabbit skin wounds and in human skin fibroblasts in culture, while a miR-29 inhibitor reciprocally regulated their expression. Biomarker expression translated to clinical proof-of-mechanism; in a double-blinded, placebo-randomized, within-subject controlled clinical trial of single and multiple ascending doses of remlarsen in normal healthy volunteers, remlarsen repressed collagen expression and the development of fibroplasia in incisional skin wounds. These results suggest that remlarsen may be an effective therapeutic to prevent formation of a fibrotic scar (hypertrophic scar or keloid) or to prevent cutaneous fibrosis, such as scleroderma.

A synthetic microRNA-92a inhibitor (MRG-110) accelerates angiogenesis and wound healing in diabetic and nondiabetic wounds.

There is a strong unmet need for new therapeutics to accelerate wound healing across both chronic and acute indications. It is well established that local tissue hypoxia, vascular insufficiency, and/or insufficient angiogenesis contribute to inadequate wound repair in the context of diabetic foot ulcers as well as to other chronic wounds such as venous stasis and pressure ulcers. microRNA-92a-3p (miR-92a) is a potent antiangiogenic miRNA whose inhibition has led to increases in angiogenesis in multiple organ systems, resulting in an improvement in function following myocardial infarction, limb ischemia, vascular injury, and bone fracture. Due to their pro-angiogenic effects, miR-92a inhibitors offer potential therapeutics to accelerate the healing process in cutaneous wounds as well. This study investigated the effect of a development stage locked nucleic acid-modified miR-92a inhibitor, MRG-110, in excisional wounds in db/db mice and in normal pigs. In both acute and chronic wounds, MRG-110 increased granulation tissue formation as assessed by histology, angiogenesis as assessed by immunohistochemistry and tissue perfusion, and wound healing as measured by time to closure and percent closure over time. The effects of MRG-110 were greater than those that were observed with the positive controls rhVEGF-165 and rhPDGF-BB, and MRG-110 was at least additive with rhPDGF-BB when co-administered in db/db mouse wounds. MRG-110 was found to up-regulate expression of the pro-angiogenic miR-92a target gene integrin alpha 5 in vitro in both human vascular endothelial cells and primary human skin fibroblasts and in vivo in mouse skin, demonstrating its on-target effects in vitro and in vivo. Additional safety endpoints were assessed in both the mouse and pig studies with no safety concerns noted. These studies suggest that MRG-110 has the potential to accelerate both chronic and acute wound healing and these data provide support for future clinical trials of MRG-110.

ΔNp63α utilizes multiple mechanisms to repress transcription in squamous cell carcinoma cells.

ΔNp63α is a potent oncogene in squamous cell carcinomas (SCCs) and a pro-proliferative factor expressed by basal epithelial cells. ΔNp63α functions both as a transcriptional repressor and activator, but it is not clear how these activities contribute to its oncogenic potential. ΔNp63α was proposed to function as a dominant negative of the related factor p53. Additionally, ΔNp63α was shown to inactivate its family member TAp73 and mediate recruitment of repressive histone deacetylase (HDAC) complexes to chromatin. Recently, we identified a new mechanism of repression involving recruitment of histone H2A/H2A.Z exchange complexes and H2A.Z deposition at ΔNp63α target genes. Here, we aimed to define the possible co-occurrence of the various repressive mechanisms. In lung SCC cells expressing ΔNp63α, p53 and TAp73, we found that ΔNp63α exerts its pro-proliferative and transcriptional repressive effects in a manner independent of p53, TAp73 and histone H3 and H4 deacetylation. Instead, ΔNp63α target genes are differentiated from non-target genes within the p53 network by incorporation and accumulation of acetylated H2A.Z. These results indicate that ΔNp63α utilizes multiple mechanisms of repression in diverse epithelial and SCC cells.

ΔNp63α represses anti-proliferative genes via H2A.Z deposition.

ΔNp63α is a member of the p53 family of transcription factors that functions as an oncogene in squamous cell carcinomas (SCCs). Because ΔNp63α and p53 bind virtually identical DNA sequence motifs, it has been proposed that ΔNp63α functions as a dominant-negative inhibitor of p53 to promote proliferation and block apoptosis. However, most SCCs concurrently overexpress ΔNp63α and inactivate p53, suggesting the autonomous action of these oncogenic events. Here we report the discovery of a novel mechanism of transcriptional repression by ΔNp63α that reconciles these observations. We found that although both proteins bind the same genomic sites, they regulate largely nonoverlapping gene sets. Upon activation, p53 binds all enhancers regardless of ΔNp63α status but fails to transactivate genes repressed by ΔNp63α. We found that ΔNp63α associates with the SRCAP chromatin regulatory complex involved in H2A/H2A.Z exchange and mediates H2A.Z deposition at its target loci. Interestingly, knockdown of SRCAP subunits or H2A.Z leads to specific induction of ΔNp63α-repressed genes. We identified SAMD9L as a key anti-proliferative gene repressed by ΔNp63α and H2A.Z whose depletion suffices to reverse the arrest phenotype caused by ΔNp63α knockdown. Collectively, these results illuminate a molecular pathway contributing to the autonomous oncogenic effects of ΔNp63α.

The p53 circuit board.

The p53 tumor suppressor is embedded in a large gene network controlling diverse cellular and organismal phenotypes. Multiple signaling pathways converge onto p53 activation, mostly by relieving the inhibitory effects of its repressors, MDM2 and MDM4. In turn, signals originating from increased p53 activity diverge into distinct effector pathways to deliver a specific cellular response to the activating stimuli. Much attention has been devoted to dissecting how the various input pathways trigger p53 activation and how the activity of the p53 protein itself can be modulated by a plethora of co-factors and post-translational modifications. In this review we will focus instead on the multiple configurations of the effector pathways. We will discuss how p53-generated signals are transmitted, amplified, resisted and eventually integrated by downstream gene circuits operating at the transcriptional, post-transcriptional and post-translational levels. We will also discuss how context-dependent variations in these gene circuits define the cellular response to p53 activation and how they may impact the clinical efficacy of p53-based targeted therapies.

Epithelial regulation of mesenchymal tissue behavior.

Fibroproliferative scars are an important clinical problem, and yet the mechanisms that regulate scar formation remain poorly understood. This study explored the hypothesis that the epithelium has a critical role in dictating scar formation, and that these interactions differ in skin and mucosa. Paired skin and vaginal mucosal wounds on New Zealand white (NZW) rabbits diverged significantly; the cutaneous epithelium exhibited a greater and prolonged response to injury when compared with the mucosa. Microarray analysis of the injured epithelium was performed, and numerous factors were identified that were more strongly upregulated in skin, including several proinflammatory cytokines and profibrotic growth factors. Analysis of the underlying mesenchymal tissue demonstrated a fibrotic response in the dermis of the skin but not the mucosal lamina propria, in the absence of a connective tissue injury. To determine if the proinflammatory factors produced by the epidermis may have a role in dermal fibrosis, an IL-1 receptor antagonist was administered locally to healing skin wounds. In the NZW rabbit model, blockade of IL-1 signaling was effective in preventing hypertrophic scar formation. These results support the idea that soluble factors produced by the epithelium in response to injury may influence fibroblast behavior and regulate scar formation in vivo.

Occlusion regulates epidermal cytokine production and inhibits scar formation.

Hypertrophic scars are a major clinical problem, yet there are few therapeutics available to prevent or treat scar formation. One of the oldest known and most effective treatments is occlusion with silicone gel. However, little is known about its mode of action. It is hypothesized that occlusion increases the hydration state of the epidermis, and that this affects the epidermal and dermal cell behavior. This study investigated this possibility. Using the rabbit hypertrophic scar model, we determined that occlusion increased the hydration state of the epidermis in a dose-dependent manner, and significantly reduced the scar hypertrophy. Quantitative reverse transcription-polymerase chain reaction and immunohistochemistry showed that occlusion altered keratinocyte behavior, including keratin expression. Furthermore, occlusion significantly decreased the epidermal expression of the profibrotic cytokine interleukin-1beta and increased the epidermal expression of the antifibrotic cytokine tumor necrosis factor alpha. These alterations in the epidermal gene expression resulted in concomitant changes in the expression of the transforming growth factor-beta family members by cells in the dermis, resulting in a decrease in profibrotic signaling within the dermis. In summary, the results of this study indicate that occlusive therapy was able to decrease dermal fibrosis by hydrating the epidermis and altering the pro- and antifibrotic signals produced following injury.

Biomechanical behavior of scar tissue and uninjured skin in a porcine model.

A new method to test axial and transverse tensile properties of skin was developed to improve our understanding of skin mechanical behavior, and how it changes following injury and formation of a scar. Skin tissue was evaluated at 70 days following full-thickness wounding in juvenile female pigs (N=14). Samples were taken in the axial (cranial-caudal) and transverse (dorsal-ventral) directions, for both scar tissue and uninjured skin, and were evaluated mechanically in vitro using a protocol of stress relaxation followed by tensile failure. Uninjured skin was more compliant, with a larger toe-in region, and faster load relaxation, in the axial direction than the transverse. Such directional differences were not present in high-load responses, such as linear stiffness or failure properties. When compared with uninjured skin, scars displayed a similar linear stiffness, with considerably reduced failure properties, and reduced low-load compliance. Scars showed no directional differences in low-load behavior, viscous response, or failure properties. These findings suggest morphological changes that may occur with injury that are consistent with the viscoelastic and directional changes observed experimentally. This improved understanding of how injury affects skin biomechanical function provides valuable information necessary for the design of successful grafting procedures and tissue-engineered skin replacements.

Expression of integrin alphavbeta6 and TGF-beta in scarless vs scar-forming wound healing.

Oral mucosal wounds heal with reduced scar formation compared with skin. The epithelial integrin alphavbeta6 is induced during wound healing, and it can activate fibrogenic transforming growth factor beta1 (TGF-beta1) and anti-fibrogenic TGF-beta3 that play key roles in scar formation. In this study, expression of beta6 integrin and members of the TGF-beta pathway were studied in experimental wounds of human gingiva and both gingiva and skin of red Duroc pigs using real-time PCR, gene microarrays, and immunostaining. Similar to human wounds, the expression of beta6 integrin was induced in the pig wounds 7 days after wounding and remained upregulated >49 days. The alphavbeta6 integrin was colocalized with both TGF-beta isoforms in the wound epithelium. Significantly higher expression levels of beta6 integrin and TGF-beta1 were observed in the pig gingival wounds compared with skin. Early gingival wounds also expressed higher levels of TGF-beta3 compared with skin. The spatio-temporal colocalization of alphavbeta6 integrin with TGF-beta1 and TGF-beta3 in the wound epithelium suggests that alphavbeta6 integrin may activate both isoforms during wound healing. Prolonged expression of alphavbeta6 integrin along with TGF-beta3 in the gingival wound epithelium may be important in protection of gingiva from scar formation.

The mast cell stabilizer ketotifen prevents development of excessive skin wound contraction and fibrosis in red Duroc pigs.

Skin wound healing in Yorkshire pigs closely approximates human wound healing. Conversely, red Duroc pigs form fibroproliferative, hypercontractile scars. As mast cells have been implicated in several fibrotic conditions, the present study used these models to evaluate the potential role of mast cells in wound contraction and fibrosis. Immediately following the creation of full-thickness excisional wounds, the mast cell stabilizer ketotifen was used to treat both Yorkshire and red Durocs. Control red Durocs showed significantly more wound contraction than Yorkshires, both before and after reepithelialization. Ketotifen treatment significantly reduced the first phase of contraction in red Duroc wounds to a level equivalent to Yorkshire wounds, but had no detectable effect on the postepithelialization phase of contraction. Cessation of drug treatment after 10 weeks did not lead to resumption of excessive contraction in red Durocs, indicating that ketotifen blocked rather than delayed such contraction during a critical phase of healing. Ketotifen treatment also reduced the deposition of collagen within the red Duroc wounds, but did not affect Yorkshire wound contraction or collagen deposition. These results suggest that ketotifen may be an effective treatment for the reduction of excessive wound contraction and fibrosis in human cutaneous injuries, without affecting the normal healing process.

Dermal fibroblasts from red Duroc and Yorkshire pigs exhibit intrinsic differences in the contraction of collagen gels.

Previous studies have shown that the Yorkshire (Y) pig is a model for normal skin wound healing, while red Duroc (RD) pigs form hypercontracted scars similar to human hypertrophic scars. In order to determine potential intrinsic differences in fibroblast phenotypes, the ability of normal dorsal and ventral dermal fibroblasts from Y and RD pigs to contract collagen gels was assessed. Cells plated in gels were cultured in media supplemented with 2% or 10% FBS +/- 1 or 10 ng/mL transforming growth factor beta1. The degree of contraction of the gels was quantified at defined time-points postrelease. Final contraction levels were dependent on cell density and serum concentration for all cell types. The rates of contraction of RD dorsal fibroblasts were significantly greater than those for Y dorsal fibroblasts. Immunocytochemical analysis revealed the presence of alpha-smooth muscle actin in contracted cells. Furthermore, mRNA levels for matrix metalloproteinase-2 and decorin showed specific increases for the RD cells during contraction. These findings have revealed intrinsically different, location-specific in vitro responses with normal dermal fibroblasts from the two breeds of pig, suggesting that the abnormal skin healing phenotype of RD pigs may be attributable in part to intrinsic genetic differences in fibroblasts between the breeds.

Genetic involvement in skin wound healing and scarring in domestic pigs: assessment of molecular expression patterns in (Yorkshire x Red Duroc) x Yorkshire backcross animals.

Yorkshire, red Duroc, and F1 (first-generation cross) pigs heal with normal, fibroproliferative/hypercontractile, and intermediate levels of scarring, respectively. The purpose of this study was to evaluate the healing phenotype of Yorkshire x F1 backcross animals, to address the molecular basis for genetic transmission of the red Duroc scarring phenotype. Macroscopically and histologically, full-thickness wounds on backcross animals followed the Yorkshire phenotype, with one exception; the backcross wounds exhibited contraction following re-epithelialization. The molecular expression patterns in the backcross animals generally correlated with the macroscopic and histologic findings. Compared to Yorkshire, red Duroc, and F1 wounds, the backcross wounds demonstrated a diminished initial inflammatory phase, followed by a prolonged expression of several relevant growth factors. Additionally, collagen expression was prolonged, expression of matrix metalloproteinases was increased, and alterations in tissue inhibitor of metalloproteinase expression were detected. Moreover, a subset of molecules still followed the red Duroc pattern of mRNA expression, a finding that allows for correlations between the scarring phenotype and the molecular expression patterns to be made in this model. The results indicate that a number of genes are likely involved in the red Duroc healing phenotype and that identification of the specific genes involved will require a more detailed genomic analysis.

Genetic analysis of skin wound healing and scarring in a porcine model.

Contraction is a normal part of skin wound healing and wound closure; however, excessive contraction and severe scarring concern patients and physicians alike. The present study has investigated the degree and kinetics of wound contraction in a porcine model of wound healing, to elucidate the genetic and molecular basis for abnormal skin wound healing and scarring. Healing of excisional skin wounds in juvenile female Yorkshire pigs closely resembled normal healing in humans. In contrast, identical wounds in female red Duroc pigs contracted significantly more, forming hypercontracted, hyperpigmented scars. Yorkshire x red Duroc F1 animals healed without hyperpigmentation, but with significantly greater wound contraction than observed in either parent breed. To examine the genetic transmission of the hypercontractile phenotype, all F1 animals were bred to a single Yorkshire boar, generating 20 backcross animals. All backcross animals healed with significantly less contraction than the normal Yorkshire animals. These findings suggest that the genetic contribution to scar phenotype in this animal model is complex, with a limited number of major genes controlling wound contraction, and an unknown number of minor genes that appear to modulate the impact of the major genes.

Comparison of in vitro disc diffusion and time kill-kinetic assays for the evaluation of antimicrobial wound dressing efficacy.

There is a plethora of new silver-containing dressings on the market today. Various manufacturers attempt to show that their dressings are the most efficacious and therefore should be preferentially employed by health care workers based on the results of their in vitro tests. However, there have been no studies that clearly identify which tests are appropriate for comparison purposes. The purpose of this study was to determine which in vitro test is most appropriate for evaluating the antimicrobial efficacy of silver-containing dressings. This was done by testing seven silver-containing dressings and two non-silver-containing topical agents against 17 clinically relevant microorganisms using zone of inhibition assays and time-kill kinetic assays in complex media. The results for the two assays were then correlated to determine whether the methods generated similar results. It was determined that the two methods do not correlate at all. This is most likely a result of the silver interacting with the media in the zone of inhibition test, thus invalidating the results of this test. We therefore conclude that zone of inhibition data generated for silver-containing dressings is of little value when assessing antimicrobial efficacy and that time-kill assays are of greater use.

Cytokine and growth factor mRNA expression patterns associated with the hypercontracted, hyperpigmented healing phenotype of red duroc pigs: a model of abnormal human scar development?

BACKGROUND: Skin wounds in red Duroc pigs heal with the formation of hypercontractile, hyperpigmented scars, similar in some respects to human hypertrophic scars. OBJECTIVE: The goal of this study was to characterize the mRNA expression patterns for a subset of relevant cytokines, growth factors, receptors, and transcription factors involved in the red Duroc scarring phenotype. METHODS: Full-thickness and deep dermal wounds were created on the backs of juvenile female red Duroc pigs. Samples were taken every two weeks postwounding and total RNA and DNA were extracted and quantified. RT-PCR was performed using porcine gene-specific primers for 15 relevant molecules. RESULTS: The majority of molecules examined exhibited a biphasic pattern of expression, with peaks of expression at days 14 and 56 postinjury. CONCLUSIONS: The molecular expression pattern observed correlates well with the gross healing phenotype and matrix molecule expression patterns previously reported in red Duroc pigs. These findings enhance our understanding of the processes associated with fibroproliferative scar-formation.