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.

Connexin 43 regulates the expression of wound healing-related genes in human gingival and skin fibroblasts.

Fibroblasts are the most abundant connective tissue cells and play an important role in wound healing. It is possible that faster and scarless wound healing in oral mucosal gingiva relative to skin may relate to the distinct phenotype of the fibroblasts residing in these tissues. Connexin 43 (Cx43) is the most ubiquitous Cx in skin (SFBLs) and gingival fibroblasts (GFBLs), and assembles into hemichannels (HCs) and gap junctions (GJs) on the cell membrane. We hypothesized that SFBLs and GFBLs display distinct expression or function of Cx43, and that this may partly underlie the different wound healing outcomes in skin and gingiva. Here we show that Cx43 distinctly formed Cx43 GJs and HCs in human skin and gingiva in vivo. However, in SFBLs, in contrast to GFBLs, only a small proportion of total Cx43 assembled into HC plaques. Using an in vivo-like 3D culture model, we further show that the GJ, HC, and channel-independent functions of Cx43 distinctly regulated wound healing-related gene expression in GFBLs and SFBLs. Therefore, the distinct wound healing outcomes in skin and gingiva may partly relate to the inherently different assembly and function of Cx43 in the resident fibroblasts.

Elevated CD26 Expression by Skin Fibroblasts Distinguishes a Profibrotic Phenotype Involved in Scar Formation Compared to Gingival Fibroblasts.

Compared to skin, wound healing in oral mucosa is faster and produces less scarring, but the mechanisms involved are incompletely understood. Studies in mice have linked high expression of CD26 to a profibrotic fibroblast phenotype, but this has not been tested in models more relevant for humans. We hypothesized that CD26 is highly expressed by human skin fibroblasts (SFBLs), and this associates with a profibrotic phenotype distinct from gingival fibroblasts (GFBLs). We compared CD26 expression in human gingiva and skin and in gingival and hypertrophic-like scar-forming skin wound healing in a pig model, and used three-dimensional cultures of human GFBLs and SFBLs. In both humans and pigs, nonwounded skin contained abundantly CD26-positive fibroblasts, whereas in gingiva they were rare. During skin wound healing, CD26-positive cells accumulated over time and persisted in forming hypertrophic-like scars, whereas few CD26-positive cells were present in the regenerated gingival wounds. Cultured human SFBLs displayed significantly higher levels of CD26 than GFBLs. This was associated with an increased expression of profibrotic genes and transforming growth factor-ß signaling in SFBLs. The profibrotic phenotype of SFBLs partially depended on expression of CD26, but was independent of its catalytic activity. Thus, a CD26-positive fibroblast population that is abundant in human skin but not in gingiva may drive the profibrotic response leading to excessive scarring.

Transgenic mice overexpressing CD109 in the epidermis display decreased inflammation and granulation tissue and improved collagen architecture during wound healing.

Transforming growth factor-ß (TGF-ß) is a multifunctional growth factor involved in all aspects of wound healing. TGF-ß accelerates wound healing, but an excess of its presence at the wound site has been implicated in pathological scar formation. Our group has recently identified CD109, a glycophosphatidylinositol-anchored protein, as a novel TGF-ß coreceptor and inhibitor of TGF-ß signaling in vitro. To determine the effects of CD109 in vivo on wound healing, we generated transgenic mice overexpressing CD109 in the epidermis. In excisional wounds, we show that CD109 transgenic mice display markedly reduced macrophage and neutrophil recruitment, granulation tissue area, and decreased Smad2 and Smad3 phosphorylation, whereas wound closure remains unaffected as compared with wild-type littermates. Futhermore, we demonstrate that the expression of the proinflammatory cytokines interleukin-1α and monocyte chemoattractant protein-1, and extracellular matrix components is markedly decreased during wound healing in CD109 transgenic mice. In incisional wounds, CD109 transgenic mice show improved dermal architecture, whereas the tensile strength of the wound remains unchanged. Taken together, our findings demonstrate that CD109 overexpression in the epidermis reduces inflammation and granulation tissue area and improves collagen organization in vivo.

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.

Exploring scarless healing of oral soft tissues.

Our research group is comparing clinical, histological and molecular healing profiles of oral and skin wounds using human and pig models. The goal is to determine the molecular cues that lead to scarless healing in the oral mucosa and use that information to develop scar prevention therapies for skin and prevent aberrant wound healing in the oral cavity. Wound healing in human and pig palatal mucosa is almost identical, and scar formation is reduced in oral wounds compared with skin. The striking difference between these tissues is transient and rapidly resolving inflammation in oral wounds compared with long-lasting inflammation in the skin wounds. Currently, we are looking at wound transcriptomes (genes differentially regulated) and proteomes (a set of proteins) to investigate how these wound healing responses in skin and oral mucosa are regulated at the molecular level.

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.

Scarless healing of oral mucosa is characterized by faster resolution of inflammation and control of myofibroblast action compared to skin wounds in the red Duroc pig model.

BACKGROUND: Scar formation following skin trauma can have devastating consequences causing physiological and psychosocial concerns. Currently, there are no accepted predictable treatments to prevent scarring which emphasizes a need for a better understanding of the wound healing and scar formation process. OBJECTIVES: Previously it was shown that healing of small experimental wounds in the oral mucosa of red Duroc pigs results in significantly reduced scar formation as compared with equivalent full-thickness skin wounds. In the present study, scar formation was assessed in 17 times larger experimental wounds in both oral mucosa and skin of the red Duroc pigs. METHODS: Equivalent experimental wounds were created in the oral mucosa and dorsal skin of red Duroc pigs, and scar formation, localization and abundance of key wound healing cells, transforming growth factor-beta (TGF-beta) and phosphorylated Smad3 (pSmad3) were assessed. RESULTS: Oral mucosal wounds displayed significantly less clinical and histological scar formation than did the corresponding skin wounds. The number of macrophages, mast cells, TGF-beta and pSmad3 immunopositive cells was significantly reduced in the oral mucosal wounds as compared with skin wounds during the maturation stage of the healing process. Although the number of myofibroblasts was significantly elevated, the oral mucosal wounds showed significantly less contraction than did the skin wounds over time. CONCLUSIONS: Earlier resolution of the inflammatory reaction and reduced wound contraction may promote scarless oral mucosal wound healing. In addition, scar formation likely depends not only on the number of myofibroblasts but also on the extracellular environment which regulates their function.

Wound healing in oral mucosa results in reduced scar formation as compared with skin: evidence from the red Duroc pig model and humans.

Scar formation is a common, unwanted result of wound healing in skin, but the mechanisms that regulate it are still largely unknown. Interestingly, wound healing in the oral mucosa proceeds faster than in skin and clinical observations have suggested that mucosal wounds rarely scar. To test this concept, we created identical experimental wounds in the oral mucosa and skin in red Duroc pigs and compared wound healing and scar development over time. We also compared the pig oral mucosal wound healing to similar experimental wounds created in human subjects. The findings showed significantly reduced scar formation at both clinical and histological level in the pig oral mucosa as compared with skin 49 days after wounding. Additionally, the skin scars contained a significantly increased number of type I procollagen immunopositive cells and an increased fibronectin content, while the oral mucosal wounds demonstrated a prolonged accumulation of tenascin-C. Furthermore, the pig oral mucosal wounds showed similar molecular composition and clinical and histological scar scores to human oral mucosal wounds. Thus, the reduced scar formation in the pig oral mucosa provides a model to study the biological processes that regulate scarless wound healing to find novel approaches to prevent scar formation in skin.

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.