The human genetic variant rs6190 unveils Foxc1 and Arid5a as novel pro-metabolic targets of the glucocorticoid receptor in muscle.

Genetic variations in the glucocorticoid receptor (GR) gene NR3C1 can impact metabolism. The single nucleotide polymorphism (SNP) rs6190 (p.R23K) has been associated in humans with enhanced metabolic health, but the SNP mechanism of action remains completely unknown. We generated a transgenic knock-in mice genocopying this polymorphism to elucidate how the mutant GR impacts metabolism. Compared to non-mutant littermates, mutant mice showed increased muscle insulin sensitivity and strength on regular chow and high-fat diet, blunting the diet-induced adverse effects on weight gain and exercise intolerance. Overlay of RNA-seq and ChIP-seq profiling in skeletal muscle revealed increased transactivation of Foxc1 and Arid5A genes by the mutant GR. Using adeno-associated viruses for in vivo overexpression in muscle, we found that Foxc1 was sufficient to transcriptionally activate the insulin response pathway genes Insr and Irs1. In parallel, Arid5a was sufficient to transcriptionally repress the lipid uptake genes Cd36 and Fabp4, reducing muscle triacylglycerol accumulation. Collectively, our findings identify a muscle-autonomous epigenetic mechanism of action for the rs6190 SNP effect on metabolic homeostasis, while leveraging a human nuclear receptor coding variant to unveil Foxc1 and Arid5a as novel epigenetic regulators of muscle metabolism.

Myofibroblasts Are Not Characteristic Features of Keloid Lesions.

Keloids are disfiguring, scar-like lesions that are challenging to treat, with low response rates to current interventions and frequent recurrence. It has been widely reported that keloids are characterized by myofibroblasts, specialized contractile fibroblasts that express alpha-smooth muscle actin (α-SMA). However, evidence supporting a role for myofibroblasts in keloid pathology is inconclusive, with conflicting reports in the literature. This complicates development of more effective therapies, as the benefit of interventions targeting myofibroblasts is unclear. This study was undertaken to determine whether myofibroblasts can be considered characteristic of keloids. Methods: Myofibroblasts in tissue sections from keloids, hypertrophic scars (HTSs), and normal skin were localized by α-SMA immunostaining. Expression of α-SMA mRNA (ACTA2 gene) in normal skin and keloid tissue, and in fibroblasts from normal skin, keloid, and HTSs, was measured using quantitative polymerase chain reaction. Results: Normal skin did not exhibit α-SMA-expressing myofibroblasts, but myofibroblasts were identified in 50% of keloids and 60% of HTSs. No significant differences in ACTA2 expression between keloid and normal skin tissue were observed. Mean ACTA2 expression was higher in HTS (2.54-fold, P = 0.005) and keloid fibroblasts (1.75-fold, P = 0.046) versus normal fibroblasts in vitro. However, α-SMA expression in keloids in vivo was not associated with elevated ACTA2 in keloid fibroblasts in vitro. Conclusions: Despite elevated ACTA2 in cultured keloid fibroblasts, myofibroblast presence is not a consistent feature of keloids. Therefore, therapies that target myofibroblasts may not be effective for all keloids. Further research is required to define the mechanisms driving keloid formation for development of more effective therapies.

Engineering redox sensors into CHO cells enables near-real-time quantification of intracellular redox in bioprocesses.

The production of biologics that treat complex diseases, such as cancer, autoimmune, and infectious disease, requires careful monitoring and control of cell cultures. While bioprocess optimizations have dramatically improved production yields, a lack of analytical tools has made it challenging to identify accompanying intracellular improvements. Intracellular redox can diminish the growth and productivity of biologics-producing cells and adversely impact product quality profiles yet characterizing redox is challenging due to its complex and highly transient nature. In this study, we integrated a fluorescent thiol-based redox biosensor to monitor intracellular redox in one bisAb- and two monoclonal antibody-producing clonal cell lines in a 14-day fed-batch bioreactor. We characterized biosensor functionality using three fluorescence measurement techniques and determined sensor oxidation correlates with the intracellular ratio of reduced (GSH) and oxidized glutathione (GSSG), an important cellular antioxidant. Our fed-batch bioreactor studies showed that sensor expression minimally affected bioprocess outcomes, including growth, productivity, product quality attributes, or intracellular redox attributes, including mitochondrial reactive oxygen species and total cellular GSH levels in all cell lines tested. Biosensor measurements taken throughout the culture revealed that the intracellular environment in these cell lines became more reduced throughout the culture, with the exception of a high pH condition which became more oxidized. Our results demonstrate the potential of using biosensors to monitor intracellular changes in near-real-time with minimal process effects, thus potentially improving future bioprocess optimizations.

Fractional CO2 laser micropatterning of cell-seeded electrospun collagen scaffolds enables rete ridge formation in 3D engineered skin.

Rete ridges are interdigitations of the epidermis and dermis of the skin that play multiple roles in homeostasis, including enhancing adhesion via increased contact area and acting as niches for epidermal stem cells. These structures, however, are generally absent from engineered skin (ES). To develop ES with rete ridges, human fibroblast-seeded dermal templates were treated with a fractional CO2 laser, creating consistently spaced wells at the surface. Constructs with and without laser treatment were seeded with keratinocytes, cultured for 10 days, and grafted onto athymic mice for four weeks. Rete-ridge like structures were observed in the laser-patterned (ridged) samples at the time of grafting and were maintained in vivo. Ridged grafts displayed improved barrier function over non-lasered (flat) grafts at the time of grafting and 4 weeks post-grafting. Presence of ridges in vivo corresponded with increased keratinocyte proliferation, epidermal area, and basement membrane length. These results suggest that this method can be utilized to develop engineered skin grafts with rete ridges, that the ridge pattern is stable for at least 4 weeks post-grafting, and that the presence of these ridges enhances epidermal proliferation and establishment of barrier function. STATEMENT OF SIGNIFICANCE: Rete ridges play a role in epidermal homeostasis, enhance epidermal-dermal adhesion and act as niches for epidermal stem cells. Despite their role in skin function, these structures are not directly engineered into synthetic skin. A new method to rapidly and reproducibly generate rete ridges in engineered skin was developed using fractional CO2 laser ablation. The resulting engineered rete ridges aided in the establishment of epidermal barrier function, basement membrane protein deposition and epidermal regeneration. This new model of engineered skin with rete ridges could be utilized as an in vitro system to study epidermal stem cells, a testbed for pharmaceutical evaluation or translated for clinical use in full-thickness wound repair.

Design and Production of Bispecific Antibodies.

With the current biotherapeutic market dominated by antibody molecules, bispecific antibodies represent a key component of the next-generation of antibody therapy. Bispecific antibodies can target two different antigens at the same time, such as simultaneously binding tumor cell receptors and recruiting cytotoxic immune cells. Structural diversity has been fast-growing in the bispecific antibody field, creating a plethora of novel bispecific antibody scaffolds, which provide great functional variety. Two common formats of bispecific antibodies on the market are the single-chain variable fragment (scFv)-based (no Fc fragment) antibody and the full-length IgG-like asymmetric antibody. Unlike the conventional monoclonal antibodies, great production challenges with respect to the quantity, quality, and stability of bispecific antibodies have hampered their wider clinical application and acceptance. In this review, we focus on these two major bispecific types and describe recent advances in the design, production, and quality of these molecules, which will enable this important class of biologics to reach their therapeutic potential.

Role of Early Application of Pressure Garments following Burn Injury and Autografting.

BACKGROUND: Pressure garment therapy, used for reduction of postburn scarring, is commonly initiated after complete healing of the wound or autograft. Although some clinicians have suggested that earlier treatment may improve outcomes, the effect of early initiation of therapy has not been studied in a controlled environment. METHODS: Full-thickness burns were created on red Duroc pigs, burn eschar was excised, and the wound bed was grafted with split-thickness autografts. Grafts were treated with pressure garments immediately, 1 week (early), or 5 weeks (delayed) after grafting with nontreated grafts as controls. Scar morphology, biomechanics, and gene expression were measured at multiple time points up to 17 weeks after grafting. RESULTS: Grafts that received pressure within 1 week after grafting exhibited no reduction in engraftment rates. Immediate and early application of pressure resulted in scars with decreased contraction, reduced scar thickness, and improved biomechanics compared with controls. Pressure garment therapy did not alter expression of collagen I, collagen III, or transforming growth factor ß1 at the time points investigated; however, expression of matrix metalloproteinase 1 was significantly elevated in the immediate pressure garment therapy group at week 3, whereas the delayed pressure garment therapy and control groups approached baseline levels at this time point. CONCLUSIONS: Early application of pressure garments is safe and effective for reducing scar thickness and contraction and improving biomechanics. This preclinical study suggests that garments should be applied as soon as possible after grafting to achieve greatest benefit, although clinical studies are needed to validate the findings in humans.

Effects of early combinatorial treatment of autologous split-thickness skin grafts in red duroc pig model using pulsed dye laser and fractional CO2 laser.

BACKGROUND AND OBJECTIVE: The use of pulsed dye laser (PDL) and fractional CO2 (FX CO2 ) laser therapy to treat and/or prevent scarring following burn injury is becoming more widespread with a number of studies reporting reduction in scar erythema and pruritus following treatment with lasers. While the majority of studies report positive outcomes following PDL or FX CO2 therapy, a number of studies have reported no benefit or worsening of the scar following treatment. The objective of this study was to directly compare the efficacy of PDL, FX CO2 , and PDL + FX CO2 laser therapy in reducing scarring post burn injury and autografting in a standardized animal model. MATERIALS AND METHODS: Eight female red Duroc pigs (FRDP) received 4 standardized, 1 in. x 1 in. third degree burns that were excised and autografted. Wound sites were treated with PDL, FX CO2 , or both at 4, 8, and 12 weeks post grafting. Grafts receiving no laser therapy served as controls. Scar appearance, morphology, size, and erythema were assessed and punch biopsies collected at weeks 4, 8, 12, and 16. At week 16, additional tissue was collected for biomechanical analyses and markers for inflammatory cytokines, extracellular matrix (ECM) proteins, re-epithelialization, pigmentation, and angiogenesis were quantified at all time points using qRT-PCR. RESULTS: Treatment with PDL, FX CO2 , or PDL + FX CO2 resulted in significantly less contraction versus skin graft only controls with no statistically significant difference among laser therapy groups. Scars treated with both PDL and FX CO2 were visually more erythematous than other groups with a significant increase in redness between two and three standard deviations above normal skin redness. Scars treated with FX CO2 were visually smoother and contained significantly fewer wrinkles. In addition, hyperpigmentation was significantly reduced in scars treated with FX CO2 . CONCLUSIONS: The use of fractional carbon dioxide or pulsed dye laser therapy within 1 month of autografting significantly reduced scar contraction versus control, though no statistically significant difference was detected between laser modalities or use of both modalities. Overall, FX CO2 therapy appears to be modestly more effective at reducing erythema, and improving scar texture and biomechanics. The current data adds to prior studies supporting the role of laser therapy in the treatment of burn scars and indicates more study is needed to optimize delivery protocols for maximum efficacy. Lasers Surg. Med. 50:78-87, 2018. © 2017 Wiley Periodicals, Inc.

Inflammatory responses, matrix remodeling, and re-epithelialization after fractional CO2 laser treatment of scars.

BACKGROUND AND OBJECTIVE: Fractional CO2 laser therapy has been used to improve scar pliability and appearance; however, a variety of treatment protocols have been utilized with varied outcomes. Understanding the relationship between laser power and extent of initial tissue ablation and time frame for remodeling could help determine an optimum power and frequency for laser treatment. The characteristics of initial injury caused by fractional CO2 laser treatment, the rates of dermal remodeling and re-epithelialization, and the extent of inflammation as a function of laser stacking were assessed in this study in a porcine scar model. MATERIALS AND METHODS: Full-thickness burn wounds were created on female Red Duroc pigs followed by immediate excision of the eschar and split-thickness autografting. Three months after injury, the resultant scars were treated with a fractional CO2 laser with 70 mJ of energy delivered as either a single pulse or stacked for three consecutive pulses. Immediately prior to laser treatment and at 1, 24, 96, and 168 hours post-laser treatment, transepidermal water loss (TEWL), erythema, and microscopic characteristics of laser injury were measured. In addition, markers for inflammatory cytokines, extracellular matrix proteins, and re-epithelialization were quantified at all time points using qRT-PCR. RESULTS: Both treatments produced erythema in the scar that peaked 24 hours after treatment then decreased to basal levels by 168 hours. TEWL increased after laser treatment and returned to normal levels between 24 and 96 hours later. Stacking of the pulses did not significantly increase the depth of ablated wells or extend the presence of erythema. Interleukin 6 and monocyte chemoattractant protein-1 were found to increase significantly 1 hour after treatment but returned to baseline by 24 hours post laser. In contrast, expression of transforming growth factor ß1 and transforming growth factor ß3 increased slowly after treatment with a more modest increase than interleukin 6 and monocyte chemoattractant protein-1. CONCLUSIONS: In the current study, the properties of the ablative zones were not directly proportional to the total amount of energy applied to the porcine scars with the use of triple stacking, resulting in only minor increases to microthermal zone (MTZ) depth and width versus a single pulse. Re-epithelialization and re-establishment of epidermal barrier function were observed in laser treated scars by 48 hours post therapy. Finally, many of the inflammatory genes up-regulated by the laser ablation returned to baseline within 1 week. As a whole, these results suggest that microthermal zones created by FXCO2 treatment re-epithelialize rapidly with the inflammatory response to the laser induced injury largely resolved within 1 week post treatment. Further study is needed to understand the relationship between laser stacking and MTZ properties in human scars in order to evaluate the clinical applicability of the stacking technique. Lasers Surg. Med. 49:675-685, 2017. © 2017 Wiley Periodicals, Inc.

Soluble Epoxide Hydrolase Inhibition and Epoxyeicosatrienoic Acid Treatment Improve Vascularization of Engineered Skin Substitutes.

BACKGROUND: Autologous engineered skin substitutes comprised of keratinocytes, fibroblasts, and biopolymers can serve as an adjunctive treatment for excised burns. However, engineered skin lacks a vascular plexus at the time of grafting, leading to slower vascularization and reduced rates of engraftment compared with autograft. Hypothetically, vascularization of engineered skin grafts can be improved by treatment with proangiogenic agents at the time of grafting. Epoxyeicosatrienoic acids (EETs) are cytochrome P450 metabolites of arachidonic acid that are inactivated by soluble epoxide hydrolase (sEH). EETs have multiple biological activities and have been shown to promote angiogenesis. Inhibitors of sEH (sEHIs) represent attractive therapeutic agents because they increase endogenous EET levels. We investigated sEHI administration, alone or combined with EET treatment, for improved vascularization of engineered skin after grafting to mice. METHODS: Engineered skin substitutes, prepared using primary human fibroblasts and keratinocytes, were grafted to full-thickness surgical wounds in immunodeficient mice. Mice were treated with the sEHI 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), which was administered in drinking water throughout the study period, with or without topical EET treatment, and were compared with vehicle-treated controls. Vascularization was quantified by image analysis of CD31-positive areas in tissue sections. RESULTS: At 2 weeks after grafting, significantly increased vascularization was observed in the TPPU and TPPU + EET groups compared with controls, with no evidence of toxicity. CONCLUSIONS: The results suggest that sEH inhibition can increase vascularization of engineered skin grafts after transplantation, which may contribute to enhanced engraftment and improved treatment of full-thickness wounds.

Deep and superficial keloid fibroblasts contribute differentially to tissue phenotype in a novel in vivo model of keloid scar.

BACKGROUND: Keloids are thick fibrous scars that are refractory to treatment and unique to humans. The lack of keloid animal models has hampered development of effective therapies. The authors' goal was to develop an animal model of keloids using grafted engineered skin substitutes composed of keloid-derived cells. To demonstrate the model's utility, differences between deep and superficial keloid fibroblasts were investigated. METHODS: Engineered skin substitutes were prepared using six combinations of cells: 1, normal keratinocytes and normal fibroblasts; 2, normal keratinocytes and deep keloid fibroblasts; 3, normal keratinocytes and superficial keloid fibroblasts; 4, keloid keratinocytes and normal fibroblasts; 5, keloid keratinocytes and deep keloid fibroblasts; and 6, keloid keratinocytes and superficial keloid fibroblasts. Engineered skin substitutes stably grafted to athymic mice were evaluated for wound area, thickness, and gene expression. RESULTS: Deep keloid fibroblasts displayed elevated expression of type 1 collagen alpha 1 (COL1A1), transforming growth factor ß-1, periostin, plasminogen activator inhibitor 2, and inhibin beta A compared with superficial keloid fibroblasts and normal fibroblasts. After grafting, engineered skin substitutes in group 5 were significantly thicker than controls and had increased COL1A1 expression. Engineered skin substitutes in group 6 showed significantly increased area. Histologic analysis revealed abnormal collagen organization in engineered skin substitutes containing deep keloid fibroblasts or superficial keloid fibroblasts. CONCLUSIONS: Aspects of the phenotypes of engineered skin substitutes prepared with keloid cells are analogous to thickening and spreading of human keloid scars. Therefore, use of keloid engineered skin substitutes is a valuable new tool for the study of keloid scarring.

Engineered human skin substitutes undergo large-scale genomic reprogramming and normal skin-like maturation after transplantation to athymic mice.

Bioengineered skin substitutes can facilitate wound closure in severely burned patients, but deficiencies limit their outcomes compared with native skin autografts. To identify gene programs associated with their in vivo capabilities and limitations, we extended previous gene expression profile analyses to now compare engineered skin after in vivo grafting with both in vitro maturation and normal human skin. Cultured skin substitutes were grafted on full-thickness wounds in athymic mice, and biopsy samples for microarray analyses were collected at multiple in vitro and in vivo time points. Over 10,000 transcripts exhibited large-scale expression pattern differences during in vitro and in vivo maturation. Using hierarchical clustering, 11 different expression profile clusters were partitioned on the basis of differential sample type and temporal stage-specific activation or repression. Analyses show that the wound environment exerts a massive influence on gene expression in skin substitutes. For example, in vivo-healed skin substitutes gained the expression of many native skin-expressed genes, including those associated with epidermal barrier and multiple categories of cell-cell and cell-basement membrane adhesion. In contrast, immunological, trichogenic, and endothelial gene programs were largely lacking. These analyses suggest important areas for guiding further improvement of engineered skin for both increased homology with native skin and enhanced wound healing.

Expression of genes encoding antimicrobial proteins and members of the toll-like receptor/nuclear factor-kappaB pathways in engineered human skin.

Skin functions as a first line of defense against microbial invasion. Tissue-engineered cultured skin substitutes (CSS) are used to aid wound closure in massively burned patients, and have been used to facilitate safe and effective wound closure in adult patients with chronic wounds. Although they contain only two cell types at grafting, they can potentially contribute to innate defense against pathogens and stimulation of adaptive immunity. Gene microarrays were used to identify expression in cultured skin of genes involved in innate and adaptive immune responses, and to evaluate the effects of cytokine stimulation on expression levels. Cultured skin expressed multiple antimicrobial protein genes, including human beta defensins 1 and 2 and S100A12. In addition, the antiviral gene APOBEC3G, which was not previously identified in skin, was expressed in CSS and up-regulated by interleukin-1alpha and tumor necrosis factor alpha. Cathelicidin was not expressed in unstimulated CSS, but was induced by cytokine treatment. Further, genes encoding several proinflammatory cytokines and members of the toll-like receptor and nuclear factor kappa B pathways were expressed in CSS, suggesting that cells in CSS can mediate activation of inflammatory responses. The observed expression patterns indicate that engineered human skin utilizes innate defense mechanisms similar to those reported for native skin. Therefore, regulation of these pathways by cytokine stimulation may offer a mechanism for increasing innate immunity in CSS to combat wound infection after grafting onto patients.