Stromal Rigidity Stress Accelerates Pancreatic Intraepithelial Neoplasia Progression and Chromosomal Instability via Nuclear Protein Tyrosine Kinase 2 Localization.

Because the mechanotransduction by stromal stiffness stimulates the rupture and repair of the nuclear envelope in pancreatic progenitor cells, accumulated genomic aberrations are under selection in the tumor microenvironment. Analysis of cell growth, micronuclei, and phosphorylated Ser-139 residue of the histone variant H2AX (γH2AX) foci linked to mechanotransduction pressure in vivo during serial orthotopic passages of mouse KrasLSL-G12D/+;Trp53flox/flox;Pdx1-Cre (KPC) cancer cells in the tumor and in migrating through the size-restricted 3-µm micropores. To search for pancreatic cancer cell-of-origin, analysis of single-cell data sets revealed that the extracellular matrix shaped an alternate route of acinar-ductal transdifferentiation of acinar cells into topoisomerase II α (TOP2A)-overexpressing cancer cells and derived subclusters with copy number amplifications in MYC-PTK2 (protein tyrosine kinase 2) locus and PIK3CA. High-PTK2 expression is associated with 171 differentially methylated CpG loci, 319 differentially expressed genes, and poor overall survival in The Cancer Genome Atlas-Pancreatic Adenocarcinoma cohort. Abolished RGD-integrin signaling by disintegrin KG blocked the PTK2 phosphorylation, increased cancer apoptosis, decreased vav guanine nucleotide exchange factor 1 (VAV1) expression, and prolonged overall survival in the KPC mice. Reduction of α-smooth muscle actin deposition in the CD248 knockout KPC mice remodeled the tissue stroma and down-regulated TOP2A expression in the epithelium. In summary, stromal stiffness induced the onset of cancer cells-of-origin by ectopic TOP2A expression, and the genomic amplification of MYC-PTK2 locus via alternative transdifferentiation of pancreatic progenitor cells is the vulnerability useful for disintegrin KG treatment.

Molecular and Cellular Characterization of Avian Reticulate Scales Implies the Evo-Devo Novelty of Skin Appendages in Foot Sole.

Among amniotic skin appendages, avian feathers and mammalian hairs protect their stem cells in specialized niches, located in the collar bulge and hair bulge, respectively. In chickens and alligators, label retaining cells (LRCs), which are putative stem cells, are distributed in the hinge regions of both avian scutate scales and reptilian overlapping scales. These LRCs take part in scale regeneration. However, it is unknown whether other types of scales, for example, symmetrically shaped reticulate scales, have a similar way of preserving their stem cells. In particular, the foot sole represents a special interface between animal feet and external environments, with heavy mechanical loading. This is different from scutate-scale-covered metatarsal feet that function as protection. Avian reticulate scales on foot soles display specialized characteristics in development. They do not have a placode stage and lack ß-keratin expression. Here, we explore the molecular and cellular characteristics of avian reticulate scales. RNAscope analysis reveals different molecular profiles during surface and hinge determination compared with scutate scales. Furthermore, reticulate scales express Keratin 15 (K15) sporadically in both surface- and hinge-region basal layer cells, and LRCs are not localized. Upon wounding, the reticulate scale region undergoes repair but does not regenerate. Our results suggest that successful skin appendage regeneration requires localized stem cell niches to guide regeneration.

Topological Distribution of Wound Stiffness Modulates Wound-Induced Hair Follicle Neogenesis.

In the large full-thickness mouse skin regeneration model, wound-induced hair neogenesis (WIHN) occurs in the wound center. This implies a spatial regulation of hair regeneration. The role of mechanotransduction during tissue regeneration is poorly understood. Here, we created wounds with equal area but different shapes to understand if perturbing mechanical forces change the area and quantity of de novo hair regeneration. Atomic force microscopy of wound stiffness demonstrated a stiffness gradient across the wound with the wound center softer than the margin. Reducing mechanotransduction signals using FAK or myosin II inhibitors significantly increased WIHN and, conversely, enhancing these signals with an actin stabilizer reduced WIHN. Here, α-SMA was downregulated in FAK inhibitor-treated wounds and lowered wound stiffness. Wound center epithelial cells exhibited a spherical morphology relative to wound margin cells. Differential gene expression analysis of FAK inhibitor-treated wound RNAseq data showed that cytoskeleton-, integrin-, and matrix-associated genes were downregulated, while hair follicular neogenesis, cell proliferation, and cell signaling genes were upregulated. Immunohistochemistry staining showed that FAK inhibition increased pSTAT3 nuclear staining in the regenerative wound center, implying enhanced signaling for hair follicular neogenesis. These findings suggest that controlling wound stiffness modulates tissue regeneration encompassing epithelial competence, tissue patterning, and regeneration during wound healing.

Successful Repigmentation of Full-Thickness Wound Healing in Fraser's Dolphins (Lagenodelphis hosei).

Fraser's dolphins (Lagenodelphis hosei) exhibit the capability to restore nearly normal pigmentation after full-thickness wounding. However, the association among melanocytes, melanin and skin pigmentation during wound healing in cetaceans has yet to be addressed. Here, the number of melanocytes and the distribution of melanocytes and melanin in different-colored skin and different wound-healing stages in Fraser's dolphins were analyzed by using Fontana-Masson staining, immunofluorescence staining and immunohistochemical staining. It was noticed that there was the highest number of melanocytes in dark skin and the lowest number of melanocytes in white skin. The appearance of functional melanocytes and full-melanized neoepidermis was observed in the early stage of wound healing in Fraser's dolphins. Furthermore, the melanocyte number and skin pigmentation and pattern in healed wounds recovered to a similar condition of unwounded skin. This study provides fundamental knowledge of skin repigmentation in cetaceans for further research, and it will be warranted to elucidate the mechanisms of the replenishment of melanocytes and the regulation of melanocyte activity that contribute to the successful repigmentation in cetacean skin wounds.

Defining Wound Healing Progression in Cetacean Skin: Characteristics of Full-Thickness Wound Healing in Fraser's Dolphins (Lagenodelphis hosei).

Cetaceans are tight-skinned mammals that exhibit an extraordinary capacity to heal deep soft tissue injuries. However, essential information of large full-thickness wound healing in cetaceans is still lacking. Here, the stages of full-thickness wound healing were characterized in Fraser's dolphins (Lagenodelphis hosei). The skin samples were collected from normal skin and full-thickness cookiecutter shark (Isistius brasiliensis)-bite wounds of stranded carcasses. We defined five stages of wound healing according to macroscopic and histopathological examinations. Wounds in Stage 1 and 2 were characterized by intercellular and intracellular edema in the epidermal cells near the wound edge, mixed inflammatory cell infiltration, and degradation of collagen fibers. In Stage 3 wounds, melanocytes, melanin granules, rete and dermal ridges were noticed in the neo-epidermis, and the adipose tissue in adjacent blubber was replaced by cells and fibers. Wounds in Stage 4 and 5 were characterized by gradual restoration of the normal skin architecture including rete and dermal ridges, collagen bundles, and adipose tissue. These phenomena were quite different from previous studies in terrestrial tight-skinned mammals, and therefore, further in-depth research into the mechanisms of dolphin wound healing would be needed to gain new insights into veterinary and human regenerative medicine.

Epigenetic silencing of AATK in acinar to ductal metaplasia in murine model of pancreatic cancer.

BACKGROUND: Cancer subtype switching, which involves unclear cancer cell origin, cell fate decision, and transdifferentiation of cells within a confined tumor microenvironment, remains a major problem in pancreatic cancer (PDA). RESULTS: By analyzing PDA subtypes in The Cancer Genome Atlas, we identified that epigenetic silencing of apoptosis-associated tyrosine kinase (AATK) inversely was correlated with mRNA expression and was enriched in the quasi-mesenchymal cancer subtype. By comparing early mouse pancreatic lesions, the non-invasive regions showed AATK co-expression in cells with acinar-to-ductal metaplasia, nuclear VAV1 localization, and cell cycle suppression; but the invasive lesions conversely revealed diminished AATK expression in those with poorly differentiated histology, cytosolic VAV1 localization, and co-expression of p63 and HNF1α. Transiently activated AATK initiates acinar differentiation into a ductal cell fate to establish apical-basal polarization in acinar-to-ductal metaplasia. Silenced AATK and ectopically expressed p63 and HNF1α allow the proliferation of ductal PanINs in mice. CONCLUSION: Epigenetic silencing of AATK regulates the cellular transdifferentiation, proliferation, and cell cycle progression in converting PDA-subtypes.

Regeneration of rete ridges in Lanyu pig (Sus scrofa): Insights for human skin wound healing.

Rete ridges are important to the mechanical function of skin in animals with minimal hair, including humans. As mice do not exhibit rete ridges, the need for a quality animal model is pertinent. Here, we develop a Lanyu pig (Sus scrofa) full-thickness wound model to explore tissue regeneration because the architecture and function are similar to humans and inbred genetic variants are available. Full- and partial-thickness wounds were generated on the dorsum. Full-thickness wounds at post-wound day 57 exhibit severe scar with no signs of wound-induced hair follicle neogenesis. Wound contraction is greater in the anterior/posterior relative to the medial/lateral axis. In wound beds, K14+ cells increased while K10+ , p63+ and PCNA+ cells decreased compared to unwounded tissue. Epithelial ß-catenin is unchanged. The wound bed expresses more ColI, less ColIII and no elastin. Rete ridges do not form after full-thickness wounding, but incompletely regenerate after partial-thickness wounding. An alkaline phosphatase (ALP)+ cell population, not associated with hair follicles, is present at the bottom of the rete ridge basal layer in pig and human unwounded skin. These K5+ /K10- /PCNA- /ALP+ epithelial cells are absent after full-thickness wounding but reappear after partial-thickness wounding, before invagination of new rete ridges. In summary, full-thickness wounding on the dorsum of Lanyu pigs results in scar formation and perturbed molecular expression while partial-thickness wounding permits limited rete ridge and papillary dermis regeneration. Future functional studies and further characterization will help contribute knowledge for the regenerative medicine field.

The tension biology of wound healing.

Following skin wounding, the healing outcome can be: regeneration, repair with normal scar tissue, repair with hypertrophic scar tissue or the formation of keloids. The role of chemical factors in wound healing has been extensively explored, and while there is evidence suggesting the role of mechanical forces, its influence is much less well defined. Here, we provide a brief review on the recent progress of the role of mechanical force in skin wound healing by comparing laboratory mice, African spiny mice, fetal wound healing and adult scar keloid formation. A comparison across different species may provide insight into key regulators. Interestingly, some findings suggest tension can induce an immune response, and this provides a new link between mechanical and chemical forces. Clinically, manipulating skin tension has been demonstrated to be effective for scar prevention and treatment, but not for tissue regeneration. Utilising this knowledge, specialists may modulate regulatory factors and develop therapeutic strategies to reduce scar formation and promote regeneration.

Msx2 Supports Epidermal Competency during Wound-Induced Hair Follicle Neogenesis.

Cutaneous wounds in adult mammals typically heal by scarring. However, large full-thickness wounds undergo wound-induced hair follicle neogenesis (WIHN), a form of regeneration. Here, we show that WIHN requires transient expression of epidermal Msx2 in two phases: the wound margin early and the wound center late. Msx2 expression is present in the migrating epithelium during early wound healing and then presents in the epithelium and mesenchyme later in the wound center. WIHN is abrogated in germline and epithelial-specific Msx2 mutant mice. Unlike the full-length Msx2 promoter, a minimal Msx2 promoter fails activation in the wound center, suggesting complex regulation of Msx2 expression. The Msx2 promoter binding sites include Tcf/Lef, Jun/Creb, Pax3, and three SMAD sites. However, basal epithelial-induced BMP suppression by noggin overexpression did not affect WIHN. We propose that Msx2 signaling is required for the epidermis to acquire spatiotemporal competence during WIHN. Topologically, hair regeneration dominates in the wound center, coinciding with late Msx2 expression. Together, these results suggest that intrinsic Msx2 expression supports epithelial competency during hair follicle neogenesis. This work provides insight into endogenous mechanisms modulating competency of adult epidermal progenitors for mammalian ectodermal appendage neogenesis, and offers the target Msx2 for future regeneration-promoting therapies.

Mechanical forces in skin disorders.

Mechanical forces are known to regulate homeostasis of the skin and play a role in the pathogenesis of skin diseases. The epidermis consists of keratinocytes that are tightly adhered to each other by cell junctions. Defects in keratins or desmosomal/hemidesmosomal proteins lead to the attenuation of mechanical strength and formation of intraepidermal blisters in the case of epidermolysis bullosa simplex. The dermis is rich in extracellular matrix, especially collagen, and provides the majority of tensile force in the skin. Keloid and hypertrophic scar, which is the result of over-production of collagen by fibroblasts during the wound healing, are associated with extrinsic tensile forces and changes of intrinsic mechanical properties of the cell. Increasing evidences shows that stiffness of the skin environment determines the regenerative ability during wound healing process. Mechanotransduction pathways are also involved in the morphogenesis and cyclic growth of hair follicles. The development of androgenetic alopecia is correlated to tensile forces generated by the fibrous tissue underlying the scalp. Acral melanoma predominantly occurs in the weight-bearing area of the foot suggesting the role of mechanical stress. Increased dermal stiffness from fibrosis might be the cause of recessive dystrophic epidermolysis bullosa associated squamous cell carcinoma. Strategies to change the mechanical forces or modify the mechanotransduction signals may lead to a new way to treat skin diseases and promote skin regeneration.

Higher plasma leptin is associated with higher risk of androgenetic alopecia in men.

In addition to genetics and androgens, novel factors could play a role in androgenetic alopecia (AGA). This study aims to investigate the association between plasma leptin level with the risk and severity of AGA in men. Forty-eight subjects were enrolled including 29 AGA and 19 non-AGA subjects. The plasma leptin level was significantly higher in AGA subjects, compared to non-AGA subjects (4.45 vs 2.76 ng/mL, P<.05). A higher plasma leptin levels were positively correlated with the risk of developing AGA in multivariate logistic analysis (odds ratio=2.77, P<.05). Leptin from the circulation might impact the development of AGA.

Novel skin chamber for rat ischemic flap studies in regenerative wound repair.

BACKGROUND: In plastic surgery, skin flap is an important approach to reconstructive wound repairs. The rat dorsal skin flap is a clinically relevant and popular animal model to investigate and evaluate flap survival and necrosis. Nonetheless, flap survival is often unstable with unpredictable outcomes, regardless of previous attempts at design modification. METHODS & RESULTS: In the present study, we report a novel flap chamber that provides stable and reproducible outcomes by separating the dorsal skin flap from its surrounding skin by in situ immobilization. The flap chamber blocks circulation that disturbs flap ischemia from both basal and lateral sides of the flap tissue. Demarcation of skin necrosis is macroscopically evident on the flap and supported by distinct changes in histological architecture under microscopic examination. The utility of the novel skin flap chamber is further proven by applying it to the examination of flap survival in streptozotocin-induced diabetic rats with an increase in skin necrosis. The flap chamber also affords size modifications where a narrower flap chamber increases ischemia and provides manipulable therapeutic windows for studying cell therapies. Accordingly, intradermal injection of endothelial cells 3 days before flap ischemia significantly increases the survival of skin flaps. CONCLUSIONS: The novel flap chamber not only may stabilize the skin flap and provide reproducible outcomes that overcome the shortfalls of the traditional ischemic flap but also may afford size modifications that support research designs and test therapeutic approaches to regenerative repair.

Molecular insight and pharmacological approaches targeting mitochondrial dynamics in skeletal muscle during obesity.

Obesity-associated insulin resistance is the major characteristic of the early stage of metabolic syndrome. A decline in mitochondrial function plays a role in the development of insulin resistance in obesity and type 2 diabetes. Accumulating data reveal that mitochondrial dynamics, the balance between mitochondrial fusion and fission, are an important factor in the maintenance of mitochondrial function. Thus, the mechanisms underlying the regulation of mitochondrial dynamics in obesity deserve further investigation. This review describes an overview of mitochondrial fusion and fission machineries, and discusses the mechanistic and functional aspects of mitochondrial dynamics, with a focus on skeletal muscle in obesity. Finally, we discuss current pharmacological approaches of targeting mitochondrial dynamics. Elucidating the role of mitochondrial dynamics in skeletal muscle afflicted by obesity may provide not only important clues in understanding muscle insulin resistance, but also new therapeutic targets.

Organ-level quorum sensing directs regeneration in hair stem cell populations.

Coordinated organ behavior is crucial for an effective response to environmental stimuli. By studying regeneration of hair follicles in response to patterned hair plucking, we demonstrate that organ-level quorum sensing allows coordinated responses to skin injury. Plucking hair at different densities leads to a regeneration of up to five times more neighboring, unplucked resting hairs, indicating activation of a collective decision-making process. Through data modeling, the range of the quorum signal was estimated to be on the order of 1 mm, greater than expected for a diffusible molecular cue. Molecular and genetic analysis uncovered a two-step mechanism, where release of CCL2 from injured hairs leads to recruitment of TNF-α-secreting macrophages, which accumulate and signal to both plucked and unplucked follicles. By coupling immune response with regeneration, this mechanism allows skin to respond predictively to distress, disregarding mild injury, while meeting stronger injury with full-scale cooperative activation of stem cells.

Emergence of differentially regulated pathways associated with the development of regional specificity in chicken skin.

BACKGROUND: Regional specificity allows different skin regions to exhibit different characteristics, enabling complementary functions to make effective use of the integumentary surface. Chickens exhibit a high degree of regional specificity in the skin and can serve as a good model for when and how these regional differences begin to emerge. RESULTS: We used developing feather and scale regions in embryonic chickens as a model to gauge the differences in their molecular pathways. We employed cosine similarity analysis to identify the differentially regulated and co-regulated genes. We applied low cell techniques for expression validation and chromatin immunoprecipitation (ChIP)-based enhancer identification to overcome limited cell availabilities from embryonic chicken skin. We identified a specific set of genes demonstrating a high correlation as being differentially expressed during feather and scale development and maturation. Some members of the WNT, TGF-beta/BMP, and Notch family known to be involved in feathering skin differentiation were found to be differentially regulated. Interestingly, we also found genes along calcium channel pathways that are differentially regulated. From the analysis of differentially regulated pathways, we used calcium signaling pathways as an example for further verification. Some voltage-gated calcium channel subunits, particularly CACNA1D, are expressed spatio-temporally in the skin epithelium. These calcium signaling pathway members may be involved in developmental decisions, morphogenesis, or epithelial maturation. We further characterized enhancers associated with histone modifications, including H3K4me1, H3K27ac, and H3K27me3, near calcium channel-related genes and identified signature intensive hotspots that may be correlated with certain voltage-gated calcium channel genes. CONCLUSION: We demonstrated the applicability of cosine similarity analysis for identifying novel regulatory pathways that are differentially regulated during development. Our study concerning the effects of signaling pathways and histone signatures on enhancers suggests that voltage-gated calcium signaling may be involved in early skin development. This work lays the foundation for studying the roles of these gene pathways and their genomic regulation during the establishment of skin regional specificity.

Leptin of dermal adipose tissue is differentially expressed during the hair cycle and contributes to adipocyte-mediated growth inhibition of anagen-phase vibrissa hair.

Adipose tissue encircles the lower portion of anagen hair follicles and may regulate hair cycle progression. As leptin is a major adipokine, its level of expression from the dermal white adipose tissue during hair cycle progression was studied. The result shows that leptin level is differentially expressed during hair cycle, the lowest in early anagen phase, upregulated in late anagen phase and the highest in the telogen phase. On the other hand, leptin receptor is detected in keratin 15-positive hair bulge epithelium of both anagen- and telogen-phase hair follicles of mice pelage and vibrissa hair, and hair from human scalp. Leptin contributes to adipocyte-mediated growth inhibition of anagen-phase vibrissa hair as demonstrated in organ culture and coculture system. Our data suggest that leptin of dermal white adipose tissue might regulate hair growth and, therefore, hair cycle progression via leptin receptor on the hair follicle epithelium.

Disrupted ectodermal organ morphogenesis in mice with a conditional histone deacetylase 1, 2 deletion in the epidermis.

Histone deacetylases (HDACs) are present in the epidermal layer of the skin, outer root sheath, and hair matrix. To investigate how histone acetylation affects skin morphogenesis and homeostasis, mice were generated with a K14 promoter-mediated reduction of Hdac1 or Hdac2. The skin of HDAC1 null (K14-Cre Hdac1(cKO/cKO)) mice exhibited a spectrum of lesions, including irregularly thickened interfollicular epidermis, alopecia, hair follicle dystrophy, claw dystrophy, and abnormal pigmentation. Hairs are sparse, short, and intermittently coiled. The distinct pelage hair types are lost. During the first hair cycle, hairs are lost and replaced by dystrophic hair follicles with dilated infundibulae. The dystrophic hair follicle epithelium is stratified and is positive for K14, involucrin, and TRP63, but negative for keratin 10. Some dystrophic follicles are K15 positive, but mature hair fiber keratins are absent. The digits form extra hyperpigmented claws on the lateral sides. Hyperpigmentation is observed in the interfollicular epithelium, the tail, and the feet. Hdac1 and Hdac2 dual transgenic mice (K14-Cre Hdac1(cKO/cKO) Hdac2(+/cKO)) have similar but more obvious abnormalities. These results show that suppression of epidermal HDAC activity leads to improper ectodermal organ morphogenesis and disrupted hair follicle regeneration and homeostasis, as well as indirect effects on pigmentation.

Nuclear topology, epigenetics, and keratinocyte differentiation.

Recent progress in epigenetics reveals dynamic chromatin interactions in the nucleus during development, regeneration, reprogramming, and in disease. Higher-order chromatin organization is manifested as changes in the topological distribution of eu-/heterochromatin and in nuclear morphology. We are now able to gain new knowledge about these changes at the genomic level.