Deep quantitative proteomics of North American Pacific coast star tunicate (Botryllus schlosseri).

Botryllus schlosseri, is a model marine invertebrate for studying immunity, regeneration, and stress-induced evolution. Conditions for validating its predicted proteome were optimized using nanoElute® 2 deep-coverage LCMS, revealing up to 4930 protein groups and 20,984 unique peptides per sample. Spectral libraries were generated and filtered to remove interferences, low-quality transitions, and only retain proteins with >3 unique peptides. The resulting DIA assay library enabled label-free quantitation of 3426 protein groups represented by 22,593 unique peptides. Quantitative comparisons of single systems from a laboratory-raised with two field-collected populations revealed (1) a more unique proteome in the laboratory-raised population, and (2) proteins with high/low individual variabilities in each population. DNA repair/replication, ion transport, and intracellular signaling processes were distinct in laboratory-cultured colonies. Spliceosome and Wnt signaling proteins were the least variable (highly functionally constrained) in all populations. In conclusion, we present the first colonial tunicate's deep quantitative proteome analysis, identifying functional protein clusters associated with laboratory conditions, different habitats, and strong versus relaxed abundance constraints. These results empower research on B. schlosseri with proteomics resources and enable quantitative molecular phenotyping of changes associated with transfer from in situ to ex situ and from in vivo to in vitro culture conditions.

The Multifaceted Functions of TRPV4 and Calcium Oscillations in Tissue Repair.

The transient receptor potential vanilloid 4 (TRPV4) specifically functions as a mechanosensitive ion channel and is responsible for conveying changes in physical stimuli such as mechanical stress, osmotic pressure, and temperature. TRPV4 enables the entry of cation ions, particularly calcium ions, into the cell. Activation of TRPV4 channels initiates calcium oscillations, which trigger intracellular signaling pathways involved in a plethora of cellular processes, including tissue repair. Widely expressed throughout the body, TRPV4 can be activated by a wide array of physicochemical stimuli, thus contributing to sensory and physiological functions in multiple organs. This review focuses on how TRPV4 senses environmental cues and thereby initiates and maintains calcium oscillations, critical for responses to organ injury, tissue repair, and fibrosis. We provide a summary of TRPV4-induced calcium oscillations in distinct organ systems, along with the upstream and downstream signaling pathways involved. In addition, we delineate current animal and disease models supporting TRPV4 research and shed light on potential therapeutic targets for modulating TRPV4-induced calcium oscillation to promote tissue repair while reducing tissue fibrosis.

Metal-Organic Frameworks and Their Composites for Chronic Wound Healing: From Bench to Bedside.

Chronic wounds are characterized by delayed and dysregulated healing processes. As such, they have emerged as an increasingly significant threat. The associated morbidity and socioeconomic toll are clinically and financially challenging, necessitating novel approaches in the management of chronic wounds. Metal-organic frameworks (MOFs) are an innovative type of porous coordination polymers, with low toxicity and high eco-friendliness. Documented anti-bacterial effects and pro-angiogenic activity predestine these nanomaterials as promising systems for the treatment of chronic wounds. In this context, the therapeutic applicability and efficacy of MOFs remain to be elucidated. It is, therefore, reviewed the structural-functional properties of MOFs and their composite materials and discusses how their multifunctionality and customizability can be leveraged as a clinical therapy for chronic wounds.

CD201+ fascia progenitors choreograph injury repair.

Optimal tissue recovery and organismal survival are achieved by spatiotemporal tuning of tissue inflammation, contraction and scar formation1. Here we identify a multipotent fibroblast progenitor marked by CD201 expression in the fascia, the deepest connective tissue layer of the skin. Using skin injury models in mice, single-cell transcriptomics and genetic lineage tracing, ablation and gene deletion models, we demonstrate that CD201+ progenitors control the pace of wound healing by generating multiple specialized cell types, from proinflammatory fibroblasts to myofibroblasts, in a spatiotemporally tuned sequence. We identified retinoic acid and hypoxia signalling as the entry checkpoints into proinflammatory and myofibroblast states. Modulating CD201+ progenitor differentiation impaired the spatiotemporal appearances of fibroblasts and chronically delayed wound healing. The discovery of proinflammatory and myofibroblast progenitors and their differentiation pathways provide a new roadmap to understand and clinically treat impaired wound healing.

Regulatory T cells in skin regeneration and wound healing.

As the body's integumentary system, the skin is vulnerable to injuries. The subsequent wound healing processes aim to restore dermal and epidermal integrity and functionality. To this end, multiple tissue-resident cells and recruited immune cells cooperate to efficiently repair the injured tissue. Such temporally- and spatially-coordinated interplay necessitates tight regulation to prevent collateral damage such as overshooting immune responses and excessive inflammation. In this context, regulatory T cells (Tregs) hold a key role in balancing immune homeostasis and mediating cutaneous wound healing. A comprehensive understanding of Tregs' multifaceted field of activity may help decipher wound pathologies and, ultimately, establish new treatment modalities. Herein, we review the role of Tregs in orchestrating the regeneration of skin adnexa and catalyzing healthy wound repair. Further, we discuss how Tregs operate during fibrosis, keloidosis, and scarring.

In vivo fluorescent labeling and tracking of extracellular matrix.

Connective tissues are essential building blocks for organ development, repair and regeneration. However, we are at the early stages of understanding connective tissue dynamics. Here, we detail a method that enables in vivo fate mapping of organ extracellular matrix (ECM) by taking advantage of a crosslinking chemical reaction between amine groups and N-hydroxysuccinimide esters. This methodology enables robust labeling of ECM proteins, which complement previous affinity-based single-protein methods. This protocol is intended for entry-level scientists and the labeling step takes between 5 and 10 min. ECM 'tagging' with fluorophores using N-hydroxysuccinimide esters enables visualization of ECM spatial modifications and is particularly useful to study connective tissue dynamics in organ fibrosis, tumor stroma formation, wound healing and regeneration. This in vivo chemical fate mapping methodology is highly versatile, regardless of the tissue/organ system, and complements cellular fate-mapping techniques. Furthermore, as the basic chemistry of proteins is highly conserved between species, this method is also suitable for cross-species comparative studies of ECM dynamics.

Fibroblasts - the cellular choreographers of wound healing.

Injuries to our skin trigger a cascade of spatially- and temporally-synchronized healing processes. During such endogenous wound repair, the role of fibroblasts is multifaceted, ranging from the activation and recruitment of innate immune cells through the synthesis and deposition of scar tissue to the conveyor belt-like transport of fascial connective tissue into wounds. A comprehensive understanding of fibroblast diversity and versatility in the healing machinery may help to decipher wound pathologies whilst laying the foundation for novel treatment modalities. In this review, we portray the diversity of fibroblasts and delineate their unique wound healing functions. In addition, we discuss future directions through a clinical-translational lens.

Immunomodulatory Nanosystems: Advanced Delivery Tools for Treating Chronic Wounds.

The increasingly aging society led to a rise in the prevalence of chronic wounds (CWs), posing a significant burden to public health on a global scale. One of the key features of CWs is the presence of a maladjusted immune microenvironment characterized by persistent and excessive (hyper)inflammation. A variety of immunomodulatory therapies have been proposed to address this condition. Yet, to date, current delivery systems for immunomodulatory therapy remain inadequate and lack efficiency. This highlights the need for new therapeutic delivery systems, such as nanosystems, to manage the pathological inflammatory imbalance and, ultimately, improve the treatment outcomes of CWs. While a plethora of immunomodulatory nanosystems modifying the immune microenvironment of CWs have shown promising therapeutic effects, the literature on the intersection of immunomodulatory nanosystems and CWs remains relatively scarce. Therefore, this review aims to provide a comprehensive overview of the pathogenesis and characteristics of the immune microenvironment in CWs, discuss important advancements in our understanding of CW healing, and delineate the versatility and applicability of immunomodulatory nanosystems-based therapies in the therapeutic management of CWs. In addition, we herein also shed light on the main challenges and future perspectives in this rapidly evolving research field.

Improved Media Formulations for Primary Cell Cultures Derived from a Colonial Urochordate.

The cultivation of marine invertebrate cells in vitro has garnered significant attention due to the availability of diverse cell types and cellular potentialities in comparison to vertebrates and particularly in response to the demand for a multitude of applications. While cells in the colonial urochordate Botryllus schlosseri have a very high potential for omnipotent differentiation, no proliferating cell line has been established in Botryllus, with results indicating that cell divisions cease 24-72 h post initiation. This research assessed how various Botryllus blood cell types respond to in vitro conditions by utilizing five different refinements of cell culture media (TGM1-TGM5). During the initial week of culture, there was a noticeable medium-dependent increase in the proliferation and viability of distinct blood cell types. Within less than one month from initiation, we developed medium-specific primary cultures, a discovery that supports larger efforts to develop cell type-specific cultures. Specific cell types were easily distinguished and classified based on their natural fluorescence properties using confocal microscopy. These results are in agreement with recent advances in marine invertebrate cell cultures, demonstrating the significance of optimized nutrient media for cell culture development and for cell selection.

Physiological changes during torpor favor association with Endozoicomonas endosymbionts in the urochordate Botrylloides leachii.

Environmental perturbations evoke down-regulation of metabolism in some multicellular organisms, leading to dormancy, or torpor. Colonies of the urochordate Botrylloides leachii enter torpor in response to changes in seawater temperature and may survive for months as small vasculature remnants that lack feeding and reproductive organs but possess torpor-specific microbiota. Upon returning to milder conditions, the colonies rapidly restore their original morphology, cytology and functionality while harboring re-occurring microbiota, a phenomenon that has not been described in detail to date. Here we investigated the stability of B. leachii microbiome and its functionality in active and dormant colonies, using microscopy, qPCR, in situ hybridization, genomics and transcriptomics. A novel lineage of Endozoicomonas, proposed here as Candidatus Endozoicomonas endoleachii, was dominant in torpor animals (53-79% read abundance), and potentially occupied specific hemocytes found only in torpid animals. Functional analysis of the metagenome-assembled genome and genome-targeted transcriptomics revealed that Endozoicomonas can use various cellular substrates, like amino acids and sugars, potentially producing biotin and thiamine, but also expressing various features involved in autocatalytic symbiosis. Our study suggests that the microbiome can be linked to the metabolic and physiological states of the host, B. leachii, introducing a model organism for the study of symbioses during drastic physiological changes, such as torpor.

Wound infiltrating adipocytes are not myofibroblasts.

The origins of wound myofibroblasts and scar tissue remains unclear, but it is assumed to involve conversion of adipocytes into myofibroblasts. Here, we directly explore the potential plasticity of adipocytes and fibroblasts after skin injury. Using genetic lineage tracing and live imaging in explants and in wounded animals, we observe that injury induces a transient migratory state in adipocytes with vastly distinct cell migration patterns and behaviours from fibroblasts. Furthermore, migratory adipocytes, do not contribute to scar formation and remain non-fibrogenic in vitro, in vivo and upon transplantation into wounds in animals. Using single-cell and bulk transcriptomics we confirm that wound adipocytes do not convert into fibrogenic myofibroblasts. In summary, the injury-induced migratory adipocytes remain lineage-restricted and do not converge or reprogram into a fibrosing phenotype. These findings broadly impact basic and translational strategies in the regenerative medicine field, including clinical interventions for wound repair, diabetes, and fibrotic pathologies.

Reactive Oxygen Species-Scavenging Nanosystems in the Treatment of Diabetic Wounds.

Diabetic wounds are characterized by drug-resistant bacterial infections, biofilm formation, impaired angiogenesis and perfusion, and oxidative damage to the microenvironment. Given their complex nature, diabetic wounds remain a major challenge in clinical practice. Reactive oxygen species (ROS), which have been shown to trigger hyperinflammation and excessive cellular apoptosis, play a pivotal role in the pathogenesis of diabetic wounds. ROS-scavenging nanosystems have recently emerged as smart and multifunctional nanomedicines with broad synergistic applicability. The documented anti-inflammatory and pro-angiogenic ability of ROS-scavenging treatments predestines these nanosystems as promising options for the treatment of diabetic wounds. Yet, in this context, the therapeutic applicability and efficacy of ROS-scavenging nanosystems remain to be elucidated. Herein, the role of ROS in diabetic wounds is deciphered, and the properties and strengths of nanosystems with ROS-scavenging capacity for the treatment of diabetic wounds are summarized. In addition, the current challenges of such nanosystems and their potential future directions are discussed through a clinical-translational lens.

Fascia Layer-A Novel Target for the Application of Biomaterials in Skin Wound Healing.

As the first barrier of the human body, the skin has been of great concern for its wound healing and regeneration. The healing of large, refractory wounds is difficult to be repaired by cell proliferation at the wound edges and usually requires manual intervention for treatment. Therefore, therapeutic tools such as stem cells, biomaterials, and cytokines have been applied to the treatment of skin wounds. Skin microenvironment modulation is a key technology to promote wound repair and skin regeneration. In recent years, a series of novel bioactive materials that modulate the microenvironment and cell behavior have been developed, showing the ability to efficiently facilitate wound repair and skin attachment regeneration. Meanwhile, our lab found that the fascial layer has an indispensable role in wound healing and repair, and this review summarizes the research progress of related bioactive materials and their role in wound healing.

Cancer-associated mesothelial cell-derived ANGPTL4 and STC1 promote the early steps of ovarian cancer metastasis.

Ovarian cancer (OvCa) preferentially metastasizes in association with mesothelial cell-lined surfaces. We sought to determine if mesothelial cells are required for OvCa metastasis and detect alterations in mesothelial cell gene expression and cytokine secretion upon interaction with OvCa cells. Using omental samples from patients with high-grade serous OvCa and mouse models with Wt1-driven GFP-expressing mesothelial cells, we validated the intratumoral localization of mesothelial cells during human and mouse OvCa omental metastasis. Removing mesothelial cells ex vivo from human and mouse omenta or in vivo using diphtheria toxin-mediated ablation in Msln-Cre mice significantly inhibited OvCa cell adhesion and colonization. Human ascites induced angiopoietin-like 4 (ANGPTL4) and stanniocalcin 1 (STC1) expression and secretion by mesothelial cells. Inhibition of STC1 or ANGPTL4 via RNAi obstructed OvCa cell-induced mesothelial cell to mesenchymal transition while inhibition of ANGPTL4 alone obstructed OvCa cell-induced mesothelial cell migration and glycolysis. Inhibition of mesothelial cell ANGPTL4 secretion via RNAi prevented mesothelial cell-induced monocyte migration, endothelial cell vessel formation, and OvCa cell adhesion, migration, and proliferation. In contrast, inhibition of mesothelial cell STC1 secretion via RNAi prevented mesothelial cell-induced endothelial cell vessel formation and OvCa cell adhesion, migration, proliferation, and invasion. Additionally, blocking ANPTL4 function with Abs reduced the ex vivo colonization of 3 different OvCa cell lines on human omental tissue explants and in vivo colonization of ID8p53-/-Brca2-/- cells on mouse omenta. These findings indicate that mesothelial cells are important to the initial stages of OvCa metastasis and that the crosstalk between mesothelial cells and the tumor microenvironment promotes OvCa metastasis through the secretion of ANGPTL4.

Therapeutic Silencing of p120 in Fascia Fibroblasts Ameliorates Tissue Repair.

Deep skin wounds rapidly heal by mobilizing extracellular matrix and cells from the fascia, deep beneath the dermal layer of the skin, to form scars. Despite wounds being an extensively studied area and an unmet clinical need, the biochemistry driving this patch-like repair remains obscure. Lacking also are efficacious therapeutic means to modulate scar formation in vivo. In this study, we identify a central role for p120 in mediating fascia mobilization and wound repair. Injury triggers p120 expression, largely within engrailed-1 lineage-positive fibroblasts of the fascia that exhibit a supracellular organization. Using adeno-associated virus‒mediated gene silencing, we show that p120 establishes the supracellular organization of fascia engrailed-1 lineage-positive fibroblasts, without which fascia mobilization is impaired. Gene silencing of p120 in fascia fibroblasts disentangles their supracellular organization, reducing the transfer of fascial cells and extracellular matrix into wounds and augmenting wound healing. Our findings place p120 as essential for fascia mobilization, opening, to our knowledge, a previously unreported therapeutic avenue for targeted intervention in the treatment of a variety of skin scar conditions.

Diversity of Fibroblasts and Their Roles in Wound Healing.

Wound healing disorders are a societal, clinical, and healthcare burden and understanding and treating them is a major challenge. A particularly important cell type in the wound healing processes is the fibroblast. Fibroblasts are not homogenous; however, there are diverse functional fibroblast subtypes coming from different embryonic origins and residing in dispersed anatomic locations including distinct classes of fibroblasts at various skin depths. In this review, we discuss the implications of fibroblast heterogeneity, with a focus on the fundamental physiological functions of the fibroblast subtypes that govern wound repair and clinical degrees of healing. A better understanding of these diverse functional fibroblast populations will likely lead to novel therapies to enhance wound healing and inhibit excessive scarring.

Continuous NPWT Regulates Fibrosis in Murine Diabetic Wound Healing.

Scarring is associated with significant morbidity. The mechanical signaling factor yes-associated protein (YAP) has been linked to Engrailed-1 (En1)-lineage positive fibroblasts (EPFs), a pro-scarring fibroblast lineage, establishing a connection between mechanotransduction and fibrosis. In this study, we investigate the impact of micromechanical forces exerted through negative pressure wound therapy (NPWT) on the pathophysiology of fibrosis. Full-thickness excisional dorsal skin wounds were created on diabetic (db/db) mice which were treated with occlusive covering (control) or NPWT (continuous, −125 mmHg, 7 days; NPWT). Analysis was performed on tissue harvested 10 days after wounding. NPWT was associated with increased YAP (p = 0.04) but decreased En1 (p = 0.0001) and CD26 (p < 0.0001). The pro-fibrotic factors Vimentin (p = 0.04), α-SMA (p = 0.04) and HSP47 (p = 0.0008) were decreased with NPWT. Fibronectin was higher (p = 0.01) and collagen deposition lower in the NPWT group (p = 0.02). NPWT increased cellular proliferation (p = 0.002) and decreased apoptosis (p = 0.03). Western blotting demonstrated increased YAP (p = 0.02) and RhoA (p = 0.03) and decreased Caspase-3 (p = 0.03) with NPWT. NPWT uncouples YAP from EPF activation, through downregulation of Caspace-3, a pro-apoptotic factor linked to keloid formation. Mechanotransduction decreases multiple pro-fibrotic factors. Through this multifactorial process, NPWT significantly decreases fibrosis and offers promising potential as a mode to improve scar appearance.

Transcriptome landscapes that signify Botrylloides leachi (Ascidiacea) torpor states.

Harsh environments enforce the expression of behavioural, morphological, physiological, and reproductive rejoinders, including torpor. Here we study the morphological, cellular, and molecular alterations in torpor architype in the colonial urochordate Botrylloides aff. leachii by employing whole organism Transmission electron (TEM) and light microscope observations, RNA sequencing, real-time polymerase chain reaction (qPCR) quantification of selected genes, and immunolocalization of WNT, SMAD and SOX2 gene expressions. On the morphological level, torpor starts with gradual regression of all zooids and buds which leaves the colony surviving as condensed vasculature remnants that may be 'aroused' to regenerate fully functional colonies upon changes in the environment. Simultaneously, we observed altered distributions of hemolymph cell types. Phagocytes doubled in number, while the number of morula cells declined by half. In addition, two new circulating cell types were observed, multi-nucleated and bacteria-bearing cells. RNA sequencing technology revealed marked differences in gene expression between different organism compartments and states: active zooids and ampullae, and between mid-torpor and naive colonies, or naive and torpid colonies. Gene Ontology term enrichment analyses further showed disparate biological processes. In torpid colonies, we observed overall 233 up regulated genes. These genes included NR4A2, EGR1, MUC5AC, HMCN2 and. Also, 27 transcription factors were upregulated in torpid colonies including ELK1, HDAC3, RBMX, MAZ, STAT1, STAT4 and STAT6. Interestingly, genes involved in developmental processes such as SPIRE1, RHOA, SOX11, WNT5A and SNX18 were also upregulated in torpid colonies. We further validated the dysregulation of 22 genes during torpor by utilizing qPCR. Immunohistochemistry of representative genes from three signaling pathways revealed high expression of these genes in circulated cells along torpor. WNT agonist administration resulted in early arousal from torpor in 80% of the torpid colonies while in active colonies WNT agonist triggered the torpor state. Abovementioned results thus connote unique transcriptome landscapes associated with Botrylloides leachii torpor.