Fibrotic remodeling of nucleus pulposus (NP) leads to structural and mechanical anomalies of intervertebral discs that prone to degeneration, leading to low back pain incidence and disability. Emergence of fibroblastic cells in disc degeneration has been reported, yet their nature and origin remain elusive. In this study, we performed an integrative analysis of multiple single-cell RNA sequencing datasets to interrogate the cellular heterogeneity and fibroblast-like entities in degenerative human NP specimens. We found that disc degeneration severity is associated with an enrichment of fibrocyte phenotype, characterized by CD45 and collagen I dual positivity, and expression of myofibroblast marker α-smooth muscle actin. Refined clustering and classification distinguished the fibrocyte-like populations as subtypes in the NP cells - and immunocytes-clusters, expressing disc degeneration markers HTRA1 and ANGPTL4 and genes related to response to TGF-β. In injury-induced mouse disc degeneration model, fibrocytes were found recruited into the NP undergoing fibrosis and adopted a myofibroblast phenotype. Depleting the fibrocytes in CD11b-DTR mice in which myeloid-derived lineages were ablated by diphtheria toxin could markedly attenuate fibrous modeling and myofibroblast formation in the NP of the degenerative discs, and prevent disc height loss and histomorphological abnormalities. Marker analysis supports that disc degeneration progression is dependent on a function of CD45+COL1A1+ and αSMA+ cells. Our findings reveal that myeloid-derived fibrocytes play a pivotal role in NP fibrosis and may therefore be a target for modifying disc degeneration and promoting its repair.

Genomic plasticity helps adapt to extreme environmental conditions. We tested the hypothesis that exposure to space environment (ESE) impacts the epigenome inducing genomic plasticity. Murine skin samples from the Rodent Research Reference Mission-1 were procured from the International Space Station (ISS) National Laboratory. Targeted RNA sequencing to test differential gene expression between the skin of ESE versus ground controls revealed upregulation of VEGF-mediated angiogenesis pathways secondary to promoter hypomethylation in responders. Methylome sequencing identified ESE-sensitive hypomethylated genes including developmental angiogenic genes Araf, Vegfb, and Vegfr1. Based on differentially expressed genes, the angiogenesis biofunction was enriched in responders. The induction of genomic plasticity in response to ESE, as reported herein, may be viewed as a mark of biological resilience that is evident in a minority of organisms, responders but not in non-responders, exposed to the same stressor. Inducible genomic plasticity may be implicated in natural resilience to ESE.

Background: Burns are a global public health issue and a major cause of disability and death around the world. Stem cells, which are the undifferentiated cells with the potential for indefinite proliferation and multilineage differentiation, have the ability to replace injured skin and facilitate the wound repair process through paracrine mechanisms. In light of this, the present study aims to conduct a bibliometric analysis in order to identify research hotspots of stem cell-related burns and assess global research tendencies. Methods: To achieve this objective, we retrieved scientific publications on burns associated with stem cells covering the period from January 1, 1978, to October 13, 2022, from the Web of Science Core Collection (WoSCC). Bibliometric analyses, including production and collaboration analyses between countries, institutions, authors, and journals, as well as keyword and topic analyses, were conducted using the bibliometrix R package, CiteSpace, and VOSviewer. Results: A total of 1648 burns associated with stem cell documents were published and listed on WOSCC. The most contributive country, institution, journal, and author were the United States, LV Prasad Eye Institute, Burns, and Scheffer C.G. Tseng, respectively. More importantly, combined with historical direct citation network, trend topic analysis, keyword co-occurrence network, and substantial literature analysis, we eventually summarized the research hotspots and frontiers on burns associated stem cell reasearch. Conclusion: The present study obtained deep insight into the developing trends and research hotspots on burns associated with stem cells, which arouses growing concerns and implies increasing clinical implications. The mechanism and therapeutics of epidermal stem cells (ESCs) for burn wounds and the mechanism of mesenchymal stem cells (MSCs) and MSC-derived exosomes for burns wounds are two research hotspots in this field.

Macrophage-myofibroblast transdifferentiation (MMT), a fibrotic process impacting diverse tissue types, has garnered recent scholarly interest. Within damaged tissues, the role of myofibroblasts is pivotal in the accumulation of excessive fibrous connective tissue, leading to persistent scarring or organ dysfunction. Consequently, the examination of MMT-related fibrosis is imperative. This review underscores MMT as a fundamental mechanism in myofibroblast generation during tissue fibrosis, and its exploration is crucial for elucidating the regulatory mechanisms underlying this process. Gaining insight into these mechanisms promises to facilitate the development of therapeutic approaches aimed at inhibiting and reversing fibrosis, thereby offering potential avenues for the treatment of fibrotic diseases.

Malignancies can compromise systemic innate immunity, but the underlying mechanisms are largely unknown. Here, we find that tumor-derived small extracellular vesicles (sEVs; TEVs) deliver PD-L1 to host macrophages, thereby impeding antibacterial immunity. Mice implanted with Rab27a-knockdown tumors are more resistant to bacterial infection than wild-type controls. Injection of TEVs into mice impairs macrophage-mediated bacterial clearance, increases systemic bacterial dissemination, and enhances sepsis score in a PD-L1-dependent manner. Mechanistically, TEV-packaged PD-L1 inhibits Bruton's tyrosine kinase/PLCγ2 signaling-mediated cytoskeleton reorganization and reactive oxygen species generation, impacting bacterial phagocytosis and killing by macrophages. Neutralizing PD-L1 markedly normalizes macrophage-mediated bacterial clearance in tumor-bearing mice. Importantly, circulating sEV PD-L1 levels in patients with tumors can predict bacterial infection susceptibility, while patients with tumors treated with αPD-1 exhibit fewer postoperative infections. These findings identify a mechanism by which cancer cells dampen host innate immunity-mediated bacterial clearance and suggest targeting TEV-packaged PD-L1 to reduce bacterial infection susceptibility in tumor-bearing conditions.

Tissue nanotransfection (TNT)-based fluorescent labeling of cell-specific exosomes has shown that exosomes play a central role in physiological keratinocyte-macrophage (mϕ) crosstalk at the wound-site. Here, we report that during the early phase of wound reepithelialization, macrophage-derived exosomes (Exomϕ), enriched with the outer mitochondrial membrane protein TOMM70, are localized in leading-edge keratinocytes. TOMM70 is a 70 kDa adaptor protein anchored in the mitochondrial outer membrane and plays a critical role in maintaining mitochondrial function and quality. TOMM70 selectively recognizes cytosolic chaperones by its tetratricopeptide repeat (TPR) domain and facilitates the import of preproteins lacking a positively charged mitochondrial targeted sequence. Exosomal packaging of TOMM70 in mϕ was independent of mitochondrial fission. TOMM70-enriched Exomϕ compensated for the hypoxia-induced depletion of epidermal TOMM70, thereby rescuing mitochondrial metabolism in leading-edge keratinocytes. Thus, macrophage-derived TOMM70 is responsible for the glycolytic ATP supply to power keratinocyte migration. Blockade of exosomal uptake from keratinocytes impaired wound closure with the persistence of proinflammatory mϕ in the wound microenvironment, pointing toward a bidirectional crosstalk between these two cell types. The significance of such bidirectional crosstalk was established by the observation that in patients with nonhealing diabetic foot ulcers, TOMM70 is deficient in keratinocytes of wound-edge tissues.

Staphylococcus aureus causes approximately 80% of skin and soft tissue infections (SSTIs). Collagen is the most abundant human extracellular matrix protein with critical roles in wound healing, and S. aureus encodes a collagen binding adhesin (Cna). The role of this protein during skin infections is unknown. Here we report that inability to bind collagen results in worsened pathology of intradermal Δcna S. aureus infection. WT/Cna+ S. aureus showed reduced infection severity, aggregate formation, and significantly improved clearance of bacteria. Cna binds to the collagen-like domain of serum C1q protein to reduce its opsonophagocytic functions. We demonstrate that infection of C1qKO mice with WT bacteria show results similar to the Δcna group. Conversely, inability to bind collagen resulted in an amplified inflammatory response caused in part by macrophage and neutrophil small molecule mediators released at the infection site (MMP-9, MMP-12, LTB4), resulting in increased immune cell infiltration and death.

Although some studies have suggested that macrophages may secrete structural collagens, and convert to fibroblast-like cells, macrophage to fibroblast transdifferentiation in infarcted and remodeling hearts remains controversial. Our study uses linage tracing approaches and single cell transcriptomics to examine whether macrophages undergo fibroblast conversion, and to characterize the extracellular matrix expression profile of myeloid cells in myocardial infarction. To examine whether infarct macrophages undergo fibroblast conversion, we identified macrophage-derived progeny using the inducible CX3CR1CreER mice crossed with the PDGFRαEGFP reporter line for reliable fibroblast identification. The abundant fibroblasts that infiltrated the infarcted myocardium after 7 and 28 days of coronary occlusion were not derived from CX3CR1+ macrophages. Infarct macrophages retained myeloid cell characteristics and did not undergo conversion to myofibroblasts, endothelial or vascular mural cells. Single cell RNA-seq of CSF1R+ myeloid cells harvested from control and infarcted hearts showed no significant expression of fibroblast identity genes by myeloid cell clusters. Moreover, infarct macrophages did not express significant levels of genes encoding structural collagens. However, infarct macrophage and monocyte clusters were the predominant source of the fibrogenic growth factors Tgfb1 and Pdgfb, and of the matricellular proteins Spp1/Osteopontin, Thbs1/Thrombospondin-1, Emilin2, and Fn1/fibronectin, while expressing significant amounts of several other matrix genes, including Vcan/versican, Ecm1 and Sparc. ScRNA-seq data suggested similar patterns of matrix gene expression in human myocardial infarction. In conclusion, infarct macrophages do not undergo fibroblast or myofibroblast conversion and do not exhibit upregulation of structural collagens but may contribute to fibrotic remodeling by producing several fibrogenic matricellular proteins.

Diabetic ulcers are one of the common complications in diabetic patients. Delayed wound healing is associated with persistent pro-inflammatory M1 polarization, reduced angiogenesis and increased reactive oxygen species (ROS) in the microenvironment. Wound healing consists of multiple phases and therefore requires treatment tailored to each phase. In this study, a biphasic drug-releasing microneedle (MN) was fabricated to achieve early ROS scavenging and late accelerated angiogenesis to promote wound healing. Vascular endothelial growth factor (VEGF) was first encapsulated in methacryloylated sulfonated chitosan (SCSMA) microspheres (V@MP), and then V@MP was loaded into hyaluronic acid (HA) microneedles along with cerium dioxide nanoparticles (CONPs). Rapid dissolution of HA rapidly releases the CONPs to clear ROS, whereas the V@MP stays in the wound. SCSMA slow degradation prolongs the release of VEGF, thereby promoting angiogenesis. In vitro and in vivo studies have shown that this biphasic drug-releasing smart microneedle improves cell proliferation and migration, effectively scavenges ROS, promotes angiogenesis and tissue regeneration, and synergistically promotes M2 macrophage polarization. It provides a new delivery mode for nano-enzymes and growth factors that could be multifunctional and synergistic in the treatment of diabetic ulcers. STATEMENT OF SIGNIFICANCE: In our study, we present a microneedle (V@MP/C@MN) that can release drugs biphasically, which showed good repair ability in diabetic ulcer model. Large amounts of CONPs were rapidly released to alleviate oxidative stress during the inflammation of the wound, and V@MP stayed in the wound for a long period of time to release VEGF and promote angiogenesis in the late stage of wound healing. The results indicated that V@MP/C@MN could promote cell proliferation and migration, effectively scavenge ROS, promote angiogenesis and tissue regeneration, and synergistically promote M2 macrophage polarization, which could play a multifunctional and synergistic role in the treatment of diabetic ulcers.

Macrophage-myofibroblast transformation (MMT) has emerged as a discovery in the field of fibrotic disease research. MMT is the process by which macrophages differentiate into myofibroblasts, leading to organ fibrosis following organ damage and playing an important role in fibrosis formation and progression. Recently, many new advances have been made in studying the mechanisms of MMT occurrence in fibrotic diseases. This article reviews some critical recent findings on MMT, including the origin of MMT in myofibroblasts, the specific mechanisms by which MMT develops, and the mechanisms and effects of MMT in the kidneys, lungs, heart, retina, and other fibrosis. By summarizing the latest research related to MMT, this paper provides a theoretical basis for elucidating the mechanisms of fibrosis in various organs and developing effective therapeutic targets for fibrotic diseases.

Chronic wounds pose a significant challenge to healthcare. Stemming from impaired wound healing, the consequences can be severe, ranging from amputation to mortality. This comprehensive review explores the multifaceted impact of chronic wounds in medicine and the roles that diet and nutritional pathologies play in the wound-healing process. It has been well established that an adequate diet is crucial to proper wound healing. Nutrients such as vitamin D, zinc, and amino acids play significant roles in cellular regeneration, immune functioning, and collagen synthesis and processing. Additionally, this review discusses how patients with chronic conditions like diabetes, obesity, and nutritional deficiencies result in the formation of chronic wounds. By integrating current research findings, this review highlights the significant impact of the genetic make-up of an individual on the risk of developing chronic wounds and the necessity for adequate personalized dietary interventions. Addressing the nutritional needs of individuals, especially those with chronic conditions, is essential for improving wound outcomes and overall patient care. With new developments in the field of genomics, there are unprecedented opportunities to develop targeted interventions that can precisely address the unique metabolic needs of individuals suffering from chronic wounds, thereby enhancing treatment effectiveness and patient outcomes.

Pulmonary hypertension (PH) is a pulmonary vascular disease characterized by elevated pulmonary vascular pressure. Long-term PH, irrespective of its etiology, leads to increased right ventricular (RV) pressure, RV hypertrophy, and ultimately, RV failure.

Research indicates that RV failure secondary to hypertrophy remains the primary cause of mortality in pulmonary arterial hypertension (PAH). However, the impact of PH on RV structure and function under increased overload remains incompletely understood. Several mechanisms have been proposed, including extracellular remodeling, RV hypertrophy, metabolic disturbances, inflammation, apoptosis, autophagy, endothelial-to-mesenchymal transition, neurohormonal dysregulation, capillary rarefaction, and ischemia.

Studies have demonstrated the significant role of oxidative stress in the development of RV failure. Understanding the interplay among these mechanisms is crucial for the prevention and management of RV failure in patients with PH.

Significance: The incidence of diabetes continues to rise throughout the world in an alarming rate. Diabetic patients often develop diabetic foot ulcers (DFUs), many of which do not heal. Non-healing DFUs are a major cause of hospitalization, amputation, and increased morbidity. Understanding the underlying mechanisms of impaired healing in DFU is crucial for its management. Recent Advances: This review focuses on the recent advancements on macrophages and neutrophils in diabetic wounds and DFUs. In particular, we discuss diabetes-induced dysregulations and dysfunctions of macrophages and neutrophils. Critical Issues: It is well established that diabetic wounds are characterized by stalled inflammation that results in impaired healing. Recent findings in the field suggest that dysregulation of macrophages and neutrophils plays a critical role in impaired healing in DFUs. The delineation of mechanisms that restore macrophage and neutrophil function in diabetic wound healing is the focus of intense investigation. Future Directions: The breadth of recently generated knowledge on the activity of macrophages and neutrophils in diabetic wound healing is impressive. Experimental models have delineated pathways that hold promise for the treatment of diabetic wounds and DFUs. These pathways may be useful targets for further clinical investigation.

Fibrosis resulting from pathological repair secondary to recurrent or persistent tissue damage often leads to organ failure and mortality. Biliary fibrosis is a crucial but easily neglected pathological feature in hepatobiliary disorders, which may promote the development and progression of benign and malignant biliary diseases through pathological healing mechanisms secondary to biliary tract injuries. Elucidating the etiology and pathogenesis of biliary fibrosis is beneficial to the prevention and treatment of biliary diseases. In this review, we emphasized the importance of biliary fibrosis in cholangiopathies and summarized the clinical manifestations, epidemiology, and aberrant cellular composition involving the biliary ductules, cholangiocytes, immune system, fibroblasts, and the microbiome. We also focused on pivotal signaling pathways and offered insights into ongoing clinical trials and proposing a strategic approach for managing biliary fibrosis-related cholangiopathies. This review will offer a comprehensive perspective on biliary fibrosis and provide an important reference for future mechanism research and innovative therapy to prevent or reverse fibrosis.

The term 'fibroblast' often serves as a catch-all for a diverse array of mesenchymal cells, including perivascular cells, stromal progenitor cells and bona fide fibroblasts. Although phenotypically similar, these subpopulations are functionally distinct, maintaining tissue integrity and serving as local progenitor reservoirs. In response to tissue injury, these cells undergo a dynamic fibroblast-myofibroblast transition, marked by extracellular matrix secretion and contraction of actomyosin-based stress fibres. Importantly, whereas transient activation into myofibroblasts aids in tissue repair, persistent activation triggers pathological fibrosis. In this Review, we discuss the roles of mechanical cues, such as tissue stiffness and strain, alongside cell signalling pathways and extracellular matrix ligands in modulating myofibroblast activation and survival. We also highlight the role of epigenetic modifications and myofibroblast memory in physiological and pathological processes. Finally, we discuss potential strategies for therapeutically interfering with these factors and the associated signal transduction pathways to improve the outcome of dysregulated healing.

Obesity is characterized by adipose tissue expansion, extracellular matrix remodelling and unresolved inflammation that contribute to insulin resistance and fibrosis. Adipose tissue macrophages represent the most abundant class of immune cells in adipose tissue inflammation and could be key mediators of adipocyte dysfunction and fibrosis in obesity. Although macrophage activation states are classically defined by the M1/M2 polarization nomenclature, novel studies have revealed a more complex range of macrophage phenotypes in response to external condition or the surrounding microenvironment. Here, we discuss the plasticity of adipose tissue macrophages (ATMs) in response to their microenvironment in obesity, with special focus on macrophage infiltration and polarization, and their contribution to adipose tissue fibrosis. A better understanding of the role of ATMs as regulators of adipose tissue remodelling may provide novel therapeutic strategies against obesity and associated metabolic diseases.

Diabetic foot ulcers (DFUs) pose a significant challenge in diabetes care. Yet, a comprehensive understanding of the underlying biological disparities between healing and non-healing DFUs remains elusive. We conducted bioinformatics analysis of publicly available transcriptome sequencing data in an attempt to elucidate these differences. Our analysis encompassed differential analysis to unveil shifts in cell composition and gene expression profiles between non-healing and healing DFUs. Cell communication alterations were explored employing the Cellchat R package. Pseudotime analysis and cytoTRACE allowed us to dissect the heterogeneity within fibroblast subpopulations. Our findings unveiled disruptions in various cell types, localized low-grade inflammation, compromised systemic antigen processing and presentation, and extensive extracellular matrix signaling disarray in non-healing DFU patients. Some of these anomalies partially reverted in healing DFUs, particularly within the abnormal ECM-receptor signaling pathway. Furthermore, we distinguished distinct fibroblast subpopulations in non-healing and healing DFUs, each with unique biological functions. Healing-associated fibroblasts exhibited heightened extracellular matrix (ECM) remodeling and a robust wound healing response, while non-healing-associated fibroblasts showed signs of cellular senescence and complement activation, among other characteristics. This analysis offers profound insights into the wound healing microenvironment, identifies pivotal cell types for DFU healing promotion, and reveals potential therapeutic targets for DFU management.

Efferocytosis is a crucial process whereby phagocytes engulf and eliminate apoptotic cells (ACs). This intricate process can be categorized into four steps: (1) ACs release "find me" signals to attract phagocytes, (2) phagocytosis is directed by "eat me" signals emitted by ACs, (3) phagocytes engulf and internalize ACs, and (4) degradation of ACs occurs. Maintaining immune homeostasis heavily relies on the efficient clearance of ACs, which eliminates self-antigens and facilitates the generation of anti-inflammatory and immunosuppressive signals that maintain immune tolerance. However, any disruptions occurring at any of the efferocytosis steps during apoptosis can lead to a diminished efficacy in removing apoptotic cells. Factors contributing to this inefficiency encompass dysregulation in the release and recognition of "find me" or "eat me" signals, defects in phagocyte surface receptors, bridging molecules, and other signaling pathways. The inadequate clearance of ACs can result in their rupture and subsequent release of self-antigens, thereby promoting immune responses and precipitating the onset of autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, type 1 diabetes, and multiple sclerosis. A comprehensive understanding of the efferocytosis process and its implications can provide valuable insights for developing novel therapeutic strategies that target this process to prevent or treat autoimmune diseases.

The potential of human umbilical cord mesenchymal stromal cell-derived extracellular vesicles (hucMSC-EVs) in wound healing is promising, yet a comprehensive understanding of how fibroblasts and keratinocytes respond to this treatment remains limited. This study utilizes single-cell RNA sequencing (scRNA-seq) to investigate the impact of hucMSC-EVs on the cutaneous wound microenvironment in mice. Through rigorous single-cell analyses, we unveil the emergence of hucMSC-EV-induced hematopoietic fibroblasts and MMP13+ fibroblasts. Notably, MMP13+ fibroblasts exhibit fetal-like expressions of MMP13, MMP9, and HAS1, accompanied by heightened migrasome activity. Activation of MMP13+ fibroblasts is orchestrated by a distinctive PIEZO1-calcium-HIF1α-VEGF-MMP13 pathway, validated through murine models and dermal fibroblast assays. Organotypic culture assays further affirm that these activated fibroblasts induce keratinocyte migration via MMP13-LRP1 interactions. This study significantly contributes to our understanding of fibroblast heterogeneities as well as intercellular interactions in wound healing and identifies hucMSC-EV-induced hematopoietic fibroblasts as potential targets for reprogramming. The therapeutic targets presented by these fibroblasts offer exciting prospects for advancing wound healing strategies.

Cardiac fibrosis is a major and complex pathophysiological process that ultimately culminates in cardiac dysfunction and heart failure. This phenomenon includes not only the replacement of the damaged tissue by a fibrotic scar produced by activated fibroblasts/myofibroblasts but also a spatiotemporal alteration of the structural, biochemical, and biomechanical parameters in the ventricular wall, eliciting a reactive remodeling process. Though mechanical stress, post-infarct homeostatic imbalances, and neurohormonal activation are classically attributed to cardiac fibrosis, emerging evidence that supports the roles of immune system modulation, inflammation, and metabolic dysregulation in the initiation and progression of cardiac fibrogenesis has been reported. Adaptive changes, immune cell phenoconversions, and metabolic shifts in the cardiac nonmyocyte population provide initial protection, but persistent altered metabolic demand eventually contributes to adverse remodeling of the heart. Altered energy metabolism, mitochondrial dysfunction, various immune cells, immune mediators, and cross-talks between the immune cells and cardiomyocytes play crucial roles in orchestrating the transdifferentiation of fibroblasts and ensuing fibrotic remodeling of the heart. Manipulation of the metabolic plasticity, fibroblast-myofibroblast transition, and modulation of the immune response may hold promise for favorably modulating the fibrotic response following different cardiovascular pathological processes. Although the immunologic and metabolic perspectives of fibrosis in the heart are being reported in the literature, they lack a comprehensive sketch bridging these two arenas and illustrating the synchrony between them. This review aims to provide a comprehensive overview of the intricate relationship between different cardiac immune cells and metabolic pathways as well as summarizes the current understanding of the involvement of immune-metabolic pathways in cardiac fibrosis and attempts to identify some of the previously unaddressed questions that require further investigation. Moreover, the potential therapeutic strategies and emerging pharmacological interventions, including immune and metabolic modulators, that show promise in preventing or attenuating cardiac fibrosis and restoring cardiac function will be discussed.

Biofilm-related infections (BRIs) present significant challenges owing to drug resistance, adverse immune responses, and implant failure; however, current approaches inadequately cater to the diverse therapeutic requirements at different stages of infection. To address this issue, a multi-immunotherapy strategy in combination with sonodynamic therapy is proposed for the chronological treatment of BRIs. Macrophage membrane-decorated targeting sonosensitive nanoadjuvants are fabricated to load cytosine-phosphate-guanine oligodeoxynucleotide (CPG-ODN) or microRNA (miR)-21-5p. In the early stages of BRI (Stage I), CPG-ODN-loaded nanoadjuvants (CPG@HMPN@M) promote the formation of neutrophil extracellular traps to capture and neutralize detached microbes. During the late stage of infection (Stage II), CPG-ODNs redirect macrophage polarization into the M1 phase to combat infections via TLR9/Myd88/TRAF6 pathway. During these stages, CPG@HMPN@M generates singlet oxygen through sonodynamic processes, eradicating the biofilms under US irradiation. Once the BRIs are eliminated, miR-21-5p-loaded nanoadjuvants (miR@HMPN@M) are delivered to the lesions to suppress excessive inflammation and promote tissue integration by evoking macrophage M2 polarization during the repair phase (Stage III) through PTEN/PI3K/Akt pathway. This innovative approach aims to provide comprehensive treatment strategies for the chronological treatment of BRI by effectively eliminating infections, promoting tissue restoration, and implementing different immune regulations at different stages, thus demonstrating promising clinical value.

The details of how macrophages control different healing trajectories (regeneration vs. scar formation) remain poorly defined. Spiny mice (Acomys spp.) can regenerate external ear pinnae tissue, whereas lab mice (Mus musculus) form scar tissue in response to an identical injury. Here, we used this dual species system to dissect macrophage phenotypes between healing modes. We identified secreted factors from activated Acomys macrophages that induce a pro-regenerative phenotype in fibroblasts from both species. Transcriptional profiling of Acomys macrophages and subsequent in vitro tests identified VEGFC, PDGFA, and Lactotransferrin (LTF) as potential pro-regenerative modulators. Examining macrophages in vivo, we found that Acomys-resident macrophages secreted VEGFC and LTF, whereas Mus macrophages do not. Lastly, we demonstrate the requirement for VEGFC during regeneration and find that interrupting lymphangiogenesis delays blastema and new tissue formation. Together, our results demonstrate that cell-autonomous mechanisms govern how macrophages react to the same stimuli to differentially produce factors that facilitate regeneration.

Diabetes causes a range of complications that can affect multiple organs. Hyperglycemia is an important driver of diabetes-associated complications, mediated by biological processes such as dysfunction of endothelial cells, fibrosis, and alterations in leukocyte number and function. Here, we dissected the transcriptional response of key cell types to hyperglycemia across multiple tissues using single-cell RNA sequencing (scRNA-seq) and identified conserved, as well as organ-specific, changes associated with diabetes complications. By studying an early time point of diabetes, we focus on biological processes involved in the initiation of the disease, before the later organ-specific manifestations had supervened. We used a mouse model of type 1 diabetes and performed scRNA-seq on cells isolated from the heart, kidney, liver, and spleen of streptozotocin-treated and control male mice after 8 weeks and assessed differences in cell abundance, gene expression, pathway activation, and cell signaling across organs and within organs. In response to hyperglycemia, endothelial cells, macrophages, and monocytes displayed organ-specific transcriptional responses, whereas fibroblasts showed similar responses across organs, exhibiting altered metabolic gene expression and increased myeloid-like fibroblasts. Furthermore, we found evidence of endothelial dysfunction in the kidney, and of endothelial-to-mesenchymal transition in streptozotocin-treated mouse organs. In summary, our study represents the first single-cell and multi-organ analysis of early dysfunction in type 1 diabetes-associated hyperglycemia, and our large-scale dataset (comprising 67 611 cells) will serve as a starting point, reference atlas, and resource for further investigating the events leading to early diabetic disease.

Macrophages assume diverse phenotypes and functions in response to cues from the microenvironment. Earlier we reported an anti-inflammatory effect of Collagenase Santyl® Ointment (CSO) and the active constituent of CSO (CS-API) on wound macrophages in resolving wound inflammation indicating roles beyond debridement in wound healing. Building upon our prior finding, this study aimed to understand the phenotypes and subsets of macrophages following treatment with CS-API. scRNA-sequencing was performed on human blood monocyte-derived macrophages (MDM) following treatment with CS-API for 24 h. Unbiased data analysis resulted in the identification of discrete macrophage subsets based on their gene expression profiles. Following CS-API treatment, clusters 3 and 4 displayed enrichment of macrophages with high expression of genes supporting extracellular matrix (ECM) function. IPA analysis identified the TGFβ-1 pathway as a key hub for the CS-API-mediated ECM-supportive phenotype of macrophages. Earlier we reported the physiological conversion of wound-site macrophages to fibroblasts in granulation tissue and impairment of such response in diabetic wounds, leading to compromised ECM and tensile strength. The findings that CSO can augment the physiological conversion of macrophages to fibroblast-like cells carry significant clinical implications. This existing clinical intervention, already employed for wound care, can be readily repurposed to improve the ECM response in chronic wounds.

Objective: Hydrolyzed collagen-based matrices are widely used as wound care dressings. Information on the mechanism of action of such dressings is scanty. The objective of this study was to test the effect of a specific hydrolyzed collagen powder (HCP), which is extensively used for wound care management in the United States. Approach: The effects of HCP on resolution of wound inflammation, perfusion, closure, and breaking strength of the repaired skin were studied in an experimental murine model. Results: In early (day 7) inflammatory phase of wound macrophages, HCP treatment boosted phagocytosis and efferocytosis of wound-site macrophages. In these cells, inducible reactive oxygen species were also higher on day (d) 7. HCP treatment potentiated the expression of anti-inflammatory interleukin (IL)-10 cytokine and proangiogenic vascular endothelial growth factor (VEGF) production. Excisional wounds dressed with HCP showed complete closure on day 21, while the control wounds remained open. HCP treatment also demonstrated improved quality of wound healing as marked by the improved breaking strength of the closed wound tissue/repaired skin. Innovation: These data represent first evidence on the mechanism of action of clinically used HCP. Conclusion: HCP dressing favorably influenced both wound inflammation and vascularization. Improved breaking strength of HCP-treated repaired skin lays the rationale for future studies testing the hypothesis that HCP-treated closed wounds would show fewer recurrences.

Cells of the monocyte/macrophage lineage are an integral component of the body's innate ability to restore tissue function after injury. In parallel to mounting an inflammatory response, clearance of monocytes/macrophages from the wound site is critical to re-establish tissue functionality and integrity during the course of healing. The role of regulated cell death in macrophage clearance from damaged tissue and its implications for the outcome of the healing response is little understood. In this study, we explored the role of macrophage-specific FADD-mediated cell death on Ripk3-/- background in a mechanical skin injury model in mice. We found that combined inhibition of RIPK3-mediated necroptosis and FADD-caspase-8-mediated apoptosis in macrophages profoundly delayed wound healing. Importantly, RIPK3 deficiency alone did not considerably alter the wound healing process and macrophage population dynamics, arguing that inhibition of FADD-caspase-8-dependent death of macrophages is primarily responsible for delayed wound closure. Notably, TNF blockade reversed the accumulation of Ly6Chigh macrophages induced by combined deficiency of FADD and RIPK3, indicating a critical dual role of TNF-mediated prosurvival and cell death signaling, particularly in this highly proinflammatory macrophage subset. Our findings reveal a previously uncharacterized cross-talk of inflammatory and cell death signaling in macrophages in regulating repair processes in the skin.

As one of the most abundant stromal cells, fibroblasts are primarily responsible for the production and remodeling of the extracellular matrix. Traditionally, fibroblasts have been viewed as quiescent cells. However, recent advances in multi-omics technologies have demonstrated that fibroblasts exhibit remarkable functional diversity at the single-cell level. Additionally, fibroblasts are heterogeneous in their origins, tissue locations, and transitions with stromal cells. The dynamic nature of fibroblasts is further underscored by the fact that disease stages can impact their heterogeneity and behavior, particularly in immune-mediated inflammatory diseases such as psoriasis, inflammatory bowel diseases, and rheumatoid arthritis, etc. Fibroblasts can actively contribute to the disease initiation, progression, and relapse by responding to local microenvironmental signals, secreting downstream inflammatory factors, and interacting with immune cells during the pathological process. Here we focus on the development, plasticity, and heterogeneity of fibroblasts in inflammation, emphasizing the need for a developmental and dynamic perspective on fibroblasts.

Exosomes, a class of extracellular vesicles of endocytic origin, play a critical role in paracrine signaling for successful cell-cell crosstalk in vivo. However, limitations in our current understanding of these circulating nanoparticles hinder efficient isolation, characterization, and downstream functional analysis of cell-specific exosomes. In this work, we sought to develop a method to isolate and characterize keratinocyte-originated exosomes (hExo κ ) from human chronic wound fluid. Furthermore, we studied the significance of hExo κ in diabetic wounds. LC-MS-MS detection of KRT14 in hExo κ and subsequent validation by Vesiclepedia and Exocarta databases identified surface KRT14 as a reliable marker of hExo κ . dSTORM nanoimaging identified KRT14+ extracellular vesicles (EV κ ) in human chronic wound fluid, 23% of which were of exosomal origin. An immunomagnetic two-step separation method using KRT14 and tetraspanin antibodies successfully isolated hExo κ from the heterogeneous pool of EV in chronic wound fluid of 15 non-diabetic and 22 diabetic patients. Isolated hExo κ (Ø75-150nm) were characterized per EV-track guidelines. dSTORM images, analyzed using online CODI followed by independent validation using Nanometrix, revealed hExo κ Ø as 80-145nm. The abundance of hExo κ was low in diabetic wound fluids and negatively correlated with patient HbA1c levels. The hExo κ isolated from diabetic wound fluid showed a low abundance of small bp RNA (<200 bp). Raman spectroscopy underscored differences in surface lipids between non-diabetic and diabetic hExo κ Uptake of hExo κ by monocyte-derived macrophages (MDM) was low for diabetics versus non-diabetics. Unlike hExo κ from non-diabetics, the addition of diabetic hExo κ to MDM polarized with LPS and INFγ resulted in sustained expression of iNOS and pro-inflammatory chemokines known to recruit macrophage (mϕ) This work provides maiden insight into the structure, composition, and function of hExo κ from chronic wound fluid thus providing a foundation for the study of exosomal malfunction under conditions of diabetic complications such as wound chronicity.

The ability of cells to sense and respond to changes in mechanical environment is vital in conditions of organ injury when the architecture of normal tissues is disturbed or lost. Among the various cellular players that respond to injury, fibroblasts take center stage in re-establishing tissue integrity by secreting and organizing extracellular matrix into stabilizing scar tissue. Activation, activity, survival, and death of scar-forming fibroblasts are tightly controlled by mechanical environment and proper mechanotransduction ensures that fibroblast activities cease after completion of the tissue repair process. Conversely, dysregulated mechanotransduction often results in fibroblast over-activation or persistence beyond the state of normal repair. The resulting pathological accumulation of extracellular matrix is called fibrosis, a condition that has been associated with over 40% of all deaths in the industrialized countries. Consequently, elements in fibroblast mechanotransduction are scrutinized for their suitability as anti-fibrotic therapeutic targets. We review the current knowledge on mechanically relevant factors in the fibroblast extracellular environment, cell-matrix and cell-cell adhesion structures, stretch-activated membrane channels, stress-regulated cytoskeletal structures, and co-transcription factors. We critically discuss the targetability of these elements in therapeutic approaches and their progress in pre-clinical and/or clinical trials to treat organ fibrosis.

Fetal skin achieves scarless wound repair. Dermal fibroblasts play a central role in extracellular matrix deposition and scarring outcomes. Both fetal and gingival wound repair share minimal scarring outcomes. We tested the hypothesis that compared to adult skin fibroblasts, human fetal skin fibroblast diversity is unique and partly overlaps with gingival skin fibroblasts. Human fetal skin (FS, n = 3), gingiva (HGG, n = 13), and mature skin (MS, n = 13) were compared at single-cell resolution. Dermal fibroblasts, the most abundant cluster, were examined to establish a connectome with other skin cells. Annexin1-FPR1 signaling pathway was dominant in both FS as well as HGG fibroblasts and related myeloid cells while scanty in MS fibroblasts. Myeloid-specific FPR1-ORF delivered in murine wound edge using tissue nanotransfection (TNT) technology significantly enhanced the quality of healing. Pseudotime analyses identified the co-existence of an HGG fibroblast subset with FPR1high myeloid cells of fetal origin indicating common underlying biological processes.

Keloids are a fibroproliferative skin disorder that develops in people of all ages. Keloids exhibit some cancer-like behaviors, with similar genetic and epigenetic modifications in the keloid microenvironment. The keloid microenvironment is composed of keratinocytes, fibroblasts, myofibroblasts, vascular endothelial cells, immune cells, stem cells and collagen fibers. Recent advances in the study of keloids have led to novel insights into cellular communication among components of the keloid microenvironment as well as potential therapeutic targets for treating keloids. In this review, we summarized the nature of genetic and epigenetic regulation in keloid-derived fibroblasts, epithelial-to-mesenchymal transition of keratinocytes, immune cell infiltration into keloids, the differentiation of keloid-derived stem cells, endothelial-to-mesenchymal transition of vascular endothelial cells, extracellular matrix synthesis and remodeling, and uncontrolled angiogenesis in keloids with the aim of identifying new targets for therapeutic benefit. Video Abstract.

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.

To date, no herpesvirus has been shown to latently persist in fibroblastic cells. Here, we show that murine cytomegalovirus, a β-herpesvirus, persists for the long term and across organs in PDGFRα-positive fibroblastic cells, with similar or higher genome loads than in the previously known sites of murine cytomegalovirus latency. Whereas murine cytomegalovirus gene transcription in PDGFRα-positive fibroblastic cells is almost completely silenced at 5 months post-infection, these cells give rise to reactivated virus ex vivo, arguing that they support latent murine cytomegalovirus infection. Notably, PDGFRα-positive fibroblastic cells also support productive virus replication during primary murine cytomegalovirus infection. Mechanistically, Stat1-deficiency promotes lytic infection but abolishes latent persistence of murine cytomegalovirus in PDGFRα-positive fibroblastic cells in vivo. In sum, fibroblastic cells have a dual role as a site of lytic murine cytomegalovirus replication and a reservoir of latent murine cytomegalovirus in vivo and STAT1 is required for murine cytomegalovirus latent persistence in vivo.

Human death marks the end of organismal life under conditions such that the components of the human body continue to be alive. Such postmortem cellular survival depends on the nature (Hardy scale of slow-fast death) of human death. Slow and expected death typically results from terminal illnesses and includes a prolonged terminal phase of life. As such organismal death process unfolds, do cells of the human body adapt for postmortem cellular survival? Organs with low energy cost-of-living, such as the skin, are better suited for postmortem cellular survival. In this work, the effect of different durations of terminal phase of human life on postmortem changes in cellular gene expression was investigated using RNA sequencing data of 701 human skin samples from the Genotype-Tissue Expression (GTEx) database. Longer terminal phase (slow-death) was associated with a more robust induction of survival pathways (PI3K-Akt signaling) in postmortem skin. Such cellular survival response was associated with the upregulation of embryonic developmental transcription factors such as FOXO1 , FOXO3 , ATF4 and CEBPD . Upregulation of PI3K-Akt signaling was independent of sex or duration of death-related tissue ischemia. Analysis of single nucleus RNA-seq of post-mortem skin tissue specifically identified the dermal fibroblast compartment to be most resilient as marked by adaptive induction of PI3K-Akt signaling. In addition, slow death also induced angiogenic pathways in the dermal endothelial cell compartment of postmortem human skin. In contrast, specific pathways supporting functional properties of the skin as an organ were downregulated following slow death. Such pathways included melanogenesis and those representing the skin extracellular matrix (collagen expression and metabolism). Efforts to understand the significance of death as a biological variable (DABV) in influencing the transcriptomic composition of surviving component tissues has far-reaching implications including rigorous interpretation of experimental data collected from the dead and mechanisms involved in transplant-tissue obtained from dead donors.

Extracellular vesicles (EVs) are nanovesicles released by all eukaryotic cells. This work reports the first nanoscale fluorescent visualization of tumor-originating vesicles bearing an angiogenic microRNA (miR)-126 cargo. In a validated experimental model of lethal murine vascular neoplasm, tumor-originating EV delivered its miR-126 cargo to tumor-associated macrophages (TAMs). Such delivery resulted in an angiogenic (LYVE+) change of state in TAM that supported tumor formation. Study of the trafficking of tumor-originating fluorescently tagged EV revealed colocalization with TAM demonstrating uptake by these cells. Ex vivo treatment of macrophages with tumor-derived EVs led to gain of tumorigenicity in these isolated cells. Single-cell RNA sequencing of macrophages revealed that EV-borne miR-126 characterized the angiogenic change of state. Unique gene expression signatures of specific macrophage clusters responsive to miR-126-enriched tumor-derived EVs were revealed. Topical tissue nanotransfection (TNT) delivery of an oligonucleotide comprising an anti-miR against miR-126 resulted in significant knockdown of miR-126 in the tumor tissue. miR-126 knockdown resulted in complete involution of the tumor and improved survival rate of tumor-affected mice. This work identifies a novel tumorigenic mechanism that relies on tumorigenic state change of TAM caused by tumor-originating EV-borne angiomiR. This disease process can be effectively targeted by topical TNT of superficial tumors.

Regeneration studies help to understand the strategies that replace a lost or damaged organ and provide insights into approaches followed in regenerative medicine and engineering. Amphibians regenerate their limbs effortlessly and are indispensable models to study limb regeneration. Xenopus and axolotl are the key models for studying limb regeneration but recent studies on non-model amphibians have revealed species specific differences in regeneration mechanisms.

The present study describes the de novo transcriptome of intact limbs and three-day post-amputation blastemas of tadpoles and froglets of the Asian tree frog Polypedates maculatus, a non-model amphibian species commonly found in India. Differential gene expression analysis between early tadpole and froglet limb blastemas discovered species-specific novel regulators of limb regeneration. The present study reports upregulation of proteoglycans, such as epiphycan, chondroadherin, hyaluronan and proteoglycan link protein 1, collagens 2,5,6, 9 and 11, several tumour suppressors and methyltransferases in the P. maculatus tadpole blastemas. Differential gene expression analysis between tadpole and froglet limbs revealed that in addition to the expression of larval-specific haemoglobin and glycoproteins, an upregulation of cysteine and serine protease inhibitors and downregulation of serine proteases, antioxidants, collagenases and inflammatory genes in the tadpole limbs were essential for creating an environment that would support regeneration. Dermal myeloid cells were GAG+, EPYC+, INMT+, LEF1+ and SALL4+ and seemed to migrate from the unamputated regions of the tadpole limb to the blastema. On the other hand, the myeloid cells of the froglet limb blastemas were few and probably contributed to sustained inflammation resulting in healing.

Studies on non-model amphibians give insights into alternate tactics for limb regeneration which can help devise a plethora of methods in regenerative medicine and engineering.

Complexity and difficulties in wound management are pressing concerns that affect patients' quality of life and may result in tissue infection, necrosis, and loss of local and systemic functions. Hence, novel approaches to accelerate wound healing are being actively explored over the last decade. Exosomes as important mediators of intercellular communications are promising natural nanocarriers due to their biocompatibility, low immunogenicity, drug loading and targeting capacities, and innate stability. More importantly, exosomes are developed as a versatile pharmaceutical engineering platform for wound repair. This review provides an overview of the biological and physiological functions of exosomes derived from a variety of biological origins during wound healing phases, strategies for exosomal engineering, and therapeutic applications in skin regeneration.

Tissue injury to skin diminishes miR-200b in dermal fibroblasts. Fibroblasts are widely reported to directly reprogram into endothelial-like cells and we hypothesized that miR-200b inhibition may cause such changes. We transfected human dermal fibroblasts with anti-miR-200b oligonucleotide, then using single cell RNA sequencing, identified emergence of a vasculogenic subset with a distinct fibroblast transcriptome and demonstrated blood vessel forming function in vivo. Anti-miR-200b delivery to murine injury sites likewise enhanced tissue perfusion, wound closure, and vasculogenic fibroblast contribution to perfused vessels in a FLI1 dependent manner. Vasculogenic fibroblast subset emergence was blunted in delayed healing wounds of diabetic animals but, topical tissue nanotransfection of a single anti-miR-200b oligonucleotide was sufficient to restore FLI1 expression, vasculogenic fibroblast emergence, tissue perfusion, and wound healing. Augmenting a physiologic tissue injury adaptive response mechanism that produces a vasculogenic fibroblast state change opens new avenues for therapeutic tissue vascularization of ischemic wounds.