IQGAP1-mediated mechanical signaling promotes the foreign body response to biomedical implants.

The aim of this study was to further elucidate the molecular mechanisms that mediate pathologic foreign body response (FBR) to biomedical implants. The longevity of biomedical implants is limited by the FBR, which leads to implant failure and patient morbidity. Since the specific molecular mechanisms underlying fibrotic responses to biomedical implants have yet to be fully described, there are currently no targeted approaches to reduce pathologic FBR. We utilized proteomics analysis of human FBR samples to identify potential molecular targets for therapeutic inhibition of FBR. We then employed a murine model of FBR to further evaluate the role of this potential target. We performed histological and immunohistochemical analysis on the murine FBR capsule tissue, as well as single-cell RNA sequencing (scRNA-seq) on cells isolated from the capsules. We identified IQ motif containing GTPase activating protein 1 (IQGAP1) as the most promising of several targets, serving as a central molecular mediator in human and murine FBR compared to control subcutaneous tissue. IQGAP1-deficient mice displayed a significantly reduced FBR compared to wild-type mice as evidenced by lower levels of collagen deposition and maturity. Our scRNA-seq analysis revealed that decreasing IQGAP1 resulted in diminished transcription of mechanotransduction, inflammation, and fibrosis-related genes, which was confirmed on the protein level with immunofluorescent staining. The deficiency of IQGAP1 significantly attenuates FBR by deactivating downstream mechanotransduction signaling, inflammation, and fibrotic pathways. IQGAP1 may be a promising target for rational therapeutic design to mitigate pathologic FBR around biomedical implants.

Macrophage Subpopulation Dynamics Shift following Intravenous Infusion of Mesenchymal Stromal Cells.

Intravenous infusion of mesenchymal stromal cells (MSCs) is thought to be a viable treatment for numerous disorders. Although the intrinsic immunosuppressive ability of MSCs has been credited for this therapeutic effect, their exact impact on endogenous tissue-resident cells following delivery has not been clearly characterized. Moreover, multiple studies have reported pulmonary sequestration of MSCs upon intravenous delivery. Despite substantial efforts to improve MSC homing, it remains unclear whether MSC migration to the site of injury is necessary to achieve a therapeutic effect. Using a murine excisional wound healing model, we offer an explanation of how sequestered MSCs improve healing through their systemic impact on macrophage subpopulations. We demonstrate that infusion of MSCs leads to pulmonary entrapment followed by rapid clearance, but also significantly accelerates wound closure. Using single-cell RNA sequencing of the wound, we show that following MSC delivery, innate immune cells, particularly macrophages, exhibit distinctive transcriptional changes. We identify the appearance of a pro-angiogenic CD9+ macrophage subpopulation, whose induction is mediated by several proteins secreted by MSCs, including COL6A1, PRG4, and TGFB3. Our findings suggest that MSCs do not need to act locally to induce broad changes in the immune system and ultimately treat disease.

Live imaging of angiogenesis during cutaneous wound healing in adult zebrafish.

Angiogenesis, the growth of new blood vessels from pre-existing vessels, is critical for cutaneous wound healing. However, it remains elusive how endothelial cells (ECs) and pericytes (PCs) establish new blood vessels during cutaneous angiogenesis. We set up a live-imaging system to analyze cutaneous angiogenesis in adult zebrafish. First, we characterized basic structures of cutaneous vasculature. In normal skin tissues, ECs and PCs remained dormant to maintain quiescent blood vessels, whereas cutaneous injury immediately induced angiogenesis through the vascular endothelial growth factor signaling pathway. Tortuous and disorganized vessel networks formed within a few weeks after the injury and subsequently normalized through vessel regression in a few months. Analyses of the repair process of injured single blood vessels revealed that severed vessels elongated upon injury and anastomosed with each other. Thereafter, repaired vessels and adjacent uninjured vessels became tortuous by increasing the number of ECs. In parallel, PCs divided and migrated to cover the tortuous blood vessels. ECs sprouted from the PC-covered tortuous vessels, suggesting that EC sprouting does not require PC detachment from the vessel wall. Thus, live imaging of cutaneous angiogenesis in adult zebrafish enables us to clarify how ECs and PCs develop new blood vessels during cutaneous angiogenesis.

Endothelial dysfunction may promote keloid growth.

Keloid is a cutaneous fibroproliferative disorder. It results from impaired wound healing that generates persistent inflammation and extensive deposition of collagen fibers in the wound/scar. Keloids tend to be worse in hypertensive patients. The present prospective cross-sectional study assessed whether endothelial dysfunction, which occurs in hypertension, associates with keloid formation and progression. This study included randomly selected patients with keloids who were assessed for surgical keloid treatment in 2013-2014. A series of nonkeloid patients admitted to the hospital was also recruited during this period. To measure endothelial function, all patients underwent digital reactive hyperemia peripheral arterial tonometry. Test results were expressed as reactive hyperemia index (RHI) and augmentation index (AI). In total, 57 patients with keloids and 19 nonkeloid controls were recruited. Keloid patients did not differ from the controls in terms of demographic or clinical variables, but had significantly worse RHI and AI values. Moreover, poor RHI and AI values associated with keloid development on binomial logistic regression. The keloid patients were then divided into four groups depending on whether their keloids started at age 0-12, 13-18, 19-29, or ≥30 years. Patients whose keloids arose before and well after puberty tended to have lower RHI than the controls, but these differences did not achieve statistical significance. However, these two groups did have significantly poorer AI values than the controls. Thus, endothelial dysfunction could cause keloid formation and/or aggravation. This indicates that vascular endothelial cells are important for wound healing.

Generation of two iPSC lines from vascular Ehlers-Danlos Syndrome (vEDS) patients carrying a missense mutation in COL3A1 gene.

Vascular Ehlers-Danlos Syndrome (vEDS) is an inherited connective tissue disorder caused by COL3A1 gene, mutations that encodes type III collagen, a crucial component of blood vessels. vEDS can be life-threatening as these patients can have severe internal bleeding due to arterial rupture. Here, we generated induced pluripotent stem cell (iPSC) lines from two vEDS patients carrying a missense mutation in the COL3A1 (c.226A > G, p.Asn76Asp) gene. These lines exhibited typical iPSC characteristics including morphology, expression of pluripotency markers, and could differentiate to all three germ layer. These iPSC lines can serve as valuable tools for elucidating the pathophysiology underlying vEDS.

Generation of induced pluripotent stem cell line from a patient suffering from arterial calcification due to deficiency of CD73 (ACDC).

Arterial calcification due to deficiency of CD73 (ACDC) is an adult onset, rare genetic vascular disorder signified by calcium deposition in lower extremity arteries and joints of hands and feet. Mutations in NT5E gene has been shown to be responsible for the inactivation of enzyme CD73 causing calcium buildup. Here, we report a iPSC line generated from a patient showing signs of ACDC and carrying a missense mutation in NT5E (c.1126A→G,p.T376A) gene. This iPSC line shows normal morphology, pluripotency, karyotype, and capability to differentiate into three germ layers, making it useful for disease modeling and investigating pathological mechanisms of ACDC.

CCL2-mediated endothelial injury drives cardiac dysfunction in long COVID.

Evidence linking the endothelium to cardiac injury in long coronavirus disease (COVID) is well documented, but the underlying mechanisms remain unknown. Here we show that cytokines released by endothelial cells (ECs) contribute to long-COVID-associated cardiac dysfunction. Using thrombotic vascular tissues from patients with long COVID and induced pluripotent stem cell-derived ECs (iPSC-ECs), we modeled endotheliitis and observed similar dysfunction and cytokine upregulation, notably CCL2. Cardiac organoids comprising iPSC-ECs and iPSC-derived cardiomyocytes showed cardiac dysfunction after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exposure, driven by CCL2. Profiling of chromatin accessibility and gene expression at a single-cell resolution linked CCL2 to 'phenotype switching' and cardiac dysfunction, validated by high-throughput proteomics. Disease modeling of cardiac organoids and exposure of human ACE2 transgenic mice to SARS-CoV-2 spike proteins revealed that CCL2-induced oxidative stress promoted post-translational modification of cardiac proteins, leading to cardiac dysfunction. These findings suggest that EC-released cytokines contribute to cardiac dysfunction in long COVID, highlighting the importance of early vascular health monitoring in patients with long COVID.

Over 90% of Childhood BCG Vaccine-Induced Keloids in Japan Occur in Women.

INTRODUCTION: Keloids are a fibroproliferative, multifactorial, cutaneous disorder whose pathophysiology is not completely understood. Various factors such as high blood pressure, pregnancy, female gender, mechanical tension of local sites, and prolonged wound healing are known to worsen keloids. Childhood-onset keloids are keloids that form before 10 years of age, before various factors in adulthood come into play, and thus studying childhood-onset keloids may provide additional insight into the underlying mechanisms that lead to keloid formation. METHODS: Retrospective chart review was performed on all patients with childhood-onset keloids who were evaluated at our plastic surgery clinic (one of the largest keloid referral centers in Japan) over a 1-year period. RESULTS: Of the 1443 patients with diagnosis of keloids, 131 patients had childhood-onset keloids. Of these, 106 patients (80.9%) were female, 38.9% of patients had family history of keloids, and 48.9% of patients had allergies or allergy-related conditions (asthma, atopic dermatitis, or allergic rhinitis). Vaccination (47.5%) and chickenpox (19.9%) were the most common triggers. Of vaccinations, BCG was the most common trigger. The majority of keloids from BCG were in female patients (92.9%). The most common location was the chest in male patients (30.0%) and the arm in female patients (41.1%). CONCLUSION: To our knowledge, this is the largest report in the literature on childhood-onset keloids. There was overall female predominance in childhood-onset keloids, and even more significant female predominance in BCG-induced keloids.

Novel regulatory mechanisms underlying angiogenesis during wound healing revealed by fluorescence-based live-imaging in zebrafish.

Angiogenesis is a dynamic morphogenetic process that refers to the growth of new blood vessels from the pre-existing vessels and is critical for tissue repair during wound healing. In adult normal tissues, quiescent endothelial cells and pericytes maintain vascular integrity, whereas angiogenesis is immediately induced upon tissue injury, thereby forming neovascular networks to maintain homeostasis. However, impaired angiogenesis results in development of chronic and non-healing wounds in various diseases such as diabetes and peripheral artery diseases. Zebrafish are a vertebrate model organism widely used for studying many medical and life science fields. Indeed, the molecular and cellular mechanisms underlying regulation of wound angiogenesis have recently been studied by performing fluorescence-based live-imaging of adult zebrafish. In this review, we describe how endothelial cells and pericytes establish neovascular networks during wound angiogenesis and also introduce a novel role of blood flow-driven intraluminal pressure in regulating angiogenesis during wound healing.

Nitric oxide-releasing gel accelerates healing in a diabetic murine splinted excisional wound model.

Introduction: According to the American Diabetes Association (ADA), 9-12 million patients suffer from chronic ulceration each year, costing the healthcare system over USD $25 billion annually. There is a significant unmet need for new and efficacious therapies to accelerate closure of non-healing wounds. Nitric Oxide (NO) levels typically increase rapidly after skin injury in the inflammatory phase and gradually diminish as wound healing progresses. The effect of increased NO concentration on promoting re-epithelization and wound closure has yet to be described in the context of diabetic wound healing. Methods: In this study, we investigated the effects of local administration of an NO-releasing gel on excisional wound healing in diabetic mice. The excisional wounds of each mouse received either NO-releasing gel or a control phosphate-buffered saline (PBS)-releasing gel treatment twice daily until complete wound closure. Results: Topical administration of NO-gel significantly accelerated the rate of wound healing as compared with PBS-gel-treated mice during the later stages of healing. The treatment also promoted a more regenerative ECM architecture resulting in shorter, less dense, and more randomly aligned collagen fibers within the healed scars, similar to that of unwounded skin. Wound healing promoting factors fibronectin, TGF-ß1, CD31, and VEGF were significantly elevated in NO vs. PBS-gel-treated wounds. Discussion: The results of this work may have important clinical implications for the management of patients with non-healing wounds.

Disrupting mechanotransduction decreases fibrosis and contracture in split-thickness skin grafting.

Burns and other traumatic injuries represent a substantial biomedical burden. The current standard of care for deep injuries is autologous split-thickness skin grafting (STSG), which frequently results in contractures, abnormal pigmentation, and loss of biomechanical function. Currently, there are no effective therapies that can prevent fibrosis and contracture after STSG. Here, we have developed a clinically relevant porcine model of STSG and comprehensively characterized porcine cell populations involved in healing with single-cell resolution. We identified an up-regulation of proinflammatory and mechanotransduction signaling pathways in standard STSGs. Blocking mechanotransduction with a small-molecule focal adhesion kinase (FAK) inhibitor promoted healing, reduced contracture, mitigated scar formation, restored collagen architecture, and ultimately improved graft biomechanical properties. Acute mechanotransduction blockade up-regulated myeloid CXCL10-mediated anti-inflammation with decreased CXCL14-mediated myeloid and fibroblast recruitment. At later time points, mechanical signaling shifted fibroblasts toward profibrotic differentiation fates, and disruption of mechanotransduction modulated mesenchymal fibroblast differentiation states to block those responses, instead driving fibroblasts toward proregenerative, adipogenic states similar to unwounded skin. We then confirmed these two diverging fibroblast transcriptional trajectories in human skin, human scar, and a three-dimensional organotypic model of human skin. Together, pharmacological blockade of mechanotransduction markedly improved large animal healing after STSG by promoting both early, anti-inflammatory and late, regenerative transcriptional programs, resulting in healed tissue similar to unwounded skin. FAK inhibition could therefore supplement the current standard of care for traumatic and burn injuries.

Inhibiting Fibroblast Mechanotransduction Modulates Severity of Idiopathic Pulmonary Fibrosis.

Objective: Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease that affects 63 in every 100,000 Americans. Its etiology remains unknown, although inflammatory pathways appear to be important. Given the dynamic environment of the lung, we examined the significance of mechanotransduction on both inflammatory and fibrotic signaling during IPF. Innovation: Mechanotransduction pathways have not been thoroughly examined in the context of lung disease, and pharmacologic approaches for IPF do not currently target these pathways. The interplay between mechanical strain and inflammation in pulmonary fibrosis remains incompletely understood. Approach: In this study, we used conditional KO mice to block mechanotransduction by knocking out Focal Adhesion Kinase (FAK) expression in fibroblasts, followed by induction of pulmonary fibrosis using bleomycin. We examined both normal human and human IPF fibroblasts and used immunohistochemistry, quantitative real-time polymerase chain reaction, and Western Blot to evaluate the effects of FAK inhibitor (FAK-I) on modulating fibrotic and inflammatory genes. Results: Our data indicate that the deletion of FAK in mice reduces expression of fibrotic and inflammatory genes in lungs. Similarly, mechanical straining in normal human lung fibroblasts activates inflammatory and fibrotic pathways. The FAK inhibition decreases these signals but has a less effect on IPF fibroblasts as compared with normal human fibroblasts. Conclusion: Administering FAK-I at early stages of fibrosis may attenuate the FAK-mediated fibrotic response pathway in IPF, potentially mediating disease progression.

Hydrogel Scaffolds to Deliver Cell Therapies for Wound Healing.

Cutaneous wounds are a growing global health burden as a result of an aging population coupled with increasing incidence of diabetes, obesity, and cancer. Cell-based approaches have been used to treat wounds due to their secretory, immunomodulatory, and regenerative effects, and recent studies have highlighted that delivery of stem cells may provide the most benefits. Delivering these cells to wounds with direct injection has been associated with low viability, transient retention, and overall poor efficacy. The use of bioactive scaffolds provides a promising method to improve cell therapy delivery. Specifically, hydrogels provide a physiologic microenvironment for transplanted cells, including mechanical support and protection from native immune cells, and cell-hydrogel interactions may be tailored based on specific tissue properties. In this review, we describe the current and future directions of various cell therapies and usage of hydrogels to deliver these cells for wound healing applications.

Adipose-Derived Stromal Cells Seeded in Pullulan-Collagen Hydrogels Improve Healing in Murine Burns.

Burn scars and scar contractures cause significant morbidity for patients. Recently, cell-based therapies have been proposed as an option for improving healing and reducing scarring after burn injury, through their known proangiogenic and immunomodulatory paracrine effects. Our laboratory has developed a pullulan-collagen hydrogel that, when seeded with mesenchymal stem cells (MSCs), improves cell viability and augments their proangiogenic capacity in vivo. Concurrently, recent research suggests that prospective isolation of cell subpopulations with desirable transcriptional profiles can be used to further improve cell-based therapies. In this study, we examined whether adipose-derived stem cell (ASC)-seeded hydrogels could improve wound healing following thermal injury using a murine contact burn model. Partial thickness contact burns were created on the dorsum of mice. On days 5 and 10 following injury, burns were debrided and received either ASC hydrogel, ASC injection alone, hydrogel alone, or no treatment. On days 10 and 25, burns were harvested for histologic and molecular analysis. This experiment was repeated using CD26+/CD55+ FACS-enriched ASCs to further evaluate the regenerative potential of ASCs in wound healing. ASC hydrogel-treated burns demonstrated accelerated time to reepithelialization, greater vascularity, and increased expression of the proangiogenic genes MCP-1, VEGF, and SDF-1 at both the mRNA and protein level. Expression of the profibrotic gene Timp1 and proinflammatory gene Tnfa was downregulated in ASC hydrogel-treated burns. ASC hydrogel-treated burns exhibited reduced scar area compared to hydrogel-treated and control wounds, with equivalent scar density. CD26+/CD55+ ASC hydrogel treatment resulted in accelerated healing, increased dermal appendage count, and improved scar quality with a more reticular collagen pattern. Here we find that ASC hydrogel therapy is effective for treating burns, with demonstrated proangiogenic, fibromodulatory, and immunomodulatory effects. Enrichment for CD26+/CD55+ ASCs has additive benefits for tissue architecture and collagen remodeling postburn injury. Research is ongoing to further facilitate clinical translation of this promising therapeutic approach. Impact statement Burns remain a significant public health burden. Stem cell therapy has gained attention as a promising approach for treating burns. We have developed a pullulan-collagen biomimetic hydrogel scaffold that can be seeded with adipose-derived stem cells (ASCs). We assessed the delivery and activity of our scaffold in a murine contact burn model. Our results suggest that localized delivery of ASC hydrogel treatment is a promising approach for the treatment of burn wounds, with the potential for rapid clinical translation. We believe our work will have broad implications for both hydrogel therapeutics and regenerative medicine and will be of interest to the general scientific community.

Disrupting biological sensors of force promotes tissue regeneration in large organisms.

Tissue repair and healing remain among the most complicated processes that occur during postnatal life. Humans and other large organisms heal by forming fibrotic scar tissue with diminished function, while smaller organisms respond with scarless tissue regeneration and functional restoration. Well-established scaling principles reveal that organism size exponentially correlates with peak tissue forces during movement, and evolutionary responses have compensated by strengthening organ-level mechanical properties. How these adaptations may affect tissue injury has not been previously examined in large animals and humans. Here, we show that blocking mechanotransduction signaling through the focal adhesion kinase pathway in large animals significantly accelerates wound healing and enhances regeneration of skin with secondary structures such as hair follicles. In human cells, we demonstrate that mechanical forces shift fibroblasts toward pro-fibrotic phenotypes driven by ERK-YAP activation, leading to myofibroblast differentiation and excessive collagen production. Disruption of mechanical signaling specifically abrogates these responses and instead promotes regenerative fibroblast clusters characterized by AKT-EGR1.

Current and Emerging Topical Scar Mitigation Therapies for Craniofacial Burn Wound Healing.

Burn injury in the craniofacial region causes significant health and psychosocial consequences and presents unique reconstructive challenges. Healing of severely burned skin and underlying soft tissue is a dynamic process involving many pathophysiological factors, often leading to devastating outcomes such as the formation of hypertrophic scars and debilitating contractures. There are limited treatment options currently used for post-burn scar mitigation but recent advances in our knowledge of the cellular and molecular wound and scar pathophysiology have allowed for development of new treatment concepts. Clinical effectiveness of these experimental therapies is currently being evaluated. In this review, we discuss current topical therapies for craniofacial burn injuries and emerging new therapeutic concepts that are highly translational.

Sex Differences in Keloidogenesis: An Analysis of 1659 Keloid Patients in Japan.

INTRODUCTION: Keloids are a cutaneous fibroproliferative disorder. Despite the fact that keloids are relatively common lesions, the statistics of patient with keloids especially sex difference remain unknown. To better understand it, we conducted an extensive cross-sectional analysis of a large cohort of patients with keloids (n = 1659). The study showed for the first time that female sex may be an inherent keloid risk factor. METHODS: This cross-sectional study of 1659 consecutive patients with keloids who attended a plastic surgery outpatient clinic in Japan in 2014 analyzed age at keloid onset, age at the first medical examination for keloid, and the influence of sex on these variables. RESULTS: In both male and female patients, the keloids were most likely to start in puberty and there was no significant difference in the mode value for age of onset (16 vs. 20 years). Though female patients were twice as prevalent as male patients at nearly all onset ages, female patients predominated over male patients with a gender ratio of 2.7:1 in cases of onset before the age of 15 years. Moreover male and female patients did not differ in terms of the mean ± SD duration between keloid onset and the first medical examination. This finding shows that female patients do not get their keloids examined earlier than male patients. These observations together suggest that female sex may promote early keloid development due to physiological, not social, reasons. CONCLUSION: This is the first report to suggest that female sex may drive keloidogenesis because of physiological reasons. Individuals were most likely to seek a medical examination almost 10 years after onset, regardless of sex. These findings provide new insight into the importance of sex in the development and progression of keloids. Future studies should address the influence of sex hormones on the keloid.