Exacerbated ischemia-reperfusion injury in fatty livers is mediated by lipid peroxidation stress and ferroptosis.

BACKGROUND: Ischemia-reperfusion injury is a common problem in liver surgery and transplantation. Although ischemia-reperfusion injury is known to be more pronounced in fatty livers, the underlying mechanisms for this difference remain poorly understood. We hypothesized that ferroptosis plays a significant role in fatty liver ischemia-reperfusion injury due to increased lipid peroxidation in the presence of stored iron in the fatty liver. To test this hypothesis, the ferroptosis pathway was evaluated in a murine fatty liver ischemia-reperfusion injury model. METHODS: C57BL6 mice were fed with a normal diet or a high fat, high sucrose diet for 12 weeks. At 22 weeks of age, liver ischemia-reperfusion injury was induced through partial (70%) hepatic pedicle clamping for 60 minutes, followed by 24 hours of reperfusion before tissue harvest. Acyl-coenzyme A synthetase long-chain family member 4 and 4-hydroxynonenal were quantified in the liver tissues. In separate experiments, liproxstatin-1 or vehicle control was administered for 7 consecutive days before liver ischemia-reperfusion injury. RESULTS: Exacerbated ischemia-reperfusion injury was observed in the livers of high fat, high sucrose diet fed mice. High fat, high sucrose diet + ischemia-reperfusion injury (HDF+IRI) livers had a significantly greater abundance of acyl-coenzyme A synthetase long-chain family member 4 and 4-hydroxynonenal compared with normal diet + ischemia-reperfusion injury (ND+IRI) livers or sham fatty livers, which indicated an increase of ferroptosis. HFD fed animals receiving liproxstatin-1 injections had a significant reduction in serum aspartate transaminase and alanine transaminase after ischemia-reperfusion injury, consistent with attenuation of ischemia-reperfusion injury in the liver. CONCLUSION: Ferroptosis plays a significant role in ischemia-reperfusion injury in fatty livers. Inhibiting ferroptotic pathways in the liver may serve as a novel therapeutic strategy to protect the fatty liver in the setting of ischemia-reperfusion injury.

Collagenase-based wound debridement agent induces extracellular matrix supporting phenotype in macrophages.

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 TGFß-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.

Tissue Nanotransfection Silicon Chip and Related Electroporation-Based Technologies for In Vivo Tissue Reprogramming.

Tissue nanotransfection (TNT), a cutting-edge technique of in vivo gene therapy, has gained substantial attention in various applications ranging from in vivo tissue reprogramming in regenerative medicine, and wound healing to cancer treatment. This technique harnesses the advancements in the semiconductor processes, facilitating the integration of conventional transdermal gene delivery methods-nanoelectroporation and microneedle technologies. TNT silicon chips have demonstrated considerable promise in reprogramming fibroblast cells of skin in vivo into vascular or neural cells in preclinical studies to assist in the recovery of injured limbs and damaged brain tissue. More recently, the application of TNT chips has been extended to the area of exosomes, which are vital for intracellular communication to track their functionality during the wound healing process. In this review, we provide an in-depth examination of the design, fabrication, and applications of TNT silicon chips, alongside a critical analysis of the electroporation-based gene transfer mechanisms. Additionally, the review discussed the existing limitations and challenges in the current technique, which may project future trajectories in the landscape of gene therapy. Through this exploration, the review aims to shed light on the prospects of TNT in the broader context of gene therapy and tissue regeneration.

Hydrolyzed Collagen Powder Dressing Improves Wound Inflammation, Perfusion, and Breaking Strength of Repaired Tissue.

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.

Bacterial Pyocyanin Inducible Keratin 6A Accelerates Closure of Epithelial Defect under Conditions of Mitochondrial Dysfunction.

Repair of epithelial defect is complicated by infection and related metabolites. Pyocyanin (PYO) is one such metabolite that is secreted during Pseudomonas aeruginosa infection. Keratinocyte (KC) migration is required for the closure of skin epithelial defects. This work sought to understand PYO-KC interaction and its significance in tissue repair. Stable Isotope Labeling by Amino acids in Cell culture proteomics identified mitochondrial dysfunction as the top pathway responsive to PYO exposure in human KCs. Consistently, functional studies showed mitochondrial stress, depletion of reducing equivalents, and adenosine triphosphate. Strikingly, despite all stated earlier, PYO markedly accelerated KC migration. Investigation of underlying mechanisms revealed, to our knowledge, a previously unreported function of keratin 6A in KCs. Keratin 6A was PYO inducible and accelerated closure of epithelial defect. Acceleration of closure was associated with poor quality healing, including compromised expression of apical junction proteins. This work recognizes keratin 6A for its role in enhancing KC migration under conditions of threat posed by PYO. Qualitatively deficient junctional proteins under conditions of defensive acceleration of KC migration explain why an infected wound close with deficient skin barrier function as previously reported.

Adult skin fibroblast state change in murine wound healing.

Wound healing is a well-organized dynamic process involving coordinated consecutive phases: homeostasis, inflammation, proliferation and resolution. Fibroblasts play major roles in skin wound healing such as in wound contraction and release of growth factors which are of importance in angiogenesis and tissue remodeling. Abnormal fibroblast phenotypes have been identified in patients with chronic wounds. In this work, we analyzed scRNA-seq datasets of normal and wounded skin from mice at day 4 post-wound to investigate fibroblast heterogeneity during the proliferative phase of wound healing. Compositional analysis revealed a specific subset of fibroblast (cluster 3) that primarily increased in wounded skin (14%) compared to normal skin (3.9%). This subset was characterized by a gene signature marked by the plasma membrane proteins Sfrp2 + Sfrp4 + Sfrp1 + and the transcription factors Ebf1 + Prrx1 + Maged1 + . Differential gene expression and enrichment analysis identified epithelial to mesenchymal transition (EMT) and angiogenesis to be upregulated in the emerging subset of fibroblasts of the wounded skin. Using two other datasets for murine wounded skin confirmed the increase in cluster 3-like fibroblasts at days 2, 7 and 14 post-wounding with a peak at day 7. By performing a similarity check between the differential gene expression profile between wounded and normal skin for this emerging fibroblast subset with drug signature from the ConnectivityMap database, we identified drugs capable of mimicking the observed gene expression change in fibroblasts during wound healing. TTNPB, verteprofin and nicotinic acid were identified as candidate drugs capable of inducing fibroblast gene expression profile necessary for wound healing. On the other hand, methocarbamol, ifosfamide and penbutolol were recognized to antagonize the identified fibroblast differential expression profile during wound healing which might cause delay in wound healing. Taken together, analysis of murine transcriptomic skin wound healing datasets suggested a subset of fibroblasts capable of inducing EMT and further inferred drugs that might be tested as potential candidates to induce wound closure.

Tissue nanotransfection causes tumor regression by its effect on nanovesicle cargo that alters microenvironmental macrophage state.

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.

Pseudomonas Aeruginosa Theft Biofilm Require Host Lipids of Cutaneous Wound.

OBJECTIVE: This work addressing complexities in wound infection, seeks to test the reliance of bacterial pathogen Pseudomonas aeruginosa (PA) on host skin lipids to form biofilm with pathological consequences. BACKGROUND: PA biofilm causes wound chronicity. Both CDC as well as NIH recognizes biofilm infection as a threat leading to wound chronicity. Chronic wounds on lower extremities often lead to surgical limb amputation. METHODS: An established preclinical porcine chronic wound biofilm model, infected with PA or Pseudomonas aeruginosa ceramidase mutant (PA ∆Cer ), was used. RESULTS: We observed that bacteria drew resource from host lipids to induce PA ceramidase expression by three orders of magnitude. PA utilized product of host ceramide catabolism to augment transcription of PA ceramidase. Biofilm formation was more robust in PA compared to PA ∆Cer . Downstream products of such metabolism such as sphingosine and sphingosine-1-phosphate were both directly implicated in the induction of ceramidase and inhibition of peroxisome proliferator-activated receptor (PPAR)δ, respectively. PA biofilm, in a ceram-idastin-sensitive manner, also silenced PPARδ via induction of miR-106b. Low PPARδ limited ABCA12 expression resulting in disruption of skin lipid homeostasis. Barrier function of the wound-site was thus compromised. CONCLUSIONS: This work demonstrates that microbial pathogens must co-opt host skin lipids to unleash biofilm pathogenicity. Anti-biofilm strategies must not necessarily always target the microbe and targeting host lipids at risk of infection could be productive. This work may be viewed as a first step, laying fundamental mechanistic groundwork, toward a paradigm change in biofilm management.

Sex as Biological Variable in Cardiac Mitochondrial Bioenergetic Responses to Acute Stress.

Cardiac dysfunction/damage following trauma, shock, sepsis, and ischemia impacts clinical outcomes. Acute inflammation and oxidative stress triggered by these injuries impair mitochondria, which are critical to maintaining cardiac function. Despite sex dimorphisms in consequences of these injuries, it is unclear whether mitochondrial bioenergetic responses to inflammation/oxidative stress are sex-dependent. We hypothesized that sex disparity in mitochondrial bioenergetics following TNFα or H2O2 exposure is responsible for reported sex differences in cardiac damage/dysfunction. Methods and Results: Cardiomyocytes isolated from age-matched adult male and female mice were subjected to 1 h TNFα or H2O2 challenge, followed by detection of mitochondrial respiration capacity using the Seahorse XF96 Cell Mito Stress Test. Mitochondrial membrane potential (ΔΨm) was analyzed using JC-1 in TNFα-challenged cardiomyocytes. We found that cardiomyocytes isolated from female mice displayed a better mitochondrial bioenergetic response to TNFα or H2O2 than those isolated from male mice did. TNFα decreased ΔΨm in cardiomyocytes isolated from males but not from females. 17ß-estradiol (E2) treatment improved mitochondrial metabolic function in cardiomyocytes from male mice subjected to TNFα or H2O2 treatment. Conclusions: Cardiomyocyte mitochondria from female mice were more resistant to acute stress than those from males. The female sex hormone E2 treatment protected cardiac mitochondria against acute inflammatory and oxidative stress.

Lysosomal acid lipase, CSF1R, and PD-L1 determine functions of CD11c+ myeloid-derived suppressor cells.

Lysosomal acid lipase (LAL) is a key enzyme in the metabolic pathway of neutral lipids. In the blood of LAL-deficient (Lal-/-) mice, increased CD11c+ cells were accompanied by upregulated programmed cell death ligand 1 (PD-L1) expression. Single-cell RNA sequencing of Lal-/- CD11c+ cells identified 2 distinctive clusters with a major metabolic shift toward glucose utilization and reactive oxygen species overproduction. Pharmacologically blocking pyruvate dehydrogenase in glycolysis not only reduced CD11c+ cells and their PD-L1 expression but also reversed their capabilities of T cell suppression and tumor growth stimulation. Colony-stimulating factor 1 receptor (CSF1R) played an essential role in controlling Lal-/- CD11c+ cell homeostasis and function and PD-L1 expression. Pharmacological inhibition of LAL activity increased CD11c, PD-L1, and CSF1R levels in both normal murine myeloid cells and human blood cells. Tumor-bearing mice and human patients with non-small cell lung cancer also showed CD11c+ cell expansion with PD-L1 and CSF1R upregulation and immunosuppression. There were positive correlations among CD11c, PD-L1, and CSF1R expression and negative correlations with LAL expression in patients with lung cancer or melanoma using The Cancer Genome Atlas database and patient samples. Therefore, CD11c+ cells switched their functions to immune suppression and tumor growth stimulation through CSF1R/PD-L1 upregulation and metabolic reprogramming.

Exosomal Transfer of DNA Methyl-Transferase mRNA Induces an Immunosuppressive Phenotype in Human Monocytes.

INTRODUCTION: Survivors of sepsis exhibit persistent immunosuppression. Epigenetic events may be responsible for some of these immunosuppressive changes. During sepsis circulating exosomes contain large quantities of DNA methyltransferase (DNMT) mRNAs. We hypothesized that exosomes directly transfer DNMT mRNAs to recipient monocytes with resultant methylation events and immunosuppression. METHODS: Exosomes containing DNMT mRNA were generated by stimulating monocytes with LPS. Confocal microscopy was used to determine uptake kinetics in the presence of pharmacologic inhibition. Expression and packaging of specific DNMT mRNA was controlled using DNMT siRNAs. Whole genome and gene specific methylation was assessed using bisulfite sequencing. Ingenuity pathway analysis was performed to determine the biological function of significance of differentially methylated regions. RESULTS: Exosomes effectively transferred DNMT mRNA to recipient monocytes. Pharmacologic inhibition of exosome uptake prevented this increase in DNMT mRNA expression. Recipient monocytes exhibited hypermethylation changes and gene suppression. siRNAs decreased the packaging of DNMT mRNAs and prevented TNFα gene suppression, restoring immunocompetence. CONCLUSION: These data support a role for exosome-mediated transfer of DNMT mRNA with resultant methylation and gene silencing. Pharmacologic uptake inhibition or targeted siRNA mediated DNMT gene silencing prevented DNMT mRNA transfer and maintained the cell's ability to express TNFα in response to LPS. This highlights the potential therapeutic value of targeting these exosome-mediated epigenetic events to maintain the host immune response during sepsis.

Endothelial Phospholipase Cγ2 Improves Outcomes of Diabetic Ischemic Limb Rescue Following VEGF Therapy.

Therapeutic vascular endothelial growth factor (VEGF) replenishment has met with limited success for the management of critical limb-threatening ischemia. To improve outcomes of VEGF therapy, we applied single-cell RNA sequencing (scRNA-seq) technology to study the endothelial cells of the human diabetic skin. Single-cell suspensions were generated from the human skin followed by cDNA preparation using the Chromium Next GEM Single-cell 3' Kit v3.1. Using appropriate quality control measures, 36,487 cells were chosen for downstream analysis. scRNA-seq studies identified that although VEGF signaling was not significantly altered in diabetic versus nondiabetic skin, phospholipase Cγ2 (PLCγ2) was downregulated. The significance of PLCγ2 in VEGF-mediated increase in endothelial cell metabolism and function was assessed in cultured human microvascular endothelial cells. In these cells, VEGF enhanced mitochondrial function, as indicated by elevation in oxygen consumption rate and extracellular acidification rate. The VEGF-dependent increase in cell metabolism was blunted in response to PLCγ2 inhibition. Follow-up rescue studies therefore focused on understanding the significance of VEGF therapy in presence or absence of endothelial PLCγ2 in type 1 (streptozotocin-injected) and type 2 (db/db) diabetic ischemic tissue. Nonviral topical tissue nanotransfection technology (TNT) delivery of CDH5 promoter-driven PLCγ2 open reading frame promoted the rescue of hindlimb ischemia in diabetic mice. Improvement of blood flow was also associated with higher abundance of VWF+/CD31+ and VWF+/SMA+ immunohistochemical staining. TNT-based gene delivery was not associated with tissue edema, a commonly noted complication associated with proangiogenic gene therapies. Taken together, our study demonstrates that TNT-mediated delivery of endothelial PLCγ2, as part of combination gene therapy, is effective in diabetic ischemic limb rescue.

Myo-Inositol in Fermented Sugar Matrix Improves Human Macrophage Function.

SCOPE: Reactive oxygen species production by innate immune cells plays a central role in host defense against invading pathogens at wound-site. A weakened host-defense results in persistent infection leading to wound chronicity. Fermented Papaya Preparation (FPP), a complex sugar matrix, bolsters respiratory burst activity and improves wound healing outcomes in chronic wound patients. The objective of the current study was to identify underlying molecular factor/s responsible for augmenting macrophage host defense mechanisms following FPP supplementation. METHODS AND RESULTS: In depth LC-MS/MS analysis of cells supplemented with FPP led to identification of myo-inositol as a key determinant of FPP activity towards improving macrophage function. Myo-inositol, in quantities that is present in FPP, significantly improved macrophage respiratory burst and phagocytosis via de novo synthesis pathway of ISYNA1. In addition, myo-inositol transporters, HMIT and SMIT1, played a significant role in such activity. Blocking these pathways using siRNA attenuated FPP-induced improved macrophage host defense activities. FPP supplementation emerged as a novel approach to increase intracellular myo-inositol levels. Such supplementation also modified wound microenvironment in chronic wound patients to augment myo-inositol levels in wound fluid. CONCLUSION: These observations indicate that myo-inositol in FPP influences multiple aspects of macrophage function critical for host defense against invading pathogens.

Immediate Lymphatic Reconstruction to Prevent Breast Cancer-Related Lymphedema: A Systematic Review.

Significance: Lymphedema is chronic limb swelling from lymphatic dysfunction. The condition affects up to 250 million people worldwide. In breast cancer patients, lymphedema occurs in 30% who undergo axillary lymph node dissection (ALND). Recent Advances: Immediate lymphatic reconstruction (ILR), also termed Lymphatic Microsurgical Preventing Healing Approach (LyMPHA), is a method to decrease the risk of lymphedema by performing prophylactic lymphovenous anastomoses at the time of ALND. The objective of this study is to assess the risk reduction of ILR in preventing lymphedema. Critical Issues: Lymphedema has significant effects on the quality of life and morbidity of patients. Several techniques have been described to manage lymphedema after development, but prophylactic treatment of lymphedema with ILR may decrease risk of development to 6.6%. Future Directions: Long-term studies that demonstrate efficacy of ILR may allow for prophylactic management of lymphedema in the patient undergoing lymph node dissection.

Bone marrow- or adipose-mesenchymal stromal cell secretome preserves myocardial transcriptome profile and ameliorates cardiac damage following ex vivo cold storage.

BACKGROUND: Heart transplantation, a life-saving approach for patients with end-stage heart disease, is limited by shortage of donor organs. While prolonged storage provides more organs, it increases the extent of ischemia. Therefore, we seek to understand molecular mechanisms underlying pathophysiological changes of donor hearts during prolonged storage. Additionally, considering mesenchymal stromal cell (MSC)-derived paracrine protection, we aim to test if MSC secretome preserves myocardial transcriptome profile and whether MSC secretome from a certain source provides the optimal protection in donor hearts during cold storage. METHODS AND RESULTS: Isolated mouse hearts were divided into: no cold storage (control), 6 h cold storage (6 h-I), 6 h-I + conditioned media from bone marrow MSCs (BM-MSC CM), and 6 h-I + adipose-MSC CM (Ad-MSC CM). Deep RNA sequencing analysis revealed that compared to control, 6 h-I led to 266 differentially expressed genes, many of which were implicated in modulating mitochondrial performance, oxidative stress response, myocardial function, and apoptosis. BM-MSC CM and Ad-MSC CM restored these gene expression towards control. They also improved 6 h-I-induced myocardial functional depression, reduced inflammatory cytokine production, decreased apoptosis, and reduced myocardial H2O2. However, neither MSC-exosomes nor exosome-depleted CM recapitulated MSC CM-ameliorated apoptosis and CM-improved mitochondrial preservation during cold ischemia. Knockdown of Per2 by specific siRNA abolished MSC CM-mediated these protective effects in cardiomyocytes following 6 h cold storage. CONCLUSIONS: Our results demonstrated that using MSC secretome (BM-MSCs and Ad-MSCs) during prolonged cold storage confers preservation of the normal transcriptional "fingerprint", and reduces donor heart damage. MSC-released soluble factors and exosomes may synergistically act for donor heart protection.

Biofilm Management in Wound Care.

LEARNING OBJECTIVES: After studying this article, the participant should be able to: 1. Understand the basics of biofilm infection and be able to distinguish between planktonic and biofilm modes of growth. 2. Have a working knowledge of conventional and emerging antibiofilm therapies and their modes of action as they pertain to wound care. 3. Understand the challenges associated with testing and marketing antibiofilm strategies and the context within which these strategies may have effective value. SUMMARY: The Centers for Disease Control and Prevention estimate for human infectious diseases caused by bacteria with a biofilm phenotype is 65 percent and the National Institutes of Health estimate is closer to 80 percent. Biofilms are hostile microbial aggregates because, within their polymeric matrix cocoons, they are protected from antimicrobial therapy and attack from host defenses. Biofilm-infected wounds, even when closed, show functional deficits such as deficient extracellular matrix and impaired barrier function, which are likely to cause wound recidivism. The management of invasive wound infection often includes systemic antimicrobial therapy in combination with débridement of wounds to a healthy tissue bed as determined by the surgeon who has no way of visualizing the biofilm. The exceedingly high incidence of false-negative cultures for bacteria in a biofilm state leads to missed diagnoses of wound infection. The use of topical and parenteral antimicrobial therapy without wound débridement have had limited impact on decreasing biofilm infection, which remains a major problem in wound care. Current claims to manage wound biofilm infection rest on limited early-stage data. In most cases, such data originate from limited experimental systems that lack host immune defense. In making decisions on the choice of commercial products to manage wound biofilm infection, it is important to critically appreciate the mechanism of action and significance of the relevant experimental system. In this work, the authors critically review different categories of antibiofilm products, with emphasis on their strengths and limitations as evident from the published literature.

Mitochondria as Target for Tumor Management of Hemangioendothelioma.

Aims: Hemangioendothelioma (HE) may be benign or malignant. Mouse hemangioendothelioma endothelial (EOMA) cells are validated to study mechanisms in HE. This work demonstrates that EOMA cells heavily rely on mitochondria to thrive. Thus, a combination therapy, including weak X-ray therapy (XRT, 0.5 Gy) and a standardized natural berry extract (NBE) was tested. This NBE is known to be effective in managing experimental HE and has been awarded with the Food and Drug Administration Investigational New Drug (FDA-IND) number 140318 for clinical studies on infantile hemangioma. Results: NBE treatment alone selectively attenuated basal oxygen consumption rate of EOMA cells. NBE specifically sensitized EOMA, but not murine aortic endothelial cells to XRT-dependent attenuation of mitochondrial respiration and adenosine triphosphate (ATP) production. Combination treatment, selectively and potently, influenced mitochondrial dynamics in EOMA cells such that fission was augmented. This was achieved by lowering of mitochondrial sirtuin 3 (SIRT3) causing increased phosphorylation of AMP-activated protein kinase (AMPK). A key role of SIRT3 in loss of EOMA cell viability caused by the combination therapy was evident when pyrroloquinoline quinone, an inducer of SIRT3, pretreatment rescued these cells. Innovation and Conclusion: Mitochondria-targeting NBE significantly extended survival of HE-affected mice. The beneficial effect of NBE in combination with weak X-ray therapy was, however, far more potent with threefold increase in murine survival. The observation that safe natural products may target tumor cell mitochondria and sharply lower radiation dosage required for tumor management warrants clinical testing.

Urolithin A augments angiogenic pathways in skeletal muscle by bolstering NAD+ and SIRT1.

Urolithin A (UA) is a natural compound that is known to improve muscle function. In this work we sought to evaluate the effect of UA on muscle angiogenesis and identify the underlying molecular mechanisms. C57BL/6 mice were administered with UA (10 mg/body weight) for 12-16 weeks. ATP levels and NAD+ levels were measured using in vivo 31P NMR and HPLC, respectively. UA significantly increased ATP and NAD+ levels in mice skeletal muscle. Unbiased transcriptomics analysis followed by Ingenuity Pathway Analysis (IPA) revealed upregulation of angiogenic pathways upon UA supplementation in murine muscle. The expression of the differentially regulated genes were validated using quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC). Angiogenic markers such as VEGFA and CDH5 which were blunted in skeletal muscles of 28 week old mice were found to be upregulated upon UA supplementation. Such augmentation of skeletal muscle vascularization was found to be bolstered via Silent information regulator 1 (SIRT1) and peroxisome proliferator-activated receptor-gamma coactivator-1-alpha (PGC-1α) pathway. Inhibition of SIRT1 by selisistat EX527 blunted UA-induced angiogenic markers in C2C12 cells. Thus this work provides maiden evidence demonstrating that UA supplementation bolsters skeletal muscle ATP and NAD+ levels causing upregulated angiogenic pathways via a SIRT1-PGC-1α pathway.

Exosome-Mediated Crosstalk between Keratinocytes and Macrophages in Cutaneous Wound Healing.

Bidirectional cell-cell communication involving exosome-borne cargo such as miRNA has emerged as a critical mechanism for wound healing. Unlike other shedding vesicles, exosomes selectively package miRNA by SUMOylation of heterogeneous nuclear ribonucleoproteinA2B1 (hnRNPA2B1). In this work, we elucidate the significance of exosome in keratinocyte-macrophage crosstalk following injury. Keratinocyte-derived exosomes were genetically labeled with GFP-reporter (Exoκ-GFP) using tissue nanotransfection (TNT), and they were isolated from dorsal murine skin and wound-edge tissue by affinity selection using magnetic beads. Surface N-glycans of Exoκ-GFP were also characterized. Unlike skin exosome, wound-edge Exoκ-GFP demonstrated characteristic N-glycan ions with abundance of low-base-pair RNA and was selectively engulfed by wound macrophages (ωmϕ) in granulation tissue. In vitro addition of wound-edge Exoκ-GFP to proinflammatory ωmϕ resulted in conversion to a proresolution phenotype. To selectively inhibit miRNA packaging within Exoκ-GFPin vivo, pH-responsive keratinocyte-targeted siRNA-hnRNPA2B1 functionalized lipid nanoparticles (TLNPκ) were designed with 94.3% encapsulation efficiency. Application of TLNPκ/si-hnRNPA2B1 to the murine dorsal wound-edge significantly inhibited expression of hnRNPA2B1 by 80% in epidermis compared to the TLNPκ/si-control group. Although no significant difference in wound closure or re-epithelialization was observed, the TLNPκ/si-hnRNPA2B1 treated group showed a significant increase in ωmϕ displaying proinflammatory markers in the granulation tissue at day 10 post-wounding compared to the TLNPκ/si-control group. Furthermore, TLNPκ/si-hnRNPA2B1 treated mice showed impaired barrier function with diminished expression of epithelial junctional proteins, lending credence to the notion that unresolved inflammation results in leaky skin. This work provides insight wherein Exoκ-GFP is recognized as a major contributor that regulates macrophage trafficking and epithelial barrier properties postinjury.

N-acetylcysteine differentially regulates the populations of bone marrow and circulating endothelial progenitor cells in mice with limb ischemia.

Endothelial progenitor cells (EPCs) are important to tissue repair and regeneration especially after ischemic injury, and very heterogeneous in phenotypes and biological features. Reactive oxygen species are involved in regulating EPC number and function. N-acetylcysteine (NAC) inhibits ischemia-induced reactive oxygen species formation and promotes ischemic limb recovery. This study was to evaluate the effect of NAC on EPC subpopulations in bone marrow (BM) and blood in mice with limb ischemia. Limb ischemia was induced by femoral artery ligation in male C57BL/6 mice with or without NAC treatment. EPC subpopulations, intracellular reactive oxygen species production, cell proliferation and apoptosis in BM and blood cells were analyzed at baseline, day 3 (acute ischemia) and 21 (chronic) after ligation. c-Kit+/CD31+, Sca-1+/Flk-1+, CD34+/CD133+, and CD34+/Flk-1+ were used to define EPC subpopulations. Limb blood flow, function, muscle structure, and capillary density were evaluated with laser Doppler perfusion imaging, treadmill test, and immunohistochemistry, respectively, at day 3, 7, 14 and 21 post ischemia. Reactive oxygen species production in circulating and BM mononuclear cells and EPCs populations were significantly increased in BM and blood in mice with acute and chronic ischemia. NAC treatment effectively blocked ischemia-induced reactive oxygen species production in circulating and BM mononuclear cells, and selectively increased EPC population in circulation, not BM, with preserved proliferation in mice with chronic ischemia, and enhanced limb blood flow and function recovery, while preventing acute ischemia-induced increase in BM and circulating EPCs. These data demonstrated that NAC selectively enhanced circulating EPC population in mice with chronic limb ischemia.