Diversity analysis of sea anemone peptide toxins in different tissues of Heteractis crispa based on transcriptomics.

Peptide toxins found in sea anemones venom have diverse properties that make them important research subjects in the fields of pharmacology, neuroscience and biotechnology. This study used high-throughput sequencing technology to systematically analyze the venom components of the tentacles, column, and mesenterial filaments of sea anemone Heteractis crispa, revealing the diversity and complexity of sea anemone toxins in different tissues. A total of 1049 transcripts were identified and categorized into 60 families, of which 91.0% were proteins and 9.0% were peptides. Of those 1049 transcripts, 416, 291, and 307 putative proteins and peptide precursors were identified from tentacles, column, and mesenterial filaments respectively, while 428 were identified when the datasets were combined. Of these putative toxin sequences, 42 were detected in all three tissues, including 33 proteins and 9 peptides, with the majority of peptides being ShKT domain, ß-defensin, and Kunitz-type. In addition, this study applied bioinformatics approaches to predict the family classification, 3D structures, and functional annotation of these representative peptides, as well as the evolutionary relationships between peptides, laying the foundation for the next step of peptide pharmacological activity research.

Venomics Reveals the Venom Complexity of Sea Anemone Heteractis magnifica.

The venoms of various sea anemones are rich in diverse toxins, which usually play a dual role in capturing prey and deterring predators. However, the complex components of such venoms have not been well known yet. Here, venomics of integrating transcriptomic and proteomic technologies was applied for the first time to identify putative protein and peptide toxins from different tissues of the representative sea anemone, Heteractis magnifica. The transcriptomic analysis of H. magnifica identified 728 putative toxin sequences, including 442 and 381 from the tentacles and the column, respectively, and they were assigned to 68 gene superfamilies. The proteomic analysis confirmed 101 protein and peptide toxins in the venom, including 91 in the tentacles and 39 in the column. The integrated venomics also confirmed that some toxins such as the ShK-like peptides and defensins are co-expressed in both the tentacles and the column. Meanwhile, a homology analysis was conducted to predict the three-dimensional structures and potential activity of seven representative toxins. Altogether, this venomics study revealed the venom complexity of H. magnifica, which will help deepen our understanding of cnidarian toxins, thereby supporting the in-depth development of valuable marine drugs.

PPARß/δ-ANGPTL4 axis mediates the promotion of mono-2-ethylhexyl phthalic acid on MYCN-amplified neuroblastoma development.

Di-2-ethylhexyl phthalic acid (DEHP) is one of the most widely used plasticizers in the industry, which can improve the flexibility and durability of plastics. It is prone to migrate from various daily plastic products through wear and leaching into the surrounding environment and decompose into the more toxic metabolite mono-2-ethylhexyl phthalic acid (MEHP) after entering the human body. However, the impacts and mechanisms of MEHP on neuroblastoma are unclear. We exposed MYCN-amplified neuroblastoma SK-N-BE(2)C cells to an environmentally related concentration of MEHP and found that MEHP increased the proliferation and migration ability of tumor cells. The peroxisome proliferator-activated receptor (PPAR) ß/δ pathway was identified as a pivotal signaling pathway in neuroblastoma, mediating the effects of MEHP through transcriptional sequencing analysis. Because MEHP can bind to the PPARß/δ protein and initiate the expression of the downstream gene angiopoietin-like 4 (ANGPTL4), the PPARß/δ-specific agonist GW501516 and antagonist GSK3787, the recombinant human ANGPTL4 protein, and the knockdown of gene expression confirmed the regulation of the PPARß/δ-ANGPTL4 axis on the malignant phenotype of neuroblastoma. Based on the critical role of PPARß/δ and ANGPTL4 in the metabolic process, a non-targeted metabolomics analysis revealed that MEHP altered multiple metabolic pathways, particularly lipid metabolites involving fatty acyls, glycerophospholipids, and sterol lipids, which may also be potential factors promoting tumor progression. We have demonstrated for the first time that MEHP can target binding to PPARß/δ and affect the progression of neuroblastoma by activating the PPARß/δ-ANGPTL4 axis. This mechanism confirms the health risks of plasticizers as tumor promoters and provides new data support for targeted prevention and treatment of neuroblastoma.

A humanized orthotopic mouse model for preclinical evaluation of immunotherapy in Ewing sarcoma.

The advent of novel cancer immunotherapy approaches is revolutionizing the treatment for cancer. Current small animal models for most cancers are syngeneic or genetically engineered mouse models or xenograft models based on immunodeficient mouse strains. These models have been limited in evaluating immunotherapy regimens due to the lack of functional human immune system. Development of animal models for bone cancer faces another challenge in the accessibility of tumor engraftment sites. Here, we describe a protocol to develop an orthotopic humanized mouse model for a bone and soft tissue sarcoma, Ewing sarcoma, by transplanting fresh human cord blood CD34+ hematopoietic stem cells into young NSG-SGM3 mice combined with subsequent Ewing sarcoma patient derived cell engraftment in the tibia of the humanized mice. We demonstrated early and robust reconstitution of human CD45+ leukocytes including T cells, B cells, natural killer cells and monocytes. Ewing sarcoma xenograft tumors successfully orthotopically engrafted in the humanized mice with minimal invasive procedures. We validated the translational utility of this orthotopic humanized model by evaluating the safety and efficacy of an immunotherapy antibody, magrolimab. Treatment with magrolimab induces CD47 blockade resulting in significantly decreased primary tumor growth, decreased lung metastasis and prolonged animal survival in the established humanized model. Furthermore, the humanized model recapitulated the dose dependent toxicity associated with the CD47 blockade as observed in patients in clinical trials. In conclusion, this orthotopic humanized mouse model of Ewing sarcoma represents an improved platform for evaluating immunotherapy in bone and soft tissue sarcoma, such as Ewing sarcoma. With careful design and optimization, this model is generalizable for other bone malignancies.

Inflammation-mediated fibroblast activation and immune dysregulation in collagen VII-deficient skin.

Inflammation is known to play a critical role in all stages of tumorigenesis; however, less is known about how it predisposes the tissue microenvironment preceding tumor formation. Recessive dystrophic epidermolysis bullosa (RDEB), a skin-blistering disease secondary to COL7A1 mutations and associated with chronic wounding, inflammation, fibrosis, and cutaneous squamous cell carcinoma (cSCC), models this dynamic. Here, we used single-cell RNA sequencing (scRNAseq) to analyze gene expression patterns in skin cells from a mouse model of RDEB. We uncovered a complex landscape within the RDEB dermal microenvironment that exhibited altered metabolism, enhanced angiogenesis, hyperproliferative keratinocytes, infiltration and activation of immune cell populations, and inflammatory fibroblast priming. We demonstrated the presence of activated neutrophil and Langerhans cell subpopulations and elevated expression of PD-1 and PD-L1 in T cells and antigen-presenting cells, respectively. Unsupervised clustering within the fibroblast population further revealed two differentiation pathways in RDEB fibroblasts, one toward myofibroblasts and the other toward a phenotype that shares the characteristics of inflammatory fibroblast subsets in other inflammatory diseases as well as the IL-1-induced inflammatory cancer-associated fibroblasts (iCAFs) reported in various cancer types. Quantitation of inflammatory cytokines indicated dynamic waves of IL-1α, TGF-ß1, TNF, IL-6, and IFN-γ concentrations, along with dermal NF-κB activation preceding JAK/STAT signaling. We further demonstrated the divergent and overlapping roles of these cytokines in inducing inflammatory phenotypes in RDEB patients as well as RDEB mouse-derived fibroblasts together with their healthy controls. In summary, our data have suggested a potential role of inflammation, driven by the chronic release of inflammatory cytokines such as IL-1, in creating an immune-suppressed dermal microenvironment that underlies RDEB disease progression.

Upregulation of Nav1.6 Mediated by the p38 MAPK Pathway in the Dorsal Root Ganglia Contributes to Cancer-Induced Bone Pain in Rats.

Cancer-induced bone pain (CIBP) occurs frequently among advanced cancer patients. Voltage-gated sodium channels (VGSCs) have been associated with chronic pain, but how VGSCs function in CIBP is poorly understood. Here, we aimed to investigate the specific role of VGSCs in the dorsal root ganglia (DRGs) in CIBP. A CIBP rat model was generated by the intratibial inoculation of MRMT-1 breast carcinoma cells. Transcriptome sequencing was conducted to assess the gene expression profiles. The expression levels of key genes and differentiated genes related to activated pathways were measured by Western blotting and qPCR. We implanted a catheter intrathecally for the administration of lentivirus and drugs. Then, the changes in the mechanical withdrawal threshold (MWT) were measured. We identified 149 differentially expressed mRNAs (DEmRNAs) in the DRGs of CIBP model rats. The expression of Nav1.6, which was among these DEmRNAs, was significantly upregulated. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the DEmRNAs showed that they were mainly enriched in the mitogen-activated protein kinase (MAPK) pathway. The decrease in MWT induced by bone cancer was attenuated by Nav1.6 knockdown. Western blot analysis revealed that a p38 inhibitor decreased the expression of Nav1.6 and attenuated pain behavior. Our study shows that the upregulation of Nav1.6 expression by p38 MAPK in the DRGs of rats contributes to CIBP.

Identification of exosomal hsa-miR-483-5p as a potential biomarker for hepatocellular carcinoma via microRNA expression profiling of tumor-derived exosomes.

To date, most studies of exosomes related to hepatocellular carcinoma (HCC) have used commercial cancer cell lines or patient plasma as source material. In this study, we isolated exosomes directly from HCC tissues to investigate the potential of exosomal contents as biomarkers for HCC. Exosomes were identified and verified using transmission electron microscopy, nano-flow cytometry analysis, and western blotting. Tissue-derived exosomal miRNA expression was profiled by high-throughput sequencing, and differential expression of miRNAs was validated by quantitative real-time polymerase chain reaction analysis. The diagnostic performance of differentially expressed exosomal miRNAs for HCC was evaluated by receiver operating characteristic curve analysis. Target genes of these miRNAs were verified using luciferase reporter assays, and their functions were studied through in vitro and rescue assays. In total, 225 differentially expressed exosomal miRNAs were identified in HCC samples compared with adjacent liver tissues, and some were associated with HCC tumorigenesis and progression. Comparison of the expression profiles of tissue-derived and plasma-derived exosomal miRNAs identified hsa-miR-483-5p as the only differentially expressed miRNA detected in both HCC tissue and plasma, and this was in a validation group of HCC patients. Analysis of the diagnostic performance of plasma exosomal hsa-miR-483-5p or plasma hsa-miR-483-5p found that both could differentiate HCC and non-HCC cases. In vitro ectopic miR-483-5p expression promoted HCC cell proliferation. CDK15 was confirmed to bind with miR-483-5p directly, and thus, miR-483-5p may function by downregulating CDK15. Hsa-miR-483-5p represents a potential specific and sensitive biomarker for HCC diagnosis.

Regional Analgesia in Video-Assisted Thoracic Surgery: A Bayesian Network Meta-Analysis.

Background: A variety of regional analgesia methods are used during video-assisted thoracic surgery (VATS). Our network meta-analysis (NMA) sought to evaluate the advantages of various methods of localized postoperative pain management in VATS patients. Methods: PubMed, the Cochrane Library, and EMBASE were searched from their date of inception to May 2021 for randomized controlled trials (RCTs) comparing two or more types of locoregional analgesia in adults using any standardized clinical criteria. This was done using Bayesian NMA. Results: A total of 3,563 studies were initially identified, and 16 RCTs with a total of 1,144 participants were ultimately included. These studies, which spanned the years 2014 to 2021 and included data from eight different countries, presented new information. There were a variety of regional analgesia techniques used, and in terms of analgesic effect, thoracic epidural anesthesia (TEA) [SMD (standard mean difference) = 1.12, CrI (Credible interval): (-0.08 to -2.33)], thoracic paravertebral block (TPVB) (SMD = 0.67, CrI: (-0.25 to 1.60) and erector spinae plane block (ESPB) (SMD = 0.34, CrI: (-0.5 to 1.17) were better than other regional analgesia methods. Conclusion: Overall, these findings show that TEA, TPVB and ESPB may be effective forms of regional analgesia in VATS. This research could be a valuable resource for future efforts regarding the use of thoracic regional analgesia and enhanced recovery after surgery. Systematic Review Registration: Identifier [PROSPERO CRD42021253218].

mTOR-mediated autophagy in the hippocampus is involved in perioperative neurocognitive disorders in diabetic rats.

INTRODUCTION: Perioperative neurocognitive disorders (PND) are common neurological complications after surgery. Diabetes mellitus (DM) has been reported to be an independent risk factor for PND, but little is known about its mechanism of action. Mammalian target of rapamycin (mTOR) signaling is crucial for neuronal growth, development, apoptosis, and autophagy, but the dysregulation of mTOR signaling leads to neurological disorders. The present study investigated whether rapamycin can attenuate PND by inhibiting mTOR and activating autophagy in diabetic rats. METHODS: Male diabetic Sprague-Dawley rats underwent tibial fracture surgery under isoflurane anesthesia to establish a PND model. Cognitive functions were examined using the Morris water maze test. The levels of phosphorylated mTOR (p-mTOR), phosphorylated tau (p-tau), autophagy-related proteins (Beclin-1, LC3), and apoptosis-related proteins (Bax, Bcl-2, cleaved caspase-3) in the hippocampus were examined on postoperative days 3, 7, and 14 by Western blot. Hippocampal amyloid ß (Aß) levels were examined by immunohistochemistry. RESULTS: The data showed that surgical trauma and/or DM impaired cognitive function, induced mTOR activation, and decreased Beclin-1 levels and the LC3-II/I ratio. The levels of Aß and p-tau and the hippocampal apoptotic responses were significantly higher in diabetic or surgery-treated rats than in control rats and were further increased in diabetic rats subjected to surgery. Pretreatment of rats with rapamycin inhibited mTOR hyperactivation and restored autophagic function, effectively decreasing tau hyperphosphorylation, Aß deposition, and apoptosis in the hippocampus. Furthermore, surgical trauma-induced neurocognitive disorders were also reversed by pretreatment of diabetic rats with rapamycin. CONCLUSION: The results demonstrate that mTOR hyperactivation regulates autophagy, playing a critical role in the mechanism underlying PND, and reveal that the modulation of mTOR signaling could be a promising therapeutic strategy for PND in patients with diabetes.

Discovery of novel peptide neurotoxins from sea anemone species.

As primitive metazoa, sea anemones are rich in various bioactive peptide neurotoxins. These peptides have been applied to neuroscience research tools or directly developed as marine drugs. To date, more than 1100 species of sea anemones have been reported, but only 5% of the species have been used to isolate and identify sea anemone peptide neurotoxins. There is an urgent need for more systematic discovery and study of peptide neurotoxins in sea anemones. In this review, we have gathered the currently available methods from crude venom purification and gene cloning to venom multiomics, employing these techniques for discovering novel sea anemone peptide neurotoxins. In addition, the three-dimensional structures and targets of sea anemone peptide neurotoxins are summarized. Therefore, the purpose of this review is to provide a reference for the discovery, development, and utilization of sea anemone peptide neurotoxins.

Dexmedetomidine Alleviates Hypoxia-Induced Synaptic Loss and Cognitive Impairment via Inhibition of Microglial NOX2 Activation in the Hippocampus of Neonatal Rats.

BACKGROUND: Perinatal hypoxia is a universal cause of death and neurological deficits in neonates worldwide. Activation of microglial NADPH oxidase 2 (NOX2) leads to oxidative stress and neuroinflammation, which may contribute to hypoxic damage in the developing brain. Dexmedetomidine has been reported to exert potent neuroprotection in several neurological diseases, but the mechanism remains unclear. We investigated whether dexmedetomidine acts through microglial NOX2 to reduce neonatal hypoxic brain damage. METHODS: The potential role of microglial NOX2 in dexmedetomidine-mediated alleviation of hypoxic damage was evaluated in cultured BV2 microglia and neonatal rats subjected to hypoxia. In vivo, neonatal rats received dexmedetomidine (25 µg/kg, i.p.) 30 min before or immediately after hypoxia (5% O2, 2 h). Apocynin-mediated NOX inhibition and lentivirus-mediated NOX2 overexpression were applied to further assess the involvement of microglial NOX2 activation. RESULTS: Pre- or posttreatment with dexmedetomidine alleviated hypoxia-induced cognitive impairment, restored damaged synapses, and increased postsynaptic density-95 and synaptophysin protein expression following neonatal hypoxia. Importantly, dexmedetomidine treatment suppressed hypoxia-induced microglial NOX2 activation and subsequent oxidative stress and the neuroinflammatory response, as reflected by reduced 4-hydroxynonenal and ROS accumulation, and decreased nuclear NF-κB p65 and proinflammatory cytokine levels in cultured BV2 microglia and the developing hippocampus. In addition, treating primary hippocampal neurons with conditioned medium (CM) from hypoxia-activated BV2 microglia resulted in neuronal damage, which was alleviated by CM from dexmedetomidine-treated microglia. Moreover, the neuroprotective effect of dexmedetomidine was reversed in NOX2-overexpressing BV2 microglia and diminished in apocynin-pretreated neonatal rats. CONCLUSION: Dexmedetomidine targets microglial NOX2 to reduce oxidative stress and neuroinflammation and subsequently protects against hippocampal synaptic loss following neonatal hypoxia.

Amitriptyline Protects Against Lidocaine-induced Neurotoxicity in SH-SY5Y Cells via Inhibition of BDNF-mediated Autophagy.

Amitriptyline (AMI) is a traditional tricyclic antidepressant that has been proven to exhibit neuroprotective effects in various neurological disorders. However, the underlying mechanism by which AMI attenuates lidocaine-induced neurotoxicity remains poorly understood. Brain-derived neurotrophic factor (BDNF) is an essential neurotrophin to neuronal development and survival in the brain, and recent studies have suggested that BDNF plays an important role in mediating lidocaine-induced neurotoxicity. The present study was performed to evaluate the protective effect of AMI against the neurotoxicity induced by lidocaine and to explore the role of BDNF-dependent autophagy in this process. The data showed that AMI pretreatment alleviated lidocaine-induced neurotoxicity, as evidenced by the restoration of cell viability, normalization of cell morphology, and reduction in the cell apoptosis index. In addition, autophagy inhibitor 3-methyladenine (3-MA) had a protective effect similar to that of AMI, but autophagy activator rapamycin eliminated the protective effect of AMI by suppressing mTOR activation. Moreover, at the molecular level, we found that AMI-mediated autophagy was involved in the expression of BDNF. The overexpression of BDNF or application of exogenous recombinant BDNF significantly suppressed autophagy and protected SH-SY5Y cells from apoptosis induced by Lido, whereas the neuroprotection of AMI was abolished by either knockdown of BDNF or use of a tropomyosin-related kinase B (TrkB) inhibitor ANA-12 in SH-SY5Y cells. Overall, our findings demonstrated that the protective effect of AMI against lidocaine-induced neurotoxicity correlated with inhibition of autophagy activity through upregulation of BDNF expression.

Using mRNA deep sequencing to analyze differentially expressed genes during Panax notoginseng saponin treatment of ischemic stroke.

Treatment with Panax notoginseng saponin (PNS) can prevent neurological damage in middle cerebral artery occlusion model rats to promote recovery after a stroke. However, the exact molecular mechanisms are unknown and require further study. In the present study, mRNA sequencing was employed to investigate differential gene expression between model and sham groups, and between model and PNS­treated groups. Enrichment of gene data was performed using Gene Ontology analysis and the Kyoto Encyclopedia of Genes and Genomes database. Hub genes were identified and networks were constructed using Cytoscape that were further verified by reverse transcription­quantitative PCR. A total of 1,104 genes of interest were found, which included 690 upregulated and 414 downregulated genes that were identified when the model was compared with the sham group. Additionally, 817 genes of interest, which included 390 upregulated and 427 downregulated genes, were identified when the PNS­treated group was compared with the model group. There were 303 overlapping genes of interest between the analysis of model to sham groups, and the analysis of model to PNS­treated groups. The top 10 genes from the 303 aberrantly expressed genes of interest included ubiquitin conjugating enzyme E2 variant 2, small ubiquitin­related modifier 1, small RNA binding exonuclease protection factor La, Finkel­Biskis­Reilly murine sarcoma virus (FBR­MuSV) ubiquitously expressed, centrosomal protein 290 kDa, DNA­directed RNA polymerase II subunit K, cullin­4B, matrin­3 and vascular endothelial growth factor receptor 2. In conclusion, these genes may be important in the underlying mechanism of PNS treatment in ischemic stroke. Additionally, the present data provided novel insight into the pathogenesis of ischemic stroke.

Exposed CendR Domain in Homing Peptide Yields Skin-Targeted Therapeutic in Epidermolysis Bullosa.

Systemic skin-selective therapeutics would be a major advancement in the treatment of diseases affecting the entire skin, such as recessive dystrophic epidermolysis bullosa (RDEB), which is caused by mutations in the COL7A1 gene and manifests in transforming growth factor-ß (TGF-ß)-driven fibrosis and malignant transformation. Homing peptides containing a C-terminal R/KXXR/K motif (C-end rule [CendR] sequence) activate an extravasation and tissue penetration pathway for tumor-specific drug delivery. We have previously described a homing peptide CRKDKC (CRK) that contains a cryptic CendR motif and homes to angiogenic blood vessels in wounds and tumors, but it cannot penetrate cells or tissues. In this study, we demonstrate that removal of the cysteine from CRK to expose the CendR sequence confers the peptide novel ability to home to normal skin. Fusion of the truncated CRK (tCRK) peptide to the C terminus of an extracellular matrix protein decorin (DCN), a natural TGF-ß inhibitor, resulted in a skin-homing therapeutic molecule (DCN-tCRK). Systemic DCN-tCRK administration in RDEB mice led to inhibition of TGF-ß signaling in the skin and significant improvement in the survival of RDEB mice. These results suggest that DCN-tCRK has the potential to be utilized as a novel therapeutic compound for the treatment of dermatological diseases such as RDEB.

Human Cord Blood-Derived Unrestricted Somatic Stem Cell Infusion Improves Neurobehavioral Outcome in a Rabbit Model of Intraventricular Hemorrhage.

Intraventricular hemorrhage (IVH) is a severe complication of preterm birth, which leads to hydrocephalus, cerebral palsy, and mental retardation. There are no available therapies to cure IVH, and standard treatment is supportive care. Unrestricted somatic stem cells (USSCs) from human cord blood have reparative effects in animal models of brain and spinal cord injuries. USSCs were administered to premature rabbit pups with IVH and their effects on white matter integrity and neurobehavioral performance were evaluated. USSCs were injected either via intracerebroventricular (ICV) or via intravenous (IV) routes in 3 days premature (term 32d) rabbit pups, 24 hours after glycerol-induced IVH. The pups were sacrificed at postnatal days 3, 7, and 14 and effects were compared to glycerol-treated but unaffected or nontreated control. Using in vivo live bioluminescence imaging and immunohistochemical analysis, injected cells were found in the injured parenchyma on day 3 when using the IV route compared to ICV where cells were found adjacent to the ventricle wall forming aggregates; we did not observe any adverse events from either route of administration. The injected USSCs were functionally associated with attenuated microglial infiltration, less apoptotic cell death, fewer reactive astrocytes, and diminished levels of key inflammatory cytokines (TNFα and IL1ß). In addition, we observed better preservation of myelin fibers, increased myelin gene expression, and altered reactive astrocyte distribution in treated animals, and this was associated with improved locomotor function. Overall, our findings support the possibility that USSCs exert anti-inflammatory effects in the injured brain mitigating many detrimental consequences associated with IVH. Stem Cells Translational Medicine 2019;8:1157-1169.

Cord Blood-Derived Stem Cells Suppress Fibrosis and May Prevent Malignant Progression in Recessive Dystrophic Epidermolysis Bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe skin fragility disorder caused by mutations in the Col7a1 gene. Patients with RDEB suffer from recurrent erosions in skin and mucous membranes and have a high risk for developing cutaneous squamous cell carcinoma (cSCCs). TGFß signaling has been associated with fibrosis and malignancy in RDEB. In this study, the activation of TGFß signaling was demonstrated in col7a1-/- mice as early as a week after birth starting in the interdigital folds of the paws, accompanied by increased deposition of collagen fibrils and elevated dermal expression of matrix metalloproteinase (MMP)-9 and MMP-13. Furthermore, human cord blood-derived unrestricted somatic stem cells (USSCs) that we previously demonstrated to significantly improve wound healing and prolong the survival of col7a1-/- mice showed the ability to suppress TGFß signaling and MMP-9 and MMP-13 expression meanwhile upregulating anti-fibrotic TGFß3 and decorin. In parallel, we cocultured USSCs in a transwell with RDEB patient-derived fibroblasts, keratinocytes, and cSCC, respectively. The patient-derived cells were constitutively active for STAT, but not TGFß signaling. Moreover, the levels of MMP-9 and MMP-13 were significantly elevated in the patient derived-keratinocytes and cSCCs. Although USSC coculture did not inhibit STAT signaling, it significantly suppressed the secretion of MMP-9 and MMP-13, and interferon (IFN)-γ from RDEB patient-derived cells. Since epithelial expression of these MMPs is a biomarker of malignant transformation and correlates with the degree of tumor invasion, these results suggest a potential role for USSCs in mitigating epithelial malignancy, in addition to their anti-inflammatory and anti-fibrotic functions. Stem Cells 2018;36:1839-12.

Efficacy of Human Placental-Derived Stem Cells in Collagen VII Knockout (Recessive Dystrophic Epidermolysis Bullosa) Animal Model.

Recessive dystrophic epidermolysis bullosa (RDEB) is a devastating inherited skin blistering disease caused by mutations in the COL7A1 gene that encodes type VII collagen (C7), a major structural component of anchoring fibrils at the dermal-epidermal junction (DEJ). We recently demonstrated that human cord blood-derived unrestricted somatic stem cells promote wound healing and ameliorate the blistering phenotype in a RDEB (col7a1-/- ) mouse model. Here, we demonstrate significant therapeutic effect of a further novel stem cell product in RDEB, that is, human placental-derived stem cells (HPDSCs), currently being used as human leukocyte antigen-independent donor cells with allogeneic umbilical cord blood stem cell transplantation in patients with malignant and nonmalignant diseases. HPDSCs are isolated from full-term placentas following saline perfusion, red blood cell depletion, and volume reduction. HPDSCs contain significantly higher level of both hematopoietic and nonhematopoietic stem and progenitor cells than cord blood and are low in T cell content. A single intrahepatic administration of HPDSCs significantly elongated the median life span of the col7a1-/- mice from 2 to 7 days and an additional intrahepatic administration significantly extended the median life span to 18 days. We further demonstrated that after intrahepatic administration, HPDSCs engrafted short-term in the organs affected by RDEB, that is, skin and gastrointestinal tract of col7a1-/- mice, increased adhesion at the DEJ and deposited C7 even at 4 months after administration of HPDSCs, without inducing anti-C7 antibodies. This study warrants future clinical investigation to determine the safety and efficacy of HPDSCs in patients with severe RDEB. Stem Cells Translational Medicine 2018;7:530-542.

Upregulation of vascular endothelial growth factor receptor-1 contributes to sevoflurane preconditioning-mediated cardioprotection.

PURPOSE: Sevoflurane preconditioning (SPC) can provide myocardial protective effects similar to ischemic preconditioning. However, the exact mechanism of SPC remains unclear. Previous studies indicate that vascular endothelial growth factor receptor 1 (VEGFR-1) is involved in ischemic preconditioning-mediated cardioprotection. This study was designed to determine the significance of VEGFR-1 signaling in SPC-mediated cardioprotection. MATERIALS AND METHODS: Myocardial ischemia-reperfusion (I/R) rat model was established using the Langendorff isolated heart perfusion apparatus. Additionally, after 15 min of baseline equilibration, the isolated hearts were pretreated with 2.5% sevoflurane, 2.5% sevoflurane+MF1 10 µmol/L, or 2.5% sevoflurane+placental growth factor 10 µmol/L, and then subjected to 30 min of global ischemia and 120 min of reperfusion. The changes in hemodynamic parameters, myocardial infarct size, and the levels of creatine kinase-MB, lactate dehydrogenase, cardiac troponin-I, tumor necrosis factor-α, and interleukin 6 in the myocardium were evaluated. RESULTS: Compared to the I/R group, pretreatment with 2.5% sevoflurane significantly improved the cardiac function, limited myocardial infarct size, reduced cardiac enzyme release, upregulated VEGFR-1 expression, and decreased inflammation. In addition, the selective VEGFR-1 agonist, placental growth factor, did not enhance the cardioprotection and anti-inflammation effects of sevoflurane, while the specific VEGFR-1 inhibitor, MF1, completely reversed these effects. CONCLUSION: Our data have demonstrated that 2.5% sevoflurane preconditioning alleviates heart I/R injury, which is probably mediated by the anti-inflammatory property and upregulation of VEGFR-1.

Rescue of the mucocutaneous manifestations by human cord blood derived nonhematopoietic stem cells in a mouse model of recessive dystrophic epidermolysis bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe skin blistering disease caused by mutations in COL7A1-encoding type VII collagen (C7). Currently, there is no curative therapy for patients with RDEB. Our previous studies demonstrated that human umbilical cord blood (HUCB) derived unrestricted somatic stem cells (USSCs) express C7 and facilitate wound healing in a murine wounding model. The primary objective of this study is to investigate the therapeutic functions of USSCs in the C7 null (Col7a1(-/-) ) C57BL6/J mice, a murine model of RDEB. We demonstrated that intrahepatic administration of USSCs significantly improved the blistering phenotype and enhanced the life span in the recipients. The injected USSCs trafficked to the sites of blistering and were incorporated in short-term in the recipients' skin and gastrointestinal tract. Consistent with an overall histological improvement in the epidermal-dermal adherence following USSC treatment, the expression of C7 at the basement membrane zone was detected and the previously disorganized integrin α6 distribution was normalized. We also demonstrated that USSCs treatment induced an infiltration of macrophages with a regenerative "M2" phenotype. Our data suggest that HUCB-derived USSCs improved the RDEB phenotype through multiple mechanisms. This study has warranted future clinical investigation of USSCs as a novel and universal allogeneic stem cell donor source in selected patients with RDEB.