The EGF Motif With CXDXXXXYXCXC Sequence Suppresses Fibrosis in a Mouse Skin Wound Model.

BACKGROUND/AIM: Fibrosis is an essential process for wound healing, but excessive fibrosis, such as keloids and hypertrophic scars, can cause cosmetic and functional problems. These lesions are caused by abnormal deposition and shrinkage of collagen fibers. The light chain of FIX, a plasma protein essential for hemostasis, has the amino acid sequence CXDXXXXYXCXC in the EGF domain. Peptides containing this sequence inhibited stromal growth in a mouse transplant tumor model. In this study, the effect of the FIX light chain on wound healing was studied. MATERIALS AND METHODS: A full-layer wound was made on the back of each mouse, and cDNA encoding the light chain of mouse FIX (F9-LC) in an expression vector was injected locally once each week using a non-viral vector. Histochemical analysis of the wound was then performed to assess the effects on wound healing. Moreover, the effect of F9-LC on fibroblasts was studied in vitro. RESULTS: Macroscopic observation showed that wounds with forced expression of F9-LC appeared flatter and had fewer wrinkles than control wounds. Tissue collagen staining and immunostaining revealed that administration of F9-LC suppressed collagen 1 and 3 deposition and decreased α-smooth muscle actin expression. Electron microscopy revealed sparse and disorganized collagen fibers in the F9-LC-treated mice. In experiments using fibroblasts, addition of a recombinant protein of the FIX light chain disrupted the typical spindle shape and alignment of fibroblasts. CONCLUSION: F9-LC is a new candidate for use in treatments to regulate excessive fibrosis and contraction in wound healing.

Differences in the stemness characteristics and molecular markers of distinct human oral tissue neural crest-derived multilineage cells.

OBJECTIVES: Although multilineage cells derived from oral tissues, especially the dental pulp, apical papilla, periodontal ligament, and oral mucosa, have neural crest-derived stem cell (NCSC)-like properties, the differences in the characteristics of these progenitor cell compartments remain unknown. The current study aimed to elucidate these differences. MATERIAL AND METHODS: Sphere-forming apical papilla-derived cells (APDCs), periodontal ligament-derived cells (PDLDCs), and oral mucosa stroma-derived cells (OMSDCs) from the same individuals were isolated from impacted developing teeth. All sphere-forming cells were characterized through biological analyses of stem cells. RESULTS: All sphere-forming cells expressed neural crest-related markers. The expression of certain tissue-specific markers such as CD24 and CD56 (NCAM1) differed among tissue-derived cells. Surprisingly, the expression of only CD24 and CD56 could be discriminated in human tissues. Although APDCs and PDLDCs exhibited greater mineralized cell differentiation than OMSDCs, they exhibited poorer differentiation into adipocytes in vitro. In immunocompromised mice, APDCs formed hard tissues better than PDLDCs and OMSDCs. CONCLUSIONS: Although cells with NCSC-like properties present the same phenotype, they differ in the expression of certain markers and differentiation abilities. This study is the first to demonstrate the differences in the differentiation ability and molecular markers among multilineage human APDCs, PDLDCs, and OMSDCs obtained from the same patients, and to identify tissue-specific markers that distinguish tissues in the developing stage of the human tooth with immature apex.

EGF domain peptide of Developmentally regulated endothelial locus1 facilitates gene expression of extracellularly applied plasmid DNA.

BACKGROUND: The successful development of messenger RNA vaccines for SARS-CoV-2 opened up venues for clinical nucleotide-based vaccinations. For development of DNA vaccines, we tested whether the EGF domain peptide of Developmentally regulated endothelial locus1 (E3 peptide) enhances uptake of extracellularly applied plasmid DNA. METHODS: DNA plasmid encoding lacZ or GFP was applied with a conditioned culture medium containing E3 peptide to cell lines in vitro or mouse soleus muscles in vivo, respectively. After 48 h incubation, gene expression was examined by ß-galactosidase (ß-gal) assay and fluorescent microscope, respectively. RESULTS: Application of E3 peptide-containing medium to cultured cell lines induced intense ß-gal activity in a dose-dependent manner. Intra-gastrocnemius injection of E3 peptide-containing medium to mouse soleus muscle succeeded in the induction of GFP fluorescence in many cells around the injection site. CONCLUSIONS: The administration of E3 peptide facilitates transmembrane uptake of extracellular DNA plasmid which induces sufficient extrinsic gene expression.

Activation peptide of coagulation factor IX improves the prognosis after traumatic brain injury.

Recently, coagulation factor IX and its activation peptide have been reported to suppress the permeability of vascular endothelial cells. In this study, the therapeutic effects of a synthesized activation peptide is investigated in traumatic brain injury model rats. In cerebral contusion, dysfunction of the blood brain barrier with increasing vascular permeability promotes the progression of neuropathy after injury. The model rats were generated by controlled cortical impact. Then, rats were intravenously injected with 350 µg/kg of the synthesized activation peptide or PBS as a control, every day for a month. Behavioral studies were conducted during a month of observation. For morphological analysis, macro- and microscopic observation were performed. Water content of brain tissue was used to assess edema. To assess the function of blood brain barrier, Evans Blue method was employed. In the neurological examinations and beam-walking, the treated rats performed significantly better than control rats. Measurements of cerebral defect volume showed that the treatment significantly reduced it by 82%. Nissl stain showed that neural cells adjacent to impacts were lost in control rats, but saved in treated rats. The treatment significantly reduced brain edema and extravascular leakage of Evans blue. Intravenous injection with a synthesized activation peptide significantly reduced damage to neural tissue and improved neural functioning in the model rats.

Combination Cancer Therapy of a Del1 Fragment and Cisplatin Enhanced Therapeutic Efficiency In Vivo.

BACKGROUND/AIM: Combination cancer therapy is currently under investigation. This study examined the effect of cancer combination therapy using the E3 and C1 (E3C1) domains of developmental endothelial locus-1 (Del1) and cisplatin (CDDP) in murine transplanted tumors. MATERIALS AND METHODS: Mice with transplanted tumors (A431, SCCKN or SCC-4 cells) were injected intraperitoneally with CDDP and injected locally with nonviral plasmid vectors encoding E3C1. Histochemical analysis of the transplanted tumors was then performed to assess the effects on prognosis. RESULTS: The CDDP+E3C1 injected group had reduced tumor growth and longer survival compared to the CDDP injected group. In addition, cell death was observed in the tumor of the CDDP+E3C1 group.. Furthermore, angiogenesis and increased blood vessels were observed together with stromal development. CONCLUSION: The CDDP+E3C1 treatment resulted in improved survival and poor tumor stromal development in mice with transplanted tumors.

An epidermal growth factor motif of developmental endothelial locus 1 protein inhibits efficient angiogenesis in explanted squamous cell carcinoma in vivo

ABSTRACT Background: Cancer gene therapy using a nonviral vector is expected to be repeatable, safe, and inexpensive, and to have long-term effectiveness. Gene therapy using the E3 and C1 (E3C1) domain of developmental endothelial locus-1 (Del1) has been shown to improve prognosis in a mouse transplanted tumor model. Objective: In this study, we examined how this treatment affects angiogenesis in mouse transplanted tumors. Materials and methods: Mouse transplanted tumors (SCCKN human squamous carcinoma cell line) were injected locally with a nonviral plasmid vector encoding E3C1 weekly. Histochemical analysis of the transplanted tumors was then performed to assess the effects of E3C1 on prognosis. Results: All mice in the control group had died or reached an endpoint within 39 days. In contrast, one of ten mice in the E3C1 group had died by day 39, and eight of ten had died or reached an endpoint by day 120 (p < 0.01). Enhanced apoptosis in tumor stroma was seen on histochemical analyses, as was inhibited tumor angiogenesis in E3C1-treated mice. In addition, western blot analysis showed decreases in active Notch and HEY1 proteins. Conclusion: These findings indicate that cancer gene therapy using a nonviral vector encoding E3C1 significantly improved life-span by inhibiting tumor angiogenesis. (REV INVEST CLIN. 2021;73(1):39-51)

An Epidermal Growth Factor Motif of Developmental Endothelial Locus 1 Protein Inhibits Efficient Angiogenesis in Explanted Squamous Cell Carcinoma In Vivo.

BACKGROUND: Cancer gene therapy using a nonviral vector is expected to be repeatable, safe, and inexpensive, and to have longterm effectiveness. Gene therapy using the E3 and C1 (E3C1) domain of developmental endothelial locus-1 (Del1) has been shown to improve prognosis in a mouse transplanted tumor model. OBJECTIVE: In this study, we examined how this treatment affects angiogenesis in mouse transplanted tumors. MATERIALS AND METHODS: Mouse transplanted tumors (SCCKN human squamous carcinoma cell line) were injected locally with a nonviral plasmid vector encoding E3C1 weekly. Histochemical analysis of the transplanted tumors was then performed to assess the effects of E3C1 on prognosis. RESULTS: All mice in the control group had died or reached an endpoint within 39 days. In contrast, one of ten mice in the E3C1 group had died by day 39, and eight of ten had died or reached an endpoint by day 120 (p < 0.01). Enhanced apoptosis in tumor stroma was seen on histochemical analyses, as was inhibited tumor angiogenesis in E3C1-treated mice. In addition, western blot analysis showed decreases in active Notch and HEY1 proteins. CONCLUSION: These findings indicate that cancer gene therapy using a nonviral vector encoding E3C1 significantly improved life-span by inhibiting tumor angiogenesis.

Oral multicentric carcinoma arising from four different sites in a female patient.

This report discusses a case of a 75-year-old female patient with metachronous multicentric carcinomas in the oral cavity at 4 different sites. In this patient, there were no generally associated characteristics, such as drinking alcohol, chewing betel quid or smoking cigarettes. However, her elder sister died due to oral carcinoma. Although well-known risk factors for oral carcinoma were not detected, a previous family history was found. These findings suggest the potential for an unknown genetic anomaly associated with oral carcinoma. This is the first report to describe a female patient with oral multicentric carcinoma arising from four different sites.

Caspase-8 Regulates Endoplasmic Reticulum Stress-Induced Necroptosis Independent of the Apoptosis Pathway in Auditory Cells.

The aim of this study is to elucidate the detailed mechanism of endoplasmic reticulum (ER) stress-induced auditory cell death based on the function of the initiator caspases and molecular complex of necroptosis. Here, we demonstrated that ER stress initiates not only caspase-9-dependent intrinsic apoptosis along with caspase-3, but also receptor-interacting serine/threonine kinase (RIPK)1-dependent necroptosis in auditory cells. We observed the ultrastructural characteristics of both apoptosis and necroptosis in tunicamycin-treated cells under transmission electron microscopy (TEM). We demonstrated that ER stress-induced necroptosis was dependent on the induction of RIPK1, negatively regulated by caspase-8 in auditory cells. Our data suggested that ER stress-induced intrinsic apoptosis depends on the induction of caspase-9 along with caspase-3 in auditory cells. The results of this study reveal that necroptosis could exist for the alternative backup cell death route of apoptosis in auditory cells under ER stress. Interestingly, our data results in a surge in the recognition that therapies aimed at the inner ear protection effect by caspase inhibitors like zVAD-fmk might arrest apoptosis but can also have the unanticipated effect of promoting necroptosis. Thus, RIPK1-dependent necroptosis would be a new therapeutic target for the treatment of sensorineural hearing loss due to ER stress.

Nonviral Gene Therapy for Cancer: A Review.

Although the development of effective viral vectors put gene therapy on the road to commercialization, nonviral vectors show promise for practical use because of their relative safety and lower cost. A significant barrier to the use of nonviral vectors, however, is that they have not yet proven effective. This apparent lack of interest can be attributed to the problem of the low gene transfer efficiency associated with nonviral vectors. The efficiency of gene transfer via nonviral vectors has been reported to be 1/10th to 1/1000th that of viral vectors. Despite the fact that new gene transfer methods and nonviral vectors have been developed, no significant improvements in gene transfer efficiency have been achieved. Nevertheless, some notable progress has been made. In this review, we discuss studies that report good results using nonviral vectors in vivo in animal models, with a particular focus on studies aimed at in vivo gene therapy to treat cancer, as this disease has attracted the interest of researchers developing nonviral vectors. We describe the conditions in which nonviral vectors work more efficiently for gene therapy and discuss how the goals might differ for nonviral versus viral vector development and use.

XBP1-FoxO1 interaction regulates ER stress-induced autophagy in auditory cells.

The purpose of this study was to clarify the relationship among X-box-binding protein 1 unspliced, spliced (XBP1u, s), Forkhead box O1 (FoxO1) and autophagy in the auditory cells under endoplasmic reticulum (ER) stress. In addition, the relationship between ER stress that causes unfolded protein response (UPR) and autophagy was also investigated. The present study reported ER stress induction by tunicamycin treatment that resulted in IRE1α-mediated XBP1 mRNA splicing and autophagy. XBP1 mRNA splicing and FoxO1 were found to be involved in ER stress-induced autophagy. This inference was based on the observation that the expression of LC3-II was suppressed by knockdown of IRE1α, XBP1 or FoxO1. In addition, XBP1u was found to interact with XBP1s in auditory cells under ER stress, functioning as a negative feedback regulator that was based on two important findings. Firstly, there was a significant inverse correlation between XBP1u and XBP1s expressions, and secondly, the expression of XBP1 protein showed different dynamics compared to the XBP1 mRNA level. Furthermore, our results regarding the relationship between XBP1 and FoxO1 by small interfering RNA (siRNA) paradoxically showed negative regulation of FoxO1 expression by XBP1. Our findings revealed that the XBP1-FoxO1 interaction regulated the ER stress-induced autophagy in auditory cells.

EGF domain of coagulation factor IX is conducive to exposure of phosphatidylserine.

Lipid rafts are an initiation site for many different signals. Recently, we reported that an EGF domain in activated coagulation factor IX (EGF-F9) increases lipid raft formation and accelerates cell migration. However, the detailed mechanism is not well understood. This study aimed to evaluate the effects of EGF-F9 on the cell membrane. A431 cells (derived from human squamous cell carcinoma) were treated with recombinant EGF-F9. Cells were immunocytochemically stained with probes for lipid rafts or phosphatidylserine (PS). After 3 min of treatment with EGF-F9, cholera toxin subunit B (CTxB) binding domains emerged at the adhesive tips of filopodia. Subsequently, CTxB staining was observed on the filopodial shaft. Finally, large clusters of CTxB domains were observed at the edge of cell bodies. Markers for lipid rafts, such as caveolin-1 and a GPI anchored protein, co-localized with CTxB. Staining with annexin V and XII revealed that PS was exposed at the tips of filopodia, translocated on filopodial shafts, and co-localized with CTxB at the rafts. Immunocytochemistry showed that scramblase-1 protein was present at the filopodial tips. Our data indicates that EGF-F9 accelerates PS exposure around the filopodial adhesion complex and induces clustering of lipid rafts in the cell body. PS exposure is thought to occur on cells undergoing apoptosis. Further study of the function of the EGF-F9 motif in mediating signal transduction is necessary because it is shared by a number of proteins.

Activation peptide of coagulation factor IX regulates endothelial permeability.

Endothelial hyperpermeability is involved in several critical illnesses, and its regulatory mechanisms have been intensively investigated. It was recently reported that the activation peptide of coagulation factor IX enhances cell matrix and intercellular adhesion. The aim of this study was to investigate the role of activation peptide of coagulation factor IX in intercellular adhesion of endothelial cells and evaluate its effects on endothelial permeability. In the presence of activation peptide, cells spread with lamellipodium-like broad protrusions multidirectionally, increasing the area of adhesion to matrix by 16% within 30 minutes. In intercellular adhesion, treatment with activation peptide induced overlapping of adjacent cell edges and remodeling of intercellular adhesion sites, with colocalization of the adherens junction proteins VE-cadherin and ß-catenin and a marker protein of the lateral border recycling compartment, PECAM. Activation peptide decreased gaps between cells by 66% in cultured endothelial cells and suppressed increased endothelial cell monolayer permeability induced by interleukin-1ß in a dose-dependent manner. Treatment with activation peptide decreased eNOS protein expression and altered its subcellular distribution, decreasing intracellular cGMP. An analogue of cGMP suppressed the effects of activation peptide on cell spreading. In addition, the effect of activation peptide on hyperpermeability was investigated in mice injected with lipopolysaccharide. Intravenous injection of lipopolysaccharide increased lung weight by 28%, and treatment with activation peptide significantly suppressed the increase in lung weight to 5%. Our results indicate that activation peptide of factor IX regulates endothelial intercellular adhesion and thus could be used in the treatment of vascular hyperpermeability.

The first EGF domain of coagulation factor IX attenuates cell adhesion and induces apoptosis.

Coagulation factor IX (FIX) is an essential plasma protein for blood coagulation. The first epidermal growth factor (EGF) motif of FIX (EGF-F9) has been reported to attenuate cell adhesion to the extracellular matrix (ECM). The purpose of the present study was to determine the effects of this motif on cell adhesion and apoptosis. Treatment with a recombinant EGF-F9 attenuated cell adhesion to the ECM within 10 min. De-adhesion assays with native FIX recombinant FIX deletion mutant proteins suggested that the de-adhesion activity of EGF-F9 requires the same process of FIX activation as that which occurs for coagulation activity. The recombinant EGF-F9 increased lactate dehydrogenase (LDH) activity release into the medium and increased the number of cells stained with annexin V and activated caspase-3, by 8.8- and 2.7-fold respectively, indicating that EGF-F9 induced apoptosis. Activated caspase-3 increased very rapidly after only 5 min of administration of recombinant EGF-F9. Treatment with EGF-F9 increased the level of phosphorylated p38 mitogen-activated protein kinase (MAPK), but not that of phosphorylated MAPK 44/42 or c-Jun N-terminal kinase (JNK). Inhibitors of caspase-3 suppressed the release of LDH. Caspase-3 inhibitors also suppressed the attenuation of cell adhesion and phosphorylation of p38 MAPK by EGF-F9. Our data indicated that EGF-F9 activated signals for apoptosis and induced de-adhesion in a caspase-3 dependent manner.

Long-term gene therapy with Del1 fragment using nonviral vectors in mice with explanted tumors.

Cancer gene therapy using nonviral vectors is useful for long periods of treatment because such vectors are both safe and inexpensive, and thus can be used repeatedly. It has been reported that gene therapy with an E3C1 fragment of Del1 in a mouse explanted tumor model improved prognosis. The present study aimed to analyze the long-term effects of repeated non-viral gene transfer of E3C1. Mice with explanted tumors of SCCKN cells, a human squamous carcinoma, were treated with a plasmid encoding E3C1. Plasmids were injected locally every week using a transfection reagent. Control mice treated with mock DNA started to be euthanized on day 18, because the tumors had grown to over 15% of the body weight, and all of them had died by day 43. On the other hand, the tumors in two of ten mice treated with E3C1 had disappeared. The other eight mice started to be euthanized on day 46 and eight of ten mice had been euthanized by day 197. After 18 days of therapy, the tumor volume of control mice was 2,804±829 mm(3) and that of the E3C1 mice was 197±159 mm(3). Histochemical studies showed enhanced apoptosis in the E3C1-treated tumors, as compared with controls. Changes in cell morphology and decreased polymerized actin induced by E3C1 indicated disturbed cell adhesion to the matrix. In in vitro studies of SCCKN cells, prolonged administration of an E3C1 recombinant protein to cultured cells reduced adhesion-independent growth of cancer cells, as compared with control cells. These data suggest that E3C1 treatment induces anoikis.

An epidermal growth factor motif from Del1 protein increases the efficiency of in vivo gene transfer with a non-viral vector.

Increasing the efficiency of gene transfer using non-viral vectors, which have the potential to be safe and economical, would improve upon available options for gene therapy. We previously reported that the third EGF motif of the extracellular matrix protein Del1 (E3) increases the transfection efficiency of non-viral vector methods. Here, we asked if E3 could increase the in vivo transfection efficiency of a polyplex-based approach. To test this, cDNA encoding a heat-stable alkaline phosphatase (AP) was first injected intravenously into mice along with recombinant E3. After 24 h, exogenous AP activity in serum was measured. We found that the introduction of E3 resulted in 50 % more AP activity as compared to the control. We next tested transfection into a tumour explant of SCCKN cells, an oral carcinoma-derived cell line. To do this, a cDNA encoding yellow fluorescent protein was locally injected into a tumour explant, followed by local injection of recombinant E3. Use of E3 increased the number of transfected cells to 2.5 times that of the control. Histochemical staining revealed that E3-induced apoptosis in a tumour explant. The data suggest that E3 might be a useful tool for cancer gene therapy using non-viral vectors.

Efficient nonviral gene therapy with FasL and Del1 fragments in mice.

BACKGROUND: The expression of FasL in cancer cells is currently being explored as a potential cancer therapy. Because high levels of FasL are necessary for effective treatment, current methods typically rely on the use of highly efficient viral vectors. However, because viral vector-based gene therapy is associated with certain risks, the development of effective nonviral routes for gene delivery would be useful. The present study aimed to improve FasL gene therapy with a nonviral vector by taking advantage of the E3 and C1 domains of Del1 protein, which induces apoptosis and localizes to the extracellular matrix. METHODS: Mouse explanted tumors derived from a human oral squamous cell carcinoma cell line, SCCKN, were treated with plasmids encoding FasL (pFasL), E3C1 (pE3C1), and a fusion of FasL and E3C1 (pFasL-E3C1). The plasmids were injected locally every 7 days along with a transfection reagent, Jet-PEI (PolyPlus-transfection, San Marcos, CA, USA). RESULTS: All mice treated with a negative control plasmid or pFasL died within 49 days. By contrast, 83% of mice treated with pFasL-E3C1 survived longer than 49 days. Histochemical studies revealed that the fusion protein is localized to the stroma and induces apoptosis in stromal cells and adjacent parenchymal cells. CONCLUSIONS: The results obtained in the present study suggest that the protein deposition-based approach described, which makes use of the E3 and C1 domains of Del1, could comprise a novel method for cancer gene therapy with nonviral vectors.

The extracellular matrix protein Del1 induces apoptosis via its epidermal growth factor motif.

Mouse Del1 is an extracellular matrix protein mainly expressed in the developing embryo. Del1 has three EGF motifs and two discoidin domains. The second EGF motif reportedly contains an RGD sequence that binds to integrin receptors. Here, we provide evidence that Del1 protein induces cell death in vitro. Chromatin condensation and DNA laddering were observed, suggestive of apoptosis. The results of analysis using the TUNEL method and annexin V staining were also consistent with apoptosis. The apoptosis-inducing activity of Del1 could be mapped to the third EGF motif, which fitted the consensus sequence CX(D/N)XXXX(F/Y)XCXC, wherein the aspartic acid residue (D) could be beta-hydroxylated. As little as twenty-five picomolar of recombinant E3 could induce apoptosis.

The Del1 deposition domain can immobilize 3alpha-hydroxysteroid dehydrogenase in the extracellular matrix without interfering with enzymatic activity.

Developing methods that result in targeting of therapeutic molecules in gene therapies to target tissues has importance, as targeting can increase efficacy and decrease off target-side-effects. Work from my laboratory previously showed that the extracellular matrix protein Del1 is organized in the extracellular matrix (ECM) via the Del1 deposition domain (DDD). In this work, a fusion protein with DDD was made to assay the ability to immobilize an enzyme without disrupting enzymatic function. A prostatic cancer-derived cell line LNCap that grows in an androgen-dependent manner was used with 3alpha-hydroxysteroid dehydrogenase (3 alphaHD), which catalyzes dihydrotestosterone (DHT). Plasmids encoding a 3alphaHD:DDD fusion were generated and transfected into cultured cells. The effects of 3alphaHD immobilized in the ECM by the DDD were evaluated by monitoring growth of LNCap cells and DHT concentrations. It was demonstrated that the DDD could immobilize an enzyme in the ECM without interfering with function.

An epidermal growth factor-like repeat of Del1 protein increases the efficiency of gene transfer in vitro.

In this paper, we found that Del1, an extracellular matrix protein secreted by embryonic endothelial cells, increases the efficiency of transfection in vitro. Conditioned medium containing Del1 increased transfection by the LacZ gene using several non-viral gene transfer systems, including lipoplex and polyplex methods. Experiments using deletion mutants and fragments of Del1 revealed that the third epidermal growth factor-like repeat (E3) of Del1 mediates the enhancement of gene transfer and, furthermore, that the motif CXDXXXFXCXC is essential. Incubation of Pro5 cells, a yolk sac-derived cell line, with as low as 16 pM recombinant E3 was sufficient to enhance transfection, and 1 nM recombinant E3 enhanced the transfection 12-fold. Inhibitors of endocytosis suppressed this activity of the recombinant E3. These results suggest that the E3 fragment of Del1 can be used as a general biological enhancer of non-viral gene transfer.