Synergistic combination of RAD51-SCR7 improves CRISPR-Cas9 genome editing efficiency by preventing R-loop accumulation.

CRISPR-Cas9 has emerged as a powerful tool for genome editing. However, Cas9 genome editing faces challenges, including low efficiency and off-target effects. Here, we report that combined treatment with RAD51, a key factor in homologous recombination, and SCR7, a DNA ligase IV small-molecule inhibitor, enhances CRISPR-Cas9-mediated genome-editing efficiency in human embryonic kidney 293T and human induced pluripotent stem cells, as confirmed by cyro- transmission electron microscopy and functional analyses. First, our findings reveal the crucial role of RAD51 in homologous recombination (HR)-mediated DNA repair process. Elevated levels of exogenous RAD51 promote a post-replication step via single-strand DNA gap repair process, ensuring the completion of DNA replication. Second, using the all-in-one CRISPR-Cas9-RAD51 system, highly expressed RAD51 improved the multiple endogenous gene knockin/knockout efficiency and insertion/deletion (InDel) mutation by activating the HR-based repair pathway in concert with SCR7. Sanger sequencing shows distinct outcomes for RAD51-SCR7 in the ratio of InDel mutations in multiple genome sites. Third, RAD51-SCR7 combination can induce efficient R-loop resolution and DNA repair by enhanced HR process, which leads to DNA replication stalling and thus is advantageous to CRISPR-Cas9-based stable genome editing. Our study suggests promising applications in genome editing by enhancing CRISPR-Cas9 efficiency through RAD51 and SCR7, offering potential advancements in biotechnology and therapeutics.

Multi-proteomic analyses of 5xFAD mice reveal new molecular signatures of early-stage Alzheimer's disease.

An early diagnosis of Alzheimer's disease is crucial as treatment efficacy is limited to the early stages. However, the current diagnostic methods are limited to mid or later stages of disease development owing to the limitations of clinical examinations and amyloid plaque imaging. Therefore, this study aimed to identify molecular signatures including blood plasma extracellular vesicle biomarker proteins associated with Alzheimer's disease to aid early-stage diagnosis. The hippocampus, cortex, and blood plasma extracellular vesicles of 3- and 6-month-old 5xFAD mice were analyzed using quantitative proteomics. Subsequent bioinformatics and biochemical analyses were performed to compare the molecular signatures between wild type and 5xFAD mice across different brain regions and age groups to elucidate disease pathology. There was a unique signature of significantly altered proteins in the hippocampal and cortical proteomes of 3- and 6-month-old mice. The plasma extracellular vesicle proteomes exhibited distinct informatic features compared with the other proteomes. Furthermore, the regulation of several canonical pathways (including phosphatidylinositol 3-kinase/protein kinase B signaling) differed between the hippocampus and cortex. Twelve potential biomarkers for the detection of early-stage Alzheimer's disease were identified and validated using plasma extracellular vesicles from stage-divided patients. Finally, integrin α-IIb, creatine kinase M-type, filamin C, glutamine γ-glutamyltransferase 2, and lysosomal α-mannosidase were selected as distinguishing biomarkers for healthy individuals and early-stage Alzheimer's disease patients using machine learning modeling with approximately 79% accuracy. Our study identified novel early-stage molecular signatures associated with the progression of Alzheimer's disease, thereby providing novel insights into its pathogenesis.

Comparative Phosphoproteomics of Neuro-2a Cells under Insulin Resistance Reveals New Molecular Signatures of Alzheimer's Disease.

Insulin in the brain is a well-known critical factor in neuro-development and regulation of adult neurogenesis in the hippocampus. The abnormality of brain insulin signaling is associated with the aging process and altered brain plasticity, and could promote neurodegeneration in the late stage of Alzheimer's disease (AD). The precise molecular mechanism of the relationship between insulin resistance and AD remains unclear. The development of phosphoproteomics has advanced our knowledge of phosphorylation-mediated signaling networks and could elucidate the molecular mechanisms of certain pathological conditions. Here, we applied a reliable phosphoproteomic approach to Neuro2a (N2a) cells to identify their molecular features under two different insulin-resistant conditions with clinical relevance: inflammation and dyslipidemia. Despite significant difference in overall phosphoproteome profiles, we found molecular signatures and biological pathways in common between two insulin-resistant conditions. These include the integrin and adenosine monophosphate-activated protein kinase pathways, and we further verified these molecular targets by subsequent biochemical analysis. Among them, the phosphorylation levels of acetyl-CoA carboxylase and Src were reduced in the brain from rodent AD model 5xFAD mice. This study provides new molecular signatures for insulin resistance in N2a cells and possible links between the molecular features of insulin resistance and AD.

Stimulation of the Migration and Expansion of Adult Mouse Neural Stem Cells by the FPR2-Specific Peptide WKYMVm.

Neural stem cells (NSCs) are multipotent cells capable of self-renewal and differentiation into different nervous system cells. Mouse NSCs (mNSCs) are useful tools for studying neurogenesis and the therapeutic applications of neurodegenerative diseases in mammals. Formyl peptide receptor 2 (FPR2), expressed in the central nervous system and brain, is involved in the migration and differentiation of murine embryonic-derived NSCs. In this study, we explored the effect of FPR2 activation in adult mNSCs using the synthetic peptide Trp-Lys-Tyr-Met-Val-D-Met-NH2 (WKYMVm), an agonist of FPR2. After isolation of NSCs from the subventricular zone of the adult mouse brain, they were cultured in two culture systems-neurospheres or adherent monolayers-to demonstrate the expression of NSC markers and phenotypes. Under different conditions, mNSCs differentiated into neurons and glial cells such as astrocytes, microglia, and oligodendrocytes. Treatment with WKYMVm stimulated the chemotactic migration of mNSCs. Moreover, WKYMVm-treated mNSCs were found to promote proliferation; this result was confirmed by the expansion of mNSCs in Matrigel and the increase in the number of Ki67-positive cells. Incubation of mNSCs with WKYMVm in a supplement-free medium enhanced the survival rate of the mNSCs. Together, these results suggest that WKYMVm-induced activation of FPR2 stimulates cellular responses in adult NSCs.

Application of Proteomics in Cancer: Recent Trends and Approaches for Biomarkers Discovery.

Proteomics has become an important field in molecular sciences, as it provides valuable information on the identity, expression levels, and modification of proteins. For example, cancer proteomics unraveled key information in mechanistic studies on tumor growth and metastasis, which has contributed to the identification of clinically applicable biomarkers as well as therapeutic targets. Several cancer proteome databases have been established and are being shared worldwide. Importantly, the integration of proteomics studies with other omics is providing extensive data related to molecular mechanisms and target modulators. These data may be analyzed and processed through bioinformatic pipelines to obtain useful information. The purpose of this review is to provide an overview of cancer proteomics and recent advances in proteomic techniques. In particular, we aim to offer insights into current proteomics studies of brain cancer, in which proteomic applications are in a relatively early stage. This review covers applications of proteomics from the discovery of biomarkers to the characterization of molecular mechanisms through advances in technology. Moreover, it addresses global trends in proteomics approaches for translational research. As a core method in translational research, the continued development of this field is expected to provide valuable information at a scale beyond that previously seen.

Emodin induces collagen type I synthesis in Hs27 human dermal fibroblasts.

Fibrillar collagen and elastic fibers are the main components of the dermal extracellular matrix (ECM), which confers mechanical strength and resilience to the skin. In particular, type I collagen produced by fibroblasts is the most abundant collagen that determines the general strength of the ECM, thereby contributing to the prevesntion of the skin-aging process. Although the natural anthraquinone derivative emodin (1,3,8-trihydroxy-6-methylanthraquinone) exerts numerous beneficial effects, including antiviral, anticancer, anti-inflammatory and wound-healing effects in diverse cells, the effect of emodin on collagen expression or skin aging is not fully understood. The present study demonstrated that exposure to emodin increased type I collagen synthesis in a concentration- and time-dependent manner in Hs27 human dermal fibroblasts. Subsequent experiments showed that emodin strongly increased collagen type I levels without altering cell proliferation or cellular matrix metalloproteinase-1 (MMP-1) expression. Additionally, it was determined that increased phosphorylation of 5' AMP-activated protein kinase, following emodin treatment, was responsible for increased type I collagen synthesis. These findings clearly indicate that emodin plays an important role in collagen type I synthesis in dermal fibroblasts, thereby making it a potential drug candidate for treating skin aging and wrinkles.

Coadministration of endothelial and smooth muscle cells derived from human induced pluripotent stem cells as a therapy for critical limb ischemia.

Critical limb ischemia is a condition in which tissue necrosis occurs due to arterial occlusion, resulting in limb amputation in severe cases. Both endothelial cells (ECs) and vascular smooth muscle cells (SMCs) are needed for the regeneration of peripheral arteries in ischemic tissues. However, it is difficult to isolate and cultivate primary EC and SMC from patients for therapeutic angiogenesis. Induced pluripotent stem cells (iPSCs) are regarded as useful stem cells due to their pluripotent differentiation potential. In this study, we explored the therapeutic efficacy of human iPSC-derived EC and iPSC-derived SMC in peripheral artery disease model. After the induction of mesodermal differentiation of iPSC, CD34+ progenitor cells were isolated by magnetic-activated cell sorting. Cultivation of the CD34+ progenitor cells in endothelial culture medium induced the expression of endothelial markers and phenotypes. Moreover, the CD34+ cells could be differentiated into SMC by cultivation in SMC culture medium. In a murine hindlimb ischemia model, cotransplantation of EC with SMC improved blood perfusion and increased the limb salvage rate in ischemic limbs compared to transplantation of either EC or SMC alone. Moreover, cotransplantation of EC and SMC stimulated angiogenesis and led to the formation of capillaries and arteries/arterioles in vivo. Conditioned medium derived from SMC stimulated the migration, proliferation, and tubulation of EC in vitro, and these effects were recapitulated by exosomes isolated from the SMC-conditioned medium. Together, these results suggest that iPSC-derived SMC enhance the therapeutic efficacy of iPSC-derived EC in peripheral artery disease via an exosome-mediated paracrine mechanism.

Mesenchymal Stem Cell-Mediated Therapy of Peripheral Artery Disease Is Stimulated by a Lamin A-Progerin Binding Inhibitor.

OBJECTIVE: Human adipose tissue-derived mesenchymal stem cells (ASCs) have been reported to promote angiogenesis and tissue repair. However, poor survival and engraftment efficiency of transplanted ASCs are the major bottlenecks for therapeutic application. The present study aims to improve the therapeutic efficacy of ASCs for peripheral artery diseases. METHODS: Hydrogen peroxide (H2O2) was used to induce apoptotic cell death in ASCs. To measure apoptosis, we used flow cytometry-based apoptosis analysis and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. A murine hindlimb ischemia model was established to measure the ASC-mediated therapeutic angiogenesis and in vivo survival ability of ASCs. RESULTS: We identified that the inhibitor of lamin A-progerin binding, JH4, protects ASCs against H2O2-induced oxidative stress and apoptosis. Co-administration of ASCs with JH4 improved ASC-mediated blood reperfusion recovery and limb salvage compared to that of the control group in a mouse hind limb ischemia model. Immunofluorescence showed that JH4 treatment potentiated ASC-mediated vascular regeneration via reducing ASC apoptosis post transplantation. CONCLUSION: JH4 exerts anti-apoptotic effects in ASCs in conditions of oxidative stress, and contributes to the repair of ischemic hind limb injury by improving cell survival.

Etv2- and Fli1-Induced Vascular Progenitor Cells Enhance Functional Recovery in Ischemic Vascular Disease Model-Brief Report.

OBJECTIVE: Vascular progenitor cells (VPCs), which are able to differentiate into both endothelial cells and smooth muscle cells, have the potential for treatment of ischemic diseases. Generated by pluripotent stem cells, VPCs carry the risk of tumorigenicity in clinical application. This issue could be resolved by direct lineage conversion, the induction of functional cells from another lineage by using only lineage-restricted transcription factors. Here, we show that induced VPCs (iVPCs) can be generated from fibroblasts by ETS (E-twenty six) transcription factors, Etv2 and Fli1. Approach and Results: Mouse fibroblasts were infected with lentivirus encoding Etv2 and Fli1. Cell colonies appeared in Fli1- and Etv2/Fli1-infected groups and were mechanically picked. The identity of cell colonies was confirmed by proliferation assay and reverse-transcription polymerase chain reaction with vascular markers. Etv2/Fli1- infected cell colonies were sorted by CD144 (also known as CDH5, VE-cadherin). We defined that CD144-positive iVPCs maintained its own population and expanded stably at multiple passages. iVPCs could differentiate into functional endothelial cells and smooth muscle cells by a defined medium. The functionalities of iVPC-derived endothelial cells and smooth muscle cells were confirmed by analyzing LDL (low-density lipoprotein) uptake, carbachol-induced contraction, and tube formation in vitro. Transplantation of iVPCs into the ischemic hindlimb model enhanced blood flow without tumor formation in vivo. Human iVPCs were generated by human ETS transcription factors ETV2 and FLI1. CONCLUSIONS: We demonstrate that ischemic disease curable iVPCs, which have self-renewal and bipotency, can be generated from mouse fibroblasts by enforced ETS family transcription factors, Etv2 and Fli1 expression. Our simple strategy opens insights into stem cell-based ischemic disease therapy.

Formyl Peptide Receptor 2 Activation Ameliorates Dermal Fibrosis and Inflammation in Bleomycin-Induced Scleroderma.

Systemic sclerosis is a profibrotic autoimmune disease mediated by the dysregulation of extracellular matrix synthesis. Formyl peptide receptor 2 (Fpr2) is a G protein-coupled receptor that modulates inflammation and host defense by regulating the activation of inflammatory cells, such as macrophages. However, the role of Fpr2 in the development and therapy of scleroderma is still unclear. The present study was conducted to investigate the effects of Fpr2 activation in the treatment of scleroderma fibrosis. We found that intradermal administration of WKYMVm, an Fpr2-specific agonist, alleviated bleomycin-induced scleroderma fibrosis in mice and decreased dermal thickness in scleroderma skin. WKYMVm-treated scleroderma skin tissues displayed reduced numbers of myofibroblasts expressing α-smooth muscle actin, Vimentin, and phosphorylated SMAD3. WKYMVm treatment attenuated macrophage infiltration in scleroderma skin and reduced the number of M2 macrophages. The therapeutic effects of WKYMVm in scleroderma-associated fibrosis and inflammation were completely abrogated in Fpr2 knockout mice. Moreover, WKYMVm treatment reduced the serum levels of inflammatory cytokines, such as tumor necrosis factor-α, and interferon-γ, in the scleroderma model of wild-type mice but not in Fpr2 knockout mice. These results suggest that WKYMVm-induced activation of Fpr2 leads to alleviation of fibrosis by stimulating immune resolution in systemic sclerosis.

Role of stem cell mobilization in the treatment of ischemic diseases.

Stem cell mobilization plays important roles in the treatment of severe ischemic diseases, including myocardial infarction, limb ischemia, ischemic stroke, and acute kidney injury. Stem cell mobilization refers to the egress of heterogeneous stem cells residing in the bone marrow into the peripheral blood. In the clinic, granulocyte colony-stimulating factor (G-CSF) is the drug most commonly used to induce stem cell mobilization. Plerixafor, a direct antagonist of CXCR4, is also frequently used alone or in combination with G-CSF to mobilize stem cells. The molecular mechanisms by which G-CSF induces stem cell mobilization are well characterized. Briefly, G-CSF activates neutrophils in the bone marrow, which then release proteolytic enzymes, such as neutrophil elastase, cathepsin G, and matrix metalloproteinase 9, which cleave a variety of molecules responsible for stem cell retention in the bone marrow, including CXCL12, VCAM-1, and SCF. Subsequently, stem cells are released from the bone marrow into the peripheral blood. The released stem cells can be collected and used in autologous or allogeneic transplantation. To identify better conditions for stem cell mobilization in the treatment of acute and chronic ischemic diseases, several preclinical and clinical studies have been conducted over the past decade on various mobilizing agents. In this paper, we are going to review methods that induce mobilization of stem cells from the bone marrow and introduce the application of stem cell mobilization to therapy of ischemic diseases.

Role of CXCR2 in the Ac-PGP-Induced Mobilization of Circulating Angiogenic Cells and its Therapeutic Implications.

Circulating angiogenic cells (CACs) have been implicated in the repair of ischemic tissues, and their mobilization from bone marrow is known to be regulated by the activations of chemokine receptors, including CXCR2 and CXCR4. This study was conducted to investigate the role of N-acetylated proline-glycine-proline (Ac-PGP; a collagen-derived chemotactic tripeptide) on CAC mobilization and its therapeutic potential for the treatment of peripheral artery diseases. Ac-PGP was administered daily to a murine hind limb ischemia model, and the effects of Ac-PGP on blood perfusion and CAC mobilization (Sca1+ Flk1+ cells) into peripheral blood were assessed. Intramuscular administration of Ac-PGP significantly improved ischemic limb perfusion and increased limb salvage rate by increasing blood vessel formation, whereas Ac-PGP-induced blood perfusion and angiogenesis in ischemic limbs were not observed in CXCR2-knockout mice. In addition, Ac-PGP-induced CAC mobilization was found to occur in wild-type mice but not in CXCR2-knockout mice. Transplantation of bone marrow from green fluorescent protein (GFP) transgenic mice to wild-type mice showed bone marrow-derived cells homed to ischemic limbs after Ac-PGP administration and that GFP-positive cells contributed to the formation of ILB4-positive capillaries and α smooth muscle actin (α-SMA)-positive arteries. These results suggest CXCR2 activation in bone marrow after Ac-PGP administration improves blood perfusion and reduces tissue necrosis by inducing CAC mobilization. These findings suggest a new pharmaceutical basis for the treatment of critical limb ischemia. Stem Cells Translational Medicine 2019;8:236&246.

TRRAP stimulates the tumorigenic potential of ovarian cancer stem cells.

Ovarian cancer is the most fatal gynecological malignancy in women and identification of new therapeutic targets is essential for the continued development of therapy for ovarian cancer. TRRAP (transformation/transcription domain-associated protein) is an adaptor protein and a component of histone acetyltransferase complex. The present study was undertaken to investigate the roles played by TRRAP in the proliferation and tumorigenicity of ovarian cancer stem cells. TRRAP expression was found to be up-regulated in the sphere cultures of A2780 ovarian cancer cells. Knockdown of TRRAP significantly decreased cell proliferation and the number of A2780 spheroids. In addition, TRRAP knockdown induced cell cycle arrest and increased apoptotic percentages of A2780 sphere cells. Notably, the mRNA levels of stemness-associated markers, that is, OCT4, SOX2, and NANOG, were suppressed in TRRAP-silenced A2780 sphere cells. In addition, TRRAP overexpression increased the mRNA level of NANOG and the transcriptional activity of NANOG promoter in these cells. Furthermore, TRRAP knockdown significantly reduced tumor growth in a murine xenograft transplantation model. Taken together, the findings of the present study suggest that TRRAP plays an important role in the regulation of the proliferation and stemness of ovarian cancer stem cells. [BMB Reports 2018; 51(10): 515-520].

Atrial natriuretic peptide accelerates human endothelial progenitor cell-stimulated cutaneous wound healing and angiogenesis.

Atrial natriuretic peptide (ANP) is a powerful vasodilating peptide secreted by cardiac muscle cells, and endothelial progenitor cells (EPCs) have been reported to stimulate cutaneous wound healing by mediating angiogenesis. To determine whether ANP can promote the EPC-mediated repair of injured tissues, we examined the effects of ANP on the angiogenic properties of EPCs and on cutaneous wound healing. In vitro, ANP treatment enhanced the migration, proliferation, and endothelial tube-forming abilities of EPCs. Furthermore, small interfering RNA-mediated silencing of natriuretic peptide receptor-1, which is a receptor for ANP, abrogated ANP-induced migration, tube formation, and proliferation of EPCs. In a murine cutaneous wound model, administration of either ANP or EPCs had no significant effect on cutaneous wound healing or angiogenesis in vivo, whereas the coadministration of ANP and EPCs synergistically potentiated wound healing and angiogenesis. In addition, ANP promoted the survival and incorporation of transplanted EPCs into newly formed blood vessels in wounds. These results suggest ANP accelerates EPC-mediated cutaneous wound healing by promoting the angiogenic properties and survival of transplanted EPCs.

3D cell printing of in vitro stabilized skin model and in vivo pre-vascularized skin patch using tissue-specific extracellular matrix bioink: A step towards advanced skin tissue engineering.

3D cell-printing technique has been under spotlight as an appealing biofabrication platform due to its ability to precisely pattern living cells in pre-defined spatial locations. In skin tissue engineering, a major remaining challenge is to seek for a suitable source of bioink capable of supporting and stimulating printed cells for tissue development. However, current bioinks for skin printing rely on homogeneous biomaterials, which has several shortcomings such as insufficient mechanical properties and recapitulation of microenvironment. In this study, we investigated the capability of skin-derived extracellular matrix (S-dECM) bioink for 3D cell printing-based skin tissue engineering. S-dECM was for the first time formulated as a printable material and retained the major ECM compositions of skin as well as favorable growth factors and cytokines. This bioink was used to print a full thickness 3D human skin model. The matured 3D cell-printed skin tissue using S-dECM bioink was stabilized with minimal shrinkage, whereas the collagen-based skin tissue was significantly contracted during in vitro tissue culture. This physical stabilization and the tissue-specific microenvironment from our bioink improved epidermal organization, dermal ECM secretion, and barrier function. We further used this bioink to print 3D pre-vascularized skin patch able to promote in vivo wound healing. In vivo results revealed that endothelial progenitor cells (EPCs)-laden 3D-printed skin patch together with adipose-derived stem cells (ASCs) accelerates wound closure, re-epithelization, and neovascularization as well as blood flow. We envision that the results of this paper can provide an insightful step towards the next generation source for bioink manufacturing.

Recent advances in stem cell therapeutics and tissue engineering strategies.

BACKGROUND: Tissue regeneration includes delivering specific types of cells or cell products to injured tissues or organs for restoration of tissue and organ function. Stem cell therapy has drawn considerable attention since transplantation of stem cells can overcome the limitations of autologous transplantation of patient's tissues; however, it is not perfect for treating diseases. To overcome the hurdles associated with stem cell therapy, tissue engineering techniques have been developed. Development of stem cell technology in combination with tissue engineering has opened new ways of producing engineered tissue substitutes. Several studies have shown that this combination of tissue engineering and stem cell technologies enhances cell viability, differentiation, and therapeutic efficacy of transplanted stem cells. MAIN BODY: Stem cells that can be used for tissue regeneration include mesenchymal stem cells, embryonic stem cells, and induced pluripotent stem cells. Transplantation of stem cells alone into injured tissues exhibited low therapeutic efficacy due to poor viability and diminished regenerative activity of transplanted cells. In this review, we will discuss the progress of biomedical engineering, including scaffolds, biomaterials, and tissue engineering techniques to overcome the low therapeutic efficacy of stem cells and to treat human diseases. CONCLUSION: The combination of stem cell and tissue engineering techniques overcomes the limitations of stem cells in therapy of human diseases, and presents a new path toward regeneration of injured tissues.

Trib2 regulates the pluripotency of embryonic stem cells and enhances reprogramming efficiency.

Embryonic stem (ES) cells are pluripotent cells characterized by self-renewability and differentiation potential. Induced pluripotent stem (iPS) cells are ES cell-equivalent cells derived from somatic cells by the introduction of core reprogramming factors. ES and iPS cells are important sources for understanding basic biology and for generating therapeutic cells for clinical applications. Tribbles homolog 2 (Trib2) functions as a scaffold in signaling pathways. However, the relevance of Trib2 to the pluripotency of ES and iPS cells is unknown. In the present study, we elucidated the importance of Trib2 in maintaining pluripotency in mouse ES cells and in generating iPS cells from somatic cells through the reprogramming process. Trib2 expression decreased as ES cells differentiated, and Trib2 knockdown in ES cells changed their colony morphology while reducing the activity of alkaline phosphatase and the expression of the pluripotency marker genes Oct4, Sox2, Nanog and Klf4. Trib2 directly interacted with Oct4 and elevated Oct4 promoter activity. During the generation of iPS cells, Trib2 knockdown decreased the reprogramming efficiency of mouse embryonic fibroblasts, whereas Trib2 overexpression significantly increased their reprogramming efficiency. In summary, our results suggest that Trib2 is important for maintaining self-renewal in ES cells and for pluripotency induction during the reprogramming process.

Identification of a novel angiogenic peptide from periostin.

Angiogenic peptides have therapeutic potential for the treatment of chronic ischemic diseases. Periostin, an extracellular matrix protein expressed in injured tissues, promotes angiogenesis and tissue repair. We previously reported that in vivo administration of both recombinant full-length protein and the first FAS I domain of periostin alleviated peripheral artery occlusive disease by stimulating the migration of humane endothelial colony forming cells (ECFCs) and subsequent angiogenesis. In the present study, we ascertained the peptide sequence responsible for the periostin-induced angiogenesis. By serial deletion mapping of the first FAS I domain, we identified a peptide sequence (amino acids 142-151) of periostin for stimulation of chemotactic migration, adhesion, proliferation and endothelial tube formation of human ECFCs in vitro. Chemotactic migration of ECFCs induced by the periostin peptide was blocked by pre-incubation with an anti-ß5 integrin neutralizing antibody. Treatment of ECFCs with the periostin peptide led to phosphorylation of both AKT and ERK, and pretreatment of ECFCs with the MEK-ERK pathway inhibitor U0126 or the PI3K-AKT pathway inhibitors, LY294002 or Wortmannin, blocked the periostin peptide-stimulated migration of ECFCs. These results suggest that the synthetic periostin peptide can be applied for stimulating angiogenic and therapeutic potentials of ECFCs.

Role of Krüppel-Like Factor 4 in the Maintenance of Chemoresistance of Anaplastic Thyroid Cancer.

BACKGROUND: Anaplastic thyroid cancer (ATC) has a very poor prognosis due to its aggressive nature and resistance to conventional treatment. Radiotherapy and chemotherapy are not fully effective because of the undifferentiated phenotype and enhanced drug resistance of ATC. The objective of this study was to evaluate the involvement of Krüppel-like factor 4 (KLF4), a stemness-associated transcription factor, in the undifferentiated phenotype and drug resistance of ATC. METHODS: ATC cells were compared to papillary thyroid cancer cells in drug resistance and gene expression. The effects of KLF4 knockdown in ATC cells on in vitro and in vivo drug resistance were measured. The effects of KLF4 overexpression and knockdown on ABC transporter activity were determined. RESULTS: ATC cells, such as HTH83, 8505C, and SW1736, exhibited higher resistance to the anticancer drug paclitaxel and higher expression of KLF4 than TPC-1 papillary thyroid cancer cells. Knockdown of KLF4 expression in ATC cells increased the expression of the thyroid-specific differentiation genes, such as thyrotropin receptor, thyroid peroxidase, thyroglobulin, and sodium-iodide symporter. Knockdown of KLF4 expression in ATC cells decreased the resistance to doxorubicin and paclitaxel, and reduced ABC transporter expression. Luciferase reporter assay results showed that KLF4 overexpression increased ABCG2 promoter activity, which was abolished by KLF4 knockdown. A tumorigenicity assay showed that the combination of paclitaxel treatment and KLF4 knockdown significantly decreased tumor mass originated from HTH83 cells in mice. CONCLUSIONS: ATC cells show high expression of KLF4, and KLF4 expression is necessary for maintaining the undifferentiated phenotype and drug resistance in vitro and in vivo. The present study identifies KLF4 as a potential therapeutic target for eliminating ATC cells.

The anti-microbial peptide SR-0379 stimulates human endothelial progenitor cell-mediated repair of peripheral artery diseases.

Ischemia is a serious disease, characterized by an inadequate blood supply to an organ or part of the body. In the present study, we evaluated the effects of the anti-microbial peptide SR-0379 on the stem cell-mediated therapy of ischemic diseases. The migratory and tube-forming abilities of human endothelial progenitor cells (EPCs) were enhanced by treatment with SR-0379 in vitro. Intramuscular administration of SR-0379 into a murine ischemic hindlimb significantly enhanced blood perfusion, decreased tissue necrosis, and increased the number of blood vessels in the ischemic muscle. Moreover, co-administration of SR-0379 with EPCs stimulated blood perfusion in an ischemic hindlimb more than intramuscular injection with either SR-0379 or EPCs alone. This enhanced blood perfusion was accompanied by a significant increase in the number of CD31- and α-SMApositive blood vessels in ischemic hindlimb. These results suggest that SR-0379 is a potential drug candidate for potentiating EPC-mediated therapy of ischemic diseases. [BMB Reports 2017; 50(10): 504-509].