Serum-induced Cell Research Articles

PBP4 is required for serum-induced cell wall thickening and antibiotic tolerance in Staphylococcus aureus.

The bacterial pathogen Staphylococcus aureus responds to the host environment by synthesizing a thick peptidoglycan cell wall, which protects the bacterium from membrane-targeting antimicrobials and the immune response. However, the proteins required for this response were previously unknown. Here, we demonstrate by three independent approaches that the penicillin-binding protein PBP4 is crucial for serum-induced cell wall thickening. First, mutants lacking various non-essential cell wall synthesis enzymes were tested, revealing that a mutant lacking pbp4 was unable to generate a thick cell wall in serum. This resulted in reduced serum-induced tolerance of the pbp4 mutant toward the last resort antibiotic daptomycin relative to wild-type cells. Second, we found that serum-induced cell wall thickening occurred in each of a panel of 134 clinical bacteremia isolates, except for one strain with a naturally occurring mutation that results in an S140R substitution in the active site of PBP4. Finally, inhibition of PBP4 with cefoxitin prevented serum-induced cell wall thickening and the resulting antibiotic tolerance in the USA300 strain and clinical MRSA isolates. Together, this provides a rationale for combining daptomycin with cefoxitin, a PBP4 inhibitor, to potentially improve treatment outcomes for patients with invasive MRSA infections.

Proteomic Analysis Reveals Oxidative Phosphorylation and JAK-STAT Pathways Mediated Pathogenesis of Pemphigus Vulgaris.

Pemphigus vulgaris (PV) stands as a rare autoimmune bullous disease, while the precise underlying mechanism remains incompletely elucidated. High-throughput proteomic methodologies, such as LC-MS/MS, have facilitated the quantification and characterisation of proteomes from clinical skin samples, enhancing our comprehension of PV pathogenesis. The objective of this study is to elucidate the signalling mechanisms underlying PV through proteomic analysis. Proteins and cell suspension were extracted from skin biopsies obtained from both PV patients and healthy volunteers and subsequently analysed using LC-MS/MS and scRNA-seq. Cultured keratinocytes were treated with PV serum, followed by an assessment of protein expression levels using immunofluorescence and western blotting. A total of 880, 605, and 586 differentially expressed proteins (DEPs) were identified between the lesion vs. control, non-lesion vs. control, and lesion vs. non-lesion groups, respectively. The oxidative phosphorylation (OXPHOS) pathway showed activation in PV. Keratinocytes are the major cell population in the epidermis and highly expressed ATP5PF, ATP6V1G1, COX6B1, COX6A1, and NDUFA9. In the cellular model, there was a notable increase in the expression levels of OXPHOS-related proteins (V-ATP5A, III-UQCRC2, II-SDHB, I-NDUFB8), along with STAT1, p-STAT1, and p-JAK1. Furthermore, both the OXPHOS inhibitor metformin and the JAK1 inhibitor tofacitinib demonstrated therapeutic effects on PV serum-induced cell separation, attenuating cell detachment. Metformin notably reduced the expression of V-ATP5A, III-UQCRC2, II-SDHB, I-NDUFB8, p-STAT1, p-JAK1, whereas tofacitinib decreased the expression of p-STAT1 and p-JAK1, with minimal impact on the expression of V-ATP5A, III-UQCRC2, II-SDHB, and I-NDUFB8. Our results indicate a potential involvement of the OXPHOS and JAK-STAT1 pathways in the pathogenesis of PV.

Ficolin-3 induces apoptosis and suppresses malignant property of hepatocellular carcinoma cells via the complement pathway

AimsFicolin 3 (FCN3) has the highest complement-activating capacity through the lectin pathway and is synthesized mainly in the liver and lung. Yet, its potential molecular mechanism in hepatocarcinogenesis is not fully understood. Materials and methodsThe expression of FCN3 in hepatocellular carcinoma (HCC) tumor and non-tumor tissues was analyzed by RT-qPCR, Western blotting and immunofluorescence staining assays. Lentivector-mediated ectopic overexpression was performed to explore the role of FCN3 in vitro and in vivo. Whether FCN3 inhibited HCC cell growth and survival via complement pathway was determined with immunocytochemical staining for C3b, membrane attack complex (MAC) formation and complement killing assay using recombinant FCN3 (rFCN3) in combination with human serum with or without heat inactivation, and with C6 blocking antibody. Key findingsThe transcript and protein of FCN3 were found to be remarkably down-regulated in HCC tumor tissues. FCN3 expression was found to be associated with better survival of HCC patients. Restoration of FCN3 expression significantly inhibited proliferation, migration and anchorage independent growth of HCC cell lines, and xenograft tumor growth. FCN3 expression induced apoptosis of HCC cells. C3 and MAC formation was stimulated by FCN3 overexpression or rFCN3 treatment. rFCN3 enhanced human serum-induced complement activation and cell death. C6 blocking antibody significantly attenuated complement-mediated cell death and restored the growth of FCN3-overexpressing HCC cells. SignificanceFCN3 has a malignant suppressor role in HCC cells. Our study provides new insights into the molecular mechanisms that drive HCC progression and potential therapeutic targets for treating HCC.

Changes of the serum properties and its effect on the endothelial cells restoration in patients with chronic venous disease treated with sulodexide

ObjectiveInflammation and endothelial dysfunction are important venous changes in patients with chronic venous disease (CVD). The use of the venoactive drugs remains an important treatment modality for patients with CVD, reducing the severity of the CVD-related symptoms and swelling but also reducing inflammation and protecting endothelial cells. In this research, the effects of the serum obtained from patients with CVD before and after sulodexide treatment were evaluated for in vivo and in vitro inflammatory markers and endothelial cell function. MethodsInflammatory markers (IL-6, matrix metalloproteinase-9 [MMP-9], vascular cell adhesion molecule-1 [VCAM-1], and von Willebrand factor [vWF]) from the incompetent great saphenous veins (GSVs) and from the systemic venous circulation were studied in 10 patients with CVD (C2s) before and after 2 months of sulodexide (2 × 500 lipasemic units/d) therapy. Serum obtained from the vein blood before and after sulodexide treatment was evaluated for in vitro cultured human umbilical vein endothelial cell function. ResultsThe serum collected from lower leg incompetent GSVs had significantly elevated levels of VCAM-1 (+29%, P < .001) compared with the serum from the systemic circulation. Endothelial cells exposed to the serum from the incompetent lower leg veins of the untreated CVD patients demonstrated higher stimulated synthesis of MMP-9 (+17%, P < .01), as well as increased markers of senescence (prolongation of population doubling time, β-galactosidase activity, and expression of p21 and p53 genes). CVD serum-induced senescent endothelial cells had a higher expression of genes regulating IL-6, MMP-9, VCAM-1, and vWF synthesis. The overall proinflammatory effect on endothelial cells by the serum collected from the incompetent GSVs was stronger as compared with the serum from the systemic circulation. Serum collected from the veins after sulodexide treatment caused lower levels of endothelial cell inflammatory markers as well as respective gene expression than serum obtained at the beginning of the study (before sulodexide treatment). Sulodexide application also reduced the inflammatory secretory activity of the senescent endothelial cells. Sulodexide treatment resulted in the decrease of the majority of the studied inflammatory parameters in both lower limb incompetent vein and systemic blood. ConclusionsIn patients with CVD, there are significant differences between circulating inflammatory markers analyzed from the lower leg incompetent GSV segments compared with the systemic circulation, indicating a higher inflammatory condition in CVD. Treatment with sulodexide reduces the proinflammatory and endothelial cell activation properties of the serum from patients with CVD. Clinical RelevanceThe study documented the significant proinflammatory human vascular endothelial cell activation when exposed to the serum collected from the varicose veins as compared with the serum from the systemic circulation in patients with chronic venous disease (CVD). The inflammatory marker expression, endothelial dysfunction, and endothelial cell senescence transformation can be successfully controlled and downregulated by patients’ exposure to the glycosaminoglycan (sulodexide) treatment. Further studies are needed to confirm if glycosaminoglycan application can prevent further CVD clinical progression due to potential CVD-related pathological processes’ modulation and their downregulation.

Soyghurt Potentially Controls the Level of sFlt1 and PLGF in Preeclampsia Maternal Serum-Induced Placental Trophoblast Cell in vitro.

To observe the effect of soya yoghurt (Soyghurt), which is high in flavonoid substance, on the expression of preeclampsia biomarkers (sFLT-1 and PLGF) on preeclampsia serum-induced trophoblast primary cell culture isolated from placental tissue. The trophoblast primary culture was induced by preeclampsia serum (10%). The Soyghurt treatment was performed with 2.5%, 5%, and 7.5% Soyghurt supernatant concentrations in culture media. The expression of preeclampsia markers, sFLT-1 and PLGF, were evaluated using ELISA. Expression of sFLT-1 on preeclampsia-induced cell culture treated with Soyghurt was significantly lowered compared to the untreated group (p<0.01). However, no significant difference was observed in the PLGF levels of all groups induced by preeclampsia serum (p>0.05). This study demonstrates the potential effect of Soyghurt's in balancing preeclampsia marker expression by inhibiting the expression of sFLT-1 in preeclampsia serum -induced trophoblast cells.

NFIL3 aggravates human coronary artery endothelial cell injury by promoting ITGAM transcription in Kawasaki disease

ABSTRACTObjectiveHigh expression of nuclear factor interleukin-3 (NFIL3) and integrin Alpha M (ITGAM) was found in serum samples from Kawasaki disease (KD) patients through bioinformatics analysis. Hence, this study aimed to explore the biological functions of NFIL3 and ITGAM in KD serum-stimulated human coronary artery endothelial cells (HCAECs).MethodsThe differentially-expressed genes in KD were analyzed through bioinformatics analysis. Serum samples were obtained from 18 KD patients and 18 healthy volunteers, followed by detection of NFIL3 and ITGAM levels in KD serum. After HCAECs were transfected with sh-NFIL3, sh-ITGAM, or sh-NFIL3 + oe-ITGAM and underwent 24-h KD serum stimulation, cell viability and apoptosis and the levels of inflammation-related factors were measured. The binding between NFIL3 and ITGAM was validated by dual-luciferase and chromatin immunoprecipitation (ChIP) assays.ResultsNFIL3 and ITGAM were up-regulated in serum from KD patients and KD serum-stimulated HCAECs. Down-regulation of NFIL3 or ITGAM inhibited KD serum-induced cell apoptosis and inflammatory response of HCAECs and promoted cell viability. Mechanistically, NFIL3 promoted ITGAM transcription level. Up-regulation of ITGAM reversed the improvement of NFIL3 down-regulation on KD serum-induced HCAEC injury.ConclusionNFIL3 aggravated KD serum-induced HCAEC injury by promoting ITGAM transcription, which provided new insights into the treatment of KD.

Effect of serum from patients with ST-segment elevation myocardial infarction on endothelial cells

Introduction and objectives Clinical and experimental studies have shown that, in patients with reperfused ST-segment elevation myocardial infarction (STEMI), abnormalities in the endothelial monolayer are initiated during ischemia but rapidly intensify upon restoration of blood perfusion to the ischemic area. We aimed to evaluate the effect of serum isolated after revascularization from STEMI patients on the degree of endothelial permeability in vitro, by promoting endothelial cell apoptosis and necrosis in vitro. We also investigated the association between the percentage of serum-induced endothelial cell apoptosis or necrosis in vitro and the extent of cardiovascular magnetic resonance (CMR)-derived parameters of reperfusion injury (edema, hemorrhage, and microvascular obstruction).Methods Human coronary artery endothelial cells were incubated with serum isolated 24 hours after revascularization from 43 STEMI patients who underwent CMR and 14 control participants. We assessed the effect of STEMI serum on activation of apoptosis and necrosis, as well as on the permeability and structure of the endothelial monolayer.Results Serum from STEMI patients increased apoptosis (P < .01) and necrosis (P < .05) in human coronary artery endothelial cells and caused increased permeability of the endothelial monolayer in vitro (P < .01), due to enlarged intercellular spaces (P < .05 vs control in all cases). Higher serum-induced necrosis was associated with greater endothelial permeability in vitro (P < .05) and with more extensive CMR-derived indices of reperfusion injury and infarct size.Conclusions Postreperfusion serum activates necrosis and apoptosis in endothelial cells and increases the degree of endothelial permeability in vitro. The more potent the necrosis-triggering effect of serum, the more deleterious the consequences in terms of the resulting cardiac structure.

Effect of serum from patients with ST-segment elevation myocardial infarction on endothelial cells

Introduction and objectivesClinical and experimental studies have shown that, in patients with reperfused ST-segment elevation myocardial infarction (STEMI), abnormalities in the endothelial monolayer are initiated during ischemia but rapidly intensify upon restoration of blood perfusion to the ischemic area. We aimed to evaluate the effect of serum isolated after revascularization from STEMI patients on the degree of endothelial permeability in vitro, by promoting endothelial cell apoptosis and necrosis in vitro. We also investigated the association between the percentage of serum-induced endothelial cell apoptosis or necrosis in vitro and the extent of cardiovascular magnetic resonance (CMR)-derived parameters of reperfusion injury (edema, hemorrhage, and microvascular obstruction). MethodsHuman coronary artery endothelial cells were incubated with serum isolated 24hours after revascularization from 43 STEMI patients who underwent CMR and 14 control participants. We assessed the effect of STEMI serum on activation of apoptosis and necrosis, as well as on the permeability and structure of the endothelial monolayer. ResultsSerum from STEMI patients increased apoptosis (P <.01) and necrosis (P <.05) in human coronary artery endothelial cells and caused increased permeability of the endothelial monolayer in vitro (P <.01), due to enlarged intercellular spaces (P <.05 vs control in all cases). Higher serum-induced necrosis was associated with greater endothelial permeability in vitro (P <.05) and with more extensive CMR-derived indices of reperfusion injury and infarct size. ConclusionsPostreperfusion serum activates necrosis and apoptosis in endothelial cells and increases the degree of endothelial permeability in vitro. The more potent the necrosis-triggering effect of serum, the more deleterious the consequences in terms of the resulting cardiac structure.

Significant expansion of the donor pool achieved by utilizing islets of variable quality in the production of allogeneic "Neo-Islets", 3-D organoids of Mesenchymal Stromal and islet cells, a novel immune-isolating biotherapy for Type I Diabetes.

Novel biotherapies for Type 1 Diabetes that provide a significantly expanded donor pool and that deliver all islet hormones without requiring anti-rejection drugs are urgently needed. Scoring systems have improved islet allotransplantation outcomes, but their use may potentially result in the waste of valuable cells for novel therapies. To address these issues, we created "Neo-Islets" (NIs), islet-sized organoids, by co-culturing in ultralow adhesion flasks culture-expanded islet (ICs) and Mesenchymal Stromal Cells (MSCs) (x 24 hrs, 1:1 ratio). The MSCs exert powerful immune- and cyto-protective, anti-inflammatory, proangiogenic, and other beneficial actions in NIs. The robust in vitro expansion of all islet hormone-producing cells is coupled to their expected progressive de-differentiation mediated by serum-induced cell cycle entry and Epithelial-Mesenchymal Transition (EMT). Re-differentiation in vivo of the ICs and resumption of their physiological functions occurs by reversal of EMT and serum withdrawal-induced exit from the cell cycle. Accordingly, we reported that allogeneic, i.p.-administered NIs engraft in the omentum, increase Treg numbers and reestablish permanent normoglycemia in autoimmune diabetic NOD mice without immunosuppression. Our FDA-guided pilot study (INAD 012-0776) in insulin-dependent pet dogs showed similar responses, and both human- and canine-NIs established normoglycemia in STZ-diabetic NOD/SCID mice even though the utilized islets would be scored as unsuitable for transplantation. The present study further demonstrates that islet gene expression profiles (α, β, γ, δ) in human "non-clinical grade" islets obtained from diverse, non-diabetic human and canine donors (n = 6 each) closely correlate with population doublings, and the in vivo re-differentiation of endocrine islet cells clearly corresponds with the reestablishment of euglycemia in diabetic mice. Conclusion: human-NIs created from diverse, "non-clinical grade" donors have the potential to greatly expand patient access to this curative therapy of T1DM, facilitated by the efficient in vitro expansion of ICs that can produce ~ 270 therapeutic NI doses per donor for 70 kg recipients.

Functional cooperation between IKCa and TRPC1 channels regulates serum-induced vascular smooth muscle cell proliferation via mediating Ca2+ influx and ERK1/2 activation.

The increased proliferation of vascular smooth muscle cells (VSMCs) contributes to the pathogenesis of vascular diseases. The intermediate conductance calcium-activated potassium (IKCa ) channel plays a critical role in VSMC proliferation by raising the intracellular calcium concentration ([Ca2+ ]i ), but the underlying mechanism is still not unclear. Here we investigated the cooperation between IKCa and transient receptor potential canonical 1 (TRPC1) channels in mediating extracellular Ca2+ entry, which in turn activates downstream Ca2+ signalling in the regulation of VSMC proliferation using serum-induced cell proliferation model. Serum-induced cell proliferation was accompanied with up-regulation of IKCa expression and an increase in [Ca2+ ]i . Serum-induced cell proliferation and increase in [Ca2+ ]i were suppressed by IKCa inhibition with TRAM-34 or IKCa knockdown. Serum-induced cell proliferation was strongly reduced by the removal of extracellular Ca2+ with EGTA or intracellular Ca2+ with BAPTA-AM and, additionally, by TRPC1 knockdown. Moreover, the increase in [Ca2+ ]i induced by serum or by IKCa activation with 1-EBIO was attenuated by TRPC1 knockdown. Finally, serum induced ERK1/2 activation, which was attenuated by treatment with TRAM-34 or BAPTA-AM, as well as TRPC1 knockdown. Consistently, serum-induced cell proliferation was suppressed by ERK1/2 inhibition with PD98059. Taken together, these results suggest that the IKCa and TRPC1 channels cooperate in mediating Ca2+ influx that activates the ERK1/2 pathway to promote cell proliferation, thus providing new mechanistic insights into VSMC proliferation.

Exogenous glycine promotes oxidation of glutathione and restores sensitivity of bacterial pathogens to serum-induced cell death

Pathogenic strains of bacteria are often highly adept at evading serum-induced cell death, which is an essential complement-mediated component of the innate immune response. This phenomenon, known as serum-resistance, is poorly understood, and as a result, no effective clinical tools are available to restore serum-sensitivity to pathogenic bacteria. Here, we provide evidence that exogenous glycine reverses defects in glycine, serine and threonine metabolism associated with serum resistance, restores susceptibility to serum-induced cell death, and alters redox balance and glutathione (GSH) metabolism. More specifically, in Vibrio alginolyticus and Escherichia coli, exogenous glycine promotes oxidation of GSH to GSH disulfide (GSSG), disrupts redox balance, increases oxidative stress and reduces membrane integrity, leading to increased binding of complement. Antioxidant or ROS scavenging agents abrogate this effect and agents that generate or potentiate oxidation stimulate serum-mediated cell death. Analysis of several clinical isolates of E. coli demonstrates that glutathione metabolism is repressed in serum-resistant bacteria. These data suggest a novel mechanism underlying serum-resistance in pathogenic bacteria, characterized by an induced shift in the GSH/GSSG ratio impacting redox balance. The results could potentially lead to novel approaches to manage infections caused by serum-resistant bacteria both in aquaculture and human health.

Metformin Inhibits Multiple Myeloma Serum-induced Endothelial Cell Thrombosis by Down-Regulating miR-532

Thrombotic complications in multiple myeloma (MM) impairs the quality of life in patients. Metformin has a certain effect on anti-thrombosis, but its role and mechanism in MM-induced thrombosis are still uncovered. Therefore, this study evaluated the effect of metformin on MM-induced thrombosis. Human umbilical vein endothelial cells (HUVECs) were exposed to normal serum (15%), MM serum (15%), metformin (0.01mmol/L), or MM serum, and metformin simultaneously. The expression of tissue factor (TF) in HUVECs was detected by flow cytometry and quantitative real-time polymerase chain reaction PCR (qRT-PCR). QRT-PCR was also used to determine the expressions of endothelial protein C receptor (EPCR) and miR-532. The generation of thrombin and activated protein C was measured by thrombin generation and protein C activation assays. EPCR, extracellular signal-regulated kinase (ERK) 1/2, p38 mitogen activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) pathway related protein expressions were detected by western blot. MM serum increased the expressions of TF and miR-532, induced thrombin generation, inhibited EPCR and protein C activation in HUVECs. And metformin could reverse the effectsof MM serum on the expressions of TF, EPCR and miR-532, thrombin generation, protein C activation in HUVECs. However, miR-532 mimic reversed the effects of metformin and promoted the levels of thrombosis-related indicators in HUVECs. Moreover, metformin activated the ERK 1/2, p38 MAPK, and NF-κB pathways but miR-532 mimic suppressed the pathway activation. Metformin played an inhibitory effect on MM serum-induced HUVEC thrombosis, suggesting that metformin could serve as a novel antithrombotic approach for MM patients.

Lamina-associated polypeptide 2\u03b1 is required for intranuclear MRTF-A activity

Myocardin-related transcription factor A (MRTF-A), a coactivator of serum response factor (SRF), regulates the expression of many cytoskeletal genes in response to cytoplasmic and nuclear actin dynamics. Here we describe a novel mechanism to regulate MRTF-A activity within the nucleus by showing that lamina-associated polypeptide 2α (Lap2α), the nucleoplasmic isoform of Lap2, is a direct binding partner of MRTF-A, and required for the efficient expression of MRTF-A/SRF target genes. Mechanistically, Lap2α is not required for MRTF-A nuclear localization, unlike most other MRTF-A regulators, but is required for efficient recruitment of MRTF-A to its target genes. This regulatory step takes place prior to MRTF-A chromatin binding, because Lap2α neither interacts with, nor specifically influences active histone marks on MRTF-A/SRF target genes. Phenotypically, Lap2α is required for serum-induced cell migration, and deregulated MRTF-A activity may also contribute to muscle and proliferation phenotypes associated with loss of Lap2α. Our studies therefore add another regulatory layer to the control of MRTF-A-SRF-mediated gene expression, and broaden the role of Lap2α in transcriptional regulation.

Wound Healing-Promoting and Melanogenesis-Inhibiting Activities of Angelica polymorpha Maxim. Flower Absolute In Vitro and Its Chemical Composition.

Angelica polymorpha Maxim. (APM) is used in traditional medicine to treat chronic gastritis, rheumatic pain, and duodenal bulbar ulcers. However, it is not known whether APM has epidermis-associated biological activities. Here, we investigated the effects of APM flower absolute (APMFAb) on responses associated with skin wound healing and whitening using epidermal cells. APMFAb was obtained by solvent extraction and its composition was analyzed by GC/MS. Water-soluble tetrazolium salt, 5-bromo-2′-deoxyuridine incorporation, Boyden chamber, sprouting, and enzyme-linked immunosorbent assays and immunoblotting were used to examine the effects of APMFAb on HaCaT keratinocytes and B16BL6 melanoma cells. APMFAb contained five compounds and induced keratinocyte migration, proliferation, and type IV collagen synthesis. APMFAb also induced the phosphorylations of ERK1/2, JNK, p38 mitogen-activated protein kinase, and AKT in keratinocytes. In addition, APMFAb decreased serum-induced B16BL6 cell proliferation and inhibited tyrosinase expression, melanin contents, and microphthalmia-associated transcription factor expression in α-melanocyte-stimulating hormone-stimulated B16BL6 cells. These findings demonstrate that APMFAb has beneficial effects on skin wound healing by promoting the proliferation, migration, and collagen synthesis of keratinocytes and on skin whitening by inhibiting melanin synthesis in melanoma cells. Therefore, we suggest that APMFAb has potential use as a wound healing and skin whitening agent.

Chemical Composition of Miscanthus sinensis var. purpurascens Flower Absolute and Its Beneficial Effects on Skin Wound Healing and Melanogenesis‐Related Cell Activities

Miscanthus sinensis var. purpurascens (MSP, flame grass) is found in Korea, Japan, and China, and its biological activities include anti-cancer, detoxifying, vasodilatory, antipyretic, and diuretic effects. However, no study has investigated the effects of MSP on skin-related biological activities. In this study, we explored the effects of the absolute extracted from the MSP flowers (MSPFAb) on skin wound healing- and whitening-related responses in keratinocytes or melanocytes. MSPFAb contained 6 components and induced the proliferation, migration, and syntheses of type I and IV collagens in keratinocytes. MSPFAb also increased the phosphorylations of serine/threonine-specific protein kinase, p38 mitogen-activated protein kinase, and extracellular signal-regulated kinase1/2 in keratinocytes. In addition, treatment with MSPFAb decreased serum-induced melanoma cell proliferation and inhibited tyrosinase activity and melanin contents in α-MSH-stimulated melanoma cells. Taken together, this study indicates MSPFAb may promote wound healing- and whitening-associated activities in dermal cells, and suggests that it has potential use as a wound healing and skin whitening agent.

Alternative Splicing of Pericentrin Contributes to Cell Cycle Control in Cardiomyocytes.

Induction of cardiomyocyte proliferation is a promising option to regenerate the heart. Thus, it is important to elucidate mechanisms that contribute to the cell cycle arrest of mammalian cardiomyocytes. Here, we assessed the contribution of the pericentrin (Pcnt) S isoform to cell cycle arrest in postnatal cardiomyocytes. Immunofluorescence staining of Pcnt isoforms combined with SiRNA-mediated depletion indicates that Pcnt S preferentially localizes to the nuclear envelope, while the Pcnt B isoform is enriched at centrosomes. This is further supported by the localization of ectopically expressed FLAG-tagged Pcnt S and Pcnt B in postnatal cardiomyocytes. Analysis of centriole configuration upon Pcnt depletion revealed that Pcnt B but not Pcnt S is required for centriole cohesion. Importantly, ectopic expression of Pcnt S induced centriole splitting in a heterologous system, ARPE-19 cells, and was sufficient to impair DNA synthesis in C2C12 myoblasts. Moreover, Pcnt S depletion enhanced serum-induced cell cycle re-entry in postnatal cardiomyocytes. Analysis of mitosis, binucleation rate, and cell number suggests that Pcnt S depletion enhances serum-induced progression of postnatal cardiomyocytes through the cell cycle resulting in cell division. Collectively, our data indicate that alternative splicing of Pcnt contributes to the establishment of cardiomyocyte cell cycle arrest shortly after birth.

Antioxidant and Anti-atherogenic Properties of Prosopis strombulifera and Tessaria absinthioides Aqueous Extracts: Modulation of NADPH Oxidase-Derived Reactive Oxygen Species.

Despite popular usage of medicinal plants, their effects as cardiovascular protective agents have not been totally elucidated. We hypothesized that treatment with aqueous extract from Prosopis strombulifera (AEPs) and Tessaria absinthioides (AETa), Argentinian native plants, produces antioxidant effects on vascular smooth muscle cells (VSMCs) and attenuates atherogenesis on apolipoprotein E-knockout (ApoE-KO) mice. In VSMCs, both extracts (5–40 μg/ml) inhibited 10% fetal calf serum-induced cell proliferation, arrested cell in G2/M phase, reduced angiotensin II-induced reactive oxygen species (ROS) generation, and decreased NADPH oxidase subunit expression. In ApoE-KO mice, extracts significantly reduced triglycerides and lipid peroxidation [plasma thiobarbituric acid reactive substances (TBARS)], increased plasma total antioxidant status (TAS), and improved glutathione peroxidase activity in the liver. Under high-fat diet (HFD), both extracts were able to inhibit O2– generation in the aortic tissue and caused a significant regression of atheroma plaques (21.4 ± 1.6% HFD group vs. 10.2 ± 1.2%∗ AEPs group and 14.3 ± 1.0%∗ AETa group; ∗p < 0.01). Consumption of AEPs and AETa produces antioxidant/antimitogenic/anti-atherosclerotic effects, and their use may be beneficial as a complementary strategy regarding cardiovascular disease therapies.

Gout-induced endothelial impairment: The role of SREBP2 transactivation of YAP.

Gout is a multifaceted inflammatory disease involving vascular impairments induced by hyperuricemia. Experiments using human umbilical vein endothelial cells treated with uric acid (UA), monosodium urate (MSU), or serum from gout patients showed increased expression of pro-inflammatory genes (ie, VCAM1, ICAM1, CYR61, CCNA1, and E2F1) with attendant increase in monocyte adhesion. Mechanistically, UA- or MSU-induced SREBP2 expression and its transcriptional activity. RNA sequencing analysis and real-time PCR showed the induction of YAP signaling and pro-inflammatory pathways in HUVECs transfected with adenovirus-SREBP2. The SREBP2 knockdown by siRNA partially abolished UA- or MSU-induced YAP activity, pro-inflammatory gene expression, and monocytes adhesion. Vascular intima from transgenic mice overexpressing SREBP2 in endothelium or mice with hyperuricemia exhibited activated YAP signaling and increased expression of pro-inflammatory genes. Betulin, an SREBP pharmacological inhibitor, attenuated the UA-, MSU-, or gout serum-induced endothelial cell inflammation and dysfunction. In the human study, endothelial cell function, assessed by EndoPAT, was negatively correlated with serum UA level among gouty patients and healthy controls. Collectively, UA or MSU causes endothelial dysfunction via SREBP2 transactivation of YAP. Betulin inhibition of SREBP2 may restrain gout-induced endothelial dysfunction.

Efficacy Assessment and Mechanism Exploration of Kruppel‐Like Factor 2‐Targeting Intervention Strategy for Non‐Small Cell Lung Cancer Therapy

Non‐small cell lung cancer (NSCLC), the most common type of lung cancer, has the problems such as less sensitivity to chemotherapy and resistance to tyrosine kinase inhibitors. Kruppel‐like factor 2 (KLF2) has been reported to have the potential activities in tumor suppression and immune regulation. The expression level of KLF2 is lower in NSCLC and negatively correlated with the stages of tumor progression and lymph node metastasis. To date, not only no KLF2‐promoting compounds have been developed, but no related studies on its anti‐tumor efficacy in vivo have been verified. Therefore, the purposes of this study were focused on the potential and related mechanisms of KLF2 applied as a targeted molecule for NSCLC therapy. Our data indicated that the intervention of lovastatin markedly up‐regulated protein expression of KLF2 in A549 human non‐small cell lung cancer cells. Moreover, KLF2 siRNA intervention could significant inhibit serum‐induced cell growth in A549 cells. The analysis result of the Ingenuity pathway analysis (IPA) showed that several intracellular molecules, such as AKT, mTOR and FOXO1, maybe the potential upstream regulators to involve in KLF2 expression. Besides, the experimental data also evidenced that lovastatin can suppress activations of AKT and mTOR proteins as well as to reduce protein expression of FOXO1. In summary, these results suggested that intensifying KLF2 expression could apply as a feasible therapeutic strategy to improve NSCLC therapy, and statinoid compounds have potential to be developed as KLF2 activator. In addition, activations or expressions of AKT‐mTOR pathway and FOXO1 may be attributed to suppression of KLF2 in NSCLC cells, such as A549 cells.

DDIS-01. NEW DRUG DISCOVERY WITH DRUG RE-POSITIONING SYSTEM TOWARD GBM TREATMENT

Abstract INTRODUCTION Invasion of Glioblastoma (GBM) cells into normal brain is the major cause of poor prognosis and requires dynamic reorganization of the actin cytoskeleton, which includes lamellipodial protrusions, focal adhesions, and stress fibers at the leading edge of GBM cells. Therefore, we hypothesized that the inhibition of actin polymerization with existing medication could lead to clinical GBM treatment directly in short time. The Drug Repositioning system is also known as drug repurposing or drug reprofiling and understood as the process of redeveloping a compound for use in a different disease. In this time, we would show the new direction of drug development with drug repositioning. MATERIALS AND METHODS We adopted a drug repositioning system for screening with a pyrene-actin-based actin polymerization assay and identified fluvoxamine, a clinically used antidepressant. Fluvoxamine, selective serotonin reuptake inhibitor, was a potent inhibitor of actin polymerization and confirmed as drug penetration through the blood–brain barrier (BBB) and accumulation of whole brain including brain tumor with no drug toxicity. RESULTS Fluvoxamine inhibited serum-induced ruffle formation, cell migration, and invasion of human GBM and glioma stem cells in vitro by suppressing FAK signaling. Fluvoxamine showed no drug toxicity. Daily treatment of athymic mice bearing human glioma-initiating cells with fluvoxamine blocked tumor cell invasion and prolonged the survival with almost same doze of anti-depressant effect. CONCLUSIONS The SSRI group has big potential for clinical GBM treatment with low cost, short time and low side effect.