RESUMO
Aerolysin-like pore-forming protein (af-PFP) superfamily members are double-edge swords that assist the bacterial infection but shied bacteria from the host by various mechanisms in some species including the toad Bombina maxima and zebrafish. While members of this family are widely expressed in all kingdoms, especially non-bacteria species, it remains unclear whether their anti-bacterial function is conserved. LIN-24 is an af-PFP that is constitutively expressed throughout the Caenorhabditis elegans lifespan. Here, we observed that LIN-24 knockdown reduced the maximum lifespan of worms. RNA-seq analysis identified 323 differentially expressed genes (DEGs) post-LIN-24 knockdown that were enriched in "immune response" and "lysosome pathway," suggesting a possible role for LIN-24 in resisting microbial infection. In line with this, we found that Pseudomonas aeruginosa 14 (PA14) infection induced LIN-24 expression, and that survival after PA14 infection was significantly reduced by LIN-24 knockdown. In contrast, LIN-24 overexpression (LIN-24-OE) conferred protection against PA14 infection, with worms showing longer survival time and reduced bacterial load. Weighted gene co-expression network analysis of LIN-24-OE worms showed that the highest correlation module was enriched in factors related to immunity and the defense response. Finally, by predicting transcription factors from RNA-seq data and knocking down candidate transcription factors in LIN-24-OE worms, we revealed that LIN-24 may protect worms against bacterial infection by stimulating DAF-16-mediated immune responses. These findings agree with our previous studies showing an anti-microbial role for the amphibian-derived af-PFP complex ßγ-CAT, suggesting that af-PFPs may play a conserved role in combatting microbial infections. Further research is needed to determine the roles this protein family plays in other physio-pathological processes, such as metabolism, longevity, and aging.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Envelhecimento , Caenorhabditis elegans/genética , Longevidade , Proteínas de Caenorhabditis elegans/genéticaRESUMO
Atrial fibrillation (AF) is the most common form of sustained clinical arrhythmia. We previously mapped an AF locus to chromosome 5p13 in an AF family with sudden death in early childhood. Here we show that the specific AF gene underlying this linkage is NUP155, which encodes a member of the nucleoporins, the components of the nuclear pore complex (NPC). We have identified a homozygous mutation, R391H, in NUP155 that cosegregates with AF, affects nuclear localization of NUP155, and reduces nuclear envelope permeability. Homozygous NUP155(-/-) knockout mice die before E8.5, but heterozygous NUP155(+/-) mice show the AF phenotype. The R391H mutation and reduction of NUP155 are associated with inhibition of both export of Hsp70 mRNA and nuclear import of Hsp70 protein. These human and mouse studies indicate that loss of NUP155 function causes AF by altering mRNA and protein transport and link the NPC to cardiovascular disease.
Assuntos
Fibrilação Atrial/genética , Morte Súbita Cardíaca , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Sequência de Aminoácidos , Animais , Feminino , Proteínas de Choque Térmico HSP72/genética , Proteínas de Choque Térmico HSP72/metabolismo , Humanos , Masculino , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Membrana Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Linhagem , Alinhamento de SequênciaRESUMO
Proprotein convertase subtilisin/kexin 6 (PCSK6) is a secreted serine protease expressed in most major organs, where it cleaves a wide range of growth factors, signaling molecules, peptide hormones, proteolytic enzymes, and adhesion proteins. Studies in Pcsk6-deficient mice have demonstrated the importance of Pcsk6 in embryonic development, body axis specification, ovarian function, and extracellular matrix remodeling in articular cartilage. In the cardiovascular system, PCSK6 acts as a key modulator in heart formation, lipoprotein metabolism, body fluid homeostasis, cardiac repair, and vascular remodeling. To date, dysregulated PCSK6 expression or function has been implicated in major cardiovascular diseases, including atrial septal defects, hypertension, atherosclerosis, myocardial infarction, and cardiac aging. In this review, we describe biochemical characteristics and posttranslational modifications of PCSK6. Moreover, we discuss the role of PCSK6 and related molecular mechanisms in cardiovascular biology and disease.
Assuntos
Sistema Cardiovascular , Infarto do Miocárdio , Animais , Camundongos , Biologia , Sistema Cardiovascular/metabolismo , Pró-Proteína Convertases/genética , Pró-Proteína Convertases/metabolismo , SubtilisinaRESUMO
Natriuretic peptide receptor 1 (NPR1) serves as a modulator of vascular endothelial homeostasis. Interactions between monocytes and endothelial cells may initiate endothelium dysfunction, which is known as an early hallmark of atherosclerosis. In this study, we performed RNA-sequencing analysis for the aorta of Npr1 knockout (Npr1+/-) mice and found that differentially expressed genes were significantly related to cell adhesion. This result was supported by an increased expression of intercellular adhesion molecule 1 (ICAM-1) in the aortic endothelium of Npr1+/- mice. Moreover, we observed that the knockdown of NPR1 increased ICAM-1 expression and promoted THP-1 monocyte adhesion to human umbilical vein endothelial cells (HUVECs). NPR1 overexpression decreased ICAM-1 expression and inhibited the adhesion of monocytes to HUVECs treated by TNF-α (a cell adhesion inducer). Further analysis showed that adhesion-related genes were enriched in the focal adhesion signaling pathway, in which integrin beta 4 (Itgb4) was determined as a key gene. Notably, ITGB4 expression increased in vascular endothelium of Npr1+/- mice and in NPR1-knockdown HUVECs. The deficiency of ITGB4 decreased ICAM-1 expression and attenuated monocyte adhesion to NPR1-knockdown endothelial cells. Additionally, a reduced NPR1 and an increased ITGB4 expression level were found in an atherosclerosis mouse model. In conclusion, our findings demonstrate that NPR1 deficiency increases vascular endothelial cell adhesion by stimulating ITGB4 expression, which may contribute to the development of atherosclerosis.
Assuntos
Aterosclerose , Molécula 1 de Adesão Intercelular , Humanos , Camundongos , Animais , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Endotélio Vascular/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Monócitos/metabolismo , Adesão Celular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Aterosclerose/genética , Aterosclerose/metabolismo , Integrinas/metabolismo , RNA/metabolismoRESUMO
Surface functionalization of polymeric porous substrates is one of the most important requirements to enhance their applications in the biomedical field. In this study, we achieved photoinduced surface modification using a highly efficient reaction of hydrophilic polymers bearing phosphorylcholine groups. Polymers composed of 2-methacryloyloxyethyl phosphorylcholine (MPC) units and 2-( N-ethylanilino)ethyl methacrylate units were synthesized with attention to the polymer architectures. The surface modification of the porous polyethylene (PE) substrates was carried out by the coating of the MPC polymers with a photochemical radical generator, followed by photoirradiation for a few minutes. Surface analysis by attenuated total reflectance Fourier transform IR spectroscopy and X-ray photoelectron spectroscopy indicated that the MPC polymer layer was generated on the PE surface. Cross-sectional confocal microscopy images showed that the MPC polymers were coated on the polymer surface, even inside the porous structure of the PE substrate. After modification, the porous PE substrates showed a significant increase in hydrophilicity and the water-penetration rate through the pores. Furthermore, the amount of protein adsorbed on the PE substrate was reduced significantly by the surface modification. These functionalities were dependent on the MPC polymer architectures. Thus, we concluded that the photoreactive polymer system developed furnished the porous substrates with antifouling properties.
RESUMO
OBJECTIVE: The genetic contribution to coronary artery disease (CAD) remains largely unclear. We combined genetic screening with functional characterizations to identify novel loci and candidate genes for CAD. APPROACH AND RESULTS: We performed genome-wide screening followed by multicenter validation in 8 cohorts consisting of 21 828 participants of Han ethnicity and identified 3 novel intragenic SNPs (single nucleotide polymorphisms), rs9486729 (SCML4 [Scm polycomb group protein-like 4]; odds ratio, 1.25; 95% CI, 1.17-1.34; P=3.51×10-11), rs17165136 (THSD7A [thrombospondin type 1 domain-containing 7A]; odds ratio 1.28; 95% CI, 1.21-1.35; P<1.00×10-25), and rs852787 (DAB1 [disabled-1]; odds ratio, 1.29; 95% CI, 1.21-1.38; P=2.02×10-14), associated with CAD with genome-wide significance. The risk allele of rs9486729 and protective allele of rs17165136 were associated with the decreased expression of their host genes, SCML4 and THSD7A, respectively, whereas rs852787 did not have transcriptional effects on any gene. Knockdown of SCML4 activated endothelial cells by increasing the expression of IL-6, E-selectin, and ICAM and weakened their antiapoptotic activity, whereas the knockdown of THSD7A had little effect on these endothelial cell functions but attenuated monocyte adhesion via decreasing the expression of ICAM, L-selectin, and ITGB2. We further showed that inhibiting the expression of SCML4 exacerbated endothelial dysfunction and vascular remodeling in a rat model with partial carotid ligation. CONCLUSIONS: We identify 3 novel loci associated with CAD and show that 2 genes, SCML4 and THSD7A, make functional contributions to atherosclerosis. How rs852787 and its host gene DAB1 are linked to CAD needs further studies.
Assuntos
Doença da Artéria Coronariana/genética , Proteínas do Grupo Polycomb/genética , Polimorfismo de Nucleotídeo Único , Trombospondinas/genética , Adulto , Idoso , Animais , Povo Asiático/genética , Artérias Carótidas/metabolismo , Artérias Carótidas/patologia , Estenose das Carótidas/genética , Estenose das Carótidas/metabolismo , Estenose das Carótidas/patologia , Células Cultivadas , China/epidemiologia , Doença da Artéria Coronariana/diagnóstico , Doença da Artéria Coronariana/etnologia , Doença da Artéria Coronariana/metabolismo , Vasos Coronários/metabolismo , Vasos Coronários/patologia , Modelos Animais de Doenças , Feminino , Frequência do Gene , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Proteínas do Grupo Polycomb/metabolismo , Ratos Sprague-Dawley , Fatores de Risco , Trombospondinas/metabolismo , Remodelação VascularRESUMO
In pregnancy, trophoblast invasion and uterine spiral artery remodelling are important for lowering maternal vascular resistance and increasing uteroplacental blood flow. Impaired spiral artery remodelling has been implicated in pre-eclampsia, a major complication of pregnancy, for a long time but the underlying mechanisms remain unclear. Corin (also known as atrial natriuretic peptide-converting enzyme) is a cardiac protease that activates atrial natriuretic peptide (ANP), a cardiac hormone that is important in regulating blood pressure. Unexpectedly, corin expression was detected in the pregnant uterus. Here we identify a new function of corin and ANP in promoting trophoblast invasion and spiral artery remodelling. We show that pregnant corin- or ANP-deficient mice developed high blood pressure and proteinuria, characteristics of pre-eclampsia. In these mice, trophoblast invasion and uterine spiral artery remodelling were markedly impaired. Consistent with this, the ANP potently stimulated human trophoblasts in invading Matrigels. In patients with pre-eclampsia, uterine Corin messenger RNA and protein levels were significantly lower than that in normal pregnancies. Moreover, we have identified Corin gene mutations in pre-eclamptic patients, which decreased corin activity in processing pro-ANP. These results indicate that corin and ANP are essential for physiological changes at the maternal-fetal interface, suggesting that defects in corin and ANP function may contribute to pre-eclampsia.
Assuntos
Serina Endopeptidases/metabolismo , Trofoblastos/citologia , Artéria Uterina/crescimento & desenvolvimento , Útero/irrigação sanguínea , Útero/metabolismo , Animais , Fator Natriurético Atrial/deficiência , Fator Natriurético Atrial/genética , Fator Natriurético Atrial/metabolismo , Pressão Sanguínea/genética , Estudos de Casos e Controles , Células Cultivadas , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Células HEK293 , Humanos , Isquemia/metabolismo , Isquemia/patologia , Rim/irrigação sanguínea , Rim/patologia , Nefropatias/genética , Nefropatias/patologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Mutação , Pré-Eclâmpsia/genética , Pré-Eclâmpsia/metabolismo , Pré-Eclâmpsia/patologia , Pré-Eclâmpsia/fisiopatologia , Gravidez , Serina Endopeptidases/química , Serina Endopeptidases/genética , Trofoblastos/metabolismoRESUMO
To overcome the thrombogenic reactions of hydrocarbon-based biomaterials in clinical blood treatment, we introduce a model study of surface zwitterionization of a polypropylene (PP) substrate using a set of well-defined copolymers for controlling the adhesion of blood cells in vitro. Random and block copolymers containing zwitterionic units of 2-methacryloyloxyethyl phosphorylcholine (MPC), [3-(methacryloylamino)propyl]dimethyl(3-sulfopropyl)ammonium hydroxide inner salt (SBAA), or nonionic units of 2-hydroxyethyl methacrylate (HEMA) with a controlled hydrophobic segment of 70% n-butyl methacrylate (BMA) units in these polymers were synthesized through reversible addition-fragmentation chain transfer polymerization. A systematic study of how zwitterionic and nonionic copolymer architectures associated with controlled chain orientation via hydration processes affect blood compatibility is reported. The surface wettability of PP substrates coated with the block copolymer with poly(MPC) (PMPC) segments was higher than that of the random copolymer poly(MPC-random-BMA). However, only the random copolymers with SBAA units demonstrate a higher surface wettability. The PP substrate coated with nonionic copolymers containing HEMA units showed relatively lower hydration capability associated with higher protein adsorption, platelet adhesion, and leukocyte attachment than those with zwitterionic copolymers. The random copolymer poly(SBAA-random-BMA) coated on the PP substrates exhibited resistance to cell adhesion in human whole blood at a level comparable to that of MPC copolymers. An ideal zwitterionic PP substrate could be obtained by coating it with a block copolymer composed of PMPC and poly(BMA) (PBMA) segments, PMPC-block-PBMA. The water contact angle decreased dramatically from approximately 100° on the original PP substrate to 11° within 30 s. The number of blood cells attached on PMPC-block-PBMA decreased significantly to less than 2.5% of that on original PP. These results prove that the rational design of zwitterionic polymers incorporated with a hydrophobic anchoring portion provides a promising approach to reduce blood cell adhesion and protein adsorption of hydrocarbon-based biomaterials applied in direct contact with human whole blood.
Assuntos
Polipropilenos/química , Materiais Biocompatíveis/química , Adesão Celular/efeitos dos fármacos , Humanos , Teste de Materiais , Metacrilatos/química , Fosforilcolina/análogos & derivados , Fosforilcolina/química , Polímeros/química , Polipropilenos/farmacologiaRESUMO
Cationic vectors are ideal candidates for gene delivery thanks to their capability to carry large gene inserts and their scalable production. However, their cationic density gives rise to high cytotoxicity. We present the proper designed core-shell polyplexes made of either poly(ethylene imine) (PEI) or poly(2-dimethylamino ethyl methacrylate) (PDMAEMA) as the core and zwitterionic poly(acrylic acid)-block-poly(sulfobetaine methacrylate) (PAA-b-PSBMA) diblock copolymer as the shell. Gel retardation and ethidium bromide displacement assays were used to determine the PEI/DNA or PDMAEMA/DNA complexation. At neutral pH, the copolymer serves as a protective shell of the complex. As PSBMA is a nonfouling block, the shell reduced the cytotoxicity and enhanced the hemocompatibility (lower hemolysis activity, longer plasma clotting time) of the gene carriers. PAA segments in the copolymer impart pH sensitivity by allowing deshielding of the core in acidic solution. Therefore, the transfection efficiency of polyplexes at pH 6.5 was better than at pH 7.0, from ß-galactosidase assay, and for all PAA-b-PSBMA tested. These results were supported by more favorable physicochemical properties in acidic solution (zeta potential, particle size, and interactions between the polymer and DNA). Thus, the results of this study offer a potential route to the development of efficient and nontoxic pH-sensitive gene carriers.
Assuntos
Polímeros/química , DNA , Técnicas de Transferência de Genes , Concentração de Íons de Hidrogênio , Iminas/química , Metacrilatos/química , Nylons/química , Polietilenos/químicaRESUMO
Corin is a membrane-bound protease essential for activating natriuretic peptides and regulating blood pressure. Human corin has 19 predicted N-glycosylation sites in its extracellular domains. It has been shown that N-glycans are required for corin cell surface expression and zymogen activation. It remains unknown, however, how N-glycans at different sites may regulate corin biosynthesis and processing. In this study, we examined corin mutants, in which each of the 19 predicted N-glycosylation sites was mutated individually. By Western analysis of corin proteins in cell lysate and conditioned medium from transfected HEK293 cells and HL-1 cardiomyocytes, we found that N-glycosylation at Asn-80 inhibited corin shedding in the juxtamembrane domain. Similarly, N-glycosylation at Asn-231 protected corin from autocleavage in the frizzled-1 domain. Moreover, N-glycosylation at Asn-697 in the scavenger receptor domain and at Asn-1022 in the protease domain is important for corin cell surface targeting and zymogen activation. We also found that the location of the N-glycosylation site in the protease domain was not critical. N-Glycosylation at Asn-1022 may be switched to different sites to promote corin zymogen activation. Together, our results show that N-glycans at different sites may play distinct roles in regulating the cell membrane targeting, zymogen activation, and ectodomain shedding of corin.
Assuntos
Glicosilação , Mutação , Serina Endopeptidases/química , Animais , Fator Natriurético Atrial/química , Membrana Celular/metabolismo , Precursores Enzimáticos/química , Citometria de Fluxo , Glicosídeo Hidrolases/química , Células HEK293 , Humanos , Camundongos , Modelos Moleculares , Miócitos Cardíacos/citologia , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
Corin is a cardiac membrane protease that activates natriuretic peptides. It is unknown how corin function is regulated. Recently, soluble corin was detected in human plasma, suggesting that corin may be shed from cardiomyocytes. Here we examined soluble corin production and activity and determined the proteolytic enzymes responsible for corin cleavage. We expressed human corin in HEK 293 cells and detected three soluble fragments of â¼180, â¼160, and â¼100 kDa, respectively, in the cultured medium by Western blot analysis. All three fragments were derived from activated corin molecules. Similar results were obtained in HL-1 cardiomyocytes. Using protease inhibitors, ionomycin and phorbol myristate acetate stimulation, small interfering RNA knockdown, and site-directed mutagenesis, we found that ADAM10 was primarily responsible for shedding corin in its juxtamembrane region to release the â¼180-kDa fragment, corresponding to the near-entire extracellular region. In contrast, the â¼160- and â¼100-kDa fragments were from corin autocleavage at Arg-164 in frizzled 1 domain and Arg-427 in LDL receptor 5 domain, respectively. In functional studies, the â¼180-kDa fragment activated atrial natriuretic peptide, whereas the â¼160- and â¼100-kDa fragments did not. Our data indicate that ADAM-mediated shedding and corin autocleavage are important mechanisms regulating corin function and preventing excessive, potentially hazardous, proteolytic activities in the heart.
Assuntos
Miocárdio/enzimologia , Miócitos Cardíacos/enzimologia , Serina Endopeptidases/metabolismo , Proteínas ADAM/antagonistas & inibidores , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAM10 , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Células HEK293 , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Estrutura Terciária de Proteína , Serina Endopeptidases/genéticaRESUMO
Corin is a protease that activates atrial natriuretic peptide, a cardiac hormone important in the control of blood pressure and salt-water balance. Here we examined the role of corin in regulating blood pressure and sodium homeostasis upon dietary salt challenge. Radiotelemetry-tracked blood pressure in corin knockout mice on a high-salt diet (4% sodium chloride) was significantly increased; however, there was no such change in similarly treated wild-type mice. In the knockout mice on the high-salt diet there was an impairment of urinary sodium excretion and an increase in body weight, but no elevation of plasma renin or serum aldosterone levels. When the knockout mice on the high-salt diet were treated with amiloride, an epithelial sodium channel blocker that inhibits renal sodium reabsorption, the impaired urinary sodium excretion and increased body weight were normalized. Amiloride treatment also reduced high blood pressure caused by the high-salt diet in these mice. Thus, the lack of corin in mice impairs their adaptive renal response to high dietary salt, suggesting that corin deficiency may represent an important mechanism underlying salt-sensitive hypertension.
Assuntos
Pressão Sanguínea , Hipertensão/enzimologia , Rim/enzimologia , Natriurese , Serina Endopeptidases/deficiência , Aldosterona/sangue , Amilorida/farmacologia , Anlodipino/farmacologia , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Animais , Anti-Hipertensivos/farmacologia , Biomarcadores/sangue , Pressão Sanguínea/efeitos dos fármacos , Monitorização Ambulatorial da Pressão Arterial/métodos , Peso Corporal , Bloqueadores dos Canais de Cálcio/farmacologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Homeostase , Hipertensão/tratamento farmacológico , Hipertensão/etiologia , Hipertensão/genética , Hipertensão/fisiopatologia , Rim/efeitos dos fármacos , Rim/fisiopatologia , Losartan/farmacologia , Masculino , Camundongos , Camundongos Knockout , Natriurese/efeitos dos fármacos , Renina/sangue , Serina Endopeptidases/genética , Bloqueadores dos Canais de Sódio/farmacologia , Cloreto de Sódio na Dieta , Telemetria , Fatores de TempoRESUMO
In this work, the hemocompatibility of zwitterionic polypropylene (PP) fibrous membranes with varying grafting coverage of poly(sulfobetaine methacrylate) (PSBMA) via plasma-induced surface polymerization was studied. Charge neutrality of PSBMA-grafted layers on PP membrane surfaces was controlled by the low-pressure and atmospheric plasma treatment in this study. The effects of grafting composition, surface hydrophilicity, and hydration capability on blood compatibility of the membranes were determined. Protein adsorption onto the different PSBMA-grafted PP membranes from human fibrinogen solutions was measured by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies. Blood platelet adhesion and plasma clotting time measurements from a recalcified platelet-rich plasma solution were used to determine if platelet activation depends on the charge bias of the grafted PSBMA layer. The charge bias of PSBMA layer deviated from the electrical balance of positively and negatively charged moieties can be well-controlled via atmospheric plasma-induced interfacial zwitterionization and was further tested with human whole blood. The optimized PSBMA surface graft layer in overall charge neutrality has a high hydration capability and keeps its original blood-inert property of antifouling, anticoagulant, and antithrmbogenic activities when it comes into contact with human blood. This work suggests that the hemocompatible nature of grafted PSBMA polymers by controlling grafting quality via atmospheric plasma treatment gives a great potential in the surface zwitterionization of hydrophobic membranes for use in human whole blood.
Assuntos
Betaína/análogos & derivados , Materiais Biocompatíveis/química , Membranas Artificiais , Plasma/química , Polipropilenos/química , Adsorção , Anticoagulantes/química , Anticoagulantes/farmacologia , Betaína/química , Materiais Biocompatíveis/farmacologia , Proteínas Sanguíneas/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Metacrilatos/química , Adesividade Plaquetária/efeitos dos fármacos , Propriedades de SuperfícieRESUMO
Poly(ether ether ketone) (PEEK) has seen increasing use in biomedical fields as a replacement for metal implants. Accordingly, the surface functionalities of PEEK are important for the development of medical devices. We have focused on the application of photoinduced reactions in PEEK to immobilize a functional polymer via radical generation on the surface, which can react with hydrocarbon groups. In this study, we used zwitterionic copolymers comprising 2-methacryloyloxyethyl phosphorylcholine (MPC) units and n-butyl methacrylate (BMA) units with various molecular architectures for surface modification. A random copolymer (poly(MPC-co-BMA) (r-PMB)), an AB-type diblock copolymer (di-PMB), and an ABA-type triblock copolymer (tri-PMB) (A segment: poly(BMA); B segment: poly(MPC)) were synthesized with the same monomer compositions. All PMBs were successfully immobilized on the PEEK surface via UV irradiation after the dip-coating process, regardless of their molecular structure. In this reaction, the alkyl group of the BMA unit functioned as a photoreactive site on the PEEK surface. This indicates that the molecular structure differences affect the surface properties. For example, compared to r-PMB and tri-PMB, di-PMB-modified surfaces exhibited an extremely low water contact angle of approximately 10°. The findings of this study demonstrate that this surface functionalization method does not require a low-molecular-weight compound, such as an initiator, and can be applied to the surface of inert PEEK through a simple photoreaction under room temperature, atmospheric pressure, and dry state conditions.
Assuntos
Cetonas , Polímeros , Éter , Cetonas/química , Metacrilatos , Fosforilcolina/análogos & derivados , Fosforilcolina/química , Polímeros/químicaRESUMO
Natriuretic peptide receptor 1 (NPR1) is conventionally known as a regulator of vascular homeostasis. Here, we generated an Npr1 knockout mouse model with CRISPR/Cas9 technology and found that homozygous mice (Npr1-/-) exhibited weight loss and poor survival rate during early postnatal stage. Careful examination revealed unexpectedly that Npr1-/- mice developed colitis characterized by shortened colon, evident colonic mucosal damage, increased histopathological score, and higher colonic expression of proinflammatory cytokines interleukin-1B (IL1B) and -6 (IL6). RNA-sequencing analysis revealed that differentially expressed genes were prominently enriched in the biological pathways related to immune response in both spleen and colon of Npr1-/- mice. Cytofluorimetric analysis demonstrated that leukocytes in the spleen were significantly increased, particularly, the populations of neutrophil and CD3+ T cell were elevated but CD4+ T cells were decreased in Npr1-/- mice. Administration of 8-Br-cGMP, a downstream activator of NPR1, restored these immune-cell populations disturbed in Npr1-/- mice and lessened the colitis-related phenotypes. To validate the involvement of Npr1 in colitis, we examined another mouse model induced by dextran sodium sulfate (DSS) and found a decreased Npr1 expression and shifted immune-cell populations as well. Importantly, 8-Br-cGMP treatment exhibited a similar effect in the restoration of immune-cell populations and attenuation of colonic inflammation in DSS mice. Our data indicate that loss of Npr1 possibly interrupts immune response, which is critical to the pathogenesis of colitis in the early life.
Assuntos
Colite , Camundongos , Animais , Sulfato de Dextrana/toxicidade , Colite/patologia , Inflamação , Camundongos Knockout , Modelos Animais de Doenças , Imunidade , Camundongos Endogâmicos C57BLRESUMO
Cardiac aging is a critical determinant of cardiac dysfunction, which contributes to cardiovascular disease in the elderly. Proprotein convertase subtilisin/kexin 6 (PCSK6) is a proteolytic enzyme important for the maintenance of cardiac function and vascular homeostasis. To date, the involvement of PCSK6 in cardiac aging remains unknown. Here we report that PCSK6 expression decreased in the hearts of aged mice, where high levels cyclin dependent kinase inhibitor 2A (P16) and cyclin dependent kinase inhibitor 1A (P21) (senescence markers) were observed. Moreover, PCSK6 protein expression was significantly reduced in senescent rat embryonic cardiomyocytes (H9c2) induced by D-galactose. Pcsk6 knockdown in H9c2 cells increased P16 and P21 expression levels and senescence-associated beta-galactosidase activity. Pcsk6 knockdown also impaired cardiomyocyte function, as indicated by increased advanced glycation end products, reactive oxygen species level, and apoptosis. Overexpression of PCSK6 blunted the senescence phenotype and cellular dysfunction. Furthermore, RNA sequencing analysis in Pcsk6-knockdown H9c2 cells identified the up-regulated DNA-damage inducible transcript 3 (Ddit3) gene involved in endoplasmic reticulum (ER) protein processing. Additionally, DDIT3 protein levels were remarkably increased in aged mouse hearts. In the presence of tunicamycin, an ER stress inducer, DDIT3 expression increased in Pcsk6-deficient H9c2 cells but reduced in PCSK6-overexpressing cells. In conclusion, our findings indicate that PCSK6 modulates cardiomyocyte senescence possibly via DDIT3-mediated ER stress.
Assuntos
Estresse do Retículo Endoplasmático , Miócitos Cardíacos , Fator de Transcrição CHOP/metabolismo , Envelhecimento , Animais , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/genética , Camundongos , Miócitos Cardíacos/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Hypertension is common in elderly population. We designed to search comprehensively for genes that are chronologically shifted in their expressions and to define their contributions to vascular aging and hypertension. RNA sequencing was conducted to search for senescence-shifted transcripts in human umbilical vein endothelial cells (HUVECs). Small interfering RNA (siRNA), small-molecule drugs, CRISPR/Cas9 techniques, and imaging were used to determine genes' function and contributions to age-related phenotypes of the endothelial cell and blood vessel. Of 25 genes enriched in the term of "regulation of blood pressure," NPRA was changed most significantly. The decreased NPRA expression was replicated in aortas of aged mice. The knockdown of NPRA promoted HUVEC senescence and it decreased expressions of protein kinase cGMP-dependent 1 (PKG), sirtuin 1 (SIRT1), and endothelial nitric oxide synthase (eNOS). Suppression of NPRA also decreased the phosphorylation of AMP-activated protein kinase (AMPK) as well as the ratio of oxidized nicotinamide adenine dinucleotide (NAD+ )/reduced nicotinamide adenine dinucleotide (NADH) but increased the production of reactive oxygen species (ROS). 8-Br-cGMP (analog of cGMP), or AICAR (AMPK activator), counteracted the observed changes in HUVECs. The Npr1+/- mice presented an elevated systolic blood pressure and their vessels became insensitive to endothelial-dependent vasodilators. Further, vessels from Npr1+/- mice increased Cdkn1a but decreased eNos expressions. These phenotypes were rescued by intravenously administrated 8-Br-cGMP and viral overexpression of human PKG, respectively. In conclusion, we demonstrate NPRA/PKG/AMPK as a novel and critical signaling axis in the modulation of endothelial cell senescence, vascular aging, and hypertension.
Assuntos
Proteínas Quinases Ativadas por AMP , Hipertensão , Proteínas Quinases Ativadas por AMP/metabolismo , Idoso , Envelhecimento , Animais , Pressão Sanguínea , Células Cultivadas , GMP Cíclico/análogos & derivados , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Hipertensão/genética , Hipertensão/metabolismo , Camundongos , NAD/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Fosforilação , RNA Interferente Pequeno/metabolismo , TionucleotídeosRESUMO
Endothelial cell senescence has a vital implication for vascular dysfunction, leading to age-related cardiovascular disease, especially hypertension and atherosclerosis. E2F transcription factor 2 (E2F2) plays a critical role in cell proliferation, differentiation, and DNA damage response. Up to date, no study has ever connected E2F2 to vascular endothelial cell senescence. Here, we demonstrate that E2F2 is involved in endothelial cellular senescence. We found that E2F2 expression is decreased during the replicative senescence of human umbilical vein endothelial cells (HUVECs) and the aortas of aged mice. The knockdown of E2F2 in young HUVECs induces premature senescence characterized by an increase in senescence-associated ß-galactosidase (SA-ß-gal) activity, a reduction in phosphorylated endothelial nitric oxide synthase (p-eNOS) and sirtuin 1 (SIRT1), and the upregulation of senescence-associated secretory phenotype (SASP) IL-6 and IL-8. The lack of E2F2 promoted cell cycle arrest, DNA damage, and cell proliferation inhibition. Conversely, E2F2 overexpression reversed the senescence phenotype and enhanced the cellular function in the senescent cells. Furthermore, E2F2 deficiency downregulated downstream target genes including CNNA2, CDK1, and FOXM1, and overexpression restored the expression of these genes. Our findings demonstrate that E2F2 plays an indispensable role in endothelial cell senescence.
Assuntos
Senescência Celular , Fator de Transcrição E2F2 , Óxido Nítrico Sintase Tipo III , Sirtuína 1 , Animais , Células Cultivadas , Senescência Celular/genética , Fator de Transcrição E2F2/genética , Fator de Transcrição E2F2/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Interleucina-6 , Interleucina-8 , Camundongos , Óxido Nítrico Sintase Tipo III/metabolismo , Sirtuína 1/genética , beta-GalactosidaseRESUMO
Atrial and brain natriuretic peptides (ANP and BNP) regulate blood pressure and cardiac function. In patients with heart failure (HF), plasma levels of pro-ANP and pro-BNP, the precursor forms of ANP and BNP, are highly elevated, but the mechanism underlying the apparent deficiency in natriuretic peptide processing is unclear. Corin is a cardiac protease that activates natriuretic peptides. In this study, we examined corin protein expression and activity in mouse and human failing hearts. Tissue samples were obtained from a mouse model of HF induced by myotrophin overexpression and from human nonfailing, hypertrophic, and failing hearts. Corin protein levels in the membrane fraction and tissue lysate were measured by Western blotting and ELISA. Corin catalytic and biological activities were measured by fluorescent substrate and pro-ANP processing assays. In mice, corin protein levels did not change with age in normal hearts but increased significantly in failing hearts. In humans, corin protein levels were similar in the atrium from nonfailing and failing hearts but were increased in the ventricle in failing hearts compared with those in nonfailing or hypertrophic hearts. Unlike the protein level, however, corin activity did not increase in failing hearts, as measured by fluorogenic substrate and pro-ANP processing assays. Our results indicate that corin activation is a rate-limiting step in failing hearts. Insufficient corin activation is expected to prevent natriuretic peptide processing and may contribute to body fluid retention and impaired cardiac function in patients with HF.
Assuntos
Insuficiência Cardíaca/metabolismo , Serina Endopeptidases/metabolismo , Adulto , Animais , Fator Natriurético Atrial/metabolismo , Pressão Sanguínea/fisiologia , Modelos Animais de Doenças , Feminino , Insuficiência Cardíaca/fisiopatologia , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , RNA Mensageiro/metabolismoRESUMO
Biofouling has long been a problem for biomaterials, so being able to control the fouling on the surface of a biomaterial would be ideal. In this study a copolymer system was designed comprising three moieties: an epoxy containing group, glycidyl methacrylate (GMA); a thermoresponsive segment, N-isopropylacrylamide (NIPAAm); and an antifouling zwitterionic unit, sulfobetaine methacrylate (SBMA). The copolymers (pGSN), synthesized via free radical polymerization with these 3 moieties, were then grafted onto polydimethylsiloxane (PDMS). The presence of a critical temperature for both the copolymers and the coated PDMS was evidenced by particle size and contact angle measurements. The coated PDMS exhibited controllable temperature-dependent antifouling behaviors and stimuli-responsive phase characteristics in the presence of salts. The interactions of the coated PDMS with biomolecules were tested via attachment of fibrinogen protein, platelets, human whole blood, and tumor cells (HT1080). The attachment and detachment of these biomolecules were studied at different temperatures. Exposed hydrophobic domains of thermoresponsive NIPAAm-rich pGSN containing NIPAAm at 56 mol% generally allows molecular and cellular attachment on the PDMS surface at 37 °C. On the other hand, the coated PDMS with a relatively high content of SBMA (>41 mol%) in the copolymer started to exhibit fouling resistance and lower the thermoresponsive properties. Interestingly, the incorporation of zwitterionic SBMA units into the copolymers was found to accelerate the hydration of the PDMS surfaces and resulted in biomolecular and cellular detachment at 25 °C, which is comparable to the detachment at 4 °C. This modified surface behavior is found to be consistent through all biofouling tests.