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Diabetic retinopathy is the most common microvascular complication caused by chronic hyperglycemia and is a leading cause of blindness; however, the underlying molecular mechanism has not been clearly elucidated. Thus, we investigated whether regulation of AMPactivated protein kinase (AMPK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by transglutaminase 2 (TGase2) is important for hyperglycemia-induced microvascular leakage in the diabetic retina. In HRECs and diabetic mouse retinas, we found that TGase2, activated by sequential elevation of intracellular Ca2+ and reactive oxygen species (ROS) levels, played an essential role in hyperglycemia-induced vascular leakage. ROS generation and TGsae2 activation were involved in hyperglycemia-induced AMPK dephosphorylation, which resulted in VE-cadherin disassembly and increased fluorescein isothiocyanate-dextran extravasation. Furthermore, high glucose-induced TGase2 activation suppressed GAPDH activity, determined by an on-chip activity assay, through inhibition of AMPK, which induced VE-cadherin disassembly and endothelial permeability in HRECs. Overall, our findings suggest that inhibition of AMPK and GAPDH by TGase2 plays a pivotal role in hyperglycemia-induced microvascular leakage in the retinas of diabetic mice.
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Targeting the hydrophobic Phe43 pocket of HIV's envelope glycoprotein gp120 is a critical strategy for antiviral interventions due to its role in interacting with the host cell's CD4. Previous inhibitors, including small molecules and CD4 mimetic peptides based on scyllatoxin, have demonstrated significant binding and neutralization capabilities but were often chemically synthesized or contained non-canonical amino acids. Microbial expression using natural amino acids offers advantages such as cost-effectiveness, scalability, and efficient production of fusion proteins. In this study, we enhanced the previous scyllatoxin-based synthetic peptide by substituting natural amino acids and successfully expressed it in E. coli. The peptide was optimized by mutating the C-terminal amidated valine to valine and glutamine, and by reducing the disulfide bonds from three to two. Circular dichroism confirmed proper secondary structure formation, and fluorescence polarization analysis revealed specific, concentration-dependent binding to HIV gp120, supported by molecular dynamics simulations. These findings indicate the potential for scalable microbial production of effective antiviral peptides, with significant applications in pharmaceutical development for HIV treatment.
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Escherichia coli , Proteína gp120 do Envelope de HIV , Peptídeos , Ligação Proteica , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/genética , Escherichia coli/metabolismo , Escherichia coli/genética , Peptídeos/química , Peptídeos/metabolismo , Peptídeos/farmacologia , Simulação de Dinâmica Molecular , Humanos , Sequência de Aminoácidos , Desenho de FármacosRESUMO
Diabetes, a severe metabolic disease characterized by chronic hypoglycemia, poses debilitating and life-threatening risks of microvascular and macrovascular complications, including blindness, kidney failure, heart attacks, and limb amputation. Addressing these complications is paramount, urging the development of interventions targeting diabetes-associated vascular dysfunctions. To effectively combat diabetes, a comprehensive understanding of the pathological mechanisms underlying complications and identification of precise therapeutic targets are imperative. Transglutaminase 2 (TGase2) is a multifunctional enzyme implicated in the pathogenesis of diverse diseases such as neurodegenerative disorders, fibrosis, and inflammatory conditions. TGase2 has recently emerged as a key player in both the pathogenesis and therapeutic intervention of diabetic complications. This review highlights TGase2 as a therapeutic target for diabetic complications and explores TGase2 inhibition as a promising therapeutic approach in their treatment.
Assuntos
Proteínas de Ligação ao GTP , Proteína 2 Glutamina gama-Glutamiltransferase , Transglutaminases , Animais , Humanos , Diabetes Mellitus , Angiopatias Diabéticas , Proteínas de Ligação ao GTP/metabolismo , Transglutaminases/metabolismo , Transglutaminases/antagonistas & inibidoresRESUMO
Proinsulin C-peptide, a biologically active polypeptide released from pancreatic ß-cells, is known to prevent hyperglycemia-induced microvascular leakage; however, the role of C-peptide in migration and invasion of cancer cells is unknown. Here, we investigated high glucose-induced migration and invasion of ovarian cancer cells and the inhibitory effects of human C-peptide on metastatic cellular responses. In SKOV3 human ovarian cancer cells, high glucose conditions activated a vicious cycle of reactive oxygen species (ROS) generation and transglutaminase 2 (TGase2) activation through elevation of intracellular Ca2+ levels. TGase2 played a critical role in high glucose-induced ovarian cancer cell migration and invasion through ß-catenin disassembly. Human C-peptide inhibited high glucose-induced disassembly of adherens junctions and ovarian cancer cell migration and invasion through inhibition of ROS generation and TGase2 activation. The preventive effect of C-peptide on high glucose-induced ovarian cancer cell migration and invasion was further demonstrated in ID8 murine ovarian cancer cells. Our findings suggest that high glucose conditions induce the migration and invasion of ovarian cancer cells, and human C-peptide inhibits these metastatic responses by preventing ROS generation, TGase2 activation, and subsequent disassembly of adherens junctions.
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Neoplasias Ovarianas , Humanos , Animais , Camundongos , Feminino , Peptídeo C/farmacologia , Espécies Reativas de Oxigênio/farmacologia , Neoplasias Ovarianas/patologia , Movimento Celular , Glucose/farmacologiaRESUMO
Rationale: Neovascularization is a hallmark of the late stages of diabetic retinopathy (DR) leading to blindness. The current anti-DR drugs have clinical disadvantages including short circulation half-lives and the need for frequent intraocular administration. New therapies with long-lasting drug release and minimal side effects are therefore needed. We explored a novel function and mechanism of a proinsulin C-peptide molecule with ultra-long-lasting delivery characteristics for the prevention of retinal neovascularization in proliferative diabetic retinopathy (PDR). Methods: We developed a strategy for ultra-long intraocular delivery of human C-peptide using an intravitreal depot of K9-C-peptide, a human C-peptide conjugated to a thermosensitive biopolymer, and investigated its inhibitory effect on hyperglycemia-induced retinal neovascularization using human retinal endothelial cells (HRECs) and PDR mice. Results: In HRECs, high glucose conditions induced oxidative stress and microvascular permeability, and K9-C-peptide suppressed those effects similarly to unconjugated human C-peptide. A single intravitreal injection of K9-C-peptide in mice resulted in the slow release of human C-peptide that maintained physiological levels of C-peptide in the intraocular space for at least 56 days without inducing retinal cytotoxicity. In PDR mice, intraocular K9-C-peptide attenuated diabetic retinal neovascularization by normalizing hyperglycemia-induced oxidative stress, vascular leakage, and inflammation and restoring blood-retinal barrier function and the balance between pro- and anti-angiogenic factors. Conclusions: K9-C-peptide provides ultra-long-lasting intraocular delivery of human C-peptide as an anti-angiogenic agent to attenuate retinal neovascularization in PDR.
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Diabetes Mellitus , Retinopatia Diabética , Hiperglicemia , Neovascularização Retiniana , Humanos , Camundongos , Animais , Neovascularização Retiniana/tratamento farmacológico , Retinopatia Diabética/tratamento farmacológico , Peptídeo C/farmacologia , Peptídeo C/uso terapêutico , Células Endoteliais , Neovascularização Patológica/tratamento farmacológico , Hiperglicemia/tratamento farmacológicoRESUMO
BACKGROUND: Hyperglycemic memory (HGM) is a pivotal phenomenon in the development of diabetic complications. Although coincident diabetic complications are reported, research on their development and treatment is limited. Thus, we investigated whether C-peptide can simultaneously inhibit HGM-induced retinal, pulmonary, and glomerular dysfunctions in diabetic mice supplemented with insulin. METHODS: Insulin-treated diabetic mice were supplemented with human C-peptide by subcutaneous implantation of K9-C-peptide depots for 4 weeks, and reactive oxygen species (ROS) generation, transglutaminase (TGase) activity, and vascular leakage were examined in the retina, lung, and kidney. RESULTS: We found hyperglycemia-induced persistent ROS generation and TGase activation after blood glucose normalization in the retina, lung, and kidney of insulin-supplemented diabetic mice. These pathological events were inhibited by systemic supplementation of human C-peptide via subcutaneous implantation of a thermosensitive biopolymer-conjugated C-peptide depot. ROS generation and TGase activation were in a vicious cycle after glucose normalization, and C-peptide suppressed the vicious cycle and subsequent endothelial permeability in human retinal endothelial cells. Moreover, C-peptide supplementation ameliorated HGM-induced retinal vascular leakage and neurodegeneration, pulmonary vascular leakage and fibrosis, and glomerular adherens junction disruption and vascular leakage. CONCLUSIONS: Overall, our findings demonstrate that C-peptide supplementation simultaneously attenuates vascular and neuronal dysfunctions in the retina, lung, and glomerulus of insulin-supplemented diabetic mice.
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Diabetes Mellitus Experimental , Retinopatia Diabética , Humanos , Camundongos , Animais , Peptídeo C , Espécies Reativas de Oxigênio , Células Endoteliais , Diabetes Mellitus Experimental/complicações , Retina , Transglutaminases/fisiologia , Insulina/farmacologia , Pulmão , Retinopatia Diabética/complicaçõesRESUMO
Diabetic retinopathy (DR) is caused by retinal vascular dysfunction and neurodegeneration. Intraocular delivery of C-peptide has been shown to be beneficial against hyperglycemia-induced microvascular leakage in the retina of diabetes; however, the effect of C-peptide on diabetes-induced retinal neurodegeneration remains unknown. Moreover, extraocular C-peptide replacement therapy against DR to avoid various adverse effects caused by intravitreal injections has not been studied. Here, we demonstrate that systemic C-peptide supplementation using osmotic pumps or biopolymer-conjugated C-peptide hydrogels ameliorates neurodegeneration by inhibiting vascular endothelial growth factor-induced pathological events, but not hyperglycemia-induced vascular endothelial growth factor expression, in the retinas of diabetic mice. C-peptide inhibited hyperglycemia-induced activation of macroglial and microglial cells, downregulation of glutamate aspartate transporter 1 expression, neuronal apoptosis, and histopathological changes by a mechanism involving reactive oxygen species generation in the retinas of diabetic mice, but transglutaminase 2, which is involved in retinal vascular leakage, is not associated with these pathological events. Overall, our findings suggest that systemic C-peptide supplementation alleviates hyperglycemia-induced retinal neurodegeneration by inhibiting a pathological mechanism, involving reactive oxygen species, but not transglutaminase 2, in diabetes.
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Diabetes Mellitus Experimental , Retinopatia Diabética , Hiperglicemia , Animais , Camundongos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Peptídeo C/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Retina/metabolismo , Fatores de Crescimento do Endotélio Vascular , Retinopatia Diabética/metabolismo , Hiperglicemia/metabolismo , Suplementos NutricionaisRESUMO
Dopamine is a neurotransmitter that mediates visual function in the retina and diabetic retinopathy (DR) is the most common microvascular complication of diabetes and the leading cause of blindness; however, the role of dopamine in retinal vascular dysfunction in DR remains unclear. Here, we report a mechanism of hyperglycemic memory (HGM)-induced retinal microvascular dysfunction and the protective effect of dopamine against the HGM-induced retinal microvascular leakage and abnormalities. We found that HGM induced persistent oxidative stress, mitochondrial membrane potential collapse and fission, and adherens junction disassembly and subsequent vascular leakage after blood glucose normalization in the mouse retinas. These persistent hyperglycemic stresses were inhibited by dopamine treatment in human retinal endothelial cells and by intravitreal injection of levodopa in the retinas of HGM mice. Moreover, levodopa supplementation ameliorated HGM-induced pericyte degeneration, acellular capillary and pericyte ghost generation, and endothelial apoptosis in the mouse retinas. Our findings suggest that dopamine alleviates HGM-induced retinal microvascular leakage and abnormalities by inhibiting persistent oxidative stress and mitochondrial dysfunction.
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Diabetes Mellitus , Retinopatia Diabética , Camundongos , Animais , Humanos , Retinopatia Diabética/tratamento farmacológico , Dopamina , Vasos Retinianos , Células Endoteliais , Levodopa/farmacologia , RetinaRESUMO
To investigate the adverse effects of clozapine on cardiovascular ion channels, we examined the inhibitory effect of clozapine on voltage-dependent K+ (Kv) channels in rabbit coronary arterial smooth muscle cells. Clozapine-induced inhibition of Kv channels occurred in a concentration-dependent manner with an half-inhibitory concentration value of 7.84 ± 4.86 µM and a Hill coefficient of 0.47 ± 0.06. Clozapine did not shift the steady-state activation or inactivation curves, suggesting that it inhibited Kv channels regardless of gating properties. Application of train pulses (1 and 2 Hz) progressively augmented the clozapine-induced inhibition of Kv channels in the presence of the drug. Furthermore, the recovery time constant from inactivation was increased in the presence of clozapine, suggesting that clozapine-induced inhibition of Kv channels is use (state)-dependent. Pretreatment of a Kv1.5 subtype inhibitor decreased the Kv current amplitudes, but additional application of clozapine did not further inhibit the Kv current. Pretreatment with Kv2.1 or Kv7 subtype inhibitors partially blocked the inhibitory effect of clozapine. Based on these results, we conclude that clozapine inhibits arterial Kv channels in a concentrationand use (state)-dependent manner. Kv1.5 is the major subtype involved in clozapine-induced inhibition of Kv channels, and Kv2.1 and Kv7 subtypes are partially involved.
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Proinsulin C-peptide has a protective effect against diabetic complications; however, its role in hyperglycemia-induced pulmonary fibrosis is unknown. In this study, we investigated the inhibitory effect of C-peptide on hyperglycemia-induced pulmonary fibrosis and the molecular mechanism of C-peptide action in the lungs of diabetic mice and in human pulmonary microvascular endothelial cells (HPMVECs). We found that, in the lungs of diabetic mice, C-peptide supplementation using osmotic pumps attenuated hyperglycemia-induced pulmonary fibrosis and expression of fibrosis-related proteins. In HPMVECs, C-peptide inhibited vascular endothelial growth factor-induced adherens junction disruption and endothelial cell permeability by inhibiting reactive oxygen species generation and transglutaminase (TGase) activation. In the lungs, C-peptide supplementation suppressed hyperglycemia-induced reactive oxygen species generation, TGase activation, and microvascular leakage. C-peptide inhibited hyperglycemia-induced inflammation and apoptosis, which are involved in the pathological process of pulmonary fibrosis. We also demonstrated the role of TGase2 in hyperglycemia-induced vascular leakage, inflammation, apoptosis, and pulmonary fibrosis in the lungs of diabetic TGase2-null (Tgm2-/-) mice. Furthermore, we demonstrated a long-term inhibitory effect of systemic delivery of C-peptide using K9-C-peptide hydrogels on hyperglycemia-induced fibrosis in diabetic lungs. Overall, our findings suggest that C-peptide alleviates hyperglycemia-induced pulmonary fibrosis by inhibiting TGase2-mediated microvascular leakage, inflammation, and apoptosis in diabetes.
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Diabetes Mellitus Experimental , Hiperglicemia , Fibrose Pulmonar , Animais , Peptídeo C/farmacologia , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Células Endoteliais/metabolismo , Hiperglicemia/complicações , Hiperglicemia/tratamento farmacológico , Hiperglicemia/metabolismo , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteína 2 Glutamina gama-Glutamiltransferase , Fibrose Pulmonar/complicações , Fibrose Pulmonar/etiologia , Espécies Reativas de Oxigênio/metabolismo , Transglutaminases/genética , Transglutaminases/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: Imaging techniques are commonly used to understand disease mechanisms and their biological features in the microenvironment of the cell. Many studies have added to our understanding of the biology of the malaria parasite Plasmodium knowlesi from functional in vitro and imaging analysis using serial block-face scanning electron microscopy (SEM). However, sample fixation and metal coating during SEM analysis can alter the parasite membrane. METHODS: In this study, we used noninvasive diffraction optical tomography (DOT), also known as holotomography, to explore the morphological, biochemical, and mechanical alterations of each stage of P. knowlesi-infected red blood cells (RBCs). Each stage of the parasite was synchronized using Nycodenz and magnetic-activated cell sorting (MACS) for P. knowlesi and P. falciparum, respectively. Holotomography was applied to measure individual three-dimensional refractive index tomograms without metal coating, fixation, or additional dye agent. RESULTS: Distinct profiles were found on the surface area and hemoglobin content of the two parasites. The surface area of P. knowlesi-infected RBCs showed significant expansion, while P. falciparum-infected RBCs did not show any changes compared to uninfected RBCs. In terms of hemoglobin consumption, P. falciparum tended to consume hemoglobin more than P. knowlesi. The observed profile of P. knowlesi-infected RBCs generally showed similar results to other studies, proving that this technique is unbiased. CONCLUSIONS: The observed profile of the surface area and hemoglobin content of malaria infected-RBCs can potentially be used as a diagnostic parameter to distinguish P. knowlesi and P. falciparum infection. In addition, we showed that holotomography could be used to study each Plasmodium species in greater depth, supporting strategies for the development of diagnostic and treatment strategies for malaria.
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Malária Falciparum , Malária , Plasmodium knowlesi , Eritrócitos/parasitologia , Humanos , Malária/parasitologia , Malária Falciparum/parasitologia , Imagem ÓpticaRESUMO
Midazolam is an anesthetic widely used for anxiolysis and sedation; however, to date, a possible role for midazolam in diabetic kidney disease remains unknown. Here, we investigated the effect of midazolam on hyperglycemia-induced glomerular endothelial dysfunction and elucidated its mechanism of action in kidneys of diabetic mice and human glomerular microvascular endothelial cells (HGECs). We found that, in diabetic mice, subcutaneous midazolam treatment for 6 weeks attenuated hyperglycemia-induced elevation in urine albumin/creatinine ratios. It also ameliorated hyperglycemia-induced adherens junction disruption and subsequent microvascular leakage in glomeruli of diabetic mice. In HGECs, midazolam suppressed high glucose-induced vascular endothelial-cadherin disruption and endothelial cell permeability via inhibition of intracellular Ca2+ elevation and subsequent generation of reactive oxygen species (ROS) and transglutaminase 2 (TGase2) activation. Notably, midazolam also suppressed hyperglycemia-induced ROS generation and TGase2 activation in glomeruli of diabetic mice and markedly improved pathological alterations in glomerular ultrastructure in these animals. Analysis of kidneys from diabetic Tgm2-/- mice further revealed that TGase2 played a critical role in microvascular leakage. Overall, our findings indicate that midazolam ameliorates hyperglycemia-induced glomerular endothelial dysfunction by inhibiting ROS-mediated activation of TGase2.
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Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/metabolismo , Células Endoteliais/metabolismo , Hiperglicemia/complicações , Glomérulos Renais/metabolismo , Midazolam/farmacologia , Proteína 2 Glutamina gama-Glutamiltransferase/antagonistas & inibidores , Animais , Biomarcadores , Cálcio/metabolismo , Permeabilidade Capilar/efeitos dos fármacos , Diabetes Mellitus Experimental , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/patologia , Gerenciamento Clínico , Modelos Animais de Doenças , Suscetibilidade a Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Glomérulos Renais/patologia , Glomérulos Renais/ultraestrutura , Masculino , Camundongos , Camundongos Knockout , Modelos Biológicos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Low-dose metronomic chemotherapy (LDMC) inhibits tumor angiogenesis and growth by targeting tumor-associated endothelial cells, but the molecular mechanism has not been fully elucidated. Here, we examined the functional role of regulated in development and DNA damage responses 1 (REDD1), an inhibitor of mammalian target of rapamycin complex 1 (mTORC1), in LDMC-mediated endothelial cell dysfunction. Low-dose doxorubicin (DOX) treatment induced REDD1 expression in cultured vascular and lymphatic endothelial cells and subsequently repressed the mRNA expression of mTORC1-dependent translation of vascular endothelial growth factor receptor (Vegfr)-2/3, resulting in the inhibition of VEGF-mediated angiogenesis and lymphangiogenesis. These regulatory effects of DOX-induced REDD1 expression were additionally confirmed by loss- and gain-of-function studies. Furthermore, LDMC with DOX significantly suppressed tumor angiogenesis, lymphangiogenesis, vascular permeability, growth, and metastasis in B16 melanoma-bearing wild-type but not Redd1-deficient mice. Altogether, our findings indicate that REDD1 is a crucial determinant of LDMC-mediated functional dysregulation of tumor vascular and lymphatic endothelial cells by translational repression of Vegfr-2/3 transcripts, supporting the potential therapeutic properties of REDD1 in highly progressive or metastatic tumors.
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Melanoma Experimental , Receptor 2 de Fatores de Crescimento do Endotélio Vascular , Animais , Regulação para Baixo , Doxorrubicina/farmacologia , Células Endoteliais/metabolismo , Linfangiogênese/fisiologia , Mamíferos/metabolismo , Melanoma Experimental/tratamento farmacológico , Camundongos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
BACKGROUND: Korean Red ginseng extract (KRGE) has beneficial effects on the cardiovascular system by improving endothelial cell function. However, its pharmacological effect on endothelial cell senescence has not been clearly elucidated. Therefore, we examined the effect and molecular mechanism of KRGE on the senescence of human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were grown in normal or KRGE-supplemented medium. Furthermore, they were transfected with heme oxygenase-1 (HO-1) gene or treated with its inhibitor, a NF-κB inhibitor, and a miR-155-5p mimic or inhibitor. Senescence-associated characteristics of endothelial cells were determined by biochemical and immunohistochemical analyses. RESULTS: Treatment of HUVECs with KRGE resulted in delayed onset and progression of senescence-associated characteristics, such as increased lysosomal acidic ß-galactosidase and decreased telomerase activity, angiogenic dysfunction, and abnormal cell morphology. KRGE preserved the levels of anti-senescent factors, such as eNOS-derived NO, MnSOD, and cyclins D and A: however, it decreased the levels of senescence-promoting factors, such as ROS, activated NF-κB, endothelial cell inflammation, and p21 expression. The beneficial effects of KRGE were due to the induction of HO-1 and the inhibition of NF-κB-dependent biogenesis of miR-155-5p that led to the downregulation of eNOS. Moreover, treatment with inhibitors of HO-1, NF-κB, and miR-155-5p abolished the anti-senescence effects of KRGE. CONCLUSION: KRGE delayed or prevented HUVEC senescence through a signaling cascade involving the induction of HO-1, the inhibition of NF-κB-dependent miR-155-5p biogenesis, and the maintenance of the eNOS/NO axis activity, suggesting that it may protect against vascular diseases associated with endothelial senescence.
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Targeting the vascular endothelial growth factor (VEGF)/its receptor-2 (VEGFR-2) system has become a mainstay of treatment for many human diseases, including retinal diseases. We examined the therapeutic effect of recently developed N-acetylated Arg-Leu-Tyr-Glu (Ac-RLYE), a human plasminogen kringle-5 domain-derived VEGFR-2 antagonists, on the pathogenesis of diabetic retinopathy. Ac-RLYE inhibited VEGF-A-mediated VEGFR-2 activation and endothelial nitric oxide synthase (eNOS)-derived NO production in the retinas of diabetic mice. In addition, Ac-RLYE prevented the disruption of adherens and tight junctions and vascular leakage by inhibiting S-nitrosylation of ß-catenin and tyrosine nitration of p190RhoGAP in the retinal vasculature of diabetic mice. Peptide treatment preserved the pericyte coverage of retinal capillaries by upregulating angiopoietin-2. These results suggest that Ac-RLYE potentially prevents blood-retinal barrier breakdown and vascular leakage by antagonizing VEGFR-2; Ac-RLYE can be used as a potential therapeutic drug for the treatment of diabetic retinopathy.
Assuntos
Inibidores da Angiogênese/farmacologia , Barreira Hematorretiniana/efeitos dos fármacos , Retinopatia Diabética/tratamento farmacológico , Oligopeptídeos/farmacologia , Vasos Retinianos/efeitos dos fármacos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Junções Aderentes/efeitos dos fármacos , Junções Aderentes/metabolismo , Junções Aderentes/patologia , Animais , Barreira Hematorretiniana/metabolismo , Barreira Hematorretiniana/patologia , Permeabilidade Capilar/efeitos dos fármacos , Células Cultivadas , Diabetes Mellitus Experimental/complicações , Retinopatia Diabética/etiologia , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Humanos , Masculino , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Vasos Retinianos/metabolismo , Vasos Retinianos/patologia , Transdução de Sinais , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Junções Íntimas/patologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Clonorchiasis caused by Clonorchis sinensis is endemic in East Asia; approximately 15 million people have been infected thus far. To diagnose the infection, serodiagnostic tests with excellent functionality should be performed. First, 607 expressed sequence tags encoding polypeptides with a secretory signal were expressed into recombinant proteins using an in vitro translation system. By protein array-based screening using C. sinensis-infected sera, 18 antigen candidate proteins were selected and assayed for cross-reactivity against Opisthorchis viverrini-infected sera. Of the six antigenic proteins selected, four were synthesized on large scale in vitro and evaluated for antigenicity against the flukes-infected human sera using ELISA. CsAg17 antigen showed the highest sensitivity (77.1%) and specificity (71.2%). The sensitivity and specificity of the bacterially produced CsAg17-28GST fusion antigen was similar to those of CsAg17 antigen. CsAg17 antigen can be used to develop point-of-care serodiagnostic tests for clonorchiasis.
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Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos/imunologia , Clonorquíase/diagnóstico , Clonorchis sinensis/imunologia , Animais , Clonorchis sinensis/genética , Reações Cruzadas/imunologia , Ensaio de Imunoadsorção Enzimática , Peixes/parasitologia , Humanos , Imunoglobulina G/sangue , Opisthorchis/imunologia , Testes Imediatos , Proteogenômica , Alimentos Crus/parasitologia , Sensibilidade e Especificidade , Testes SorológicosRESUMO
Due to their short half-lives, repeated administration of anti-hyperglycemic drugs can cause pain, discomfort, tissue damage, and infection in diabetic patients. Therefore, there is a need to develop long-term drug delivery systems to treat diabetes and its complications. C-peptide can prevent diabetic complications, including diabetic vasculopathy, but its clinical application is limited by its short half-life. Here, we developed K9-C-peptide (human C-peptide conjugated to an elastin-like biopolymer) and investigated its long-term influence on hyperglycemia-induced vascular dysfunction using an aortic endothelium model in diabetic mice. Using pharmacokinetics and in vivo imaging, we found that subcutaneously injected K9-C-peptide formed a hydrogel depot that slowly released human C-peptide into the blood circulation for 19 days. Administration of K9-C-peptide, human C-peptide, or K8 polypeptide had no effect on body weight or blood glucose levels. The slow release of C-peptide from K9-C-peptide hydrogels provided prolonged prevention of oxidative stress, inflammatory responses, and endothelial apoptosis in a hyperglycemia-induced vascular dysfunction model using the diabetic mouse aorta. Subcutaneous administration of unbound human C-peptide and K8 polypeptide were used as negative controls and had no effects. These results suggest that K9-C-peptide is suitable for the long-term delivery of human C-peptide for treating vascular dysfunction in diabetic patients.
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Diabetes Mellitus Experimental , Elastina , Animais , Aorta , Biopolímeros , Peptídeo C , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/tratamento farmacológico , Endotélio Vascular , Humanos , Hidrogéis/farmacologia , CamundongosRESUMO
Preeclampsia (PE) is a pregnancy-specific disorder associated with hypertension and proteinuria. Since there is no proven method to treat PE, early prediction and accurate diagnosis are essential for appropriate management of the disease. Thus, reliable biomarkers for diagnosing PE need to be identified and evaluated. We analyzed serum-soluble factors and miRNAs in 92 patients with PE and an equal number of healthy controls to identify new useful biomarkers for PE. Serum miR-31-5p, miR-155-5p, and miR-214-3p levels were significantly elevated in these patients and highly correlated with clinical symptoms of hypertension and proteinuria, whereas the miR-1290-3p level was decreased. The increased miRNAs were upregulated in an NF-κB-dependent manner and suppressed endothelial nitric oxide synthase (eNOS) and placental growth factor (PlGF) expression. The level of each miRNA had greater than 90% diagnostic accuracy, which was further increased by analyzing its ratio relative to that of miR-1290-3p. Taken together, the ratios of miR-31-5p, miR-155-5p, or miR-214-3p to miR-1290-3p may serve as reliable diagnostic or prognostic tools for PE, thereby providing evidence that these miRNAs are promising mechanism-based targets for designing therapeutic and preventive strategies for the clinical management of PE.
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Biomarcadores/metabolismo , MicroRNAs/genética , Pré-Eclâmpsia/sangue , Trofoblastos/metabolismo , Adulto , Feminino , Humanos , Masculino , GravidezRESUMO
Endothelial progenitor cell (EPC) dysfunction impairs vascular function and remodeling in inflammation-associated diseases, including preeclampsia. However, the underlying mechanism of this inflammation-induced dysfunction remains unclear. In the present study, we found increases in TNF-α and miR-31/155 levels and reduced numbers of circulating EPCs in patients with preeclampsia. Patient-derived mononuclear cells (MNCs) cultured in autologous serum had decreased endothelial nitric oxide synthase (eNOS) expression, nitric oxide production, and differentiation into EPCs with angiogenic potential, and these effects were inhibited by a TNF-α-neutralizing antibody and miR-31/155 inhibitors. Moreover, TNF-α treatment of normal MNCs increased miR-31/155 biogenesis, decreased eNOS expression, reduced EPC differentiation, and impaired angiogenic potential. The TNF-α-induced impairment of EPC differentiation and function was rescued by NF-κB p65 knockdown or miR-31/155 inhibitors. In addition, treatment of MNCs with synthetic miR-31/155 or an eNOS inhibitor mimicked the inhibitory effects of TNF-α on eNOS expression and EPC functions. Moreover, transplantation of EPCs that had been differentiated from TNF-α-treated MNCs decreased neovascularization and blood perfusion in ischemic mouse hindlimbs compared with those of normally differentiated EPCs. These findings suggest that NF-κB activation is required for TNF-α-induced impairment of EPC mobilization, differentiation, and function via miR-31/155 biogenesis and eNOS downregulation. Our data provide a new role for NF-κB-dependent miR-31/155 in EPC dysfunction under the pathogenic conditions of inflammation-associated vascular diseases, including preeclampsia.
Assuntos
Células Progenitoras Endoteliais/metabolismo , MicroRNAs/metabolismo , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Diferenciação Celular/genética , Regulação para Baixo/genética , Células Progenitoras Endoteliais/patologia , Feminino , Membro Posterior/irrigação sanguínea , Humanos , Isquemia/patologia , Masculino , Camundongos Nus , MicroRNAs/sangue , MicroRNAs/genética , Neovascularização Fisiológica/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Pré-Eclâmpsia/sangue , Pré-Eclâmpsia/genética , Gravidez , Fator de Necrose Tumoral alfa/sangueRESUMO
We investigated the effect of ziprasidone, a widely used treatment for schizophrenia, on voltage-dependent K+ (Kv) channels of coronary arterial smooth muscle cells using the patch-clamp technique. Ziprasidone dose-dependently inhibited Kv channels with an IC50 value of 0.39 ± 0.06 µM and a Hill coefficient of 0.62 ± 0.03. Although ziprasidone had no effect on the steady-state inactivation kinetics of the Kv channels, the steady-state activation curve shifted towards a more positive potential. These results suggest that ziprasidone inhibits Kv channels by targeting their voltage sensors. The recovery time constant of Kv channel inactivation was increased in the presence of ziprasidone. Furthermore, application of train steps (of 1 and 2 Hz) in the presence of ziprasidone led to a progressive increase in the blockade of Kv currents, suggesting that ziprasidone-induced inhibition of Kv channels is use (state)-dependent. Pretreatment with Kv1.5, Kv2.1, and Kv7 subtype inhibitors partially suppressed the ziprasidone-induced inhibition of Kv currents. These results suggest that ziprasidone inhibits vascular Kv channels through its effect on gating properties. The Kv channel-inhibiting action of ziprasidone is concentration- and use (state)-depedent.