RESUMO
Chronic kidney disease (CKD) progression is associated with persisting oxidative stress, which impairs the NO-sGC-cGMP signaling cascade through the formation of oxidized and heme-free apo-sGC that cannot be activated by NO. Runcaciguat (BAY 1101042) is a novel, potent, and selective sGC activator that binds and activates oxidized and heme-free sGC and thereby restores NO-sGC-cGMP signaling under oxidative stress. Therefore, runcaciguat might represent a very effective treatment option for CKD/DKD. The potential kidney-protective effects of runcaciguat were investigated in ZSF1 rats as a model of CKD/DKD, characterized by hypertension, hyperglycemia, obesity, and insulin resistance. ZSF1 rats were treated daily orally for up to 12 weeks with runcaciguat (1, 3, 10 mg/kg/bid) or placebo. The study endpoints were proteinuria, kidney histopathology, plasma, urinary biomarkers of kidney damage, and gene expression profiling to gain information about relevant pathways affected by runcaciguat. Furthermore, oxidative stress was compared in the ZSF1 rat kidney with kidney samples from DKD patients. Within the duration of the 12-week treatment study, kidney function was significantly decreased in obese ZSF1 rats, indicated by a 20-fold increase in proteinuria, compared to lean ZSF1 rats. Runcaciguat dose-dependently and significantly attenuated the development of proteinuria in ZSF1 rats with reduced uPCR at the end of the study by -19%, -54%, and -70% at 1, 3, and 10 mg/kg/bid, respectively, compared to placebo treatment. Additionally, average blood glucose levels measured as HbA1C, triglycerides, and cholesterol were increased by five times, twenty times, and four times, respectively, in obese ZSF1 compared to lean rats. In obese ZSF1 rats, runcaciguat reduced HbA1c levels by -8%, -34%, and -76%, triglycerides by -42%, -55%, and -71%, and cholesterol by -16%, -17%, and -34%, at 1, 3, and 10 mg/kg/bid, respectively, compared to placebo. Concomitantly, runcaciguat also reduced kidney weights, morphological kidney damage, and urinary and plasma biomarkers of kidney damage. Beneficial effects were accompanied by changes in gene expression that indicate reduced fibrosis and inflammation and suggest improved endothelial stabilization. In summary, the sGC activator runcaciguat significantly prevented a decline in kidney function in a DKD rat model that mimics common comorbidities and conditions of oxidative stress of CKD patients. Thus, runcaciguat represents a promising treatment option for CKD patients, which is in line with recent phase 2 clinical study data, where runcaciguat showed promising efficacy in CKD patients (NCT04507061).
Assuntos
Rim , Insuficiência Renal Crônica , Animais , Ratos , GMP Cíclico , Hemoglobinas Glicadas , Heme , Obesidade , Proteinúria , Insuficiência Renal Crônica/tratamento farmacológico , Ensaios Clínicos Fase II como AssuntoRESUMO
Bone morphogenetic protein-9 (BMP-9) is a circulating cytokine that is known to play an essential role in the endothelial homeostasis and the binding of BMP-9 to the receptor activin-like kinase 1 (ALK-1) promotes endothelial cell quiescence. Previously, using an unbiased screen, we identified ALK-1 as a high-capacity receptor for low-density lipoprotein (LDL) in endothelial cells that mediates its transcytosis in a nondegradative manner. Here we examine the crosstalk between BMP-9 and LDL and how it influences their interactions with ALK-1. Treatment of endothelial cells with BMP-9 triggers the extensive endocytosis of ALK-1, and it is mediated by caveolin-1 (CAV-1) and dynamin-2 (DNM2) but not clathrin heavy chain. Knockdown of CAV-1 reduces BMP-9-mediated internalization of ALK-1, BMP-9-dependent signaling and gene expression. Similarly, treatment of endothelial cells with LDL reduces BMP-9-induced SMAD1/5 phosphorylation and gene expression and silencing of CAV-1 and DNM2 diminishes LDL-mediated ALK-1 internalization. Interestingly, BMP-9-mediated ALK-1 internalization strongly re-duces LDL transcytosis to levels seen with ALK-1 deficiency. Thus, BMP-9 levels can control cell surface levels of ALK-1, via CAV-1, to regulate both BMP-9 signaling and LDL transcytosis.
Assuntos
Receptores de Activinas Tipo II/metabolismo , Caveolina 1/metabolismo , Membrana Celular/metabolismo , Endocitose , Endotélio Vascular/fisiologia , Fator 2 de Diferenciação de Crescimento/metabolismo , Lipoproteínas LDL/metabolismo , Receptores de Activinas Tipo II/genética , Caveolina 1/genética , Células Cultivadas , Endotélio Vascular/citologia , Fator 2 de Diferenciação de Crescimento/genética , Humanos , Fosforilação , Transdução de SinaisRESUMO
BACKGROUND: Atherosclerosis is driven by synergistic interactions between pathological, biomechanical, inflammatory, and lipid metabolic factors. Our previous studies demonstrated that absence of caveolin-1 (Cav1)/caveolae in hyperlipidemic mice strongly inhibits atherosclerosis, which was attributed to activation of endothelial nitric oxide (NO) synthase (eNOS) and increased production of NO and reduced inflammation and low-density lipoprotein trafficking. However, the contribution of eNOS activation and NO production in the athero-protection of Cav1 and the exact mechanisms by which Cav1/caveolae control the pathogenesis of diet-induced atherosclerosis are still not clear. METHODS: Triple-knockout mouse lacking expression of eNOS, Cav1, and Ldlr were generated to explore the role of NO production in Cav1-dependent athero-protective function. The effects of Cav1 on lipid trafficking, extracellular matrix remodeling, and vascular inflammation were studied both in vitro and in vivo with a mouse model of diet-induced atherosclerosis. The expression of Cav1 and distribution of caveolae regulated by flow were analyzed by immunofluorescence staining and transmission electron microscopy. RESULTS: We found that absence of Cav1 significantly suppressed atherogenesis in Ldlr-/-eNOS-/- mice, demonstrating that athero-suppression is independent of increased NO production. Instead, we find that the absence of Cav1/caveolae inhibited low-density lipoprotein transport across the endothelium and proatherogenic fibronectin deposition and disturbed flow-mediated endothelial cell inflammation. Consistent with the idea that Cav1/caveolae may play a role in early flow-dependent inflammatory priming, distinct patterns of Cav1 expression and caveolae distribution were observed in athero-prone and athero-resistant areas of the aortic arch even in wild-type mice. CONCLUSIONS: These findings support a role for Cav1/caveolae as a central regulator of atherosclerosis that links biomechanical, metabolic, and inflammatory pathways independently of endothelial eNOS activation and NO production.
Assuntos
Aterosclerose/metabolismo , Caveolina 1/fisiologia , Endotélio Vascular/metabolismo , Lipoproteínas LDL/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Transcitose/fisiologia , Animais , Aterosclerose/patologia , Aterosclerose/prevenção & controle , Células Cultivadas , Cães , Endotélio Vascular/patologia , Ativação Enzimática/fisiologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos TransgênicosRESUMO
Endothelial cells lining the vessel wall control important aspects of vascular homeostasis. In particular, the production of endothelium-derived nitric oxide and activation of soluble guanylate cyclase promotes endothelial quiescence and governs vasomotor function and proportional remodeling of blood vessels. Here, we discuss novel approaches to improve endothelial nitric oxide generation and preserve its bioavailability. We also discuss therapeutic opportunities aimed at activation of soluble guanylate cyclase for multiple cardiovascular indications.
Assuntos
Doenças Cardiovasculares/metabolismo , GMP Cíclico/metabolismo , Endotélio Vascular/efeitos dos fármacos , Guanilato Ciclase/antagonistas & inibidores , Óxido Nítrico/metabolismo , Animais , Endotélio Vascular/metabolismo , Humanos , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Inibidores de Fosfodiesterase/farmacologiaRESUMO
OBJECTIVE: The importance of PI3K/Akt signaling in the vasculature has been demonstrated in several models, as global loss of Akt1 results in impaired postnatal ischemia- and VEGF-induced angiogenesis. The ubiquitous expression of Akt1, however, raises the possibility of cell-type-dependent Akt1-driven actions, thereby necessitating tissue-specific characterization. APPROACH AND RESULTS: Herein, we used an inducible, endothelial-specific Akt1-deleted adult mouse model (Akt1iECKO) to characterize the endothelial cell autonomous functions of Akt1 in the vascular system. Endothelial-targeted ablation of Akt1 reduces eNOS (endothelial nitric oxide synthase) phosphorylation and promotes both increased vascular contractility in isolated vessels and elevated diastolic blood pressures throughout the diurnal cycle in vivo. Furthermore, Akt1iECKO mice subject to the hindlimb ischemia model display impaired blood flow and decreased arteriogenesis. CONCLUSIONS: Endothelial Akt1 signaling is necessary for ischemic resolution post-injury and likely reflects the consequence of NO insufficiency critical for vascular repair.
Assuntos
Aorta Torácica/enzimologia , Células Endoteliais/enzimologia , Isquemia/enzimologia , Músculo Esquelético/irrigação sanguínea , Neovascularização Fisiológica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Vasoconstrição , Animais , Velocidade do Fluxo Sanguíneo , Pressão Sanguínea , Modelos Animais de Doenças , Membro Posterior , Isquemia/genética , Isquemia/patologia , Isquemia/fisiopatologia , Masculino , Camundongos Knockout , Músculo Esquelético/enzimologia , Músculo Esquelético/patologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/deficiência , Proteínas Proto-Oncogênicas c-akt/genética , Fluxo Sanguíneo Regional , Transdução de SinaisRESUMO
cis-Prenyltransferases (cis-PTs) constitute a large family of enzymes conserved during evolution and present in all domains of life. In eukaryotes and archaea, cis-PT is the first enzyme committed to the synthesis of dolichyl phosphate, an obligate lipid carrier in protein glycosylation reactions. The homodimeric bacterial enzyme, undecaprenyl diphosphate synthase, generates 11 isoprene units and has been structurally and mechanistically characterized in great detail. Recently, we discovered that unlike undecaprenyl diphosphate synthase, mammalian cis-PT is a heteromer consisting of NgBR (Nus1) and hCIT (dehydrodolichol diphosphate synthase) subunits, and this composition has been confirmed in plants and fungal cis-PTs. Here, we establish the first purification system for heteromeric cis-PT and show that both NgBR and hCIT subunits function in catalysis and substrate binding. Finally, we identified a critical RXG sequence in the C-terminal tail of NgBR that is conserved and essential for enzyme activity across phyla. In summary, our findings show that eukaryotic cis-PT is composed of the NgBR and hCIT subunits. The strong conservation of the RXG motif among NgBR orthologs indicates that this subunit is critical for the synthesis of polyprenol diphosphates and cellular function.
Assuntos
Alquil e Aril Transferases/química , Dimetilaliltranstransferase/química , Receptores de Superfície Celular/química , Transferases/química , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Motivos de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Dimetilaliltranstransferase/genética , Dimetilaliltranstransferase/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Transferases/genética , Transferases/metabolismoRESUMO
RATIONALE: Caveolin-1 (Cav-1) negatively regulates endothelial nitric oxide (NO) synthase-derived NO production, and this has been mapped to several residues on Cav-1, including F92. Herein, we reasoned that endothelial expression of an F92ACav-1 transgene would let us decipher the mechanisms and relationships between caveolae structure and intracellular signaling. OBJECTIVE: This study was designed to separate caveolae formation from its downstream signaling effects. METHODS AND RESULTS: An endothelial-specific doxycycline-regulated mouse model for the expression of Cav-1-F92A was developed. Blood pressure by telemetry and nitric oxide bioavailability by electron paramagnetic resonance and phosphorylation of vasodilator-stimulated phosphoprotein were determined. Caveolae integrity in the presence of Cav-1-F92A was measured by stabilization of caveolin-2, sucrose gradient, and electron microscopy. Histological analysis of heart and lung, echocardiography, and signaling were performed. CONCLUSIONS: This study shows that mutant Cav-1-F92A forms caveolae structures similar to WT but leads to increases in NO bioavailability in vivo, thereby demonstrating that caveolae formation and downstream signaling events occur through independent mechanisms.
Assuntos
Cavéolas/metabolismo , Caveolina 1/biossíntese , Caveolina 1/genética , Líquido Intracelular/metabolismo , Transdução de Sinais/fisiologia , Animais , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Cavéolas/efeitos dos fármacos , Doxiciclina/farmacologia , Humanos , Líquido Intracelular/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Óxido Nítrico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Desacopladores/farmacologiaRESUMO
Chronic Kidney Disease (CKD) is a highly prevalent disease with a substantial medical need for new and more efficacious treatments. The Nitric Oxide (NO), soluble guanylyl cyclase (sGC), cyclic guanosine monophosphate (cGMP) signaling cascade regulates various kidney functions. cGMP directly influences renal blood flow, renin secretion, glomerular function, and tubular exchange processes. Downregulation of NO/sGC/cGMP signaling results in severe kidney pathologies such as CKD. Therefore, treatment strategies aiming to maintain or increase cGMP might have beneficial effects for the treatment of progressive kidney diseases. Within this article, we review the NO/sGC/cGMP signaling cascade and its major pharmacological intervention sites. We specifically focus on the currently known effects of cGMP on kidney function parameters. Finally, we summarize the preclinical evidence for kidney protective effects of NO-donors, PDE inhibitors, sGC stimulators, and sGC activators.
Assuntos
Nefropatias/patologia , Rim/patologia , Óxido Nítrico/metabolismo , Transdução de Sinais , Guanilil Ciclase Solúvel/metabolismo , Animais , GMP Cíclico/metabolismo , Humanos , Nefropatias/terapiaRESUMO
Increasing cGMP is a unique therapeutic principle, and drugs inhibiting cGMP-degrading enzymes or stimulating cGMP production are approved for the treatment of various diseases such as erectile dysfunction, coronary artery disease, pulmonary hypertension, chronic heart failure, irritable bowel syndrome, or achondroplasia. In addition, cGMP-increasing therapies are preclinically profiled or in clinical development for quite a broad set of additional indications, e.g., neurodegenerative diseases or different forms of dementias, bone formation disorders, underlining the pivotal role of cGMP signaling pathways. The fundamental understanding of the signaling mediated by nitric oxide-sensitive (soluble) guanylyl cyclase and membrane-associated receptor (particulate) guanylyl cyclase at the molecular and cellular levels, as well as in vivo, especially in disease models, is a key prerequisite to fully exploit treatment opportunities and potential risks that could be associated with an excessive increase in cGMP. Furthermore, human genetic data and the clinical effects of cGMP-increasing drugs allow back-translation into basic research to further learn about signaling and treatment opportunities. The biannual international cGMP conference, launched nearly 20 years ago, brings all these aspects together as an established and important forum for all topics from basic science to clinical research and pivotal clinical trials. This review summarizes the contributions to the "10th cGMP Conference on cGMP Generators, Effectors and Therapeutic Implications," which was held in Augsburg in 2022 but will also provide an overview of recent key achievements and activities in the field of cGMP research.
Assuntos
GMP Cíclico , Guanilato Ciclase , Masculino , Humanos , Guanilato Ciclase/metabolismo , Guanilil Ciclase Solúvel/metabolismo , GMP Cíclico/metabolismo , Transdução de Sinais , Pesquisa , Óxido Nítrico/metabolismoAssuntos
Células Progenitoras Endoteliais/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Longevidade/genética , Músculo Esquelético/irrigação sanguínea , Neovascularização Fisiológica , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Animais , Feminino , Humanos , MasculinoRESUMO
BACKGROUND AND PURPOSE: Generation of cGMP via NO-sensitive soluble guanylyl cyclase (sGC) has been implicated in the regulation of renal functions. Chronic kidney disease (CKD) is associated with decreased NO bioavailability, increased oxidative stress and oxidation of sGC to its haem-free form, apo-sGC. Apo-sGC cannot be activated by NO, resulting in impaired cGMP signalling that is associated with chronic kidney disease progression. We hypothesised that sGC activators, which activate apo-sGC independently of NO, increase renal cGMP production under conditions of oxidative stress, thereby improving renal blood flow (RBF) and kidney function. EXPERIMENTAL APPROACH: Two novel sGC activators, runcaciguat and BAY-543, were tested on murine kidney. We measured cGMP levels in real time in kidney slices of cGMP sensor mice, vasodilation of pre-constricted glomerular arterioles and RBF in isolated perfused kidneys. Experiments were performed at baseline conditions, under L-NAME-induced NO deficiency, and in the presence of oxidative stress induced by ODQ. KEY RESULTS: Mouse glomeruli showed NO-induced cGMP increases. Under baseline conditions, sGC activator did not alter glomerular cGMP concentration or NO-induced cGMP generation. In the presence of ODQ, NO-induced glomerular cGMP signals were markedly reduced, whereas sGC activator induced strong cGMP increases. L-NAME and ODQ pretreated isolated glomerular arterioles were strongly dilated by sGC activator. sGC activator also increased cGMP and RBF in ODQ-perfused kidneys. CONCLUSION AND IMPLICATION: sGC activators increase glomerular cGMP, dilate glomerular arterioles and improve RBF under disease-relevant oxidative stress conditions. Therefore, sGC activators represent a promising class of drugs for chronic kidney disease treatment. LINKED ARTICLES: This article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc.
Assuntos
Insuficiência Renal Crônica , Vasodilatação , Animais , GMP Cíclico , Feminino , Guanilato Ciclase , Humanos , Rim , Masculino , Camundongos , NG-Nitroarginina Metil Éster , Óxido Nítrico , Insuficiência Renal Crônica/tratamento farmacológico , Guanilil Ciclase SolúvelRESUMO
Chronic kidney diseaQueryse (CKD) is associated with oxidative stress which can interrupt the nitric oxide (NO)/soluble guanylyl cyclase (sGC) signaling and decrease cyclic guanosine monophosphate (cGMP) production. Low cGMP concentrations can cause kidney damage and progression of CKD. The novel sGC activator runcaciguat targets the oxidized and heme-free form of sGC, restoring cGMP production under oxidative stress. The purpose of this study is to investigate if runcaciguat could provide an effective treatment for CKD. Runcaciguat was used for the treatment not only in rat CKD models with different etiologies and comorbidities, namely of hypertensive rats, the renin transgenic (RenTG) rat, and angiotensin-supplemented (ANG-SD) rat, but also in rats with diabetic and metabolic CKD, the Zucker diabetic fatty (ZDF) rat. The treatment duration was 2 to 42 weeks and runcaciguat was applied orally in doses from 1 to 10 mg/kg/bid. In these different rat CKD models, runcaciguat significantly reduced proteinuria (urinary protein to creatinine ratio; uPCR). These effects were also significant at doses which did not or only moderately decrease systemic blood pressure. Moreover, runcaciguat significantly decreased kidney injury biomarkers and attenuated morphological kidney damages. In RenTG rats, runcaciguat improved survival rates and markers of heart injury. These data demonstrate that the sGC activator runcaciguat exhibits cardio-renal protection at doses which did not reduce blood pressure and was effective in hypertensive as well as diabetic and metabolic CKD models. These data, therefore, suggest that runcaciguat, with its specific mode of action, represents an efficient treatment approach for CKD and associated CV diseases.
Assuntos
Ciclopropanos , Diabetes Mellitus Experimental , Hipertensão , Insuficiência Renal Crônica , Animais , Masculino , Ratos , Pressão Sanguínea/efeitos dos fármacos , GMP Cíclico/metabolismo , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/tratamento farmacológico , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Ativadores de Enzimas/administração & dosagem , Ativadores de Enzimas/farmacologia , Hipertensão/complicações , Hipertensão/tratamento farmacológico , Ratos Sprague-Dawley , Ratos Transgênicos , Ratos Zucker , Insuficiência Renal Crônica/etiologia , Insuficiência Renal Crônica/prevenção & controle , Guanilil Ciclase Solúvel/efeitos dos fármacos , Guanilil Ciclase Solúvel/metabolismo , Fatores de Tempo , Ciclopropanos/farmacologia , Ciclopropanos/uso terapêuticoRESUMO
Low rates of arteriovenous fistula (AVF) maturation prevent optimal fistula use for hemodialysis; however, the mechanism of venous remodeling in the fistula environment is not well understood. We hypothesized that the embryonic venous determinant Eph-B4 mediates AVF maturation. In human AVF and a mouse aortocaval fistula model, Eph-B4 protein expression increased in the fistula vein; expression of the arterial determinant Ephrin-B2 also increased. Stimulation of Eph-B-mediated signaling with Ephrin-B2/Fc showed improved fistula patency with less wall thickness. Mutagenesis studies showed that tyrosine-774 is critical for Eph-B4 signaling and administration of inactive Eph-B4-Y774F increased fistula wall thickness. Akt1 expression also increased in AVF; Akt1 knockout mice showed reduced fistula diameter and wall thickness. In Akt1 knockout mice, stimulation of Eph-B signaling with Ephrin-B2/Fc showed no effect on remodeling. These results show that AVF maturation is associated with acquisition of dual arteriovenous identity; increased Eph-B activity improves AVF patency. Inhibition of Akt1 function abolishes Eph-B-mediated venous remodeling suggesting that Eph-B4 regulates AVF venous adaptation through an Akt1-mediated mechanism.
Assuntos
Derivação Arteriovenosa Cirúrgica , Grau de Desobstrução Vascular , Remodelação Vascular , Animais , Masculino , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor EphB2/genética , Receptor EphB2/metabolismo , Receptor EphB4/genéticaRESUMO
Ex vivo normothermic machine perfusion (NMP) is a new clinical strategy to assess and resuscitate organs likely to be declined for transplantation, thereby increasing the number of viable organs available. Short periods of NMP provide a window of opportunity to deliver therapeutics directly to the organ and, in particular, to the vascular endothelial cells (ECs) that constitute the first point of contact with the recipient's immune system. ECs are the primary targets of both ischemia-reperfusion injury and damage from preformed antidonor antibodies, and reduction of perioperative EC injury could have long-term benefits by reducing the intensity of the host's alloimmune response. Using NMP to administer therapeutics directly to the graft avoids many of the limitations associated with systemic drug delivery. We have previously shown that polymeric nanoparticles (NPs) can serve as depots for long-term drug release, but ensuring robust NP accumulation within a target cell type (graft ECs in this case) remains a fundamental challenge of nanomedicine. We show that surface conjugation of an anti-CD31 antibody enhances targeting of NPs to graft ECs of human kidneys undergoing NMP. Using a two-color quantitative microscopy approach, we demonstrate that targeting can enhance EC accumulation by about 5- to 10-fold or higher in discrete regions of the renal vasculature. In addition, our studies reveal that NPs can also nonspecifically accumulate within obstructed regions of the vasculature that are poorly perfused. These quantitative preclinical human studies demonstrate the therapeutic potential for targeted nanomedicines delivered during ex vivo NMP.
Assuntos
Endotélio/citologia , Endotélio/metabolismo , Rim/citologia , Rim/metabolismo , Células Endoteliais/citologia , Células Endoteliais/fisiologia , Humanos , Nanopartículas , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismoRESUMO
Activin receptor-like kinase 1 (ALK1) is an endothelial serine-threonine kinase receptor for bone morphogenetic proteins (BMPs) 9 and 10. Inactivating mutations in the ALK1 gene cause hereditary haemorrhagic telangiectasia type 2 (HHT2), a disabling disease characterized by excessive angiogenesis with arteriovenous malformations (AVMs). Here we show that inducible, endothelial-specific homozygous Alk1 inactivation and BMP9/10 ligand blockade both lead to AVM formation in postnatal retinal vessels and internal organs including the gastrointestinal (GI) tract in mice. VEGF and PI3K/AKT signalling are increased on Alk1 deletion and BMP9/10 ligand blockade. Genetic deletion of the signal-transducing Vegfr2 receptor prevents excessive angiogenesis but does not fully revert AVM formation. In contrast, pharmacological PI3K inhibition efficiently prevents AVM formation and reverts established AVMs. Thus, Alk1 deletion leads to increased endothelial PI3K pathway activation that may be a novel target for the treatment of vascular lesions in HHT2.
Assuntos
Inibidores de Fosfoinositídeo-3 Quinase , Telangiectasia Hemorrágica Hereditária/complicações , Malformações Vasculares/complicações , Malformações Vasculares/enzimologia , Receptores de Ativinas Tipo I/metabolismo , Receptores de Activinas Tipo II , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Modelos Animais de Doenças , Deleção de Genes , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Camundongos , Modelos Biológicos , Neovascularização Patológica/tratamento farmacológico , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Retina/patologia , Transdução de Sinais/efeitos dos fármacos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
In humans and animals lacking functional LDL receptor (LDLR), LDL from plasma still readily traverses the endothelium. To identify the pathways of LDL uptake, a genome-wide RNAi screen was performed in endothelial cells and cross-referenced with GWAS-data sets. Here we show that the activin-like kinase 1 (ALK1) mediates LDL uptake into endothelial cells. ALK1 binds LDL with lower affinity than LDLR and saturates only at hypercholesterolemic concentrations. ALK1 mediates uptake of LDL into endothelial cells via an unusual endocytic pathway that diverts the ligand from lysosomal degradation and promotes LDL transcytosis. The endothelium-specific genetic ablation of Alk1 in Ldlr-KO animals leads to less LDL uptake into the aortic endothelium, showing its physiological role in endothelial lipoprotein metabolism. In summary, identification of pathways mediating LDLR-independent uptake of LDL may provide unique opportunities to block the initiation of LDL accumulation in the vessel wall or augment hepatic LDLR-dependent clearance of LDL.
Assuntos
Receptores de Activinas Tipo II/metabolismo , LDL-Colesterol/metabolismo , Células Endoteliais/metabolismo , Receptores de Ativinas Tipo I/genética , Receptores de Ativinas Tipo I/metabolismo , Receptores de Activinas Tipo II/genética , Animais , Apolipoproteínas B/genética , Apolipoproteínas B/metabolismo , Transporte Biológico , Células Cultivadas , LDL-Colesterol/genética , Clonagem Molecular , Técnicas de Silenciamento de Genes , Estudo de Associação Genômica Ampla , Humanos , Masculino , Camundongos , Interferência de RNARESUMO
Heme oxygenase (HO) degrades heme in concert with NADPH cytochrome P450 reductase (CPR) which donates electrons to the reaction. Earlier studies reveal the importance of the hydrophobic carboxy-terminus of HO-1 for anchorage to the endoplasmic reticulum (ER) which facilitates the interaction with CPR. In addition, HO-1 has been shown to undergo regulated intramembrane proteolysis of the carboxy-terminus during hypoxia and subsequent translocation to the nucleus. Translocated nuclear HO-1 was demonstrated to alter binding of transcription factors and to alter gene expression. Little is known about the homologous membrane anchor of the HO-2 isoform. The current work is the first systematic analysis in a eukaryotic system that demonstrates the crucial role of the membrane anchor of HO-2 for localization at the endoplasmic reticulum, oligomerization and interaction with CPR. We show that although the carboxy-terminal deletion mutant of HO-2 is found in the nucleus, translocation of HO-2 to the nucleus does not occur under conditions of hypoxia. Thus, we demonstrate that proteolytic regulation and nuclear translocation under hypoxic conditions is specific for HO-1. In addition we show for the first time that CPR prevents this translocation and promotes oligomerization of HO-1. Based on these findings, CPR may modulate gene expression via the amount of nuclear HO-1. This is of particular relevance as CPR is a highly polymorphic gene and deficiency syndromes of CPR have been described in humans.
Assuntos
Heme Oxigenase (Desciclizante)/análise , Heme Oxigenase (Desciclizante)/metabolismo , Heme Oxigenase-1/análise , Heme Oxigenase-1/metabolismo , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Animais , Hipóxia Celular , Núcleo Celular/metabolismo , Retículo Endoplasmático/metabolismo , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Heme Oxigenase (Desciclizante)/genética , Heme Oxigenase (Desciclizante)/isolamento & purificação , Heme Oxigenase-1/genética , Heme Oxigenase-1/isolamento & purificação , Humanos , Mutação , Isoformas de Proteínas/análise , Isoformas de Proteínas/genética , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/metabolismo , Multimerização Proteica , Transporte ProteicoRESUMO
BACKGROUND: Nitric oxide sensitive guanylyl cyclase (NOsGC) is a heterodimeric enzyme formed by an α- and a ß1-subunit. A splice variant (C-α1) of the α1-subunit, lacking at least the first 236 amino acids has been described by Sharina et al. 2008 and has been shown to be expressed in differentiating human embryonic cells. Wagner et al. 2005 have shown that the amino acids 61-128 of the α1-subunit are mandatory for quantitative heterodimerization implying that the C-α1-splice variant should lose its capacity to dimerize quantitatively. METHODOLOGY/PRINCIPAL FINDINGS: In the current study we demonstrate preserved quantitative dimerization of the C-α1-splice by co-purification with the ß1-subunit. In addition we used fluorescence resonance energy transfer (FRET) based on fluorescence lifetime imaging (FLIM) using fusion proteins of the ß1-subunit and the α1-subunit or the C-α1 variant with ECFP or EYFP. Analysis of the respective combinations in HEK-293 cells showed that the fluorescence lifetime was significantly shorter (≈0.3 ns) for α1/ß1 and C-α1/ß1 than the negative control. In addition we show that lack of the amino-terminus in the α1 splice variant directs it to a more oxidized subcellular compartment. CONCLUSIONS/SIGNIFICANCE: We conclude that the amino-terminus of the α1-subunit is dispensable for dimerization in-vivo and ex-vivo, but influences the subcellular trafficking.
Assuntos
Guanilato Ciclase/química , Guanilato Ciclase/metabolismo , Linhagem Celular , Transferência Ressonante de Energia de Fluorescência , Guanilato Ciclase/genética , Humanos , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismoRESUMO
Nitric oxide sensitive guanylyl cyclase (NOsGC) is a heterodimeric enzyme consisting of an α and a ß subunit. Two heterodimeric enzymes are known to be important for NO-signalling in humans: α(1)/ß(1) and α(2)/ß(1). No difference had so far been detected with respect to their pharmacological properties, but as we show in the present paper the new drugs cinaciguat and ataciguat activate the α(1)/ß(1) form more effectively. Recent evidence suggests that homodimeric complexes of α and ß subunits exist in vivo and that these non-heterodimerizing subunits have a separate function from cGMP signaling. To isolate the effect of the α(1)/ß(1) or α(2)/ß(1) heterodimeric enzyme in overexpression experiments from potential effects of non-heterodimerizing α(1), ß(1) or α(2) subunits, we cloned constructs that guarantee a 1:1 stochiometry between α and ß subunits and rule out the presence of homodimers. The carboxy-terminus of the ß(1) subunit was directly fused to the amino-terminus of either the α(1) or α(2) subunit. The two different "conjoined" NOsGCs faithfully reproduced the biochemical and pharmacological properties of the α(1)/ß(1) and α(2)/ß(1) heterodimeric enzymes including the differential activation by ciguat-activators. Conjoined NOsGCs can be used for isoform specific overexpression in transgenic animals and therapeutic overexpression may be an application in the future. In both cases possible side effects of homodimeric α or ß subunits are avoided. Crystallization with the goal of structure determination may also be easier for conjoined NOsGCs because enzyme preparations are more homogenous and are free of "contaminating" homodimers.
Assuntos
Benzoatos/farmacologia , Guanilato Ciclase/genética , Óxido Nítrico/fisiologia , Multimerização Proteica/genética , Subunidades Proteicas/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Guanilato Ciclase/química , Guanilato Ciclase/metabolismo , Guanilato Ciclase/fisiologia , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Ligação Proteica/genética , Multimerização Proteica/efeitos dos fármacos , Multimerização Proteica/fisiologia , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Ratos , Receptores Citoplasmáticos e Nucleares/química , Guanilil Ciclase Solúvel , SpodopteraRESUMO
BACKGROUND: To examine the structural organisation of heterodimeric soluble guanylyl cyclase (sGC) Förster resonance energy transfer (FRET) was measured between fluorescent proteins fused to the amino- and carboxy-terminal ends of the sGC beta1 and alpha subunits. METHODOLOGY/PRINCIPAL FINDINGS: Cyan fluorescent protein (CFP) was used as FRET donor and yellow fluorescent protein (YFP) as FRET acceptor. After generation of recombinant baculovirus, fluorescent-tagged sGC subunits were co-expressed in Sf9 cells. Fluorescent variants of sGC were analyzed in vitro in cytosolic fractions by sensitized emission FRET. Co-expression of the amino-terminally tagged alpha subunits with the carboxy-terminally tagged beta1 subunit resulted in an enzyme complex that showed a FRET efficiency of 10% similar to fluorescent proteins separated by a helix of only 48 amino acids. Because these findings indicated that the amino-terminus of the alpha subunits is close to the carboxy-terminus of the beta1 subunit we constructed fusion proteins where both subunits are connected by a fluorescent protein. The resulting constructs were not only fluorescent, they also showed preserved enzyme activity and regulation by NO. CONCLUSIONS/SIGNIFICANCE: Based on the ability of an amino-terminal fragment of the beta1 subunit to inhibit activity of an heterodimer consisting only of the catalytic domains (alphacatbetacat), Winger and Marletta (Biochemistry 2005, 44:4083-90) have proposed a direct interaction of the amino-terminal region of beta1 with the catalytic domains. In support of such a concept of "trans" regulation of sGC activity by the H-NOX domains our results indicate that the domains within sGC are organized in a way that allows for direct interaction of the amino-terminal regulatory domains with the carboxy-terminal catalytic region. In addition, we constructed "fluorescent-conjoined" sGC's by fusion of the alpha amino-terminus to the beta1 carboxy-terminus leading to a monomeric, fluorescent and functional enzyme complex. To our knowledge this represents the first example where a fluorescent protein links two different subunits of a higher ordered complex to yield a stoichometrically fixed functionally active monomer.