ABSTRACT
BACKGROUND: Increased circulating uric acid (UA) concentration may disrupt cardiac function in heart failure patients, but the specific mechanism remains unclear. Here, we postulate that hyperuremia induces sterol regulatory element binding protein 1 (SREBP1), which in turn activate hepatic fatty acid biosynthesis response, leading to cardiac dysfunction. METHODS AND RESULTS: Increased circulating uric acid was observed in heart failure patients and inversely correlated to cardiac function. Besides, uric acid correlated to circulating lipids profile based on metabolomics in heart failure patients. Using cultured human hepatoellular carcinomas (HepG2) and Tg(myl7:egfp) zebrafish, we demonstrated that UA regulated fatty acid synthase (FASN) via SREBP1 signaling pathway, leading to FFA accumulation and impaired energy metabolism, which could be rescued via SREBP1 knockdown. In ISO treated zebrafish, UA aggravated heart failure via increased cardiovascular cavity size, decreased heart beats, pericardial edema and long-stretched heart deformation. CONCLUSIONS: Our findings suggest that UA-SREBP1-FASN signaling exacerbates cardiac dysfunction during FFA accumulation. Identification of this mechanism may help in treatment and prevention of heart failure.
Subject(s)
Heart Diseases , Heart Failure , Animals , Humans , Uric Acid , Zebrafish/metabolism , Fatty Acid Synthases/metabolism , Fatty Acids/metabolism , Heart Failure/complicationsABSTRACT
BACKGROUND AND AIMS: In patients with acute liver failure (ALF) who suffer from massive hepatocyte loss, liver progenitor cells (LPCs) take over key hepatocyte functions, which ultimately determines survival. This study investigated how the expression of hepatocyte nuclear factor 4α (HNF4α), its regulators, and targets in LPCs determines clinical outcome of patients with ALF. APPROACH AND RESULTS: Clinicopathological associations were scrutinized in 19 patients with ALF (9 recovered and 10 receiving liver transplantation). Regulatory mechanisms between follistatin, activin, HNF4α, and coagulation factor expression in LPC were investigated in vitro and in metronidazole-treated zebrafish. A prospective clinical study followed up 186 patients with cirrhosis for 80 months to observe the relevance of follistatin levels in prevalence and mortality of acute-on-chronic liver failure. Recovered patients with ALF robustly express HNF4α in either LPCs or remaining hepatocytes. As in hepatocytes, HNF4α controls the expression of coagulation factors by binding to their promoters in LPC. HNF4α expression in LPCs requires the forkhead box protein H1-Sma and Mad homolog 2/3/4 transcription factor complex, which is promoted by the TGF-ß superfamily member activin. Activin signaling in LPCs is negatively regulated by follistatin, a hepatocyte-derived hormone controlled by insulin and glucagon. In contrast to patients requiring liver transplantation, recovered patients demonstrate a normal activin/follistatin ratio, robust abundance of the activin effectors phosphorylated Sma and Mad homolog 2 and HNF4α in LPCs, leading to significantly improved coagulation function. A follow-up study indicated that serum follistatin levels could predict the incidence and mortality of acute-on-chronic liver failure. CONCLUSIONS: These results highlight a crucial role of the follistatin-controlled activin-HNF4α-coagulation axis in determining the clinical outcome of massive hepatocyte loss-induced ALF. The effects of insulin and glucagon on follistatin suggest a key role of the systemic metabolic state in ALF.
Subject(s)
Activins/genetics , Follistatin/metabolism , Hepatocyte Nuclear Factor 4/metabolism , Liver Failure, Acute/metabolism , Activins/metabolism , Acute-On-Chronic Liver Failure/blood , Adult , Aged , Animals , Blood Coagulation , Cell Line , Factor V/genetics , Female , Follistatin/blood , Follow-Up Studies , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Gene Expression , Hepatocyte Nuclear Factor 4/genetics , Hepatocytes/metabolism , Humans , Liver Failure, Acute/chemically induced , Liver Failure, Acute/pathology , Liver Failure, Acute/surgery , Liver Regeneration , Liver Transplantation , Male , Metronidazole , Mice , Middle Aged , Prognosis , Promoter Regions, Genetic , Prospective Studies , Prothrombin/genetics , Signal Transduction , Smad2 Protein/genetics , Smad2 Protein/metabolism , Smad3 Protein/genetics , Smad3 Protein/metabolism , Smad4 Protein/genetics , Stem Cells/metabolism , Transforming Growth Factor beta1/genetics , ZebrafishABSTRACT
The gene CNDP1 was associated with the development of diabetic nephropathy. Its enzyme carnosinase 1 (CN1) primarily hydrolyzes the histidine-containing dipeptide carnosine but other organ and metabolic functions are mainly unknown. In our study we generated CNDP1 knockout zebrafish, which showed strongly decreased CN1 activity and increased intracellular carnosine levels. Vasculature and kidneys of CNDP1-/- zebrafish were not affected, except for a transient glomerular alteration. Amino acid profiling showed a decrease of certain amino acids in CNDP1-/- zebrafish, suggesting a specific function for CN1 in the amino acid metabolisms. Indeed, we identified a CN1 activity for Ala-His and Ser-His. Under diabetic conditions increased carnosine levels in CNDP1-/- embryos could not protect from respective organ alterations. Although, weight gain through overfeeding was restrained by CNDP1 loss. Together, zebrafish exhibits CN1 functions, while CNDP1 knockout alters the amino acid metabolism, attenuates weight gain but cannot protect organs from diabetic complications.
Subject(s)
Amino Acids/metabolism , Diabetes Complications/metabolism , Dipeptidases/metabolism , Weight Gain/physiology , Animals , Carnosine/metabolism , Diabetes Mellitus, Type 2/metabolism , Gene Knockout Techniques/methods , Kidney/metabolism , ZebrafishABSTRACT
Diabetes mellitus (DM) is a crucial metabolic disease that leads to severe disorders. These include macrovascular complications such as myocardial infarction, stroke, and peripheral artery disease and microvascular complications including diabetic nephropathy, neuropathy, and retinopathy. Diabetes mellitus, along with its associated organ pathologies, is one of the key problems in today's medicine. Zebrafish is an upcoming disease model organism in diabetes research. Its glucose metabolism and the pathways of reactive metabolite formation are very similar to those of humans. Moreover, several physiological and pathophysiological pathways that also exist in humans and other mammals have been identified in this species or are currently under intense investigation. Zebrafish offer sophisticated imaging techniques and allow simple and fast genetic and pharmacological approaches with a high throughput. In this review, we highlight achievements and mechanisms concerning microvascular complications discovered in zebrafish, and we discuss the advantages and disadvantages of zebrafish as a model for studying diabetic complications.
Subject(s)
Diabetic Angiopathies/genetics , Disease Models, Animal , Zebrafish/genetics , Animals , Diabetic Angiopathies/pathology , Microvessels/metabolism , Microvessels/pathology , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolismABSTRACT
Engulfment and cell motility 1/dedicator of cytokinesis 180 (Elmo1/Dock180) is a bipartite guanine nucleotide exchange factor for the monomeric GTPase Ras-related C3 botulinum toxin substrate 1 (Rac1). Elmo1/Dock180 regulates Rac1 activity in a specific spatiotemporal manner in endothelial cells (ECs) during zebrafish development and acts downstream of the Netrin-1/Unc5-homolog B (Unc5B) signaling cascade. However, mechanistic details on the pathways by which Elmo1/Dock180 regulates endothelial function and vascular development remained elusive. In this study, we aimed to analyze the vascular function of Elmo1 and Dock180 in human ECs and during vascular development in zebrafish embryos. In vitro overexpression of Elmo1 and Dock180 in ECs reduced caspase-3/7 activity and annexin V-positive cell number upon induction of apoptosis. This protective effect of Elmo1 and Dock180 is mediated by activation of Rac1, p21-activated kinase (PAK) and AKT/protein kinase B (AKT) signaling. In zebrafish, Elmo1 and Dock180 overexpression reduced the total apoptotic cell and apoptotic EC number and promoted the formation of blood vessels during embryogenesis. In conclusion, Elmo1 and Dock180 protect ECs from apoptosis by the activation of the Rac1/PAK/AKT signaling cascade in vitro and in vivo. Thus, Elmo1 and Dock180 facilitate blood vessel formation by stabilization of the endothelium during angiogenesis.
Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Blood Vessels/metabolism , Neovascularization, Physiologic , rac GTP-Binding Proteins/genetics , rac1 GTP-Binding Protein/metabolism , Adaptor Proteins, Signal Transducing/biosynthesis , Adaptor Proteins, Signal Transducing/metabolism , Animals , Apoptosis/genetics , Blood Vessels/growth & development , Endothelial Cells/metabolism , Gene Expression Regulation, Developmental , Guanine Nucleotide Exchange Factors/genetics , Human Umbilical Vein Endothelial Cells , Humans , Zebrafish/genetics , Zebrafish/growth & development , rac GTP-Binding Proteins/biosynthesis , rac GTP-Binding Proteins/metabolism , rac1 GTP-Binding Protein/geneticsABSTRACT
Blood vessels have been described a long time ago as passive circuits providing sufficient blood supply to ensure proper distribution of oxygen and nutrition. Blood vessels are mainly formed during embryonic development and in the early postnatal period. In the adult, blood vessels are quiescent, but can be activated and subsequently induced under pathophysiological conditions, such as ischemia and tumor growth. Surprisingly, recent data have suggested an active function for blood vessels, named angiocrine signaling, releasing trophogens which regulate organ development and organ regeneration including in the pancreas, lung, tumor cells, liver and bone. Lung development is driven by hypoxia as well as an intense endothelial-epithelial interaction, and important mechanisms contributing to these processes have recently been identified. This review aims to summarize recent developments and concepts about embryonic pulmonary vascular development and lung regeneration. We discuss hypoxia-inducible factor HIF-2α and vascular endothelial growth factor VEGF as important mediators in lung development and focus on endothelial-epithelial interactions and angiocrine signaling mechanisms.
Subject(s)
Lung/physiology , Regeneration , Basic Helix-Loop-Helix Transcription Factors/metabolism , Basic Helix-Loop-Helix Transcription Factors/physiology , Humans , Lung/embryology , Lung/growth & development , Neovascularization, Physiologic , Signal Transduction , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor A/physiologyABSTRACT
Histone deacetylases (HDACs) deacetylate histones and non-histone proteins, thereby affecting protein activity and gene expression. The regulation and function of the cytoplasmic class IIb HDAC6 in endothelial cells (ECs) is largely unexplored. Here, we demonstrate that HDAC6 is upregulated by hypoxia and is essential for angiogenesis. Silencing of HDAC6 in ECs decreases sprouting and migration in vitro and formation of functional vascular networks in matrigel plugs in vivo. HDAC6 regulates zebrafish vessel formation, and HDAC6-deficient mice showed a reduced formation of perfused vessels in matrigel plugs. Consistently, overexpression of wild-type HDAC6 increases sprouting from spheroids. HDAC6 function requires the catalytic activity but is independent of ubiquitin binding and deacetylation of α-tubulin. Instead, we found that HDAC6 interacts with and deacetylates the actin-remodelling protein cortactin in ECs, which is essential for zebrafish vessel formation and which mediates the angiogenic effect of HDAC6. In summary, we show that HDAC6 is necessary for angiogenesis in vivo and in vitro, involving the interaction and deacetylation of cortactin that regulates EC migration and sprouting.
Subject(s)
Cell Movement , Cortactin/metabolism , Histone Deacetylases/metabolism , Neovascularization, Physiologic , Tubulin/metabolism , Zebrafish Proteins/metabolism , Acetylation , Animals , Carcinoma, Lewis Lung/enzymology , Cells, Cultured , Endothelial Cells/metabolism , Female , Histone Deacetylase 6 , Humans , Lung Neoplasms/metabolism , Male , Mice , Mice, KnockoutABSTRACT
OBJECTIVE: Nucleoside diphosphate kinase B (NDPKB) participates in the activation of heterotrimeric and monomeric G proteins, which are pivotal mediators in angiogenic signaling. The role of NDPKB in angiogenesis has to date not been defined. Therefore, we analyzed the contribution of NDPKB to angiogenesis and its underlying mechanisms in well-characterized in vivo and in vitro models. APPROACH AND RESULTS: Zebrafish embryos were depleted of NDPKB by morpholino-mediated knockdown. These larvae displayed severe malformations specifically in vessels formed by angiogenesis. NDPKB-deficient (NDPKB(-/-)) mice were subjected to oxygen-induced retinopathy. In this model, the number of preretinal neovascularizations in NDPKB(-/-) mice was strongly reduced in comparison with wild-type littermates. In accordance, a delayed blood flow recovery was detected in the NDPKB(-/-) mice after hindlimb ligation. In in vitro studies, a small interfering RNA-mediated knockdown of NDPKB was performed in human umbilical endothelial cells. NDPKB depletion impaired vascular endothelial growth factor (VEGF)-induced sprouting and hampered the VEGF-induced spatial redistributions of the VEGF receptor type 2 and VE-cadherin at the plasma membrane. Concomitantly, NDPKB depletion increased the permeability of the human umbilical endothelial cell monolayer. CONCLUSIONS: This is the first report to show that NDPKB is required for VEGF-induced angiogenesis and contributes to the correct localization of VEGF receptor type 2 and VE-cadherin at the endothelial adherens junctions. Therefore, our data identify NDPKB as a novel molecular target to modulate VEGF-dependent angiogenesis.
Subject(s)
Antigens, CD/metabolism , Cadherins/metabolism , Endothelial Cells/enzymology , Muscle, Skeletal/blood supply , NM23 Nucleoside Diphosphate Kinases/metabolism , Neovascularization, Physiologic , Vascular Endothelial Growth Factor Receptor-2/metabolism , Zebrafish Proteins/metabolism , Zebrafish/metabolism , Animals , Cells, Cultured , Disease Models, Animal , Gene Expression Regulation, Developmental , Gene Knockdown Techniques , Hindlimb , Human Umbilical Vein Endothelial Cells/enzymology , Humans , Ischemia/enzymology , Ischemia/genetics , Ischemia/physiopathology , Mice , Mice, Inbred C57BL , Mice, Knockout , NM23 Nucleoside Diphosphate Kinases/deficiency , NM23 Nucleoside Diphosphate Kinases/genetics , RNA Interference , Recovery of Function , Regional Blood Flow , Retinal Neovascularization/enzymology , Retinal Neovascularization/genetics , Retinal Neovascularization/physiopathology , Signal Transduction , Time Factors , Transfection , Zebrafish/embryology , Zebrafish/genetics , Zebrafish Proteins/deficiency , Zebrafish Proteins/geneticsABSTRACT
ß1-Integrins are essential for angiogenesis. The mechanisms regulating integrin function in endothelial cells (EC) and their contribution to angiogenesis remain elusive. Brag2 is a guanine nucleotide exchange factor for the small Arf-GTPases Arf5 and Arf6. The role of Brag2 in EC and angiogenesis and the underlying molecular mechanisms remain unclear. siRNA-mediated Brag2-silencing reduced EC angiogenic sprouting and migration. Brag2-siRNA transfection differentially affected α5ß1- and αVß3-integrin function: specifically, Brag2-silencing increased focal/fibrillar adhesions and adhesion on ß1-integrin ligands (fibronectin and collagen), while reducing the adhesion on the αVß3-integrin ligand, vitronectin. Consistent with these results, Brag2-silencing enhanced surface expression of α5ß1-integrin, while reducing surface expression of αVß3-integrin. Mechanistically, Brag2-mediated αVß3-integrin-recycling and ß1-integrin endocytosis and specifically of the active/matrix-bound α5ß1-integrin present in fibrillar/focal adhesions (FA), suggesting that Brag2 contributes to the disassembly of FA via ß1-integrin endocytosis. Arf5 and Arf6 are promoting downstream of Brag2 angiogenic sprouting, ß1-integrin endocytosis and the regulation of FA. In vivo silencing of the Brag2-orthologues in zebrafish embryos using morpholinos perturbed vascular development. Furthermore, in vivo intravitreal injection of plasmids containing Brag2-shRNA reduced pathological ischemia-induced retinal and choroidal neovascularization. These data reveal that Brag2 is essential for developmental and pathological angiogenesis by promoting EC sprouting through regulation of adhesion by mediating ß1-integrin internalization and link for the first time the process of ß1-integrin endocytosis with angiogenesis.
Subject(s)
Cell Adhesion/physiology , Guanine Nucleotide Exchange Factors/genetics , Integrin beta1/metabolism , Integrin beta3/metabolism , Neovascularization, Pathologic/physiopathology , Retinopathy of Prematurity/physiopathology , ADP-Ribosylation Factor 6 , ADP-Ribosylation Factors/metabolism , Animals , Animals, Genetically Modified , COS Cells , Cell Movement/physiology , Chlorocebus aethiops , Choroidal Neovascularization/genetics , Choroidal Neovascularization/metabolism , Choroidal Neovascularization/physiopathology , Disease Models, Animal , Guanine Nucleotide Exchange Factors/metabolism , Human Umbilical Vein Endothelial Cells , Humans , Integrin alphaVbeta3/genetics , Integrin alphaVbeta3/metabolism , Mice, Inbred C57BL , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/metabolism , Neovascularization, Physiologic/genetics , Neovascularization, Physiologic/physiology , RNA, Small Interfering/genetics , Receptors, Vitronectin/genetics , Receptors, Vitronectin/metabolism , Retinopathy of Prematurity/genetics , Retinopathy of Prematurity/metabolism , Vascular Endothelial Growth Factor A/metabolism , ZebrafishABSTRACT
MicroRNAs (miRs) are small RNAs that regulate gene expression at the posttranscriptional level. miR-27 is expressed in endothelial cells, but the specific functions of miR-27b and its family member miR-27a are largely unknown. Here we demonstrate that overexpression of miR-27a and miR-27b significantly increased endothelial cell sprouting. Inhibition of both miR-27a and miR-27b impaired endothelial cell sprout formation and induced endothelial cell repulsion in vitro. In vivo, inhibition of miR-27a/b decreased the number of perfused vessels in Matrigel plugs and impaired embryonic vessel formation in zebrafish. Mechanistically, miR-27 regulated the expression of the angiogenesis inhibitor semaphorin 6A (SEMA6A) in vitro and in vivo and targeted the 3'-untranslated region of SEMA6A. Silencing of SEMA6A partially reversed the inhibition of endothelial cell sprouting and abrogated the repulsion of endothelial cells mediated by miR-27a/b inhibition, indicating that SEMA6A is a functionally relevant miR-27 downstream target regulating endothelial cell repulsion. In summary, we show that miR-27a/b promotes angiogenesis by targeting the angiogenesis inhibitor SEMA6A, which controls repulsion of neighboring endothelial cells.
Subject(s)
Endothelial Cells/metabolism , MicroRNAs/genetics , Neovascularization, Physiologic/genetics , Semaphorins/genetics , 3' Untranslated Regions/genetics , Animals , Blood Vessels/embryology , Blood Vessels/metabolism , Blotting, Western , Cell Survival/genetics , Cell Survival/physiology , Cells, Cultured , Embryo, Nonmammalian/blood supply , Embryo, Nonmammalian/embryology , Embryo, Nonmammalian/metabolism , Endothelial Cells/physiology , Gene Expression , HEK293 Cells , Human Umbilical Vein Endothelial Cells/metabolism , Human Umbilical Vein Endothelial Cells/physiology , Humans , Mice , Mice, Inbred C57BL , MicroRNAs/metabolism , Neovascularization, Physiologic/physiology , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Semaphorins/metabolism , Transfection , Zebrafish/embryology , Zebrafish/geneticsABSTRACT
RATIONALE: Formation and remodeling of the vasculature during development and disease involve a highly conserved and precisely regulated network of attractants and repellants. Various signaling pathways control the behavior of endothelial cells, but their posttranscriptional dose titration by microRNAs is poorly understood. OBJECTIVE: To identify microRNAs that regulate angiogenesis. METHODS AND RESULTS: We show that the highly conserved microRNA family encoding miR-10 regulates the behavior of endothelial cells during angiogenesis by positively titrating proangiogenic signaling. Knockdown of miR-10 led to premature truncation of intersegmental vessel growth in the trunk of zebrafish larvae, whereas overexpression of miR-10 promoted angiogenic behavior in zebrafish and cultured human umbilical venous endothelial cells. We found that miR-10 functions, in part, by directly regulating the level of fms-related tyrosine kinase 1 (FLT1), a cell-surface protein that sequesters vascular endothelial growth factor, and its soluble splice variant sFLT1. The increase in FLT1/sFLT1 protein levels upon miR-10 knockdown in zebrafish and in human umbilical venous endothelial cells inhibited the angiogenic behavior of endothelial cells largely by antagonizing vascular endothelial growth factor receptor 2 signaling. CONCLUSIONS: Our study provides insights into how FLT1 and vascular endothelial growth factor receptor 2 signaling is titrated in a microRNA-mediated manner and establishes miR-10 as a potential new target for the selective modulation of angiogenesis.
Subject(s)
Endothelial Cells/metabolism , MicroRNAs/genetics , Neovascularization, Physiologic/genetics , Vascular Endothelial Growth Factor Receptor-1/genetics , Vascular Endothelial Growth Factor Receptor-2/genetics , Animals , Animals, Genetically Modified , Base Sequence , Cell Proliferation/drug effects , Cells, Cultured , Female , Gene Knockdown Techniques , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Immunoblotting , Larva/genetics , Larva/metabolism , Male , Mice , Microscopy, Confocal , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Nucleic Acid , Signal Transduction/genetics , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor A/pharmacology , Vascular Endothelial Growth Factor Receptor-1/metabolism , Vascular Endothelial Growth Factor Receptor-2/metabolism , ZebrafishABSTRACT
OBJECTIVE: Histone deacetylases (HDACs) modulate gene expression by deacetylation of histone and nonhistone proteins. Several HDACs control angiogenesis, but the role of HDAC9 is unclear. METHODS AND RESULTS: Here, we analyzed the function of HDAC9 in angiogenesis and its involvement in regulating microRNAs. In vitro, silencing of HDAC9 reduces endothelial cell tube formation and sprouting. Furthermore, HDAC9 silencing decreases vessel formation in a spheroid-based Matrigel plug assay in mice and disturbs vascular patterning in zebrafish embryos. Genetic deletion of HDAC9 reduces retinal vessel outgrowth and impairs blood flow recovery after hindlimb ischemia. Consistently, overexpression of HDAC9 increases endothelial cell sprouting, whereas mutant constructs lacking the catalytic domain, the nuclear localization sequence, or sumoylation site show no effect. To determine the mechanism underlying the proangiogenic effect of HDAC9, we measured the expression of the microRNA (miR)-17-92 cluster, which is known for its antiangiogenic activity. We demonstrate that silencing of HDAC9 in endothelial cells increases the expression of miR-17-92. Inhibition of miR-17-20a rescues the sprouting defects induced by HDAC9 silencing in vitro and blocking miR-17 expression partially reverses the disturbed vascular patterning of HDAC9 knockdown in zebrafish embryos. CONCLUSIONS: We found that HDAC9 promotes angiogenesis and transcriptionally represses the miR-17-92 cluster.
Subject(s)
Histone Deacetylases/metabolism , Human Umbilical Vein Endothelial Cells/enzymology , Ischemia/enzymology , MicroRNAs/metabolism , Muscle, Skeletal/blood supply , Neovascularization, Physiologic , Repressor Proteins/metabolism , Retinal Neovascularization/enzymology , Zebrafish Proteins/metabolism , Animals , Disease Models, Animal , Gene Knockdown Techniques , HEK293 Cells , Hindlimb , Histone Deacetylases/deficiency , Histone Deacetylases/genetics , Humans , Ischemia/genetics , Ischemia/physiopathology , Mice , Mice, Knockout , MicroRNAs/genetics , Mutation , Neovascularization, Physiologic/genetics , RNA Interference , RNA, Long Noncoding , Regional Blood Flow , Repressor Proteins/deficiency , Repressor Proteins/genetics , Retinal Neovascularization/genetics , Retinal Neovascularization/physiopathology , Transfection , Zebrafish/embryology , Zebrafish/genetics , Zebrafish Proteins/geneticsABSTRACT
The angiogenic cascade is a multi-step process essential for embryogenesis and other physiological and pathological processes. Rho family GTPases are binary molecular switches and serve as master regulators of various basic cellular processes. Rho GTPases are known to exert important functions in angiogenesis and vascular physiology. These functions demand a tight and context-specific control of cellular processes requiring superordinate control by a multitude of guanine nucleotide exchange factors (GEFs). GEFs display various features enabling them to fine-tune the actions of Rho GTPases in the vasculature: (1) GEFs regulate specific steps of the angiogenic cascade; (2) GEFs show a spatio-temporally specific expression pattern; (3) GEFs differentially regulate endothelial function depending on their subcellular location; (4) GEFs mediate crosstalk between complex signaling cascades and (5) GEFs themselves are regulated by another layer of interacting proteins. The aim of this review is to provide an overview about the role of GEFs in regulating angiogenesis and vascular function and to point out current limitations as well as clinical perspectives.
Subject(s)
Blood Vessels/physiology , Guanine Nucleotide Exchange Factors/physiology , Neovascularization, Physiologic , Animals , Endothelium, Vascular/physiology , Humans , Muscle, Smooth, Vascular/physiology , Organ Specificity , rho GTP-Binding Proteins/metabolismABSTRACT
Omega-6 fatty acids are the primary polyunsaturated fatty acids in most Western diets, while their role in diabetes remains controversial. Exposure of omega-6 fatty acids to an oxidative environment results in the generation of a highly reactive carbonyl species known as trans, trans-2,4-decadienal (tt-DDE). The timely and efficient detoxification of this metabolite, which has actions comparable to other reactive carbonyl species, such as 4-hydroxynonenal, acrolein, acetaldehyde, and methylglyoxal, is essential for disease prevention. However, the detoxification mechanism for tt-DDE remains elusive. In this study, the enzyme Aldh9a1b is identified as having a key role in the detoxification of tt-DDE. Loss of Aldh9a1b increased tt-DDE levels and resulted in an abnormal retinal vasculature and glucose intolerance in aldh9a1b-/- zebrafish. Transcriptomic and metabolomic analyses revealed that tt-DDE and aldh9a1b deficiency in larval and adult zebrafish induced insulin resistance and impaired glucose homeostasis. Moreover, alterations in hyaloid vasculature is induced by aldh9a1b knockout or by tt-DDE treatment can be rescued by the insulin receptor sensitizers metformin and rosiglitazone. Collectively, these results demonstrated that tt-DDE is the substrate of Aldh9a1b which causes microvascular damage and impaired glucose metabolism through insulin resistance.
Subject(s)
Aldehydes , Insulin Resistance , Insulin , Animals , Zebrafish , Gluconeogenesis , Fatty Acids, Omega-6ABSTRACT
The actinomycete Amycolatopsis sp. strain ATCC 39116 is capable of synthesizing large amounts of vanillin from ferulic acid, which is a natural cell wall component of higher plants. The desired intermediate vanillin is subject to undesired catabolism caused by the metabolic activity of a hitherto unknown vanillin dehydrogenase (VDH(ATCC 39116)). In order to prevent the oxidation of vanillin to vanillic acid and thereby to obtain higher yields and concentrations of vanillin, the responsible vanillin dehydrogenase in Amycolatopsis sp. ATCC 39116 was investigated for the first time by using data from our genome sequence analysis and further bioinformatic approaches. The vdh gene was heterologously expressed in Escherichia coli, and the encoded vanillin dehydrogenase was characterized in detail. VDH(ATCC 39116) was purified to apparent electrophoretic homogeneity and exhibited NAD(+)-dependent activity toward vanillin, coniferylaldehyde, cinnamaldehyde, and benzaldehyde. The enzyme showed its highest level of activity toward vanillin at pH 8.0 and at a temperature of 44°C. In a next step, a precise vdh deletion mutant of Amycolatopsis sp. ATCC 39116 was generated. The mutant lost its ability to grow on vanillin and did not show vanillin dehydrogenase activity. A 2.3-times-higher vanillin concentration and a substantially reduced amount of vanillic acid occurred with the Amycolatopsis sp. ATCC 39116 Δvdh::Km(r) mutant when ferulic acid was provided for biotransformation in a cultivation experiment on a 2-liter-bioreactor scale. Based on these results and taking further metabolic engineering into account, the Amycolatopsis sp. ATCC 39116 Δvdh::Km(r) mutant represents an optimized and industrially applicable platform for the biotechnological production of natural vanillin.
Subject(s)
Actinomycetales/enzymology , Actinomycetales/metabolism , Aldehyde Oxidoreductases/genetics , Aldehyde Oxidoreductases/metabolism , Benzaldehydes/metabolism , Biotechnology/methods , Metabolic Engineering/methods , Actinomycetales/genetics , Actinomycetales/growth & development , Coenzymes/metabolism , Coumaric Acids/metabolism , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Escherichia coli/genetics , Gene Deletion , Gene Expression , Hydrogen-Ion Concentration , Industrial Microbiology/methods , Molecular Sequence Data , NAD/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Analysis, DNA , Substrate Specificity , Temperature , Vanillic Acid/metabolismABSTRACT
RATIONALE: The transcription factor HOXC9 belongs to the homeobox gene family acting as developmental morphogen in several species. HOXC9 is EXPRESSED in different vascular beds in vivo. Yet vascular functions of HOXC9 have not been studied. OBJECTIVE: This study was aimed at characterizing HOXC9 functions in human vascular endothelial cells and in zebrafish vascular development. METHODS AND RESULTS: HOXC9 was abundantly expressed in resting human umbilical vein endothelial cells and was downregulated by hypoxia. Overexpression of HOXC9 inhibited endothelial cell proliferation, migration, and tube formation in vitro. Expression profiling and chromatin immunoprecipitation experiments in human umbilical vein endothelial cells identified interleukin 8 as the major HOXC9 target and demonstrated the direct binding of HOXC9 to the interleukin 8 promotor. HOXC9 overexpression led to reduced endothelial interleukin 8 production, whereas HOXC9 silencing increased interleukin 8. The antimigratory and antiangiogenic effect of HOXC9 overexpression could be rescued by external interleukin 8 administration. Corresponding to the cellular experiments, endothelial-specific overexpression of HOXC9 and morpholino-based interleukin 8 loss-of-function experiments inhibited zebrafish vascular development. CONCLUSION: The data identify HOXC9 as an endothelial cell active transcriptional repressor promoting the resting, antiangiogenic endothelial cell phenotype in an interleukin 8-dependent manner.
Subject(s)
Endothelial Cells/physiology , Homeodomain Proteins/genetics , Hypoxia/physiopathology , Interleukin-8/physiology , Neovascularization, Physiologic/physiology , Zebrafish Proteins/genetics , Animals , Capillaries/cytology , Capillaries/physiology , Cell Division/physiology , Cell Movement/physiology , Cells, Cultured , Down-Regulation/physiology , Endothelial Cells/cytology , Homeodomain Proteins/metabolism , Humans , Umbilical Veins/cytology , Zebrafish , Zebrafish Proteins/metabolismABSTRACT
Methylglyoxal (MG), the major dicarbonyl substrate of the enzyme glyoxalase 1 (GLO1), is a reactive metabolite formed via glycolytic flux. Decreased GLO1 activity in situ has been shown to result in an accumulation of MG and increased formation of advanced glycation endproducts, both of which can accumulate during physiological aging and at an accelerated rate in diabetes and other chronic degenerative diseases. To determine the physiological consequences which result from elevated MG levels and the role of MG and GLO1 in aging, wound healing in young (≤12 weeks) and old (≥52 weeks) wild-type mice was studied. Old mice were found to have a significantly slower rate of wound healing compared to young mice (74.9 ± 2.2 vs. 55.4 ± 1.5% wound closure at day 6; 26% decrease; p < 0.0001). This was associated with decreases in GLO1 transcription, expression and activity. The importance of GLO1 was confirmed in mice by inhibition of GLO1. Direct application of MG to the wounds of young mice, decreased wound healing by 24% compared to untreated mice, whereas application of BSA modified minimally by MG had no effect. Treatment of either young or old mice with aminoguanidine, a scavenger of free MG, significantly increased wound closure by 16% (66.8 ± 1.6 vs. 77.2 ± 3.1%; p < 0.05) and 64% (40.4 ± 7.9 vs. 66.4 ± 5.2%; p < 0.05), respectively, by day 6. As a result of the aminoguanidine treatment, the overall rate of wound healing in the old mice was restored to the level observed in the young mice. These findings were confirmed in vitro, as MG reduced migration and proliferation of fibroblasts derived from young and old, wild-type mice. The data demonstrate that the balance between MG and age-dependent GLO1 downregulation contributes to delayed wound healing in old mice.
Subject(s)
Aging/physiology , Lactoylglutathione Lyase/physiology , Wound Healing/physiology , Aging/genetics , Aging/metabolism , Animals , Cells, Cultured , Down-Regulation , Fibroblasts/drug effects , Fibroblasts/physiology , Guanidines/pharmacology , Lactoylglutathione Lyase/antagonists & inhibitors , Lactoylglutathione Lyase/genetics , Male , Mice , Mice, Inbred C57BL , Pyruvaldehyde/metabolism , Pyruvaldehyde/pharmacology , Wound Healing/drug effects , Wound Healing/geneticsABSTRACT
Background: Zebrafish (Danio rerio) have been established in recent years as a model organism to study Diabetic Retinopathy (DR). Loss of endothelial cells and pericytes is an early hallmark sign of developing DR in the mammalian retina. However, morphology, numbers, ratios, and distributions of different vascular cells in the retinal compartment in zebrafish have not yet been analyzed and compared with the mammalian retina. Methods: The retinal trypsin digest protocol was established on the zebrafish retina. Cell types were identified using the Tg(nflk:EGFP)-reporter line. Cells were quantified using quantitative morphometry. Results: Vascular cells in the zebrafish retina have distinct morphologies and locations. Nuclei of vascular mural cells appear as long and flat nuclei located near the vessel wall. Round nuclei within the vessel walls can be identified as endothelial cells. The vessel diameter decreases from central to peripheral parts of the retina. Additionally, the numbers of vascular cells decrease from central to peripheral parts of the retina. Discussion: The retinal trypsin digest protocol, which can be applied to the zebrafish retina, provides novel insights into the zebrafish retinal vascular architecture. Quantification of the different cell types shows that, in comparison to the mammalian retina, zebrafish have higher numbers of mural cells and an increased mural cell to endothelial cell ratio. This protocol enables to quantify mural cell and endothelial cell numbers, is easily adaptable to different transgenic and mutant zebrafish lines and will enable investigators to compare novel models on a single cell level.
ABSTRACT
Glyoxalase 2 is the second enzyme of the glyoxalase system, catalyzing the detoxification of methylglyoxal to d-lactate via SD-Lactoylglutathione. Recent in vitro studies have suggested Glo2 as a regulator of glycolysis, but if Glo2 regulates glucose homeostasis and related organ specific functions in vivo has not yet been evaluated. Therefore, a CRISPR-Cas9 knockout of glo2 in zebrafish was created and analyzed. Consistent with its function in methylglyoxal detoxification, SD-Lactoylglutathione, but not methylglyoxal accumulated in glo2-/- larvae, without altering the glutathione metabolism or affecting longevity. Adult glo2-/- livers displayed a reduced hexose concentration and a reduced postprandial P70-S6 kinase activation, but upstream postprandial AKT phosphorylation remained unchanged. In contrast, glo2-/- skeletal muscle remained metabolically intact, possibly compensating for the dysfunctional liver through increased glucose uptake and glycolytic activity. glo2-/- zebrafish maintained euglycemia and showed no damage of the retinal vasculature, kidney, liver and skeletal muscle. In conclusion, the data identified Glo2 as a regulator of cellular energy metabolism in liver and skeletal muscle, but the redox state and reactive metabolite accumulation were not affected by the loss of Glo2.
Subject(s)
Lactoylglutathione Lyase , Zebrafish , Animals , Zebrafish/genetics , Zebrafish/metabolism , Lactoylglutathione Lyase/genetics , Lactoylglutathione Lyase/metabolism , Pyruvaldehyde/metabolism , Lactic Acid , Glucose , Thiolester Hydrolases/metabolismABSTRACT
RATIONALE: Angiogenesis is regulated by the small GTPase Rac1. The ELMO1/DOCK180 complex forms a guanine nucleotide exchange factor for Rac1, regulating its activation during cell migration in different biological systems. OBJECTIVE: To investigate the function of ELMO1/DOCK180 in vascular development. METHODS AND RESULTS: In situ hybridization studies for elmo1 identified a vascular and neuronal expression in zebrafish. Morpholino-based expression silencing of elmo1 severely impaired the formation of the vasculature, including intersomitic vessels, the dorsal longitudinal anastomotic vessel, the parachordal vessel, and the development of the thoracic duct in tg(fli1:EGFP) embryos. Mechanistically, we identified Netrin-1 and its receptor Unc5B as upstream activators of the ELMO1/DOCK180 complex, regulating its functional interaction and leading to Rac1 activation in endothelial cells and vessel formation in zebrafish. CONCLUSIONS: Our data have identified a novel signaling cascade regulating vasculature formation in zebrafish.