ABSTRACT
BACKGROUND: Neutrophil extracellular traps (NETs) are composed of DNA, enzymes, and citrullinated histones that are expelled by neutrophils in the process of NETosis. NETs accumulate in the aorta and kidneys in hypertension. PAD4 (protein-arginine deiminase-4) is a calcium-dependent enzyme that is essential for NETosis. TRPV4 (transient receptor potential cation channel subfamily V member 4) is a mechanosensitive calcium channel expressed in neutrophils. Thus, we hypothesize that NETosis contributes to hypertension via NET-mediated endothelial cell (EC) dysfunction. METHODS: NETosis-deficient Padi4-/- mice were treated with Ang II (angiotensin II). Blood pressure was measured by radiotelemetry, and vascular reactivity was measured with wire myography. Neutrophils were cultured with or without ECs and exposed to normotensive or hypertensive uniaxial stretch. NETosis was measured by flow cytometry. ECs were treated with citrullinated histone H3, and gene expression was measured by quantitative reverse transcription PCR. Aortic rings were incubated with citrullinated histone H3, and wire myography was performed to evaluate EC function. Neutrophils were treated with the TRPV4 agonist GSK1016790A. Calcium influx was measured using Fluo-4 dye, and NETosis was measured by immunofluorescence. RESULTS: Padi4-/- mice exhibited attenuated hypertension, reduced aortic inflammation, and improved EC-dependent vascular relaxation in response to Ang II. Coculture of neutrophils with ECs and exposure to hypertensive uniaxial stretch increased NETosis and accumulation of neutrophil citrullinated histone H3. Histone H3 and citrullinated histone H3 exposure attenuates EC-dependent vascular relaxation. Treatment of neutrophils with the TRPV4 agonist GSK1016790A increases intracellular calcium and NETosis. CONCLUSIONS: These observations identify a role of NETosis in the pathogenesis of hypertension. Moreover, they define an important role of EC stretch and TRPV4 as initiators of NETosis. Finally, they define a role of citrullinated histones as drivers of EC dysfunction in hypertension.
Subject(s)
Extracellular Traps , Hypertension , Mice, Knockout , Protein-Arginine Deiminase Type 4 , TRPV Cation Channels , Animals , Extracellular Traps/metabolism , Hypertension/metabolism , Hypertension/physiopathology , TRPV Cation Channels/metabolism , TRPV Cation Channels/genetics , Mice , Protein-Arginine Deiminase Type 4/metabolism , Neutrophils/metabolism , Mice, Inbred C57BL , Male , Angiotensin II/pharmacology , Humans , Histones/metabolism , Blood Pressure , Cells, Cultured , Endothelial Cells/metabolismABSTRACT
BACKGROUND: Hypertension is characterized by CD8+ (cluster differentiation 8) T cell activation and infiltration into peripheral tissues. CD8+ T cell activation requires proteasomal processing of antigenic proteins. It has become clear that isoLG (isolevuglandin)-adduced peptides are antigenic in hypertension; however, IsoLGs inhibit the constitutive proteasome. We hypothesized that immunoproteasomal processing of isoLG-adducts is essential for CD8+ T cell activation and inflammation in hypertension. METHODS: IsoLG adduct processing was studied in murine dendritic cells (DCs), endothelial cells (ECs), and B8 fibroblasts. The role of the proteasome and the immunoproteasome in Ang II (angiotensin II)-induced hypertension was studied in C57BL/6 mice treated with bortezomib or the immunoproteasome inhibitor PR-957 and by studying mice lacking 3 critical immunoproteasome subunits (triple knockout mouse). We also examined hypertension in mice lacking the critical immunoproteasome subunit LMP7 (large multifunctional peptidase 7) specifically in either DCs or ECs. RESULTS: We found that oxidant stress increases the presence of isoLG adducts within MHC-I (class I major histocompatibility complex), and immunoproteasome overexpression augments this. Pharmacological or genetic inhibition of the immunoproteasome attenuated hypertension and tissue inflammation. Conditional deletion of LMP7 in either DCs or ECs attenuated hypertension and vascular inflammation. Finally, we defined the role of the innate immune receptors STING (stimulator of interferon genes) and TLR7/8 (toll-like receptor 7/8) as drivers of LMP7 expression in ECs. CONCLUSIONS: These studies define a previously unknown role of the immunoproteasome in DCs and ECs in CD8+ T cell activation. The immunoproteasome in DCs and ECs is critical for isoLG-adduct presentation to CD8+ T cells, and in the endothelium, this guides homing and infiltration of T cells to specific tissues.
Subject(s)
Bortezomib , CD8-Positive T-Lymphocytes , Dendritic Cells , Hypertension , Proteasome Endopeptidase Complex , Animals , Male , Mice , Angiotensin II , Bortezomib/pharmacology , CD8-Positive T-Lymphocytes/immunology , Cells, Cultured , Dendritic Cells/immunology , Dendritic Cells/metabolism , Endothelial Cells/metabolism , Endothelial Cells/immunology , Fibroblasts/metabolism , Histocompatibility Antigens Class I/metabolism , Histocompatibility Antigens Class I/genetics , Hypertension/metabolism , Hypertension/immunology , Lymphocyte Activation , Mice, Inbred C57BL , Mice, Knockout , Oligopeptides , Oxidative Stress , Proteasome Endopeptidase Complex/metabolism , Proteasome Inhibitors/pharmacologyABSTRACT
Heart failure continues to be a significant global health concern, causing substantial morbidity and mortality. The limited ability of the adult heart to regenerate has posed challenges in finding effective treatments for cardiac pathologies. While various medications and surgical interventions have been used to improve cardiac function, they are not able to address the extensive loss of functioning cardiomyocytes that occurs during cardiac injury. As a result, there is growing interest in understanding how the cell cycle is regulated and exploring the potential for stimulating cardiomyocyte proliferation as a means of promoting heart regeneration. This review aims to provide an overview of current knowledge on cell cycle regulation and mechanisms underlying cardiomyocyte proliferation in cases of heart failure, while also highlighting established and novel therapeutic strategies targeting this area for treatment purposes.
Subject(s)
Cell Cycle , Cell Proliferation , Heart Failure , Myocytes, Cardiac , Heart Failure/physiopathology , Heart Failure/metabolism , Heart Failure/pathology , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , Humans , Animals , RegenerationABSTRACT
After myocardial infarction in the adult heart the remaining, non-infarcted tissue adapts to compensate the loss of functional tissue. This adaptation requires changes in gene expression networks, which are mostly controlled by transcription regulating proteins. Long non-coding transcripts (lncRNAs) are taking part in fine-tuning such gene programs. We describe and characterize the cardiomyocyte specific lncRNA Sweetheart RNA (Swhtr), an approximately 10 kb long transcript divergently expressed from the cardiac core transcription factor coding gene Nkx2-5. We show that Swhtr is dispensable for normal heart development and function but becomes essential for the tissue adaptation process after myocardial infarction in murine males. Re-expressing Swhtr from an exogenous locus rescues the Swhtr null phenotype. Genes that depend on Swhtr after cardiac stress are significantly occupied and therefore most likely regulated by NKX2-5. The Swhtr transcript interacts with NKX2-5 and disperses upon hypoxic stress in cardiomyocytes, indicating an auxiliary role of Swhtr for NKX2-5 function in tissue adaptation after myocardial injury.
Subject(s)
Heart Injuries , Myocardial Infarction , RNA, Long Noncoding , Male , Mice , Animals , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Myocytes, Cardiac/metabolism , Cardiomegaly/genetics , Cardiomegaly/metabolism , Myocardial Infarction/metabolismABSTRACT
Aging is a major risk factor for impaired cardiovascular health. Because the aging myocardium is characterized by microcirculatory dysfunction, and because nerves align with vessels, we assessed the impact of aging on the cardiac neurovascular interface. We report that aging reduces nerve density in the ventricle and dysregulates vascular-derived neuroregulatory genes. Aging down-regulates microRNA 145 (miR-145) and derepresses the neurorepulsive factor semaphorin-3A. miR-145 deletion, which increased Sema3a expression or endothelial Sema3a overexpression, reduced axon density, mimicking the aged-heart phenotype. Removal of senescent cells, which accumulated with chronological age in parallel to the decline in nerve density, rescued age-induced denervation, reversed Sema3a expression, preserved heart rate patterns, and reduced electrical instability. These data suggest that senescence-mediated regulation of nerve density contributes to age-associated cardiac dysfunction.
Subject(s)
Aging , Cellular Senescence , Heart , MicroRNAs , Microvascular Density , Myocardium , Semaphorin-3A , Heart/innervation , Microcirculation , MicroRNAs/genetics , MicroRNAs/metabolism , Semaphorin-3A/genetics , Animals , Mice , Aging/genetics , Aging/pathology , Male , Mice, Inbred C57BL , Cellular Senescence/genetics , Myocardium/pathology , AxonsABSTRACT
BACKGROUND: Cardiovascular diseases (CVDs) are the leading cause of death worldwide. Genome-wide association studies (GWAS) have identified many single nucleotide polymorphisms (SNPs) appearing in non-coding genomic regions in CVDs. The SNPs may alter gene expression by modifying transcription factor (TF) binding sites and lead to functional consequences in cardiovascular traits or diseases. To understand the underlying molecular mechanisms, it is crucial to identify which variations are involved and how they affect TF binding. METHODS: The SNEEP (SNP exploration and analysis using epigenomics data) pipeline was used to identify regulatory SNPs, which alter the binding behavior of TFs and link GWAS SNPs to their potential target genes for six CVDs. The human-induced pluripotent stem cells derived cardiomyocytes (hiPSC-CMs), monoculture cardiac organoids (MCOs) and self-organized cardiac organoids (SCOs) were used in the study. Gene expression, cardiomyocyte size and cardiac contractility were assessed. RESULTS: By using our integrative computational pipeline, we identified 1905 regulatory SNPs in CVD GWAS data. These were associated with hundreds of genes, half of them non-coding RNAs (ncRNAs), suggesting novel CVD genes. We experimentally tested 40 CVD-associated non-coding RNAs, among them RP11-98F14.11, RPL23AP92, IGBP1P1, and CTD-2383I20.1, which were upregulated in hiPSC-CMs, MCOs and SCOs under hypoxic conditions. Further experiments showed that IGBP1P1 depletion rescued expression of hypertrophic marker genes, reduced hypoxia-induced cardiomyocyte size and improved hypoxia-reduced cardiac contractility in hiPSC-CMs and MCOs. CONCLUSIONS: IGBP1P1 is a novel ncRNA with key regulatory functions in modulating cardiomyocyte size and cardiac function in our disease models. Our data suggest ncRNA IGBP1P1 as a potential therapeutic target to improve cardiac function in CVDs.
Subject(s)
Cardiovascular Diseases , Polymorphism, Single Nucleotide , Humans , Polymorphism, Single Nucleotide/genetics , Genome-Wide Association Study , Cardiovascular Diseases/genetics , Genomics , GenomeABSTRACT
Isolevuglandins (isoLGs) are lipid aldehydes that form in the presence of reactive oxygen species (ROS) and drive immune activation. We found that isoLG-adducts are presented within the context of major histocompatibility complexes (MHC-I) by an immunoproteasome dependent mechanism. Pharmacologic inhibition of LMP7, the chymotrypsin subunit of the immunoproteasome, attenuates hypertension and tissue inflammation in the angiotensin II (Ang II) model of hypertension. Genetic loss of function of all immunoproteasome subunits or conditional deletion of LMP7 in dendritic cell (DCs) or endothelial cells (ECs) attenuated hypertension, reduced aortic T cell infiltration, and reduced isoLG-adduct MHC-I interaction. Furthermore, isoLG adducts structurally resemble double-stranded DNA and contribute to the activation of STING in ECs. These studies define a critical role of the immunoproteasome in the processing and presentation of isoLG-adducts. Moreover they define a role of LMP7 as a regulator of T cell activation and tissue infiltration in hypertension.
ABSTRACT
It has become clear that circadian clocks in peripheral tissues play important functions. Disruption of the circadian clock in skeletal muscle, for example, results in insulin resistance, sarcomere disorganization, and muscle weakness1. Interestingly, cavefish, which exhibit a disrupted central clock, exhibit similar muscle phenotypes2,3,4, raising the question of whether they are caused by alterations to central or peripheral clocks. Here, we demonstrate a loss in clock function in the skeletal muscle of the Mexican Cavefish Astyanax mexicanus that is associated with reduced rhythmicity of a large number of genes and disrupted nocturnal protein catabolism. Some of the identified genes are associated with metabolic dysfunction in humans.
Subject(s)
Characidae , Circadian Clocks , Animals , Humans , Circadian Rhythm/physiology , Circadian Clocks/genetics , Muscle, Skeletal/physiology , MexicoABSTRACT
The tetra fish species Astyanax mexicanus comprises two morphotypes: cavefish that live in caves and surface fish that inhabit rivers and lakes. Because cavefish have adapted to the nutrient-poor conditions in their habitat whereas the surface fish populations can be used as a proxy for the ancestral condition, this species has become a powerful model system for understanding genetic variation underlying metabolic adaptation. The liver plays a critical role in glucose and fat metabolism in the body and hence is an important tissue for studying altered metabolism in health and disease. Cavefish morphs of A. mexicanus have been shown to develop fatty livers and exhibit massive differences in gene expression and chromatin architecture. Primary cell lines from various tissues have become invaluable tools for biochemical, toxicology, and cell biology experiments, as well as genetic and genomic analyses. To enhance the utility of the model system by enabling an expanded set of biochemical and in vitro experiments, we developed protocols for the isolation and maintenance of primary liver cells from A. mexicanus surface fish and cavefish. We also describe methods that can be used for primary cell characterization, including cloning, characterization of cell growth pattern, and lentivirus transduction. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Primary culture of liver cells Support Protocol 1: Maintenance of A. mexicanus primary liver cells Support Protocol 2: Banking of A. mexicanus primary liver cells Support Protocol 3: Recovery of A. mexicanus primary liver cells Support Protocol 4: Primary liver cell cloning Support Protocol 5: Characterization of A. mexicanus primary liver cell growth pattern Basic Protocol 2: Lentiviral transduction of A. mexicanus primary liver cells.
Subject(s)
Characidae , Animals , Characidae/genetics , Genome , Adaptation, Physiological , LiverABSTRACT
Circadian control of physiology and metabolism is pervasive throughout nature, with circadian disruption contributing to premature aging, neurodegenerative disease, and type 2 diabetes (Musiek et al. 2016; Panda, 2016). It has become increasingly clear that peripheral tissues, such as skeletal muscle, possess cell-autonomous clocks crucial for metabolic homeostasis (Gabriel et al. 2021). In fact, disruption of the skeletal muscle circadian rhythm results in insulin resistance, sarcomere disorganization, and muscle weakness in both vertebrates and non-vertebrates - indicating that maintenance of a functional muscle circadian rhythm provides an adaptive advantage. We and others have found that cavefish possess a disrupted central circadian rhythm and, interestingly, a skeletal muscle phenotype strikingly similar to circadian knock-out mutants; namely, muscle loss, muscle weakness, and insulin resistance (Olsen et al. 2022; Riddle et al. 2018; Mack et al. 2021). However, whether the cavefish muscle phenotype results from muscle-specific circadian disruption remains untested. To this point, we investigated genome-wide, circadian-regulated gene expression within the skeletal muscle of the Astyanax mexicanus - comprised of the river-dwelling surface fish and troglobitic cavefish - providing novel insights into the evolutionary consequence of circadian disruption on skeletal muscle physiology.
ABSTRACT
Long non-coding RNAs (lncRNAs) can act as regulatory RNAs which, by altering the expression of target genes, impact on the cellular phenotype and cardiovascular disease development. Endothelial lncRNAs and their vascular functions are largely undefined. Deep RNA-Seq and FANTOM5 CAGE analysis revealed the lncRNA LINC00607 to be highly enriched in human endothelial cells. LINC00607 was induced in response to hypoxia, arteriosclerosis regression in non-human primates, post-atherosclerotic cultured endothelial cells from patients and also in response to propranolol used to induce regression of human arteriovenous malformations. siRNA knockdown or CRISPR/Cas9 knockout of LINC00607 attenuated VEGF-A-induced angiogenic sprouting. LINC00607 knockout in endothelial cells also integrated less into newly formed vascular networks in an in vivo assay in SCID mice. Overexpression of LINC00607 in CRISPR knockout cells restored normal endothelial function. RNA- and ATAC-Seq after LINC00607 knockout revealed changes in the transcription of endothelial gene sets linked to the endothelial phenotype and in chromatin accessibility around ERG-binding sites. Mechanistically, LINC00607 interacted with the SWI/SNF chromatin remodeling protein BRG1. CRISPR/Cas9-mediated knockout of BRG1 in HUVEC followed by CUT&RUN revealed that BRG1 is required to secure a stable chromatin state, mainly on ERG-binding sites. In conclusion, LINC00607 is an endothelial-enriched lncRNA that maintains ERG target gene transcription by interacting with the chromatin remodeler BRG1 to ultimately mediate angiogenesis.
Subject(s)
RNA, Long Noncoding , Animals , Humans , Mice , Chromatin , DNA Helicases/genetics , DNA Helicases/metabolism , Endothelial Cells/metabolism , Mice, SCID , Nuclear Proteins/metabolism , RNA, Long Noncoding/genetics , Neovascularization, PhysiologicABSTRACT
In healthy vessels, endothelial cells maintain a stable, differentiated, and growth-arrested phenotype for years. Upon injury, a rapid phenotypic switch facilitates proliferation to restore tissue perfusion. Here we report the identification of the endothelial cell-enriched long non-coding RNA (lncRNA) PCAT19, which contributes to the proliferative switch and acts as a safeguard for the endothelial genome. PCAT19 is enriched in confluent, quiescent endothelial cells and binds to the full replication protein A (RPA) complex in a DNA damage- and cell-cycle-related manner. Our results suggest that PCAT19 limits the phosphorylation of RPA2, primarily on the serine 33 (S33) residue, and thereby facilitates an appropriate DNA damage response while slowing cell cycle progression. Reduction in PCAT19 levels in response to either loss of cell contacts or knockdown promotes endothelial proliferation and angiogenesis. Collectively, PCAT19 acts as a dynamic guardian of the endothelial genome and facilitates rapid switching from quiescence to proliferation.
Subject(s)
RNA, Long Noncoding , Phosphorylation , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Endothelial Cells/metabolism , DNA/metabolism , Replication Protein A/genetics , Replication Protein A/metabolismABSTRACT
BACKGROUND: Salt sensitivity of blood pressure is an independent predictor of cardiovascular morbidity and mortality. The exact mechanism by which salt intake increases blood pressure and cardiovascular risk is unknown. We previously found that sodium entry into antigen-presenting cells (APCs) via the amiloride-sensitive epithelial sodium channel EnaC (epithelial sodium channel) leads to the formation of IsoLGs (isolevuglandins) and release of proinflammatory cytokines to activate T cells and modulate salt-sensitive hypertension. In the current study, we hypothesized that ENaC-dependent entry of sodium into APCs activates the NLRP3 (NOD [nucleotide-binding and oligomerization domain]-like receptor family pyrin domain containing 3) inflammasome via IsoLG formation leading to salt-sensitive hypertension. METHODS: We performed RNA sequencing on human monocytes treated with elevated sodium in vitro and Cellular Indexing of Transcriptomes and Epitopes by Sequencing analysis of peripheral blood mononuclear cells from participants rigorously phenotyped for salt sensitivity of blood pressure using an established inpatient protocol. To determine mechanisms, we analyzed inflammasome activation in mouse models of deoxycorticosterone acetate salt-induced hypertension as well as salt-sensitive mice with ENaC inhibition or expression, IsoLG scavenging, and adoptive transfer of wild-type dendritic cells into NLRP3 deficient mice. RESULTS: We found that high levels of salt exposure upregulates the NLRP3 inflammasome, pyroptotic and apoptotic caspases, and IL (interleukin)-1ß transcription in human monocytes. Cellular Indexing of Transcriptomes and Epitopes by Sequencing revealed that components of the NLRP3 inflammasome and activation marker IL-1ß dynamically vary with changes in salt loading/depletion. Mechanistically, we found that sodium-induced activation of the NLRP3 inflammasome is ENaC and IsoLG dependent. NLRP3 deficient mice develop a blunted hypertensive response to elevated sodium, and this is restored by the adoptive transfer of NLRP3 replete APCs. CONCLUSIONS: These findings reveal a mechanistic link between ENaC, inflammation, and salt-sensitive hypertension involving NLRP3 inflammasome activation in APCs. APC activation via the NLRP3 inflammasome can serve as a potential diagnostic biomarker for salt sensitivity of blood pressure.
Subject(s)
Hypertension , Inflammasomes , Animals , Epithelial Sodium Channels/genetics , Epitopes , Humans , Hypertension/chemically induced , Hypertension/genetics , Inflammasomes/metabolism , Interleukin-1beta/metabolism , Leukocytes, Mononuclear/metabolism , Mice , Mice, Inbred C57BL , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Sodium/metabolism , Sodium Chloride/metabolism , Sodium Chloride, Dietary/adverse effectsABSTRACT
Insights from organisms, which have evolved natural strategies for promoting survivability under extreme environmental pressures, may help guide future research into novel approaches for enhancing human longevity. The cave-adapted Mexican tetra, Astyanax mexicanus, has attracted interest as a model system for metabolic resilience, a term we use to denote the property of maintaining health and longevity under conditions that would be highly deleterious in other organisms (Figure 1). Cave-dwelling populations of Mexican tetra exhibit elevated blood glucose, insulin resistance and hypertrophic visceral adipocytes compared to surface-dwelling counterparts. However, cavefish appear to avoid pathologies typically associated with these conditions, such as accumulation of advanced-glycation-end-products (AGEs) and chronic tissue inflammation. The metabolic strategies underlying the resilience properties of A. mexicanus cavefish, and how they relate to environmental challenges of the cave environment, are poorly understood. Here, we provide an untargeted metabolomics study of long- and short-term fasting in two A. mexicanus cave populations and one surface population. We find that, although the metabolome of cavefish bears many similarities with pathological conditions such as metabolic syndrome, cavefish also exhibit features not commonly associated with a pathological condition, and in some cases considered indicative of an overall robust metabolic condition. These include a reduction in cholesteryl esters and intermediates of protein glycation, and an increase in antioxidants and metabolites associated with hypoxia and longevity. This work suggests that certain metabolic features associated with human pathologies are either not intrinsically harmful, or can be counteracted by reciprocal adaptations. We provide a transparent pipeline for reproducing our analysis and a Shiny app for other researchers to explore and visualize our dataset.
Subject(s)
Antioxidants , Characidae , Aging , Animals , Biological Evolution , Humans , Metabolome , SugarsABSTRACT
BACKGROUND: IsoLGs (isolevuglandins) are electrophilic products of lipid peroxidation formed in the presence of reactive oxygen species. IsoLGs contribute to hypertension by an unknown mechanism. Studies have shown that reactive oxygen species production drives the formation of neutrophil extracellular traps (NETs) and that NETs accumulate within the aorta and kidneys of patients with hypertension. The purpose of this study was to determine the role of isoLGs in neutrophil migration and NET formation (NETosis) in hypertension. METHODS: Mice were treated with Ang II (angiotensin II) and the specific isoLG scavenger 2-hydroxybenzylamine and examined for tissue neutrophil and NET accumulation by single-cell sequencing and flow cytometry. Isolated human neutrophils were studied to determine the role of isoLGs in NETosis and neutrophil chromatin expansion by immunofluorescence and live cell confocal microscopy. RESULTS: Single-cell sequencing performed on sham, Ang II, and Ang II+2-hydroxybenzylamine treated mice revealed neutrophils as a primary target of 2-hydroxybenzylamine. Peripheral neutrophil migration, aortic NET accumulation, and renal NET accumulation is blocked with 2-hydroxybenzylamine treatment. In isolated human neutrophils, isoLGs accumulate during NETosis and scavenging of isoLGs prevents NETosis. IsoLGs drive neutrophil chromatin expansion during NETosis and disrupt nucleosome structure. CONCLUSIONS: These observations identified a critical role of isoLGs in neutrophil migration and NETosis in hypertension and provide a potential therapy for NET-associated diseases including hypertension and associated end organ damage.
Subject(s)
Extracellular Traps , Hypertension , Animals , Chromatin , Humans , Lipids , Mice , Neutrophils , Reactive Oxygen SpeciesABSTRACT
Cell lines have become an integral resource and tool for conducting biological experiments ever since the Hela cell line was first developed (Scherer et al. in J Exp Med 97:695-710, 1953). They not only allow detailed investigation of molecular pathways but are faster and more cost-effective than most in vivo approaches. The last decade saw many emerging model systems strengthening basic science research. However, lack of genetic and molecular tools in these newer systems pose many obstacles. Astyanax mexicanus is proving to be an interesting new model system for understanding metabolic adaptation. To further enhance the utility of this system, we developed liver-derived cell lines from both surface-dwelling and cave-dwelling morphotypes. In this study, we provide detailed methodology of the derivation process along with comprehensive biochemical and molecular characterization of the cell lines, which reflect key metabolic traits of cavefish adaptation. We anticipate these cell lines to become a useful resource for the Astyanax community as well as researchers investigating fish biology, comparative physiology, and metabolism.
Subject(s)
Characidae , Adaptation, Physiological/genetics , Animals , Biological Evolution , Caves , Characidae/physiology , HeLa Cells , Humans , LiverABSTRACT
We describe a mechanism responsible for systemic lupus erythematosus (SLE). In humans with SLE and in 2 SLE murine models, there was marked enrichment of isolevuglandin-adducted proteins (isoLG adducts) in monocytes and dendritic cells. We found that antibodies formed against isoLG adducts in both SLE-prone mice and humans with SLE. In addition, isoLG ligation of the transcription factor PU.1 at a critical DNA binding site markedly reduced transcription of all C1q subunits. Treatment of SLE-prone mice with the specific isoLG scavenger 2-hydroxybenzylamine (2-HOBA) ameliorated parameters of autoimmunity, including plasma cell expansion, circulating IgG levels, and anti-dsDNA antibody titers. 2-HOBA also lowered blood pressure, attenuated renal injury, and reduced inflammatory gene expression uniquely in C1q-expressing dendritic cells. Thus, isoLG adducts play an essential role in the genesis and maintenance of systemic autoimmunity and hypertension in SLE.
Subject(s)
Hypertension , Lupus Erythematosus, Systemic , Animals , Antibodies, Antinuclear , Autoimmunity , Complement C1q/genetics , Lipids , MiceABSTRACT
Cis-regulatory changes are key drivers of adaptative evolution. However, their contribution to the metabolic adaptation of organisms is not well understood. Here, we used a unique vertebrate model, Astyanax mexicanus-different morphotypes of which survive in nutrient-rich surface and nutrient-deprived cave waters-to uncover gene regulatory networks underlying metabolic adaptation. We performed genome-wide epigenetic profiling in the liver tissues of Astyanax and found that many of the identified cis-regulatory elements (CREs) have genetically diverged and have differential chromatin features between surface and cave morphotypes, while retaining remarkably similar regulatory signatures between independently derived cave populations. One such CRE in the hpdb gene harbors a genomic deletion in cavefish that abolishes IRF2 repressor binding and derepresses enhancer activity in reporter assays. Selection of this mutation in multiple independent cave populations supports its importance in cave adaptation, and provides novel molecular insights into the evolutionary trade-off between loss of pigmentation and adaptation to food-deprived caves.
Subject(s)
Characidae , Acclimatization , Adaptation, Physiological/genetics , Animals , Biological Evolution , Caves , Characidae/genetics , Characidae/metabolism , MutationABSTRACT
Nutrient availability varies seasonally and spatially in the wild. While many animals, such as hibernating animals or migrating birds, evolved strategies to overcome periods of nutrient scarcity,1,2 the cellular mechanisms of these strategies are poorly understood. Cave environments represent an example of nutrient-deprived environments, since the lack of sunlight and therefore primary energy production drastically diminishes the nutrient availability.3 Here, we used Astyanax mexicanus, which includes river-dwelling surface fish and cave-adapted cavefish populations, to study the genetic adaptation to nutrient limitations.4-9 We show that cavefish populations store large amounts of fat in different body regions when fed ad libitum in the lab. We found higher expression of lipogenesis genes in cavefish livers when fed the same amount of food as surface fish, suggesting an improved ability of cavefish to use lipogenesis to convert available energy into triglycerides for storage into adipose tissue.10-12 Moreover, the lipid metabolism regulator, peroxisome proliferator-activated receptor γ (Pparγ), is upregulated at both transcript and protein levels in cavefish livers. Chromatin immunoprecipitation sequencing (ChIP-seq) showed that Pparγ binds cavefish promoter regions of genes to a higher extent than surface fish and inhibiting Pparγ in vivo decreases fat accumulation in A. mexicanus. Finally, we identified nonsense mutations in per2, a known repressor of Pparγ, providing a possible regulatory mechanism of Pparγ in cavefish. Taken together, our study reveals that upregulated Pparγ promotes higher levels of lipogenesis in the liver and contributes to higher body fat accumulation in cavefish populations, an important adaptation to nutrient-limited environments.