ABSTRACT
Source tracking of fecal pollution is an emerging component in water quality monitoring. It may be implemented in a tiered approach involving Escherichia coli and/or Enterococcus spp. as the standard fecal indicator bacteria (FIB) and the 16S rRNA gene markers of Bacteroidales as source identifiers. The relative population dynamics of the source identifiers and the FIB may strongly influence the implementation of such approach. Currently, the relative performance of DNA and RNA as detection targets of Bacteroidales markers in the tiered approach is not known. We compared the decay of the DNA and RNA of the total (AllBac) and ruminant specific (CF128) Bacteroidales markers with those of the FIB in seawater spiked with cattle feces. Four treatments of light and oxygen availability simulating the subtropical seawater of Hong Kong were tested. All Bacteroidales markers decayed significantly slower than the FIB in all treatments. Nonetheless, the concentrations of the DNA and RNA markers and E. coli correlated significantly in normoxic seawater independent of light availability, and in hypoxic seawater only under light. In hypoxic seawater without light, the concentrations of RNA but not DNA markers correlated with that of E. coli. Generally, the correlations between Enterococcus spp. and Bacteroidales were insignificant. These results suggest that either DNA or RNA markers may complement E. coli in the tiered approach for normoxic or hypoxic seawater under light. When light is absent, either DNA or RNA markers may serve for normoxic seawater, but only the RNA markers are suitable for hypoxic seawater.
Subject(s)
Bacteroidetes/isolation & purification , Enterococcus/isolation & purification , Escherichia coli/isolation & purification , Genetic Markers , Seawater/microbiology , Water Pollution , Animals , Bacteroidetes/genetics , Cattle , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Ribosomal/chemistry , DNA, Ribosomal/genetics , Enterococcus/genetics , Escherichia coli/genetics , Hong Kong , Polymerase Chain Reaction , RNA, Bacterial/genetics , RNA, Ribosomal, 16S/geneticsABSTRACT
BACKGROUND: The peptide hormone relaxin plays a key role in the systemic hemodynamic and renovascular adaptive changes that occur during pregnancy, which is linked to its antiremodelling effects. Serelaxin (a recombinant form of human gene-2 relaxin) has been shown to inhibit lung fibrosis in various disease models and reverse airway remodelling and airway hyperresponsiveness (AHR) in allergic airways disease (AAD). OBJECTIVE: Although continuous systemic delivery of exogenous serelaxin alleviates allergic fibrosis and AHR, more direct routes for administration into the lung have not been investigated. Thus, intranasal administration of serelaxin was evaluated for its ability to reverse airway remodelling and AHR associated with AAD. METHODS: Female Balb/c mice were subjected to a 9-week model of chronic AAD. Subgroups of animals (n = 12/group) were then treated intranasally with serelaxin (0.8 mg/mL) or vehicle once daily for 14 days (from weeks 9-11). Saline-sensitized/challenged mice treated with intranasal saline served as additional controls. Differential bronchoalveolar lavage (BAL) cell counts, ovalbumin (OVA)-specific IgE levels, tissue inflammation, parameters of airway remodelling and AHR were then assessed. RESULTS: Chronic AAD was associated with significant increases in differential BAL cell counts, OVA-specific IgE levels, inflammation, epithelial thickening, goblet cell metaplasia, TGF-ß1 expression, epithelial Smad2 phosphorylation (pSmad2), subepithelial collagen thickness, total lung collagen concentration and AHR (all P < 0.05 vs. respective measurements from saline-treated mice). Daily intranasal delivery of serelaxin significantly diminished AAD-induced epithelial thickening, epithelial pSmad2, subepithelial and total lung collagen content (fibrosis) and AHR (all P < 0.05 vs. vehicle-treated AAD mice). CONCLUSIONS AND CLINICAL RELEVANCE: Intranasal delivery of serelaxin can effectively reduce airway remodelling and AHR, when administered once daily. Respirable preparations of serelaxin may have therapeutic potential for the prevention and/or reversal of established airway remodelling and AHR in asthma.
Subject(s)
Airway Remodeling/drug effects , Recombinant Proteins/administration & dosage , Relaxin/administration & dosage , Respiratory Hypersensitivity/immunology , Respiratory Hypersensitivity/pathology , Administration, Intranasal , Animals , Bronchoalveolar Lavage Fluid/immunology , Disease Models, Animal , Female , Fibrosis , Goblet Cells/pathology , Immunoglobulin E/immunology , Lung/immunology , Lung/pathology , Metaplasia , Mice , Ovalbumin/adverse effects , Ovalbumin/immunology , Respiratory Hypersensitivity/drug therapy , Respiratory Mucosa/immunology , Respiratory Mucosa/pathologyABSTRACT
Tonic γ-aminobutyric acid (GABA)A receptor-mediated signalling controls neuronal network excitability in the hippocampus. Although the extracellular concentration of GABA (e[GABA]) is critical in determining tonic conductances, knowledge on how e[GABA] is regulated by different GABA transporters (GATs) in vivo is limited. Therefore, we studied the role of GATs in the regulation of hippocampal e[GABA] using in vivo microdialysis in freely moving rats. Here we show that GAT-1, which is predominantly presynaptically located, is the major GABA transporter under baseline, quiescent conditions. Furthermore, a significant contribution of GAT-3 in regulating e[GABA] was revealed by administration of the GAT-3 inhibitor SNAP-5114 during simultaneous blockade of GAT-1 by NNC-711. Thus, the GABA transporting activity of GAT-3 (the expression of which is confined to astrocytes) is apparent under conditions in which GAT-1 is blocked. However, sustained neuronal activation by K(+)-induced depolarization caused a profound spillover of GABA into the extrasynaptic space and this increase in e[GABA] was significantly potentiated by sole blockade of GAT-3 (i.e. even when uptake of GAT-1 is intact). Furthermore, experiments using tetrodotoxin to block action potentials revealed that GAT-3 regulates extrasynaptic GABA levels from action potential-independent sources when GAT-1 is blocked. Importantly, changes in e[GABA] resulting from both GAT-1 and GAT-3 inhibition directly precipitate changes in tonic conductances in dentate granule cells as measured by whole-cell patch-clamp recording. Thus, astrocytic GAT-3 contributes to the regulation of e[GABA] in the hippocampus in vivo and may play an important role in controlling the excitability of hippocampal cells when network activity is increased.
Subject(s)
GABA Plasma Membrane Transport Proteins/physiology , Hippocampus/physiology , gamma-Aminobutyric Acid/physiology , Action Potentials , Animals , Astrocytes/physiology , Male , Potassium/physiology , Rats , Rats, Sprague-DawleyABSTRACT
Acute myocardial ischemia and reperfusion injury (IRI) underly the detrimental effects of coronary heart disease on the myocardium. Despite the ongoing advances in reperfusion therapies, there remains a lack of effective therapeutic strategies for preventing IRI. Growth hormone secretagogues (GHS) have been demonstrated to improve cardiac function, attenuate inflammation and modulate the autonomic nervous system (ANS) in models of cardiovascular disease. Recently, we demonstrated a reduction in infarct size after administration of hexarelin (HEX), in a murine model of myocardial infarction. In the present study we employed a reperfused ischemic (IR) model, to determine whether HEX would continue to have a cardioprotective influence in a model of higher clinical relevance. Myocardial ischemia was induced by transient ligation of the left descending coronary artery (tLAD) in C57BL/6 J mice followed by HEX (0.3 mg/kg/day; n = 20) or vehicle (VEH) (n = 18) administration for 21 days, first administered immediately prior-to reperfusion. IR-injured and sham mice were subjected to high-field magnetic resonance imaging to assess left ventricular (LV) function, with HEX-treated mice demonstrating a significant improvement in LV function compared with VEH-treated mice. A significant decrease in interstitial collagen, TGF-ß1 expression and myofibroblast differentiation was also seen in the HEX-treated mice after 21 days. HEX treatment shifted the ANS balance towards a parasympathetic predominance; combined with a significant decrease in cardiac troponin-I and TNF-α levels, these findings were suggestive of an anti-inflammatory action on the myocardium mediated via HEX. In this model of IR, HEX appeared to rebalance the deregulated ANS and activate vagal anti-inflammatory pathways to prevent adverse remodelling and LV dysfunction. There are limited interventions focusing on IRI that have been successful in improving clinical outcome in acute myocardial infarction (AMI) patients, this study provides compelling evidence towards the translational potential of HEX where all others have largely failed.
Subject(s)
Myocardial Infarction/drug therapy , Myocardial Reperfusion Injury/drug therapy , Oligopeptides/pharmacology , Ventricular Function, Left/drug effects , Animals , Anti-Inflammatory Agents/pharmacology , Disease Models, Animal , Inflammation/drug therapy , Inflammation/pathology , Male , Mice , Mice, Inbred C57BL , Myocardial Infarction/physiopathology , Myocardial Reperfusion Injury/physiopathology , Troponin I/metabolism , Ventricular Dysfunction, Left/prevention & controlABSTRACT
We have evaluated the effectiveness of systemic adenovirally delivered mouse relaxin on reversing fibrosis in a transgenic murine model of fibrotic cardiomyopathy due to beta(2)-adrenergic receptor (beta(2)AR) overexpression. Recombinant adenoviruses expressing green fluorescent protein (Ad-GFP), rat relaxin (Ad-rRLN) and mouse relaxin (Ad-mRLN) were generated and Ad-rRLN and Ad-mRLN were demonstrated to direct the expression of bioactive relaxin peptides in vitro. A single systemic injection of Ad-mRLN resulted in transgene expression in the liver and bioactive relaxin peptide in the plasma. Ad-mRLN, but not Ad-GFP, treatment reversed the increased left ventricular collagen content in beta(2)AR mice to control levels without affecting collagen levels in other heart chambers or in the lung and kidney. Hence a single systemic injection of adenovirus producing mouse relaxin reverses cardiac fibrosis without adversely affecting normal collagen levels in other organs and establishes the potential for the use of relaxin gene therapy for the treatment of cardiac fibrosis.
Subject(s)
Adenoviridae/genetics , Cardiomyopathies/therapy , Gene Transfer Techniques , Genetic Therapy/methods , Genetic Vectors , Relaxin/metabolism , Animals , Cardiomyopathies/genetics , Cardiomyopathies/metabolism , Collagen/metabolism , Disease Models, Animal , Feasibility Studies , Female , Fibrosis , Heart Ventricles/metabolism , Liver/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Rats , Receptors, Adrenergic, beta-2/genetics , Receptors, Adrenergic, beta-2/metabolism , Relaxin/blood , Relaxin/geneticsABSTRACT
Fibrosis refers to the hardening or scarring of tissues that usually results from aberrant wound healing in response to organ injury, and its manifestations in various organs have collectively been estimated to contribute to around 45-50% of deaths in the Western world. Despite this, there is currently no effective cure for the tissue structural and functional damage induced by fibrosis-related disorders. Relaxin meets several criteria of an effective anti-fibrotic based on its specific ability to inhibit pro-fibrotic cytokine and/or growth factor-mediated, but not normal/unstimulated, fibroblast proliferation, differentiation and matrix production. Furthermore, relaxin augments matrix degradation through its ability to up-regulate the release and activation of various matrix-degrading matrix metalloproteinases and/or being able to down-regulate tissue inhibitor of metalloproteinase activity. Relaxin can also indirectly suppress fibrosis through its other well-known (anti-inflammatory, antioxidant, anti-hypertrophic, anti-apoptotic, angiogenic, wound healing and vasodilator) properties. This review will outline the organ-specific and general anti-fibrotic significance of exogenously administered relaxin and its mechanisms of action that have been documented in various non-reproductive organs such as the cardiovascular system, kidney, lung, liver, skin and tendons. In addition, it will outline the influence of sex on relaxin's anti-fibrotic actions, highlighting its potential as an emerging anti-fibrotic therapeutic. LINKED ARTICLES: This article is part of a themed section on Recent Progress in the Understanding of Relaxin Family Peptides and their Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.10/issuetoc.
Subject(s)
Fibrosis/drug therapy , Relaxin/pharmacology , Cytokines/antagonists & inhibitors , Cytokines/metabolism , Fibrosis/metabolism , Humans , Intercellular Signaling Peptides and Proteins/metabolism , Relaxin/administration & dosageABSTRACT
Leucine-rich repeat-containing G-protein-coupled receptor 8 (LGR8, or RXFP2) is a member of the type C leucine-rich repeat-containing G protein-coupled receptor family, and its endogenous ligand is insulin-like peptide-3 (INSL3). Although LGR8 expression has been demonstrated in various human tissues, including testis, ovary, brain and kidney, the precise roles of this receptor in many of these tissues are unknown. In an effort to better understand INSL3-LGR8 systems in the rat, we cloned the full-length Lgr8 cDNA and investigated the presence and cellular localization of Lgr8 mRNA expression in adult and developing rat kidney. On the basis of these findings, we investigated the presence and distribution of renal 125I-labelled human INSL3-binding sites and the nature of INSL3-LGR8 signalling in cultured renal cells. Thus, using in situ hybridization histochemistry, cells expressing Lgr8 mRNA were observed in glomeruli of renal cortex from adult rats and were tentatively identified as mesangial cells. Quantitative, real-time PCR analysis of the developmental profile of Lgr8 mRNA expression in kidney revealed highest relative levels at late stage gestation (embryonic day 18), with a sharp decrease after birth and lowest levels in the adult. During development, silver grains associated with Lgr8 mRNA hybridization were observed overlying putative mesangial cells in mature glomeruli, with little or no signal associated with less-mature glomeruli. In adult and developing kidney, specific 125I-INSL3-binding sites were associated with glomeruli throughout the renal cortex. In primary cultures of glomerular cells, synthetic human INSL3 specifically and dose-dependently inhibited cell proliferation over a 48 h period, further suggesting the presence of functional LGR8 (receptors) on these cells (mesangial and others). These findings suggest INSL3-LGR8 signalling may be involved in the genesis and/or developmental maturation of renal glomeruli and possibly in regulating mesangial cell density in adult rat kidney.
Subject(s)
Insulin/metabolism , Kidney Glomerulus/chemistry , Proteins/metabolism , Receptors, G-Protein-Coupled/analysis , Animals , Binding Sites , Cell Division/physiology , Cells, Cultured , Cloning, Molecular/methods , Female , In Situ Hybridization/methods , Kidney/cytology , Kidney Glomerulus/embryology , Kidney Glomerulus/physiology , Male , RNA, Messenger/analysis , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction/methods , Sequence Analysis, DNA/methods , Signal Transduction/physiologyABSTRACT
BACKGROUND AND PURPOSE: We evaluated the extent to which individual versus combination treatments that specifically target airway epithelial damage [trefoil factor-2 (TFF2)], airway fibrosis [serelaxin (RLX)] or airway inflammation [dexamethasone (DEX)] reversed the pathogenesis of chronic allergic airways disease (AAD). EXPERIMENTAL APPROACH: Following induction of ovalbumin (OVA)-induced chronic AAD in 68 week female Balb/c mice, animals were i.p. administered naphthalene (NA) on day 64 to induce epithelial damage, then received daily intranasal administration of RLX (0.8 mg·mL(−1)), TFF2 (0.5 mg·mL(−1)), DEX (0.5 mg·mL(−1)), RLX + TFF2 or RLX + TFF2 + DEX from days 6774. On day 75, lung function was assessed by invasive plethysmography, before lung tissue was isolated for analyses of various measures. The control group was treated with saline + corn oil (vehicle for NA). KEY RESULTS: OVA + NA-injured mice demonstrated significantly increased airway inflammation, airway remodelling (AWR) (epithelial damage/thickness; subepithelial myofibroblast differentiation, extracellular matrix accumulation and fibronectin deposition; total lung collagen concentration), and significantly reduced airway dynamic compliance (cDyn). RLX + TFF2 markedly reversed several measures of OVA + NA-induced AWR and normalized the reduction in cDyn. The combined effects of RLX + TFF2 + DEX significantly reversed peribronchial inflammation score, airway epithelial damage, subepithelial extracellular matrix accumulation/fibronectin deposition and total lung collagen concentration (by 5090%) and also normalized the reduction of cDyn. CONCLUSIONS AND IMPLICATIONS: Combining an epithelial repair factor and anti-fibrotic provides an effective means of treating the AWR and dysfunction associated with AAD/asthma and may act as an effective adjunct therapy to anti-inflammatory corticosteroids
Subject(s)
Asthma/drug therapy , Dexamethasone/therapeutic use , Hypersensitivity/drug therapy , Animals , Asthma/complications , Drug Therapy, Combination , Female , Fibrosis/prevention & control , Hypersensitivity/complications , Mice , Mice, Inbred BALB C , Trefoil Factor-2/drug effectsABSTRACT
BACKGROUND AND PURPOSE: In the phase III clinical trial, RELAX-AHF, serelaxin caused rapid and long-lasting haemodynamic changes. However, the cellular mechanisms involved are unclear in humans. EXPERIMENTAL APPROACH: This study examined the effects of serelaxin in co-cultures of human primary endothelial cells (ECs) and smooth muscle cells (SMCs) on cAMP and cGMP signalling. KEY RESULTS: Stimulation of HUVECs or human coronary artery endothelial cells (HCAECs) with serelaxin, concentration-dependently increased cGMP accumulation in co-cultured SMCs to a greater extent than in monocultures of either cell type. This was not observed in human umbilical artery endothelial cells (HUAECs) that do not express the relaxin receptor, RXFP1. Treatment of ECs with l-N(G) -nitro arginine (NOARG; 30 µM, 30 min) inhibited serelaxin-mediated (30 nM) cGMP accumulation in HUVECs, HCAECs and co-cultured SMCs. In HCAECs, but not HUVECs, pre-incubation with indomethacin (30 µM, 30 min) also inhibited cGMP accumulation in SMCs. Pre-incubation of SMCs with the guanylate cyclase inhibitor ODQ (1 µM, 30 min) had no effect on serelaxin-mediated (30 nM) cGMP accumulation in HUVECs and HCAECs but inhibited cGMP accumulation in SMCs. Serelaxin stimulation of HCAECs, but not HUVECs, increased cAMP accumulation concentration-dependently in SMCs. Pre-incubation of HCAECs with indomethacin, but not l-NOARG, abolished cAMP accumulation in co-cultured SMCs, suggesting involvement of prostanoids. CONCLUSIONS AND IMPLICATIONS: In co-cultures, treatment of ECs with serelaxin caused marked cGMP accumulation in SMCs and with HCAEC also cAMP accumulation. Responses involved EC-derived NO and with HCAEC prostanoid production. Thus, serelaxin differentially modulates vascular tone in different vascular beds.
Subject(s)
Coronary Vessels/cytology , Endothelial Cells/metabolism , Myocytes, Smooth Muscle/metabolism , Relaxin/pharmacology , Umbilical Arteries/cytology , Umbilical Veins/cytology , Coculture Techniques , Cyclic AMP/metabolism , Cyclic GMP/metabolism , Humans , Indomethacin/pharmacology , Nitroarginine/pharmacology , Oxadiazoles/pharmacology , Quinoxalines/pharmacology , Receptors, G-Protein-Coupled/metabolism , Receptors, Peptide/metabolism , Recombinant Proteins/pharmacology , Signal TransductionABSTRACT
BACKGROUND AND PURPOSE: Inflammasomes are multimeric complexes that facilitate caspase-1-mediated processing of the pro-inflammatory cytokines IL-1ß and IL-18. Clinical hypertension is associated with renal inflammation and elevated circulating levels of IL-1ß and IL-18. Therefore, we investigated whether hypertension in mice is associated with increased expression and/or activation of the inflammasome in the kidney, and if inhibition of inflammasome activity reduces BP, markers of renal inflammation and fibrosis. EXPERIMENTAL APPROACH: Wild-type and inflammasome-deficient ASC(-/-) mice were uninephrectomized and received deoxycorticosterone acetate and saline to drink (1K/DOCA/salt). Control mice were uninephrectomized but received a placebo pellet and water. BP was measured by tail cuff; renal expression of inflammasome subunits and inflammatory markers was measured by real-time PCR and immunoblotting; macrophage and collagen accumulation was assessed by immunohistochemistry. KEY RESULTS: 1K/DOCA/salt-induced hypertension in mice was associated with increased renal mRNA expression of inflammasome subunits NLRP3, ASC and pro-caspase-1, and the cytokine, pro-IL-1ß, as well as protein levels of active caspase-1 and mature IL-1ß. Following treatment with 1K/DOCA/salt, ASC(-/-) mice displayed blunted pressor responses and were also protected from increases in renal expression of IL-6, IL-17A, CCL2, ICAM-1 and VCAM-1, and accumulation of macrophages and collagen. Finally, treatment with a novel inflammasome inhibitor, MCC950, reversed hypertension in 1K/DOCA/salt-treated mice. CONCLUSIONS AND IMPLICATIONS: Renal inflammation, fibrosis and elevated BP induced by 1K/DOCA/salt treatment are dependent on inflammasome activity, highlighting the inflammasome/IL-1ß pathway as a potential therapeutic target in hypertension.
Subject(s)
Hypertension/metabolism , Inflammasomes/metabolism , Kidney Diseases/metabolism , Animals , Apoptosis Regulatory Proteins/deficiency , Apoptosis Regulatory Proteins/metabolism , CARD Signaling Adaptor Proteins , Desoxycorticosterone/administration & dosage , Hypertension/chemically induced , Inflammasomes/antagonists & inhibitors , Kidney Diseases/chemically induced , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Salts/administration & dosageABSTRACT
BACKGROUND AND PURPOSE: In a recently conducted phase III clinical trial, RELAX-AHF, serelaxin infusion over 48 h improved short- and long-term clinical outcomes in patients with acute heart failure. In this study we used human primary cells from the umbilical vasculature to better understand the signalling mechanisms activated by serelaxin. EXPERIMENTAL APPROACH: We examined the acute effects of serelaxin on signal transduction mechanisms in primary human umbilical vascular cells and its chronic actions on markers of cardiovascular function and disease. KEY RESULTS: The RXFP1 receptor, the cognate serelaxin receptor, was expressed at the cell surface in HUVECs and human umbilical vein smooth muscle cells (HUVSMCs), human umbilical artery smooth muscle cells (HUASMCs) and human cardiac fibroblasts (HCFs), but not human umbilical artery endothelial cells. In HUVECs and HUVSMCs, serelaxin increased cAMP, cGMP accumulation and pERK1/2, and the concentration-response curves (CRCs) were bell-shaped. Similar bell-shaped CRCs for cGMP and pERK1/2 were observed in HCFs, whereas in HUASMCs, serelaxin increased cAMP, cGMP and pERK1/2 with sigmoidal CRCs. Gαi/o and lipid raft disruption, but not Gαs inhibition, altered the serelaxin CRC for cAMP and cGMP accumulation in HUVSMC but not HUASMC. Longer term serelaxin exposure increased the expression of neuronal NOS, VEGF, ETß receptors and MMPs (gelatinases) in RXFP1 receptor-expressing cells. CONCLUSIONS AND IMPLICATIONS: Serelaxin caused acute and chronic changes in human umbilical vascular cells that were cell background dependent. Bell-shaped CRCs that were observed only in venous cells and fibroblasts involved Gαi/o located within membrane lipid rafts.
Subject(s)
Endothelial Cells/drug effects , Fibroblasts/drug effects , GTP-Binding Proteins/metabolism , Myocytes, Smooth Muscle/drug effects , Relaxin/pharmacology , Cells, Cultured , Dose-Response Relationship, Drug , Endothelial Cells/metabolism , Fibroblasts/metabolism , Humans , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Myocytes, Smooth Muscle/metabolism , Nitric Oxide Synthase Type I/metabolism , Receptor, Endothelin B/metabolism , Receptors, G-Protein-Coupled/metabolism , Receptors, Peptide/metabolism , Recombinant Proteins/pharmacology , Signal Transduction , Umbilical Arteries/cytology , Umbilical Veins/cytology , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Nicotinamide adenine dinucleotide phosphate oxidases (NOX) are enzymes that generate reactive oxygen species (ROS). NOX2 activity in the vascular wall is elevated in hypercholesterolemia, and contributes to oxidative stress and atherogenesis. Here we examined the role of another NOX isoform, NOX1, in atherogenesis in apolipoprotein E-knockout (APOE(-/-)) mice fed a Western diet for 14 weeks. Although NOX1 mRNA expression was unchanged in aortas from APOE(-/-) versus wild-type mice, expression of the NOX1-specific organizer, NOXO1, was diminished, consistent with an overall reduction in NOX1 activity in APOE(-/-) mice. To examine the impact of a further reduction in NOX1 activity, APOE(-/-) mice were crossed with NOX1(-/y) mice to generate NOX1(-/y)/APOE(-/-) double-knockouts. NOX1 deficiency in APOE(-/-) mice was associated with 30-50% higher plasma very-low-density lipoprotein (VLDL)/LDL and triglyceride levels (P < 0.01). Vascular ROS levels were also elevated by twofold in NOX1(-/y)/APOE(-/-) versus APOE(-/-) mice (P < 0.05), despite no changes in expression of other NOX subunits. Although en face analysis of the descending aorta revealed no differences in plaque area between NOX1(-/y)/APOE(-/-) and APOE(-/-) mice, intimal thickening in the aortic sinus was increased by 40% (P < 0.05) in the double-knockouts. Moreover, NOX1 deficiency was associated with a less stable plaque phenotype; aortic sinus lesions contained 60% less collagen (P < 0.01), 40% less smooth muscle (P < 0.01), and 2.5-fold higher levels of matrix metalloproteinase-9 (P < 0.001) than lesions in APOE(-/-) mice. Thus, these data, which suggest a protective role for NOX1 against hyperlipidemia and atherosclerosis in APOE(-/-) mice, highlight the complex and contrasting roles of different NOX isoforms (e.g., NOX2 versus NOX1) in vascular pathology.
Subject(s)
Apolipoproteins E/genetics , Atherosclerosis/genetics , Lipoproteins, LDL/blood , Lipoproteins, VLDL/blood , NADH, NADPH Oxidoreductases/genetics , Triglycerides/blood , Animals , Atherosclerosis/blood , Lipoproteins, LDL/genetics , Lipoproteins, VLDL/genetics , Mice , Mice, Knockout , NADPH Oxidase 1 , Triglycerides/geneticsABSTRACT
The aim of this study was to examine the effects of relaxin on collagen content, solubility, and composition in the rat pubic symphysis. Nonpregnant, female Sprague-Dawley rats were bilaterally ovariectomized and either unprimed or primed with estrogen or progesterone alone, or a combination of estrogen and progesterone. One week later these animals were given increasing doses of a synthetic human (gene-2) relaxin (0-100 micrograms) before being killed 16 h later. Their pubic symphysial tissues were then removed and analyzed for collagen content and solubility, whereas collagen composition was determined by SDS-PAGE. Relaxin administration significantly increased the length (140 +/- 6%) and weight (170 +/- 9%) of the interpubic fibrocartilage in estrogen-primed rats (n = 15). At the same time, it decreased the total collagen content by 68 +/- 6%, without altering the proportions of collagen types, which were predominantly type I (85%) and type II collagen (15%). Relaxin administered alone reduced the total collagen content by 64 +/- 4% but had no effect on collagen solubility or composition. Progesterone abolished the effects of relaxin in estrogen-primed rats. It is concluded that relaxin has a potent effect on the amount of collagen in the rat pubic symphysis that is enhanced by estrogen and antagonized by progesterone. The changes in the extracellular matrix within the pubic symphysis induced by relaxin may be important in the modifications that this tissue undergoes during pregnancy.
Subject(s)
Collagen/drug effects , Estrogens/physiology , Progesterone/physiology , Pubic Symphysis/drug effects , Relaxin/metabolism , Relaxin/pharmacology , Animals , Body Water/metabolism , Cartilage, Articular/metabolism , Collagen/chemistry , Collagen/metabolism , Female , Humans , Ovariectomy , Pubic Symphysis/metabolism , Rats , Rats, Sprague-Dawley , Recombinant Proteins , SolubilityABSTRACT
The aim of this study was to examine the changes in collagen metabolism that occur during pregnancy and parturition and upon relaxin administration to the rat pubic symphysial interpubic tissue. Pubic symphyses were collected from non-pregnant, and intact and ovariectomised pregnant Sprague-Dawley rats at days 15, 18 and 21 of pregnancy as well as during and after delivery, and analysed for collagen content and solubility. SDS-PAGE was used to determine collagen composition. During pregnancy and particularly during birth, there was a significant reduction in both the tissue wet (57+/-3%) and dry (43+/-3%) weight (n=7), which coincided with a significant increase in water content (to 80%) and was attributed to a significant (P<0.05) reduction in overall tissue collagen content (by 47+/-2%). This resulted in both soluble (10%) and insoluble (90%) collagen levels being reduced, but gel electrophoresis demonstrated the presence of types I, II and V collagen in all samples. Western blot analysis confirmed the presence of type II collagen throughout pregnancy, confirming that the rat pubic symphysis remained a fibrocartilaginous tissue throughout gestation. In the absence of the ovaries and hence relaxin, tissue collagen content and solubility were not significantly different from control measurements. However, tissues of ovariectomised animals treated with oestrogen and progesterone (pellets) and relaxin (injection) contained collagen levels that mimicked those of late pregnancy and parturition. These results suggest that relaxin plays an important role in regulating collagen catabolism during gestation in the rat.
Subject(s)
Collagen/metabolism , Labor, Obstetric/metabolism , Pregnancy, Animal/metabolism , Pubic Symphysis/metabolism , Relaxin/pharmacology , Analysis of Variance , Animals , Blotting, Western , Collagen/analysis , Electrophoresis, Polyacrylamide Gel , Estradiol/pharmacology , Female , Ovariectomy , Pregnancy , Progesterone/pharmacology , Pubic Symphysis/chemistry , Pubic Symphysis/drug effects , RatsABSTRACT
The tensile strength of fetal membranes is largely due to their collagen content. In this study we have examined the changes in collagen in the amniotic and allantoic membranes of the sheep over a wide gestational range (27-142 days of gestation; term, 145-150 days). The results have been correlated with volume changes in normal development, and in particular, the changes in allantois have been studied after a rapid and extensive increase in allantoic volume, as a result of maternal dexamethasone treatment (0.76 mg h-1 for 48 h) from Day 60 of gestation. Electron microscopy and immunohistochemistry were used to delineate collagen distribution, and gel electrophoresis was used to assess the relative proportions of each type. In the amnion, collagen content increased from 37 +/- 4% to 50 +/- 1% dry weight of the tissue from 41-102 days and declined slightly thereafter. In the allantois, collagen content increased from 20 +/- 1% at Day 27 to 50 +/- 6% at Day 142, significantly correlated with a volume increase from 25 +/- 3 mL to 813 +/- 274 mL. Collagen types I (>85%), III (10%) and small amounts of types IV and V (<5%) were identified in both membranes at all ages. When allantoic fluid volume was increased rapidly by maternal dexamethasone infusion, there was a significant decrease in collagen content from 38 +/- 6% to 25 +/- 2% (P < 0.05). By immunohistochemistry it was observed that both epithelial cells and fibroblasts were synthesizing collagen.
Subject(s)
Allantois/drug effects , Amnion/drug effects , Collagen/metabolism , Dexamethasone/pharmacology , Allantois/metabolism , Amnion/metabolism , Animals , Body Water , Embryonic and Fetal Development/drug effects , Gestational Age , Immunoenzyme Techniques , Microscopy, Electron , SheepABSTRACT
The gastric ulcerogenic effect of the glucocorticoid, prednisolone, was studied in albino rats. Shay operation in forestomachectomized rats with intact adrenals induced acute gastric ulcers in the corpus, whereas adrenalectomy prevented the development of gastric ulcers in another group of forestomachectomized Shay rats. Only superficial erosion of gastric epithelium was noticed in 66.67% of these rats. Administration of prednisolone to another group of similarly operated rats increased the number and severity of gastric ulceration, whether the adrenals were ablated or otherwise. From the observation of acid and mucous content of the stomach, it is postulated that the reduced mucous secretion may be an important predisposing factor in gastric ulcerogenic effect of steroids.
Subject(s)
Adrenal Glands/physiology , Gastric Mucosa/physiology , Prednisolone/pharmacology , Stomach Ulcer/etiology , Adrenalectomy , Animals , Gastrectomy , Gastric Acid/metabolism , Male , N-Acetylneuraminic Acid , Pepsin A/metabolism , Rats , Sialic Acids/metabolism , Stomach Ulcer/pathologyABSTRACT
To study the specific actions of relaxin through RXFP1 in human cells, it would be advantageous to develop cell populations with permanent RXFP1 knockdown (KD). We have developed and assessed four microRNA against human RXFP1. One of the four designed microRNA displayed significant RXFP1 KD as assessed by reduced relaxin binding when co-transfected with human RXFP1 into HEK-293T cells. The selected microRNA sequence was subsequently retrovirally delivered into the human dermal fibroblast cell line BJ3 which natively expresses RXFP1. The RXFP1 KD BJ3 cells displayed diminished RXFP1 mRNA expression and complete loss of ability of relaxin treatment to reduce collagen deposition after TGF-beta1 stimulation. The retroviral expression of miRNA to successfully silence RXFP1 expression is an invaluable tool to investigate receptor specificity, signalling and possible off-target effects of newly developed relaxin analogs.
Subject(s)
MicroRNAs/genetics , Receptors, G-Protein-Coupled/genetics , Receptors, Peptide/genetics , Retroviridae/genetics , Fibroblasts/cytology , Fibroblasts/physiology , Gene Knockdown Techniques , Genetic Vectors/genetics , HEK293 Cells , HumansABSTRACT
The peptide hormone relaxin is emerging as a multi-functional factor in a broad range of target tissues including several non-reproductive organs, in addition to its historical role as a hormone of pregnancy. This review discusses the evidence that collectively demonstrates the many diverse and vital roles of relaxin: the homeostatic role of endogenous relaxin in mammalian pregnancy and ageing; its gender-related effects; the therapeutic effects of relaxin in the treatment of fibrosis, inflammation, cardioprotection, vasodilation and wound healing (angiogenesis) amongst other pathophysiological conditions, and its potential mechanism of action. Furthermore, translational issues using experimental models (to humans) and its use in various clinical trials, are described, each with important lessons for the design of future trials involving relaxin. The diverse physiological and pathological roles for relaxin have led to the search for its significance in humans and highlight its potential as a drug of the future.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/administration & dosage , Cardiotonic Agents/therapeutic use , Relaxin/physiology , Relaxin/therapeutic use , Vasodilator Agents/therapeutic use , Animals , Clinical Trials as Topic , Homeostasis , Humans , Mice , Rats , Relaxin/chemistry , Signal Transduction , Wound HealingABSTRACT
Fibrosis (organ scarring) is a hallmark of many forms of cardiovascular and renal disease, and causes organ dysfunction and structural changes when normal tissue is replaced with scar tissue; the accumulation of scar tissue being a leading cause of death around the world. Despite deep organ scarring potentially existing in many forms (including myocardial and vascular sclerosis, renal interstitial fibrosis, and glomerulosclerosis), current therapies have only had limited success in delaying end-stage disease. The peptide hormone relaxin is emerging as a potent antifibrotic therapy with rapid-occurring efficacy. Recent studies have demonstrated the antifibrotic actions of relaxin in experimental models of cardiac and renal disease in vivo, and the various levels at which relaxin acts to inhibit fibroblast-induced collagen overproduction leading to fibrosis, in vitro. Separate studies using relaxin gene-knockout mice have demonstrated the significance of endogenous relaxin as a naturally occurring and protective moderator of collagen turnover, while the therapeutic potential of relaxin has been enhanced by its ability to promote vasodilation and renal hyperfiltration. This review will summarize these coherent findings as a means of highlighting the clinical potential of relaxin in cardiovascular and renal disease.