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There is growing interest in the beneficial effects of immune system boosting through the administration of adjuvants, not only in acute infections such as COVID but also in chronic degenerative disorders that are potentially associated with infection. The best-known immunopotentiators are Freund’s complete adjuvant (FCA) and its relative Bacille Calmete–Guérin (BCG), both based onMycobacteriumspecies. The key pathogen-associated molecular patterns (PAMPs) in both FCA and BCG are muramyl dipeptides (MDPs and glucosaminyl-MDP, GMDP). We previously identified the evolutionarily conserved protein Y-box factor YB-1/YBX1 as a primary target for MDP/GMDP. Unlike other host receptors for PAMPs, YB-1 is a diffusible molecule, and we therefore explored whetherin vivoadministration of YB-1, rather than its PAMP ligands, might enhance the immune response to a bacterial antigen and/or influence survival in the face of bacterial infection. We report that mice receiving YB-1 plus GMDPin vivomount a significantly increased B cell response versus GMDP alone against a test antigen (Yersinia pestisV antigen), and that YB-1 administration alone significantly promotes survival in the face of lethal bacterial (Escherichia coli) challengein vivo. Independent confirmation is warranted because recombinant YB-1 and its ligands could hold great promise both as adjuvants and as therapeutics.

Elevated plasma levels of hyaluronic acid (HA) is a disease marker in liver pathology and other inflammatory disorders. Inhibition of HA synthesis with coumarin 4-methylumbelliferone (4MU) has a beneficial effect in animal models of fibrosis, inflammation, cancer and metabolic syndrome. 4MU is an active compound of approved choleretic drug hymecromone with low bioavailability and a broad spectrum of action. New, more specific and efficient inhibitors of hyaluronan synthases (HAS) are required. We have tested several newly synthesized coumarin compounds and commercial chitin synthesis inhibitors to inhibit HA production in cell culture assay. Coumarin derivative compound VII (10′-methyl-6′-phenyl-3′H-spiro[piperidine-4,2′-pyrano[3,2-g]chromene]-4′,8′-dione) demonstrated inhibition of HA secretion by NIH3T3 cells with the half-maximal inhibitory concentration (IC50) = 1.69 ± 0.75 μΜ superior to 4MU (IC50 = 8.68 ± 1.6 μΜ). Inhibitors of chitin synthesis, etoxazole, buprofezin, triflumuron, reduced HA deposition with IC50 of 4.21 ± 3.82 μΜ, 1.24 ± 0.87 μΜ and 1.48 ± 1.44 μΜ, respectively. Etoxazole reduced HA production and prevented collagen fibre formation in the CCl4 liver fibrosis model in mice similar to 4MU. Bioinformatics analysis revealed homology between chitin synthases and HAS enzymes, particularly in the pore-forming domain, containing the proposed site for etoxazole binding.

The Prussian blue (PB) based nanostructure is a mixed-valence coordination network with excellent biosafety, remarkable photothermal effect and multiple enzyme-mimicking behaviours. Compared with other nanomaterials, PB-based nanoparticles (NPs) exhibit several unparalleled advantages in biomedical applications. This review begins with the chemical composition and physicochemical properties of PB-based NPs. The tuning strategies of PB-based NPs and their biomedical properties are systemically demonstrated. Afterwards, the biomedical applications of PB-based NPs are comprehensively recounted, mainly focusing on treatment of tumors, bacterial infection and inflammatory diseases. Finally, the challenges and future prospects of PB-based NPs and their application in disease treatment are discussed.

AAV-delivered microdystrophin genes hold great promise for Duchenne muscular dystrophy (DMD) treatment. It is anticipated that the optimization of engineered dystrophin genes will be required to increase the efficacy and reduce the immunogenicity of transgenic proteins. An in vitro system is required for the efficacy testing of genetically engineered dystrophin genes. We report here on the proof of concept for an in vitro assay based on the assessment of sarcolemma damage after repetitively applied electrical stimuli. The primary cell culture of myoblasts was established from wild-type C57BL/10ScSnJ and dystrophin-deficient mdx mice. The preparation parameters and the differentiation of contractile myotubes were optimized. DAPI and TO-PRO-3 dyes were used to assess myotubular membrane permeability in response to electrical pulse stimulation (EPS). Myotubes derived from mdx mice exhibited a greater increase in membrane damage, as assessed by TO-PRO-3-measured permeability after EPS, than was exhibited by the healthy control myotubes. AAV-DJ particles carrying the microdystrophin gene were used to transduce mdx-derived differentiated myotubes. Microdystrophin delivery ameliorated the disease phenotype and reduced the EPS-induced membrane damage to a level comparable to that of the healthy controls. Thus, the in vitro system was shown to be capable of supporting studies on DMD gene therapy.

4-methylumbelliferone (4MU) is an inhibitor of hyaluronan deposition and an active substance of hymecromone, a choleretic and antispasmodic drug. 4MU reported to be anti-fibrotic in mouse models, however, precise mechanism of action still requires further investigation. Here we describe the cellular and molecular mechanisms of 4MU action on CCl4-induced liver fibrosis in mice using NGS transcriptome, Q-PCR and immunohistochemical analysis. Collagen and hyaluronan deposition were prevented by 4MU. The CCl4 stimulated expression of Col1a and &alpha, SMA were reduced, while the expression of the ECM catabolic gene Hyal1 was increased in the presence of 4MU. Bioinformatic analysis identified an activation of TGF-beta and Wnt/beta-catenin signaling pathways, and inhibition of the genes associated with lipid metabolism by CCL4 treatment, while 4MU restored key markers of these pathways to the control level. Immunohistochemical analysis reveals the suppression of hepatic stellate cells (HSCs) transdifferentiation to myofibroblasts by 4MU treatment. The drug affected the localization of HSCs and macrophages in the sites of fibrogenesis. CCl4 treatment induced the expression of FSTL1, which was downregulated by 4MU. Our results support the hypothesis that 4MU alleviates CCl4-induced liver fibrosis by reducing hyaluronan deposition and downregulating FSTL1 expression, accompanied by the suppression of HSC trans-differentiation and altered macrophage localization.

Experience and changes in neuronal activity can alter CNS myelination, but the signalling pathways responsible remain poorly understood. Here we define a pathway in which endothelin, signalling through the G protein-coupled receptor endothelin receptor B and PKC epsilon, regulates the number of myelin sheaths formed by individual oligodendrocytes in mouse and zebrafish. We show that this phenotype is also observed in the prefrontal cortex of mice following social isolation, and is associated with reduced expression of vascular endothelin. Additionally, we show that increasing endothelin signalling rescues this myelination defect caused by social isolation. Together, these results indicate that the vasculature responds to changes in neuronal activity associated with experience by regulating endothelin levels, which in turn affect the myelinating capacity of oligodendrocytes. This pathway may be employed to couple the metabolic support function of myelin to activity-dependent demand and also represents a novel mechanism for adaptive myelination.

Cortisol is a steroid hormone that regulates a wide range of vital processes. Its level changes with diurnal rhythm and reacts to stress. Measurement of cortisol levels is still a complex multi-step process. A reversible washing-free registration method is required. Here we describe metal-enhanced fluorescence assay based on a displacement of a dye labeled BSA-cortisol conjugate from the immune complex immobilized on the golden islands by free cortisol. This competitive approach allows time-resolved monitoring of the fluorescent signal, surface-enhanced by the gold film, and provides the possibility of continuous real-time cortisol monitoring based on the implantable surface-enhanced immunosensor, which was not demonstrated so far even in vitro.

Long non-coding RNAs constitute the most abundant part of the transcribed mammalian genome. lncRNAs affect all essential processes in the living cell including transcription, splicing, translation, replication, shaping of chromatin and post translational modification of proteins. Alterations in lncRNA expression have been linked to a number of diseases; thus, modulation of lncRNA expression holds a huge potential for gene-based therapy. In this review we summarize published data about lncRNAs in the context of hepatic carcinogenesis and liver fibrosis, and the corresponding potential targets for gene therapy. Recent advancements in targeted delivery to the liver made RNA interference an invaluable tool to decipher hepatic lncRNA function and to develop lncRNA-oriented therapies for liver-involved diseases in the future. Different approaches for RNA delivery that can be used for functional studies in the lab and for clinical lncRNA based applications are critically discussed in this review.

Protein assisted-, DNA hybridized- plasmonic nanostructures for the visible, fractal shell core-less for the extremely broad band VIS-IR and magnetic nanoparticles for the deep UV involved in our efforts on biosensing including cortisol probe.

This review covers the basic aspects of small interfering RNA delivery by lipid nano-particles (LNPs) and elaborates on the current status of clinical trials for these systems. We briefly describe the roles of all LNP components and possible strategies for their improvement. We also focus on the current clinical trials using LNP-formulated RNA and the possible outcomes for therapy in the near future. Also, we present a critical analysis of selected clinical trials that reveals the common logic behind target selection. We address this review to a wide audience, especially to medical doctors who are interested in the application of RNA interference-based treatment platforms. We anticipate that this review may spark interest in this particular audience and generate new ideas in target selection for the disorders they are dealing with.

During the last two decades numerous research teams demonstrated that skeletal muscles function as an exercise-dependent endocrine organ secreting dozens of myokines. Variety of physiological and pathophysiological implications of skeletal muscle myokines secretion has been described; however, upstream signals and sensing mechanisms underlying this phenomenon remain poorly understood. It is well documented that in skeletal muscles intensive exercise triggers dissipation of transmembrane gradient of monovalent cations caused by permanent activation of voltage-gated Na+ and K+ channels. Recently, we demonstrated that sustained elevation of the [Na+]i/[K+]i ratio triggers expression of dozens ubiquitous genes including several canonical myokines, such as interleukin-6 and cyclooxygenase 2, in the presence of intra- and extracellular Ca2+ chelators. These data allowed us to suggest a novel [Na+]i/[K+]i-sensitive, Ca2+i-independent mechanism of excitation-transcription coupling which triggers myokine production. This pathway exists in parallel with canonical signaling mediated by Ca2+i, AMP-activated protein kinase and hypoxia-inducible factor 1α (HIF-1α). In our mini-review we briefly summarize data supporting this hypothesis as well as unresolved issues aiming to forthcoming studies.

Cascade nanoreactors (NRs) which can be activated in tumor microenvironment and catalyze the local substance to toxic species for killing cancer cells is an appealing emerging agent for cancer treatment in recent years. Peroxidase-like nanozymes are often integrated into such kind of systems for cancer therapy. In this study, magnetic metal-organic framework (MOF: zeolitic imidazolate framework 8, ZIF-8) nanoparticles (NPs) with ferrimagnetic structures inside are fabricated via the mineralization of iron during the growth of ZIF-8. The Fe-base magnetic structures can serve as peroxidase-like nanozyme. After the loading of GOx on the magnetic MOF NPs, magnetic cascade MOF/enzyme hybrid NRs (Fe@ZIF-8@GOx NRs) are successfully constructed. In the presence of glucose, the NRs can perform high cascade reaction activity to produce hydroxyl radicals (•OH) under mild acidic condition at physiological temperature. The NRs can be more internalized by cancer cells under a magnetic field and take advantage of acidic environment in lysosomes to produce high toxic •OH by cascade reaction to kill cancer cells. Magnetic targeting to kill cancer cells at the specified location can be achieved by applying a magnetic field to the targeted area and then damage to normal cells will be minimized.

Cells are always living in a dynamic microenvironment and are able to respond to stimuli and modulate their functions. Cell adhesion is the fundamental of almost all cell behaviors, such as cell proliferation, migration, differentiation etc. Therefore, it is essential to provide a platform for studying and regulating cell adhesion in dynamic environment. Owing to the controllability of surface properties, materials with dynamic surface stand out as ideal candidates of this platform. This review summarized the recent advances of design strategies of dynamic surface, and their applications in regulating cell adhesion and related functions.

International audience; The endothelin system has emerged as a novel target for the treatment of diabetic nephropathy. Endothelin-1 promotes mesangial cell proliferation and sclerosis. However, no direct pathogenic effect of endothelin-1 on podocytes has been shown in vivo and endothelin-1 signaling in podocytes has not been investigated. This study investigated endothelin effects in podocytes during experimental diabetic nephropathy. Stimulation of primary mouse podocytes with endothelin-1 elicited rapid calcium transients mediated by endothelin type A receptors (ETARs) and endothelin type B receptors (ETBRs). We then generated mice with a podocyte-specific double deletion of ETAR and ETBR (NPHS2-Cre3Ednra lox/lox 3Ednrb lox/lox [Pod-ETRKO]). In vitro, treatment with endothelin-1 increased total b-catenin and phospho-NF-kB expression in wild-type glomeruli, but this effect was attenuated in Pod-ETRKO glomeruli. After streptozotocin injection to induce diabetes, wild-type mice developed mild diabetic nephropathy with microalbuminuria, mesangial matrix expansion, glomerular basement membrane thickening, and podocyte loss, whereas Pod-ETRKO mice presented less albuminuria and were completely protected from glomerulosclerosis and podocyte loss, even when uninephrectomized. Moreover, glomeruli from normal and diabetic Pod-ETRKO mice expressed substantially less total b-catenin and phospho-NF-kB compared with glomeruli from counterpart wild-type mice. This evidence suggests that endothelin-1 drives development of glomerulosclerosis and podocyte loss through direct activation of endo-thelin receptors and NF-kB and b-catenin pathways in podocytes. Notably, both the expression and function of the ETBR subtype were found to be important. Furthermore, these results indicate that activation of the endothelin-1 pathways selectively in podocytes mediates pathophysiologic crosstalk that influences mesangial architecture and sclerosis.

Steroid/sterol-binding receptors and enzymes are remarkably promiscuous in the range of ligands they can bind to and, in the case of enzymes, modify - raising the question of how specific receptor activation is achieved in vivo. Estrogen receptors (ER) are modulated by 27-hydroxycholesterol and 5α-androstane-3β,17β-diol (Adiol), in addition to estradiol (E2), and respond to diverse small molecules such as bisphenol A. Steroid-modifying enzymes are also highly promiscuous in ligand binding and metabolism. The specificity problem is compounded by the fact that the steroid core (hydrogenated cyclopentophenanthrene ring system) has several planes of symmetry. Ligand binding can be in symmetrical East-West (rotation) and North-South (inversion) orientations. Hydroxysteroid dehydrogenases (HSDs) can modify symmetrical 7 and 11, also 3 and 17/20, positions, exemplified here by yeast 3α,20β-HSD and mammalian 11β-HSD and 17β-HSD enzymes. Faced with promiscuity and symmetry, other strategies are clearly necessary to promote signaling selectivity in vivo. Gating regulates hormone access via enzymes that preferentially inactivate (or activate) a subclass of ligands, thereby governing which ligands gain receptor access - exemplified by 11β-HSD gating cortisol access to the mineralocorticoid receptor, and P450 CYP7B1 gating Adiol access to ER. Counter-intuitively, the specificity of steroid/sterol action is achieved not by intrinsic binding selectivity but by the combination of local metabolism and binding affinity.

After liver injury, regeneration occurs through self-replication of hepatocytes. In severe liver injury, hepatocyte proliferation is impaired—a feature of human chronic liver disease. It is unclear whether other liver cell types can regenerate hepatocytes. Here we use two independent systems to impair hepatocyte proliferation during liver injury to evaluate the contribution of non-hepatocytes to parenchymal regeneration. First, loss of β1-integrin in hepatocytes with liver injury triggered a ductular reaction of cholangiocyte origin, with approximately 25% of hepatocytes being derived from a non-hepatocyte origin. Second, cholangiocytes were lineage traced with concurrent inhibition of hepatocyte proliferation by β1-integrin knockdown or p21 overexpression, resulting in the significant emergence of cholangiocyte-derived hepatocytes. We describe a model of combined liver injury and inhibition of hepatocyte proliferation that causes physiologically significant levels of regeneration of functional hepatocytes from biliary cells.

Aims The potent vasoconstrictor endothelin-1 (ET-1), acting on the endothelin-A (ETA) receptor, promotes intimal lesion formation following vascular injury. The endothelin-B (ETB) receptor, which mediates nitric oxide release and ET-1 clearance in endothelial cells, may moderate lesion formation, but this is less clear. We used selective ET receptor antagonists and cell-specific deletion to address the hypothesis that ETB receptors in the endothelium inhibit lesion formation following arterial injury. Methods and results Neointimal proliferation was induced by wire or ligation injury to the femoral artery in mice treated with selective ETA (ABT-627) and/or ETB antagonists (A192621). Measurement of lesion formation by optical projection tomography and histology indicated that ETA blockade reduced lesion burden in both models. Although ETB blockade had little effect on ligation injury-induced lesion formation, after wire injury, blockade of the ETB receptor increased lesion burden (184% of vehicle; P < 0.05) and reversed the protective effects of an ETA antagonist. Selective deletion of ETB receptors from the endothelium, however, had no effect on neointimal lesion size. Conclusion These results are consistent with ETB receptor activation playing an important role in limiting neointimal lesion formation following acute vascular injury, but indicate that this protective effect is not mediated by those ETB receptors expressed by endothelial cells. These data support the proposal that selective ETA antagonists may be preferable to mixed ETA/ETB antagonists for targeting the arterial response to injury.

Salt sensitivity of blood pressure is an independent risk factor for cardiovascular morbidity. Mechanistically, abnormal mineralocorticoid action and subclinical renal impairment may blunt the natriuretic response to high sodium intake, causing blood pressure to rise. 11β-Hydroxysteroid dehydrogenase type 2 (11βHSD2) controls ligand access to the mineralocorticoid receptor, and ablation of the enzyme causes severe hypertension. Polymorphisms in HSD11B2 are associated with salt sensitivity of blood pressure in normotensives. In this study, we used mice heterozygote for a null mutation in Hsd11b2 ( Hsd11b2 +/− ) to define the mechanisms linking reduced enzyme activity to salt sensitivity of blood pressure. A high-sodium diet caused a rapid and sustained increase in blood pressure in Hsd11b2 +/− mice but not in wild-type littermates. During the adaptation to high-sodium diet, heterozygotes displayed impaired sodium excretion, a transient positive sodium balance, and hypokalemia. After 21 days of high-sodium feeding, Hsd11b2 +/− mice had an increased heart weight. Mineralocorticoid receptor antagonism partially prevented the increase in heart weight but not the increase in blood pressure. Glucocorticoid receptor antagonism prevented the rise in blood pressure. In Hsd11b2 +/− mice, high-sodium feeding caused suppression of aldosterone and a moderate but sustained increase in corticosterone. This study demonstrates an inverse relationship among 11βHSD2 activity, heart weight, and blood pressure in a clinically important context. Reduced activity causes salt sensitivity of blood pressure, but this does not reflect illicit activation of mineralocorticoid receptors by glucocorticoids. Instead, we have identified a novel interaction among 11βHSD2, dietary salt, and circulating glucocorticoids.

Inactivation of endothelin B receptors (ETB), either through selective pharmacological antagonism or genetic mutation, increases the circulating concentration of endothelin-1 (ET-1), suggesting ETB plays an important role in clearance of this peptide. However, the cellular site of ETB-mediated clearance has not yet been determined. We have used a novel mouse model of endothelial cell-specific knockout (KO) of ETB (EC ETB–/–) to evaluate the relative contribution of EC-ETB to the clearance of ET-1. Phenotypic evidence of EC-specific ETB KO was confirmed by immunocytochemistry and autoradiography. Binding of the radiolabelled selective ETB ligand BQ3020 was significantly and selectively decreased in EC-rich tissues of EC ETB–/– mice, including the lung, liver, and kidney. By contrast, ETA binding was unaltered. RT-PCR confirmed equal expression of ET-1 in tissue from EC ETB–/– mice and controls, despite increased concentration of plasma ET-1 in EC ETB–/–. Clearance of an intravenous bolus of [125I]ET-1 was impaired in EC ETB–/– mice. Pretreatment with the selective ETB antagonist A192621 impaired [125I]ET-1 clearance in control animals to a similar extent, but did not further impair clearance in EC ETB–/– mice. These studies suggest that EC-ETB are largely responsible for the clearance of ET-1 from the circulation.

Nature 547, 350–354 (2017); doi:10.1038/nature23015 In Extended Data Fig. 10b of this Letter, the axes of the single-cell gating (middle panel) FACS plots were mislabelled. Single cells were gated using forward scatter area (FSC-A) against height (FSC-H) on a linear scale, instead of side scatter area (SSC-A) against height (SSC-H) on a log scale.

Background: We hypothesised that the potential protective effects of endothelial ETB are important in limiting pulmonary vascular muscularisation, vasoconstriction and the development of pulmonary arterial hypertension in response to hypoxia. Methods: EC-specific ETB knockout mice (EC ETB–/–) and control mice (ETBf/f) were subjected to hypobaric hypoxic (10% FiO2) or normoxic conditions for 14 days before assessment of right ventricular pressure and pulmonary vascular morphology and function. Results: During normoxia, no difference in right ventricular pressure was detected between EC ETB–/– (23.7 ± 1.7 mm Hg) and ETBf/f mice (20.2 ± 1.5 mm Hg). Hypoxia induced an exaggerated increase in right ventricular pressure in EC ETB–/– mice (34.4 ± 1.2 mm Hg vs. 24.6 ± 1.4 mm Hg), accompanied by an increase in right ventricular mass. No effect was observed in ETBf/f mice. Endothelin-1 constricted pulmonary arteries from both groups, although maximum response was similar irrespective of inspired oxygen or genotype. Hypoxia increased the percentage of muscularised vessels in both groups of mice, but the percentage increase was significantly greater in EC ETB–/– mice. Conclusions: The potential protective effects of endothelial ETB are important in limiting pulmonary vascular muscularisation and the development of pulmonary arterial hypertension in response to hypoxia.

The bacterial cell wall muramyl dipeptides MDP and glucosaminyl-MDP (GMDP) are powerful immunostimulators but their binding target remains controversial. We previously reported expression cloning of GMDP-binding polypeptides and identification of Y-box protein 1 (YB-1) as their sole target. Here we show specific binding of GMDP to recombinant YB-1 protein and subcellular colocalization of YB-1 and GMDP. GMDP binding to YB-1 upregulated gene expression levels of NF-κB2, a mediator of innate immunity. Furthermore, YB-1 knockdown abolished GMDP-induced Nfkb2 expression. GMDP/YB-1 stimulation led to NF-κB2 cleavage, transport of activated NF-κB2 p52 to the nucleus, and upregulation of NF-κB2-dependent chemokine Cxcr4 gene expression. Therefore, our findings identify YB-1 as new target for muramyl peptide signaling.

Endothelin B receptors in different tissues regulate diverse physiological responses including vasoconstriction, vasodilatation, clearance of endothelin-1, and renal tubular sodium reabsorption. To examine the role of endothelial cell endothelin B receptors in these processes, we generated endothelial cell-specific endothelin B receptor knockout mice using a Cre- loxP approach. We have demonstrated loss of endothelial cell endothelin B receptor expression and function and preservation of nonendothelial endothelin B receptor-mediated responses through binding and functional assays. Ablation of endothelin B receptors exclusively from endothelial cells produces endothelial dysfunction in the absence of hypertension, with evidence of decreased endogenous release of NO and increased plasma endothelin-1. In contrast to models of total endothelin B receptor ablation, the blood pressure response to a high-salt diet is unchanged in endothelial cell–specific endothelin B receptor knockouts compared with control floxed mice. These findings suggest that the endothelial cell endothelin B receptor mediates a tonic vasodilator effect and that nonendothelial cell endothelin B receptors are important for the regulation of blood pressure.

The number of cardiovascular publications featuring gene expression profiling technologies is growing rapidly. This article introduces four profiling techniques; serial analysis of gene expression, differential display, subtractive hybridisation and DNA microarrays. Illustrations of their application towards cardiovascular research are given and their potential for gene discovery and improving our understanding of gene function is discussed.

Background Aging is accompanied by increasing vulnerability to pathologies such as atherosclerosis (ATH) and Alzheimer disease (AD). Are these different pathologies, or different presentations with a similar underlying pathoetiology? Discussion Both ATH and AD involve inflammation, macrophage infiltration, and occlusion of the vasculature. Allelic variants in common genes including APOE predispose to both diseases. In both there is strong evidence of disease association with viral and bacterial pathogens including herpes simplex and Chlamydophila. Furthermore, ablation of components of the immune system (or of bone marrow-derived macrophages alone) in animal models restricts disease development in both cases, arguing that both are accentuated by inflammatory/immune pathways. We discuss that amyloid β, a distinguishing feature of AD, also plays a key role in ATH. Several drugs, at least in mouse models, are effective in preventing the development of both ATH and AD. Given similar age-dependence, genetic underpinnings, involvement of the vasculature, association with infection, Aβ involvement, the central role of macrophages, and drug overlap, we conclude that the two conditions reflect different manifestations of a common pathoetiology. Mechanism Infection and inflammation selectively induce the expression of cholesterol 25-hydroxylase (CH25H). Acutely, the production of ‘immunosterol’ 25-hydroxycholesterol (25OHC) defends against enveloped viruses. We present evidence that chronic macrophage CH25H upregulation leads to catalyzed esterification of sterols via 25OHC-driven allosteric activation of ACAT (acyl-CoA cholesterol acyltransferase/SOAT), intracellular accumulation of cholesteryl esters and lipid droplets, vascular occlusion, and overt disease. Summary We postulate that AD and ATH are both caused by chronic immunologic challenge that induces CH25H expression and protection against particular infectious agents, but at the expense of longer-term pathology.

A novel inbred rat model with inducible hypertension has been generated using a renin transgene under the transcriptional control of the cytochrome P450, Cyp1a1 promoter. The degree and duration of hypertension are regulated tightly by administration of the natural xenobiotic indole-3 carbinol and can be readily reversed. Induction experiments reveal distinct temporal and mechanistic responses to hypertensive injury in different vascular beds, which is indicative of differential susceptibility of organs to a hypertensive stimulus. The mesentery and heart exhibited the greatest sensitivity to damage, and the kidney showed an adaptive response prior to the development of malignant hypertensive injury. Quantitative analysis of morphological changes induced in mesenteric resistance arteries suggest eutrophic remodeling of the vessels. Kinetic evidence suggests that locally activated plasma prorenin may play a critical role in mediating vascular injury. This model will facilitate studies of the cellular and genetic mechanisms underlying vascular injury and repair and provide a basis for the identification of novel therapeutic targets for vascular disease.

Time for primary review 26 days. There is a widely held view that function of the kidney is the key to understanding human essential hypertension. Indeed, all of the currently recognised human monogenic causes of hypertension reflect abnormalities in the genes that determine ion transport in the distal portion of the nephron [1]. It is also recognised that permanent elevation of blood pressure in experimental models of hypertension, and both primary and secondary hypertension in man, requires the shifting of the natriuretic response curve of the kidney towards higher pressures [2]. However, the mechanism underlying pressure-natriuresis, and its resetting in hypertension, remains poorly understood. In this review we have analysed experiments indicating a role for endothelin (ET)-1 as a key natriuretic hormone and propose a new and testable hypothesis for a paracrine action of ET-1 through NO that accommodates the existing experimental findings. ET-1 is a 21 amino acid peptide, synthesised mainly by endothelial cells (ECs) and produced from an inactive precursor peptide by several rounds of specific proteolysis. The last step is performed by endothelin converting enzyme (ECE), which releases ET-1 from big ET-1. ET-1 acts in an autocrine and paracrine fashion on two subtypes of ET receptors, termed the ETA (ETAR) and ETB receptors (ETBR). Both ETAR and ETBR are located on vascular smooth muscle cells and binding of ET-1 to these receptors results in sustained vasoconstriction. ETBR are also present on ECs where their activation results in the production of NO and dilator prostanoids, and subsequent vasodilatation. The ET system is now known to play an important role in the regulation of vascular tone and blood pressure [3,4]. However, the role of ET-1 produced and secreted in the kidney may be independent of the systemic vascular effects. Analysis of different tissues found the highest … *Corresponding author. Tel.: +44-131-651-11-94; fax: +44-131-650-65-27 Yuri.Kotelevtsev{at}ed.ac.uk

11β-hydroxysteroid dehydrogenase type 1 (11β-HSD-1) intracellularly regenerates active corticosterone from circulating inert 11-dehydrocorticosterone (11-DHC) in specific tissues. The hippocampus is a brain structure particularly vulnerable to glucocorticoid neurotoxicity with aging. In intact hippocampal cells in culture, 11β-HSD-1 acts as a functional 11β-reductase reactivating inert 11-DHC to corticosterone, thereby potentiating kainate neurotoxicity. We examined the functional significance of 11β-HSD-1 in the central nervous system by using knockout mice. Aged wild-type mice developed elevated plasma corticosterone levels that correlated with learning deficits in the watermaze. In contrast, despite elevated plasma corticosterone levels throughout life, this glucocorticoid-associated learning deficit was ameliorated in aged 11β-HSD-1 knockout mice, implicating lower intraneuronal corticosterone levels through lack of 11-DHC reactivation. Indeed, aged knockout mice showed significantly lower hippocampal tissue corticosterone levels than wild-type controls. These findings demonstrate that tissue corticosterone levels do not merely reflect plasma levels and appear to play a more important role in hippocampal functions than circulating blood levels. The data emphasize the crucial importance of local enzymes in determining intracellular glucocorticoid activity. Selective 11β-HSD-1 inhibitors may protect against hippocampal function decline with age.

11β-Hydroxysteroid dehydrogenases (11β-HSDs) catalyze interconversion of active corticosterone and inert 11-dehydrocorticosterone, thus regulating glucocorticoid access to intracellular receptors in vivo. 11β-HSD type 1 is a reductase, locally regenerating active glucocorticoids. To explore the role of this isozyme in the brain, we examined hypothalamic-pituitary-adrenal axis (HPA) regulation in mice homozygous for a targeted disruption of the 11β-HSD-1 gene. 11β-HSD-1-deficient mice showed elevated plasma corticosterone and ACTH levels at the diurnal nadir, with a prolonged corticosterone peak, suggesting abnormal HPA control and enhanced circadian HPA drive. Despite elevated corticosterone levels, several hippocampal and hypothalamic glucocorticoid-sensitive messenger RNAs were normally expressed in 11β-HSD-1-deficient mice, implying reduced effective glucocorticoid activity within neurons. 11β-HSD-1-deficient mice showed exaggerated ACTH and corticosterone responses to restraint stress, with a delayed fall after stress, suggesting diminished glucocorticoid feedback. Indeed, 11β-HSD-1-deficient mice were less sensitive to exogenous cortisol suppression of HPA activation. Thus 11β-HSD-1 amplifies glucocorticoid feedback on the HPA axis and is an important regulator of neuronal glucocorticoid exposure under both basal and stress conditions in vivo.

This Special Issue on the topic of Steroid and Sterol Signaling: Promiscuity and Diversity , dwells on the growing realization that the ‘one ligand, one binding site’ and ‘one enzyme, one reaction’ concepts are out of date. Focusing on cytochromes P450 (CYP), hydroxysteroid dehydrogenases (HSDs), and related enzymes, the Special Issue highlights that a single enzyme can bind to diverse substrates, and in different conformations, and can catalyze multiple different conversions (and in different directions), thereby, generating an unexpectedly wide spectrum of ligands that can have subtly different biological actions. This article is part of a Special Issue entitled ‘Steroid/Sterol Signaling’ .

Glucocorticoid hormones, acting via nuclear receptors, regulate many metabolic processes, including hepatic gluconeogenesis. It recently has been recognized that intracellular glucocorticoid concentrations are determined not only by plasma hormone levels, but also by intracellular 11β-hydroxysteroid dehydrogenases (11β-HSDs), which interconvert active corticosterone (cortisol in humans) and inert 11-dehydrocorticosterone (cortisone in humans). 11β-HSD type 2, a dehydrogenase, thus excludes glucocorticoids from otherwise nonselective mineralocorticoid receptors in the kidney. Recent data suggest the type 1 isozyme (11β-HSD-1) may function as an 11β-reductase, regenerating active glucocorticoids from circulating inert 11-keto forms in specific tissues, notably the liver. To examine the importance of this enzyme isoform in vivo , mice were produced with targeted disruption of the 11β-HSD-1 gene. These mice were unable to convert inert 11-dehydrocorticosterone to corticosterone in vivo . Despite compensatory adrenal hyperplasia and increased adrenal secretion of corticosterone, on starvation homozygous mutants had attenuated activation of the key hepatic gluconeogenic enzymes glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, presumably, because of relative intrahepatic glucocorticoid deficiency. The 11β-HSD-1 −/− mice were found to resist hyperglycamia provoked by obesity or stress. Attenuation of hepatic 11β-HSD-1 may provide a novel approach to the regulation of gluconeogenesis.

By inactivating potent glucocorticoid hormones (cortisol and corticosterone), 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta-HSD2) plays an important role in the placenta by controlling fetal exposure to maternal glucocorticoids, and in aldosterone target tissues by controlling ligand access to co-localized glucocorticoid and mineralocorticoid receptors. Amino acid sequence from homogeneous human placental 11 beta-HSD2 was used to isolate a 1897 bp cDNA encoding this enzyme (predicted M(r) 44126; predicted pI 9.9). Transfection into mammalian (CHO) cells produces 11 beta-HSD2 activity which is NAD(+)-dependent, is without reductase activity, avidly metabolizes glucocorticoids (Km values for corticosterone, cortisol and dexamethasone of 12.4 +/- 1.5, 43.9 +/- 8.5 and 119 +/- 15 nM respectively) and is inhibited by glycyrrhetinic acid and carbenoxolone (IC50 values 10-20 nM). Rabbit antisera recognizing 11 beta-HSD2 have been raised to an 11 beta-HSD2-(370--383)-peptide-carrier conjugate. Recombinant 11 beta-HSD2, like native human placental 11 beta-HSD2, is detectable with affinity labelling and anti-11 beta-HSD2 antisera, and appears to require little post-translational processing for activity. 11 beta-HSD2 mRNA (approximately 1.9 kb transcript) is expressed in placenta, aldosterone target tissues (kidney, parotid, colon and skin) and pancreas. In situ hybridization and immunohistochemistry localize abundant 11 beta-HSD2 expression to the distal nephron in human adult kidney and to the trophoblast in the placenta. 11 beta-HSD2 transcripts are expressed in fetal kidney (but not lung, liver or brain) at 21-26 weeks, suggesting that an 11 beta-HSD2 distribution resembling that in the adult is established by this stage in human development.

The use of reporter genes such as beta-galactosidase and green fluorescent protein is a powerful molecular tool for the visualization of in-vivo gene expression. In this paper, we describe the generation of mice that possess a beta-galactosidase reporter gene introduced into the endothelin-B receptor locus by homologous recombination in embryonic stem cells. These mice express beta- galactosidase wherever endothelin-B is expressed and therefore provide a precise in-vivo localization profile of endothelin-B expression.

Most of the known effects of aldosterone are mediated by the mineralocorticoid receptor, an intracellular receptor belonging to the steroid/thyroid hormone/retinoic acid receptor superfamily. We determined the genomic structure of the human MR (hMR) and identified 10 exons in the gene, including two exons (1 alpha and 1 beta) that encode different 5'-untranslated sequence. Expression of the two different hMR variants is under the control of two different promoters that contain no obvious TATA element, but multiple GC boxes. Our results indicate that hMR expression is regulated by alternative promoters perhaps in a tissue- or developmental-specific manner.

A genetic model of malignant phase hypertension in rats. A genetic model of malignant phase hypertension in rats is described which closely parallels the natural history of untreated human malignant phase hypertension. Although the factors initiating transition from essential hypertension to the accelerated phase in humans remain unknown, we report the characteristics of a genetically determined and reproducible phenotype which was found to result from a cross between hypertensive transgenic Ren-2 rats and normotensive Sprague-Dawley (Edinburgh) rats. Male F1 hybrids developed malignant phase hypertension with a penetrance of 73.5% (95% confidence limits 65.7 to 81.3%) by 100 days of age. Phenotypic features included an accelerated rise in blood pressure, fibrinoid necrosis, activation of the renal renin-angiotensin system and microangiopathic hemolytic anemia. In an analytical cross no significant difference in blood pressure was observed between malignant phase and non-malignant phase animals prior to transition, implying that a factor in addition to hypertension appears necessary for inducing transition to the malignant phase phenotype. Segregation of the malignant phenotype suggested that susceptibility is determined by at most two genetic loci.

Several genetic loci involved in blood pressure regulation have recently been localized in experimental models of hypertension, but the manner in which they influence blood pressure remains unknown. Here, we report a study of the Lyon hypertensive rat strain showing that different loci are involved in the regulation of steady-state (diastolic pressure) and pulsatile (systolic – diastolic, or pulse pressure) components of blood pressure. Significant linkage was established between diastolic blood pressure and a microsatellite marker of the renin gene (REN) on rat chromosome 13, and between pulse pressure and the carboxypeptidase B gene (CPB) on chromosome 2. These findings show that two independent loci influence different haemodynamic components of blood pressure, and that pulse pressure has a specific genetic determination.

Mineralocorticoid receptor (MR) activation is proinflammatory and proatherogenic. Antagonism of MR improves survival in humans with congestive heart failure caused by atherosclerotic disease. In animal models, activation of MR exacerbates atherosclerosis. The enzyme 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) prevents inappropriate activation of the MR by inactivating glucocorticoids in mineralocorticoid-target tissues. To determine whether glucocorticoid-mediated activation of MR increases atheromatous plaque formation, we generated Apoe−/−/11β-HSD2−/− double-knockout (E/b2) mice. On chow diet, E/b2 mice developed atherosclerotic lesions by 3 months of age, whereas Apolipoprotein E (Apoe−/−) mice remained lesion free. Brachiocephalic plaques in 3-month-old E/b2 mice showed increased macrophage and lipid content and reduced collagen content compared with similar sized brachiocephalic plaques in 6-month-old Apoe−/− mice. Crucially, treatment of E/b2 mice with eplerenone, an MR antagonist, reduced plaque development and macrophage infiltration while increasing collagen and smooth muscle cell content without any effect on systolic blood pressure. In contrast, reduction of systolic blood pressure in E/b2 mice using the epithelial sodium channel blocker amiloride produced a less-profound atheroprotective effect. Vascular cell adhesion molecule 1 expression was increased in the endothelium of E/b2 mice compared with Apoe−/− mice. Similarly, aldosterone increased vascular cell adhesion molecule 1 expression in mouse aortic endothelial cells, an effect mimicked by corticosterone only in the presence of an 11β-HSD2 inhibitor. Thus, loss of 11β-HSD2 leads to striking atherogenesis associated with activation of MR, stimulating proinflammatory processes in the endothelium of E/b2 mice.

This study characterized the actions of the newly synthesized PAF precursor 1-hexadecyl-2-alkylcarbamoyl-glycerol (HAG) on blood pressure (BP) in male spontaneously hypertensive rats (SHR), SHR-stroke prone (SHRSP) and Wistar rats with 1-kidney 1-clip (1K1C) renovascular hypertension used as experimental models of human primary and secondary hypertension. Systolic blood pressure (SBP) in the tail artery and mean arterial pressure (MAP) in the abdominal aorta were measured by tail plethysmography and invasive pressure transducer, respectively. Intravenous treatment with 1mg/kg HAG in SHR resulted in a rapid decline of MAP from 151±4 to 127±4mmHg in 50min (p0.001) that was maintained for 24h after injection (128±5mmHg, p0.01). We also observed a profound hypotensive effect of HAG in SHRSP but not in normotensive Wistar rats. In 1K1C rats, the magnitude of the BP decline evoked by HAG was correlated with MAP measured before drug administration (R=0.74, p0.005). In 1K1C rats with SBP140mmHg, 5mg/kg/48h HAG, given orally for 14 days, decreased SBP by 20-30mmHg without an increase in the death rate and other adverse effects. Thus, our results show that intravenous and oral administration of HAG led to a long-lasting reduction of BP in experimental models of primary and secondary hypertension. In contrast to PAF and its derivatives, the hypotensive action of HAG was preserved for 24h after a single administration, was absent in normotensive animals, and was not accompanied by visible side-effects, at least during 2 weeks of treatment.

Knockout (KO) models have provided important insights into the function of many receptors and signalling molecules. However, analysis of endothelin (ET) receptor knockouts has been complicated by the development of lethal phenotypes. In this paper, we present our strategy for examining endothelin-B- (ET(B)) receptor function in the context of other strategies for rescuing the lethal phenotype of ET(B) knockout mice.

The collecting duct (CD) endothelin (ET) system regulates blood pressure (BP) and Na excretion. CD-specific knockout (KO) of ET-1 causes hypertension, CD-specific KO of the ETA receptor does not alter BP, while CD-specific KO of the ETB receptor increases BP to a lesser extent than CD ET-1 KO. These findings suggest a paracrine role for CD-derived ET-1; however, they do not exclude compensation for the loss of one ET receptor by the other. To examine this, mice with CD-specific KO of both ETA and ETB receptors were generated (CD ETA/B KO). CD ETA/B KO mice excreted less urinary Na than controls during acute or chronic Na loading. Urinary aldosterone excretion and plasma renin concentration were similar during Na intake and both fell comparably during Na loading. On a normal sodium diet, CD ETA/B KO mice had increased BP, which increased further with high salt intake. The degree of BP elevation during normal Na intake was similar to CD ET-1 KO mice and higher than CD ETB KO animals. During 1 wk of Na loading, CD ETA/B KO mice had higher BPs than CD ETB KO, while BP was less than CD ET-1 KOs until the latter days of Na loading. These studies suggest that 1) CD ETA/B deficiency causes salt-sensitive hypertension, 2) CD ETA/B KO-associated Na retention is associated with failure to suppress the renin-angiotensin-aldosterone system, and 3) CD ETA and ETB receptors exerts a combined hypotensive effect that exceeds that of either receptor alone.

Collecting duct (CD)-derived endothelin-1 (ET-1) inhibits renal Na reabsorption and its deficiency increases blood pressure (BP). The role of CD endothelin B (ETB) receptors in mediating these effects is unknown. CD-specific knockout of the ETB receptor was achieved using an aquaporin-2 promoter-Cre recombinase transgene and the loxP-flanked ETB receptor gene (CD ETB KO). Systolic BP in mice with CD-specific knockout of the ETB receptor, ETA receptor (CD ETA KO) and ET-1 (CD ET-1 KO), and their respective controls were compared during normal- and high-salt diet. On a normal-sodium diet, CD ETB KO mice had elevated BP, which increased further during high salt feeding. However, the degree of hypertension in CD ETB KO mice and the further increase in BP during salt feeding were lower than that of CD ET-1 KO mice, whereas CD ETA KO mice were normotensive. CD ETB KO mice had impaired sodium excretion following acute sodium loading. Aldosterone and plasma renin activity were decreased in CD ETB KO mice on normal- and high-sodium diets, while plasma and urinary ET-1 levels did not differ from controls. In conclusion, the CD ETB receptor partially mediates the antihypertensive and natriuretic effects of ET-1. CD ETA and ETB receptors do not fully account for the antihypertensive and natriuretic effects of CD-derived ET-1, suggesting paracrine effects of this peptide.

The resolution of inflammation is dependent on recognition and phagocytic removal of apoptotic cells by macrophages. Receptors for apoptotic cells are sensitive to degradation by human neutrophil elastase (HNE). We show in the present study that HNE cleaves macrophage cell surface CD14 and in so doing, reduces phagocytic recognition of apoptotic lymphocytic cells (Mutu 1). Using an improved method of adenovirus-mediated transfection of macrophages with the HNE inhibitor elafin, we demonstrate that elafin overexpression prevents CD14 cleavage and restores apoptotic cell recognition by macrophages. This approach of genetic modification of macrophages could be used to restore apoptotic cell recognition in inflammatory conditions.

Atherosclerosis is a chronic inflammatory disease affecting arterial vessels. Strategies to reduce the inflammatory responses of endothelial cells and macrophages may slow lesion development and prevent complications such as plaque rupture. The human protease human neutrophil elastase (HNE), oxidized low density lipoprotein, LPS, and TNF-α were chosen as model stimuli of arterial wall inflammation and led to production of the chemokine IL-8 in endothelial cells. To counteract the activity of HNE, we have examined the effects of adenoviral gene delivery of the anti-elastases elafin, previously demonstrated within human atheroma, and murine secretory leukocyte protease inhibitor (SLPI), a related molecule, on the inflammatory responses of human endothelial cells and macrophages to atherogenic stimuli. We developed a technique of precomplexing adenovirus with cationic lipid to augment adenoviral infection efficiency in endothelial cells and to facilitate infection in macrophages. Elafin overexpression protected endothelial cells from HNE-induced IL-8 production and cytotoxicity. Elafin and murine SLPI also reduced endothelial IL-8 release in response to oxidized low density lipoprotein, LPS, and TNF-α and macrophage TNF-α production in response to LPS. This effect was associated with reduced activation of the inflammatory transcription factor NF-κB, through up-regulation of IκBα, in both cell types. Our work suggests a novel and extended anti-inflammatory role for these HNE inhibitors working as effectors of innate immunity to protect tissues against maladaptive inflammatory responses. Our findings indicate that elafin and SLPI may be gene therapy targets for the treatment of atheroma.

Glucocorticoids affect a wide range of processes in the brain, altering neurotransmission, electrophysiological activity, metabolism, cell division, and death. These actions are mediated by corticosteroid receptors (glucocorticoid and mineralocorticoid) that modify transcriptional activity of target genes. The amount of steroid available to activate these receptors is not only dependent on the circulating levels but also on pre-receptor metabolism of glucocorticoids occurring intracellularly. This metabolism is carried out by the enzymes 11beta-hydroxysteroid dehydrogenases (11beta-HSDs). There are two distinct isozymes, the products of distantly related genes. 11beta-HSD type 2 inactivates glucocorticoids to its inert 11-keto derivative, while 11alpha-HSD type 1 elevates intracellular glucocorticoid levels by regenerating active glucocorticoids from circulating 11-dehydrocorticosterone or cortisone. This review highlights the important and very different roles the two enzymes play in the brain, outlining recent results obtained from studying mice with a targeted gene deletion in the 11beta-HSD1 or 11beta-HSD2 genes.