Conditional KCa3.1-transgene induction in murine skin produces pruritic eczematous dermatitis with severe epidermal hyperplasia and hyperkeratosis.

Ion channels have recently attracted attention as potential mediators of skin disease. Here, we explored the consequences of genetically encoded induction of the cell volume-regulating Ca2+-activated KCa3.1 channel (Kcnn4) for murine epidermal homeostasis. Doxycycline-treated mice harboring the KCa3.1+-transgene under the control of the reverse tetracycline-sensitive transactivator (rtTA) showed 800-fold channel overexpression above basal levels in the skin and solid KCa3.1-currents in keratinocytes. This overexpression resulted in epidermal spongiosis, progressive epidermal hyperplasia and hyperkeratosis, itch and ulcers. The condition was accompanied by production of the pro-proliferative and pro-inflammatory cytokines, IL-ß1 (60-fold), IL-6 (33-fold), and TNFα (26-fold) in the skin. Treatment of mice with the KCa3.1-selective blocker, Senicapoc, significantly suppressed spongiosis and hyperplasia, as well as induction of IL-ß1 (-88%) and IL-6 (-90%). In conclusion, KCa3.1-induction in the epidermis caused expression of pro-proliferative cytokines leading to spongiosis, hyperplasia and hyperkeratosis. This skin condition resembles pathological features of eczematous dermatitis and identifies KCa3.1 as a regulator of epidermal homeostasis and spongiosis, and as a potential therapeutic target.

KCa3.1 Transgene Induction in Murine Intestinal Epithelium Causes Duodenal Chyme Accumulation and Impairs Duodenal Contractility.

Abstract: The epithelial intermediate-conductance calcium/calmodulin-regulated KCa3.1 channel is considered to be a regulator of intestine function by controlling chloride secretion and water/salt balance. Yet, little is known about the functional importance of KCa3.1 in the intestinal epithelium in vivo. Our objective was to determine the impact of epithelial-specific inducible overexpression of a KCa3.1 transgene (KCa3.1+) and of inducible suppression (KCa3.1-) on intestinal homeostasis and function in mice. KCa3.1 overexpression in the duodenal epithelium of doxycycline (DOX)-treated KCa3.1+ mice was 40-fold above the control levels. Overexpression caused an inflated duodenum and doubling of the chyme content. Histology showed conserved architecture of crypts, villi, and smooth muscle. Unaltered proliferating cell nuclear antigen (PCNA) immune reactivity and reduced amounts of terminal deoxynucleotide transferase mediated X-dUTP nick end labeling (TUNEL)-positive apoptotic cells in villi indicated lower epithelial turnover. Myography showed a reduction in the frequency of spontaneous propulsive muscle contractions with no change in amplitude. The amount of stool in the colon was increased and the frequency of colonic contractions was reduced in KCa3.1+ animals. Senicapoc treatment prevented the phenotype. Suppression of KCa3.1 in DOX-treated KCa3.1- mice caused no overt intestinal phenotype. In conclusion, inducible KCa3.1 overexpression alters intestinal functions by increasing the chyme content and reducing spontaneous contractions and epithelial apoptosis. Induction of epithelial KCa3.1 can play a mechanistic role in the process of adaptation of the intestine.

Pharmacological activation of TRPV4 produces immediate cell damage and induction of apoptosis in human melanoma cells and HaCaT keratinocytes.

BACKGROUND: TRPV4 channels are calcium-permeable cation channels that are activated by several physicochemical stimuli. Accordingly, TRPV4 channels have been implicated in the regulation of osmosensing, mechanotransduction, thermosensation, and epithelial/endothelial barrier functions. Whether TRPV4 is also mechanistically implicated in melanoma cell proliferation is not clear. Here, we hypothesized that TRPV4 is expressed in human melanoma and that pharmacological activation interferes with cell proliferation. METHODOLOGY/PRINCIPAL FINDINGS: TRPV4 functions were studied in melanoma cell lines (A375, SK-MEL-28, MKTBR), immortalized non-cancer keratinocytes (HaCaT), and murine 3T3 fibroblasts by patch-clamp, qRT-PCR, intracellular calcium measurements, cell proliferation, and flow cytometric assays of apoptosis and cell cycle. The selective TRPV4-activator, GSK1016790A, elicited non-selective cation currents with TRPV4-typical current-voltage-relationship in all cell lines. GSK1016790A-induced currents were blocked by the TRPV4-blocker, HC067047. TRPV4 mRNA expression was demonstrated by qRT-PCR. In A375 cells, TRPV4 activation was frequently paralleled by co-activation of calcium/calmodulin-regulated KCa3.1 channels. Light microscopy showed that TRPV4-activation produced rapid cellular disarrangement, nuclear densification, and detachment of a large fraction of all melanoma cell lines and HaCaT cells. TRPV4-activation induced apoptosis and drastically inhibited A375 and HaCaT proliferation that could be partially prevented by HC067047. CONCLUSIONS/SIGNIFICANCE: Our study showed that TRPV4 channels were functionally expressed in human melanoma cell lines and in human keratinocytes. Pharmacological TRPV4 activation in human melanoma cells and keratinocytes caused severe cellular disarrangement, necrosis and apoptosis. Pharmacological targeting of TRPV4 could be an alternative or adjuvant therapeutic strategy to treat melanoma progression and other proliferative skin disorders.

ImageJ-based semiautomatic method to analyze senescence in cell culture.

ß-Galactosidase accumulates in the lysosomes of senescent cells of certain tissues. Cell staining with X-gal is a common procedure to detect senescent cells in culture. However, the organelle nature of the staining makes automatic count impossible, requiring time-consuming manual counting or expensive alternative techniques such as flow cytometry to effectively determine the amount of stained cells. Here we present an analysis strategy for images of X-gal stained cells which can be implemented into a macro for the ImageJ software overcoming some of the drawbacks of computational analysis of organelle staining.

Inhibition of Intermediate-Conductance Calcium-Activated K Channel (KCa3.1) and Fibroblast Mitogenesis by α-Linolenic Acid and Alterations of Channel Expression in the Lysosomal Storage Disorders, Fabry Disease, and Niemann Pick C.

The calcium/calmodulin-gated KCa3.1 channel regulates normal and abnormal mitogenesis by controlling K+-efflux, cell volume, and membrane hyperpolarization-driven calcium-entry. Recent studies suggest modulation of KCa3.1 by omega-3 fatty acids as negative modulators and impaired KCa3.1 functions in the inherited lysosomal storage disorder (LSD), Fabry disease (FD). In the first part of present study, we characterize KCa3.1 in murine and human fibroblasts and test the impact of omega-3 fatty acids on fibroblast proliferation. In the second, we study whether KCa3.1 is altered in the LSDs, FD, and Niemann-Pick disease type C (NPC). Our patch-clamp and mRNA-expression studies on murine and human fibroblasts show functional expression of KCa3.1. KCa currents display the typical pharmacological fingerprint of KCa3.1: Ca2+-activation, potentiation by the positive-gating modulators, SKA-31 and SKA-121, and inhibition by TRAM-34, Senicapoc (ICA-17043), and the negative-gating modulator, 13b. Considering modulation by omega-3 fatty acids we found that α-linolenic acid (α-LA) and docosahexanenoic acid (DHA) inhibit KCa3.1 currents and strongly reduce fibroblast growth. The α-LA-rich linseed oil and γ-LA-rich borage oil at 0.5% produce channel inhibition while α-LA/γ-LA-low oils has no anti-proliferative effect. Concerning KCa3.1 in LSD, mRNA expression studies, and patch-clamp on primary fibroblasts from FD and NPC patients reveal lower KCa3.1-gene expression and membrane expression than in control fibroblasts. In conclusion, the omega-3 fatty acid, α-LA, and α-LA/γ-LA-rich plant oils, inhibit fibroblast KCa3.1 channels and mitogenesis. Reduced fibroblast KCa3.1 functions are a feature and possible biomarker of cell dysfunction in FD and NPC and supports the concept that biased lipid metabolism is capable of negatively modulating KCa3.1 expression.

Vascular Reactivity Profile of Novel KCa 3.1-Selective Positive-Gating Modulators in the Coronary Vascular Bed.

Opening of intermediate-conductance calcium-activated potassium channels (KC a 3.1) produces membrane hyperpolarization in the vascular endothelium. Here, we studied the ability of two new KC a 3.1-selective positive-gating modulators, SKA-111 and SKA-121, to (1) evoke porcine endothelial cell KC a 3.1 membrane hyperpolarization, (2) induce endothelium-dependent and, particularly, endothelium-derived hyperpolarization (EDH)-type relaxation in porcine coronary arteries (PCA) and (3) influence coronary artery tone in isolated rat hearts. In whole-cell patch-clamp experiments on endothelial cells of PCA (PCAEC), KC a currents evoked by bradykinin (BK) were potentiated ≈7-fold by either SKA-111 or SKA-121 (both at 1 µM) and were blocked by a KC a 3.1 blocker, TRAM-34. In membrane potential measurements, SKA-111 and SKA-121 augmented bradykinin-induced hyperpolarization. Isometric tension measurements in large- and small-calibre PCA showed that SKA-111 and SKA-121 potentiated endothelium-dependent relaxation with intact NO synthesis and EDH-type relaxation to BK by ≈2-fold. Potentiation of the BK response was prevented by KC a 3.1 inhibition. In Langendorff-perfused rat hearts, SKA-111 potentiated coronary vasodilation elicited by BK. In conclusion, our data show that positive-gating modulation of KC a 3.1 channels improves BK-induced membrane hyperpolarization and endothelium-dependent relaxation in small and large PCA as well as in the coronary circulation of rats. Positive-gating modulators of KC a 3.1 could be therapeutically useful to improve coronary blood flow and counteract impaired coronary endothelial dysfunction in cardiovascular disease.

Novel phenolic inhibitors of small/intermediate-conductance Ca²âº-activated K⁺ channels, KCa3.1 and KCa2.3.

BACKGROUND: KCa3.1 channels are calcium/calmodulin-regulated voltage-independent K(+) channels that produce membrane hyperpolarization and shape Ca(2+)-signaling and thereby physiological functions in epithelia, blood vessels, and white and red blood cells. Up-regulation of KCa3.1 is evident in fibrotic and inflamed tissues and some tumors rendering the channel a potential drug target. In the present study, we searched for novel potent small molecule inhibitors of KCa3.1 by testing a series of 20 selected natural and synthetic (poly)phenols, synthetic benzoic acids, and non-steroidal anti-inflammatory drugs (NSAIDs), with known cytoprotective, anti-inflammatory, and/or cytostatic activities. METHODOLOGY/PRINCIPAL FINDINGS: In electrophysiological experiments, we identified the natural phenols, caffeic acid (EC50 1.3 µM) and resveratrol (EC50 10 µM) as KCa3.1 inhibitors with moderate potency. The phenols, vanillic acid, gallic acid, and hydroxytyrosol had weak or no blocking effects. Out of the NSAIDs, flufenamic acid was moderately potent (EC50 1.6 µM), followed by mesalamine (EC50≥10 µM). The synthetic fluoro-trivanillic ester, 13b ([3,5-bis[(3-fluoro-4-hydroxy-benzoyl)oxymethyl]phenyl]methyl 3-fluoro-4-hydroxy-benzoate), was identified as a potent mixed KCa2/3 channel inhibitor with an EC50 of 19 nM for KCa3.1 and 360 pM for KCa2.3, which affected KCa1.1 and Kv channels only at micromolar concentrations. The KCa3.1/KCa2-activator SKA-31 antagonized the 13b-blockade. In proliferation assays, 13b was not cytotoxic and reduced proliferation of 3T3 fibroblasts as well as caffeic acid. In isometric vessel myography, 13b increased contractions of porcine coronary arteries to serotonin and antagonized endothelium-derived hyperpolarization-mediated vasorelaxation to pharmacological KCa3.1/KCa2.3 activation. CONCLUSIONS/SIGNIFICANCE: We identified the natural phenols, caffeic acid and resveratrol, the NSAID, flufenamic acid, and the polyphenol 13b as novel KCa3.1 inhibitors. The high potency of 13b with pan-activity on KCa3.1/KCa2 channels makes 13b a new pharmacological tool to manipulate inflammation and cancer growth through KCa3.1/KCa2 blockade and a promising template for new drug design.

Expression and purification of recombinant apolipoprotein A-I Zaragoza (L144R) and formation of reconstituted HDL particles.

Apolipoprotein A-I Zaragoza (L144R) (apo A-I Z), has been associated with severe hypoalphalipoproteinemia and an enhanced effect of high density lipoprotein (HDL) reverse cholesterol transport. In order to perform further studies with this protein we have optimized an expression and purification method of recombinant wild-type apo A-I and apo A-I Z and produced mimetic HDL particles with each protein. An pET-45 expression system was used to produce N-terminal His-tagged apo A-I, wild-type or mutant, in Escherichia coli BL21 (DE3) which was subsequently purified by affinity chromatography in non-denaturing conditions. HDL particles were generated via a modified sodium cholate method. Expression and purification of both proteins was verified by SDS-PAGE, MALDI-TOF MS and immunochemical procedures. Yield was 30mg of purified protein (94% purity) per liter of culture. The reconstituted HDL particles checked via non-denaturing PAGE showed high homogeneity in their size when reconstituted both with wild-type apo A-I and apo A-I Z. An optimized system for the expression and purification of wild-type apo A-I and apo A-I Z with high yield and purity grade has been achieved, in addition to their use in reconstituted HDL particles, as a basis for further studies.

Proteomic study of macrophages exposed to oxLDL identifies a CAPG polymorphism associated with carotid atherosclerosis.

OBJECTIVE: Macrophages play a key role in the development of atherosclerosis. The objective of this observational study was to characterize the proteome of macrophages to identify proteins implicated in atherosclerosis. METHODS: The proteome of macrophage exposed to oxidized low-density lipoprotein (LDL) was studied in a sample of 12 subjects with autosomal dominant hypercholesterolemia and analyzed according to carotid atherosclerosis. Carotid intima-media thickness (IMT), genotyping of the polymorphisms responsible for the amino acid change present in the identified proteins, and an association study was performed in a sample of 320 subjects with autosomal dominant hypercholesterolemia and 145 normolipemic controls. RESULTS: Mass spectroscopy identified two proteins, gelsolin like capping protein (CapG) and glutathione-S-transferase omega 1 (GSTO1), with large variability among subjects which corresponded with two common genetic variants. The rs6886 polymorphism in CAPG was significantly associated with carotid IMT. Carriers of the minor allele in CAPG polymorphism presented less carotid IMT than noncarriers in the hypercholesterolemia group (mean and maximum internal carotid IMT p=0.016 and p=0.032, respectively). This effect was more important in subjects below 50 years old (mean and maximum internal carotid IMT p<0.001). CONCLUSIONS: Association analysis revealed rs6886 polymorphism in CAPG to be associated with carotid IMT, suggesting that this polymorphism could modulate macrophages' response to oxidized LDL in subjects with hypercholesterolemia.

Overexpression of the CXCL3 gene in response to oxidized low-density lipoprotein is associated with the presence of tendon xanthomas in familial hypercholesterolemia.

To examine if overexpression of certain chemokines and proinflammatory cytokines in response to oxidized low-density lipoprotein could be involved in the onset and development of tendon xanthomas (TX), we quantified IL-1beta, TNF-alpha, and IL-8 and compared gene expression of PPAR-gamma, NF-kappaBIA, IL-8, IL-1beta, CXCL3, tryptase, and TNF-alpha in macrophages of familial hypercholesterolemia subjects with and without TX stimulated with oxidized low-density lipoprotein at 1, 3, 6, and 18 h of incubation. We propose that chemokines belonging to the CXC family could play an important role in the etiology of TX, with CXCL3 being a possible biological marker of onset and development of TX.

Atorvastatin decreases stearoyl-CoA desaturase gene expression in THP-1 macrophages incubated with oxidized LDL.

Statins, inhibitors of HMG-CoA reductase, reduce plasma low-density lipoprotein (LDL) cholesterol levels decreasing the incidence of coronary events. However, the observed benefit of statins appears to extend beyond their lipid-lowering effects. Previous studies by our group have demonstrated that atorvastatin in oxidized LDL incubated macrophages modifies the gene expression profile of certain enzymes involved in fatty acid metabolism, mainly stearoyl-CoA desaturase (SCD). SCD is a rate-limiting enzyme in the biosynthesis of monounsaturated fatty acids and its expression is mediated by sterol regulatory element-binding protein-1 (SREBP-1). The aim of this study was to determine whether atorvastatin might affect the fatty acid composition in macrophages and if their SCD gene expression profile could explain this effect. Therefore, THP-1 macrophages were treated with atorvastatin and native or oxidized LDL, their fatty acid composition was determined by gas-chromatography, and the SCD and SREBP-1 gene expression profile was analysed using quantitative RT-PCR. We found that atorvastatin reduces the percentage of palmitoleic and oleic acids in THP-1 cells incubated with oxLDL, which could be explained by the inhibition of SCD and SREBP-1 gene expression. The observed results were reversed when mevalonate was added to THP-1 macrophages. This would suggest that inhibition of SCD in THP-1 macrophages incubated with oxLDL and the change in fatty acid composition is an important effect of atorvastatin.

Association and linkage disequilibrium analyses of APOE polymorphisms in atherosclerosis.

BACKGROUND: Apolipoprotein E (apo E) plays a major role in lipid metabolism, and its genetic variations have been associated with cardiovascular risk. The objective of this study was to investigate the influence of the APOE promoter (-491 A/T, -427 T/C and -219 G/T) and coding region (APOE epsilon2/epsilon3/epsilon4) polymorphisms in atherosclerosis disease by association and linkage disequilibrium analyses. MATERIALS AND METHODS: We analyzed these polymorphisms in a sample of 286 subjects with atherosclerosis disease: 153 subjects with atherothrombotic stroke (ATS) and 133 subjects with ischemic heart disease (IHD); and in two control groups, 103 newborns and 114 elderly subjects. RESULTS: The epsilon4 allele was associated with more severe carotid stenosis in the ATS group, being the percentages of epsilon4 carriers 26.7% and 11.4% for the higher and lower carotid stenosis groups, respectively (p=0,066). The -491 T/T IHD subjects presented higher vessel scores than subjects A/A and A/T genotypes at that position (p=0,041), and the frequencies of -2 (5.1% versus 14.1%, p=0,060) and -427C (10.3% versus 24.4%, p=0,019) alleles were lower in IHD subjects with higher extent score versus lower extent score. The epsilon2 allele was in linkage disequilibrium with the -427C allele in all studied groups, and the -219T allele was associated with the epsilon4 allele in the IHD group. CONCLUSION: In summary, the epsilon2 allele was in linkage disequilibrium with the -427C allele in all studied groups, and only slight associations between the analyzed APOE polymorphisms in the promoter and in the coding region and carotid and coronary vascular disease have been observed.

Individual variation of scavenger receptor expression in human macrophages with oxidized low-density lipoprotein is associated with a differential inflammatory response.

Atherosclerosis is an inflammatory disease in which oxidized low-density lipoprotein (oxLDL) plays important roles. Scavenger receptors (SR) CD36, SR-A, and LOX-1 uptake over 90% of the oxLDL leading to foam cell formation and secretion of inflammatory cytokines. To investigate whether the interindividual differences in macrophage SR gene expression could determine the inflammatory variability in response to oxLDL, we quantified the gene and protein expression of SR and inflammatory molecules from macrophages isolated from 18 volunteer subjects and incubated with oxLDL for 1, 3, 6, and 18 h. The individual gene expression profile of the studied SR at 1 h of incubation was highly variable, showing a wide fold-change range: CD36: -3.57-4.22, SR-A: -5.0-4.43, and LOX-1: -1.56-75.32. We identified subjects as high and low responders depending on whether their SR gene expression was above or below the median, showing a different inflammation response pattern. CD36 and LOX-1 gene expression correlated positively with IL-1beta; SR-A correlated negatively with IL-8 and positively with PPARgamma and NF-kappaBIotaA. These results were confirmed in the same subjects 3 mo after the first sampling. Furthermore, a negative correlation existed between CD36 and SR-A at protein level after 18 h of oxLDL incubation (R = -0.926, p = 0.024). These data would suggest that the type of SR could determine the macrophage activation: more proinflammatory when associated to CD36 and LOX-1 than when associated with SR-A.

[Sensitivity and specificity of metabolic syndrome criteria for insulin resistance diagnosis in Spanish population]. / Sensibilidad y especificidad de los criterios del síndrome metabólico para el diagnóstico de insulinorresistencia en la población española.

BACKGROUND AND OBJECTIVE: Metabolic syndrome (MS) is an abdominal obesity and insulin resistance (IR)-related syndrome associated with a high cardiovascular risk. Recently, the International Diabetes Federation (IDF) has proposed a modification of the Adult Treatment Panel III (ATP III) diagnostic criteria. However, the sensitivity of these new criteria has not been established. The aim of the present study was to define the sensitivity and specificity of the different criteria used for the diagnosis of the MS in our population. SUBJECTS AND METHOD: We studied in 177 healthy subjects, 68 men and 109 women, the body mass index, waist circumference (WC), blood pressure, glucose, insulin, lipids and apolipoproteins A1 and B. The HOMA index was used as an IR indicator. IR was considered with an HOMA index > or = 3.8. RESULTS: Subjects with IR showed higher age, systolic blood pressure, triglycerides and apo B, and lower HDL cholesterol. A WC > or = 102 cm in men and > or = 88 cm in women (ATP III criteria) had a low sensitivity for IR (29.4% and 44.7% respectively), with high specificity (81% and 90%). A WC > or = 94 cm in men and > or = 80 cm in women (IDF criteria) showed good sensitivity (73.5% and 73.7% respectively) but less specificity (57.1% and 53.3%). The IDF criteria showed better sensitivity than ATP III, without substantial change in the specificity for the different HOMA cut-off points. CONCLUSIONS: ATP III criteria had low sensitivity in our population. The new criteria (WC > or = 94 cm in men and > or = 80 cm in women, and blood glucose > or = 100 mg/dL) improve three-fold the diagnostic sensitivity and, therefore, seems to be more useful for detecting IR in our country.

Oligodendrocyte differentiation is increased in transferrin transgenic mice.

Transferrin (Tf), the iron transport glycoprotein found in biological fluids of vertebrates, is synthesized mainly by hepatocytes. Tf is also synthesized by oligodendrocytes (Ol), and several lines of evidence indicate that brain Tf could be involved in myelinogenesis. Because Tf is postnatally expressed in the brain, we sought to investigate whether Tf could intervene in Ol differentiation. For this purpose, we analyzed transgenic mice overexpressing the complete human Tf gene in Ol. We show that the hTf transgene was expressed only from 5 days postpartum onward. In the brain of 14-day-old transgenic mice, the DM-20 mRNA level was decreased, whereas the PLP, MBP, CNP, and MAG mRNA levels were increased. We counted a higher proportion of Ol expressing the O4 (Ol-specific antigens) and PLP in brain cells cultured from transgenic mice. These results support the idea that overexpressing Tf in the brain accelerates the oligodendrocyte lineage maturation. Accordingly, by NMR imaging acquisition of diffusion tensor in hTf transgenic mice, we observed early maturation of the cerebellum and spinal cord and more myelination in the corpus callosum. In addition, hTf overexpression led to an increase in Sox10 mRNA and protein. Increases in Sox10 and in Tf expression occur simultaneously during brain development. The Olig1 mRNA level also increased, but long after the rise of hTf and Sox10. The Olig2 mRNA level remained unchanged in the brain of transgenic mice. Our findings suggest that Tf could influence oligodendrocyte progenitor differentiation in the CNS.

Mammalian genome targeting using site-specific recombinases.

Nowadays, a wide array of procedures in mouse technology has been made available to researchers in order to establish valuable models for the study of gene function. The efficiency of gene transfer and gene targeting as methods for producing genetic changes in mice, in addition to continuous advances in molecular biology tools, has converted the mouse into the major experimental model for the study of mammalian physiology. In recent years, the emergence of site-specific recombinases as tools to engineer mammalian genomes has opened new avenues into the design of genetically modified mouse models. The original Cre and FLP recombinases have demonstrated their utility in developing conditional gene targeting, and now other analogous recombinases are also ready to be used, in the same way or in combined strategies, to achieve more sophisticated experimental schemes for addressing complex biological questions. The properties of site-specific recombinases in combination with other biotechnological tools (tet on/off system, siRNA mediated gene silencing, fluorescent proteins, et al.) make them useful instruments to induce precise mutations in specific cells or tissues in a time-controlled manner. This ability can be applied in functional genomics in several ways: from conditional and inducible gene targeting to controlled expression of transgenes and recombination-mediated cassette exchange in mouse models for the study of development or disease phenotypes. This review focuses on the use of site-specific recombinases for mouse genome manipulation. A historical perspective of site-specific recombinases is considered and a number of strategies for achieving inducible or conditional genomic manipulations are contemplated in the context of current techniques for producing genetically modified mice. Finally, several model generation approaches from recent examples in the literature are revised.

Human apolipoprotein A-IV reduces secretion of proinflammatory cytokines and atherosclerotic effects of a chronic infection mimicked by lipopolysaccharide.

OBJECTIVE: Expression of human apolipoprotein (h-apo) A-IV in apoE-deficient (apoE(0)) mice (h-apoA-IV/E(0)) reduces susceptibility to atherosclerosis. Chronic infection mimicked by exposure to lipopolysaccharide (LPS) increases the size of atherosclerosis lesions in apoE(0) mice. Thus, we used h-apoA-IV/E(0) mice to determine whether h-apoA-IV plays a protective role after LPS administration. METHODS AND RESULTS: We injected apoE(0), h-apoA-IV/E(0), and C57Bl/6 (wild-type) mice intraperitoneally with either LPS or phosphate-buffered saline (PBS) every week for 10 weeks. Atherosclerotic lesions were significantly smaller in h-apoA-IV/E(0) mice treated with LPS than in their apoE(0) counterparts. The titers of IgG2a and IgG2b autoantibodies to oxidized low-density lipoprotein (LDL) were higher in the LPS-group of h-apoA-IV/E(0) mice than in apoE(0) mice, suggesting that the Th1 response is stronger in the presence of h-apoA-IV. Lymphocytes from the blood, liver, spleen, and thymus of h-apoA-IV/E(0) mice treated with LPS produced less IL-4, INF-gamma, and TNF-alpha proinflammatory cytokines than their apoE(0) counterparts. Furthermore, we demonstrated that recombinant h-apoA-IV blocks the LPS-induced stimulation of monocytes. CONCLUSIONS: The expression of h-apoA-IV in apoE(0) mice reduces the susceptibility to atherogenesis and decreases the secretion of proinflammatory cytokines after LPS administration.

Myelination and motor coordination are increased in transferrin transgenic mice.

Myelin deficiency in the central nervous system (CNS) can cause severe disabling conditions. Most of the transgenic mice models overexpressing myelin components have limitations for investigators of myelin deficiency and myelin therapy as they severely alter CNS architecture. It has been postulated that transferrin (Tf) is involved in oligodendrocyte (OL) maturation and myelinogenesis. Because Tf is not an intrinsic myelin constituent, we decided to investigate if its overexpression could have an impact on the myelination process without affecting myelin integrity. We generated transgenic mice containing the complete human Tf gene specifically overexpressed in OLs. This overexpression leads to more than a 30% increase in myelin components, such as galactolipids, phospholipids, and proteins. Electron microscopy showed that myelin is structurally normal in terms of thickness and compaction. Behavior analysis showed that mice do not display significant modifications in their locomotion and cognitive and emotional abilities. Furthermore, in one of the genetic background, animals presented a significant increase in motor coordination. We did not find any modification in OL number during early postnatal development, suggesting that Tf does not act on OL proliferation. In addition, the levels of iron and ferritin remained unchanged in the brain of transgenic mice compared to control mice. Our findings indicate that, besides its known iron transport function, Tf is able to influence myelination process and induce behavioral improvements in mice.

Analysis of apolipoprotein A-I, lecithin:cholesterol acyltransferase and glucocerebrosidase genes in hypoalphalipoproteinemia.

Hypoalphalipoproteinemia (HALP) is a dyslipidemia characterized by low HDL-cholesterol (HDL-C) levels with important genetic contribution. However, no common genetic mutations have been found to be associated with this disorder. We screened the promoter and coding sequence of apolipoprotein (apo) A-I and lecithin:cholesterol acyltransferase (LCAT) genes and the 5' apo C-III region by SSCP and heteroduplex analysis, and DNA sequencing in 66 unrelated subjects with recurrent low HDL-C levels. We also analyzed the N370S and L444P variants, in the glucocerebrosidase (GBA) gene by restriction fragment analysis. Three mutations in the apo A-I gene (L144R, W108R, g.1833C>T) and 3 mutations in the LCAT gene (S208T, I178T, IVS3-23C>A) were detected, in six heterozygous subjects. In addition, a novel polymorphic site in LCAT gene (g.4886C>T) has been identified. Allelic frequencies of polymorphisms g.(-636)C>A, g.(-625)G>A, g.(-620)T>del, g.(-479C>T and g.(-452)T>C, located upstream of the apo C-III gene, were in normal range, and no other mutation was found in this region. Two HALP subjects were found to carry the N370S mutation at GBA locus. In conclusion, 12% of HALP subjects were found to carry mutations in apo A-I, LCAT, or GBA genes, which could explain this phenotype. Our results confirm the molecular, genetic and phenotypic heterogeneity of HALP.