A Novel Murine Model of Marfan Syndrome Accelerates Aortopathy and Cardiomyopathy.

BACKGROUND: Marfan syndrome (MFS) represents a genetic disorder with variable phenotypic expression. The main cardiovascular sequelae of MFS include aortic aneurysm/dissection and cardiomyopathy. Although significant advances in the understanding of transforming growth factor beta signaling have led to promising therapeutic targets for the treatment of aortopathy, clinical studies have tempered this optimism. In particular, these studies suggest additional signaling pathways that play a significant role in disease progression. To date, studies aimed at elucidating molecular mechanisms involved in MFS-induced disease progression have been hampered by the lack of an accelerated disease model. METHODS: Wild-type B6.129 mice and MFS Fbn1C1039G/+ mice underwent subcutaneous, cervical osmotic minipump installation with sodium chloride (wild-type mice, n = 39; MFS mice, n = 12) or angiotensin II, 4.5 mg/kg daily (wild-type mice, n = 11; MFS mice; n = 35) for as long as 28 days. Hemodynamic measurements were obtained throughout the experiment. Aortas and hearts were analyzed by transthoracic echocardiography and histopathology study. RESULTS: This accelerated murine MFS model replicates increased mortality from MFS-related maladies (20.0%, 39.3%, and 52.9% at 10, 14, and 28 days, respectively). Aortic diameters in accelerated MFS mice were significantly enlarged at 10 days after minipump implantation and correlated with a higher degree of elastin fragmentation. Accelerated MFS mice also demonstrated dilated cardiomyopathy at 14 days, even without aortic insufficiency, suggesting an intrinsic etiology. CONCLUSIONS: A novel in vivo model consisting of subcutaneously delivered angiotensin II in MFS mice reproducibly causes accelerated aortic aneurysm formation and cardiomyopathy. This model allows for better investigation of MFS sequelae by rapid experimental processes.

Mechanisms for autophagy modulation by isoprenoid biosynthetic pathway inhibitors in multiple myeloma cells.

Multiple myeloma (MM) is characterized by the production of monoclonal protein (MP). We have shown previously that disruption of the isoprenoid biosynthetic pathway (IBP) causes a block in MP secretion through a disruption of Rab GTPase activity, leading to an enhanced unfolded protein response and subsequent apoptosis in MM cells. Autophagy is induced by cellular stressors including nutrient deprivation and ER stress. IBP inhibitors have been shown to have disparate effects on autophagy. Here we define the mechanisms underlying the differential effects of IBP inhibitors on autophagic flux in MM cells utilizing specific pharmacological inhibitors. We demonstrate that IBP inhibition induces a net increase in autophagy as a consequence of disruption of isoprenoid biosynthesis which is not recapitulated by direct geranylgeranyl transferase inhibition. IBP inhibitor-induced autophagy is a cellular defense mechanism as treatment with the autophagy inhibitor bafilomycin A1 enhances the cytotoxic effects of GGPP depletion, but not geranylgeranyl transferase inhibition. Immunofluorescence microscopy studies revealed that IBP inhibitors disrupt ER to Golgi trafficking of monoclonal light chain protein and that this protein is not a substrate for alternative degradative pathways such as aggresomes and autophagosomes. These studies support further development of specific GGTase II inhibitors as anti-myeloma agents.

N-Oxide derivatives of 3-(3-pyridyl)-2-phosphonopropanoic acids as potential inhibitors of Rab geranylgeranylation.

The N-oxide derivatives of [2-(3-pyridinyl)-1-hydroxyethylidene-1,1-phosphonocarboxylic acid (or PEHPC) and [2-(3-pyridinyl)-1-ethylidene-1,1-phosphonocarboxylic acid (or PEPC) have been prepared and evaluated for their activity against several enzymes which utilize isoprenoids. The parent pyridines are known inhibitors of GGTase II, but the N-oxide derivatives show no improvement in biological activity in assays with the isolated enzyme. However, the PEHPC N-oxide did induce significant accumulation of intracellular light chain in myeloma cells, consistent with inhibition of Rab geranylgeranylation.

Geranyl and neryl triazole bisphosphonates as inhibitors of geranylgeranyl diphosphate synthase.

When inhibitors of enzymes that utilize isoprenoid pyrophosphates are based on the natural substrates, a significant challenge can be to achieve selective inhibition of a specific enzyme. One element in the design process is the stereochemistry of the isoprenoid olefins. We recently reported preparation of a series of isoprenoid triazoles as potential inhibitors of geranylgeranyl transferase II but these compounds were obtained as a mixture of olefin isomers. We now have accomplished the stereoselective synthesis of these triazoles through the use of epoxy azides for the cycloaddition reaction followed by regeneration of the desired olefin. Both geranyl and neryl derivatives have been prepared as single olefin isomers through parallel reaction sequences. The products were assayed against multiple enzymes as well as in cell culture studies and surprisingly a Z-olefin isomer was found to be a potent and selective inhibitor of geranylgeranyl diphosphate synthase.

Triazole-based inhibitors of geranylgeranyltransferase II.

A small set of triazole bisphosphonates has been prepared and tested for the ability to inhibit geranylgeranyltransferase II (GGTase II). The compounds were prepared through use of click chemistry to assemble a central triazole that links a polar head group to a hydrophobic tail. The resulting compounds were tested for their ability to inhibit GGTase II in an in vitro enzyme assay and also were tested for cytotoxic activity in an MTT assay with the human myeloma RPMI-8226 cell line. The most potent enzyme inhibitor was the triazole with a geranylgeranyl tail, which suggests that inhibitors that can access the enzyme region that holds the isoprenoid tail will display greater activity.

Cardiovascular consequences of genetic variation at -6/235 in human angiotensinogen using "humanized" gene-targeted mice.

Genetic and functional data support a role for angiotensinogen in blood pressure control, and many population studies have suggested that polymorphisms in the angiotensinogen gene contribute to hypertension. Two common haplotypes of the human angiotensinogen gene are -6A/235T and -6G/235M. To study their contributions to blood pressure regulation in a controlled model system, we developed triple-transgenic mice expressing either -6A/235T or -6G/235M human angiotensinogen, expressing either an overexpressed and poorly regulated (REN9) or a tightly regulated (PAC160) human renin, and all carrying a null mutation in the endogenous murine angiotensinogen gene. These humanized mice were then examined for blood pressure differences at baseline and after a high-salt diet, changes in cardiovascular organ weight, and differences in angiotensinogen and renin gene expression. Mice expressing the -6G/235M haplotype on the PAC160 background exhibited increased blood pressure and cardiac hypertrophy at baseline. In contrast, all of the mice with the REN9 background had equivalent baseline blood pressures. On the REN9 background, there was a greater increase in blood pressure in -6A/235T in response to a high-salt diet, providing evidence it may be a susceptibility allele. There were no differences in angiotensinogen expression between haplotypes on either background strain. The data suggest that the impact of angiotensinogen haplotypes on cardiovascular end points may be dependent on renin status and environmental influences, such as dietary sodium. These insights may help explain the discrepancies among observational studies that have examined roles for the -6A/235T and -6G/235M angiotensinogen haplotypes in varied human populations.

Regulation of renin gene expression by oxidative stress.

Increased arterial pressure, angiotensin II, and cytokines each result in feedback inhibition of renin gene expression. Because angiotensin II and cytokines can stimulate reactive oxygen species production, we tested the hypothesis that oxidative stress may be a mediator of this inhibition. Treatment of renin-expressing As4.1 cells with the potent cytokine tumor necrosis factor-alpha caused an increase in the steady-state levels of cellular reactive oxygen species, which was reversed by the antioxidant N-acetylcysteine. Exogenous H(2)O(2) caused a dose- and time-dependent decrease in the level of endogenous renin mRNA and decreased the transcriptional activity of a 4.1-kb renin promoter fused to luciferase, which was maximal when the renin enhancer was present. The effect of H(2)O(2) appeared to be specific to renin, because there was no change in the expression of beta-actin or cyclophilin mRNA or transcriptional activity of the SV40 promoter. The tumor necrosis factor-alpha-induced decrease in renin mRNA was partially reversed by either N-acetylcysteine or panepoxydone, a nuclear factor kappaB (NFkappaB) inhibitor. Interestingly, H(2)O(2) did not induce NFkappaB in As4.1 cells, and panepoxydone had no effect on the downregulation of renin mRNA by H(2)O(2). The transcriptional activity of a cAMP response element-luciferase construct was decreased by both tumor necrosis factor-alpha and H(2)O(2). These data suggest that cellular reactive oxygen species can negatively regulate renin gene expression via an NFkappaB-independent mechanism involving the renin enhancer and inhibiting cAMP response element-mediated transcription. Our data further suggest that tumor necrosis factor-alpha decreases renin expression through both NFkappaB-dependent and NFkappaB-independent mechanisms, the latter involving the production of reactive oxygen species.

Dysregulated human renin expression in transgenic mice carrying truncated genomic constructs: evidence supporting the presence of insulators at the renin locus.

We previously generated transgenic mice carrying a large P1 artificial chromosome (PAC160) encompassing a 160-kb segment containing the human renin gene, two upstream genes, and one downstream gene. We also previously generated mutant PAC160 constructs lacking the distal enhancer and concluded it is required to maintain baseline expression of human renin, but is not required for tissue-specific, cell-specific, and regulated expression of renin in vivo. We now report two additional transgenic lines carrying random truncations of PAC160 upstream of the renin gene. Southern and PCR mapping studies indicate that the truncation break points in the two lines are located approximately 10.4 and 2.5 kb upstream of the renin gene causing a deletion of all DNA upstream of the break. We tested the hypothesis that large-scale deletion of DNA upstream of the human renin gene including the enhancer would cause dysregulation of human renin expression. Phenotypically, these truncations cause a severe dysregulation of human renin expression, but remarkably, a preservation of the normal tissue-specific expression of the human ethanolamine kinase 2 (ETNK2) gene which lies immediately downstream of renin. Several functional binding sites for CTCF, a mammalian insulator protein, were identified in and around the renin and ETNK2 loci by gel shift and chromatin immunoprecipitation. We conclude that there are sequences in and around the renin and ETNK2 loci which act as boundaries between neighboring genes which insulate them from each other. The study illustrates the value of taking a much wider genomic perspective when studying mechanisms regulating gene expression.

Stanol esters decrease plasma cholesterol independently of intestinal ABC sterol transporters and Niemann-Pick C1-like 1 protein gene expression.

Possible mechanisms for the cholesterol-lowering effects of plant stanol esters were addressed by feeding hamsters diets containing stanol esters, cholesterol, or cholestyramine/lovastatin. ABCA1, ATP binding cassette G1 (ABCG1), ABCG5, ABCG8, and Niemann-Pick C1-like 1 (NPC1L1) mRNA levels were then estimated in duodenum, jejunum, and ileum. Plasma cholesterol was decreased by 36% and 94% in animals fed stanol esters and cholestyramine/lovastatin, respectively. Cholesterol feeding increased plasma cholesterol by 2.5-fold. Plasma plant sterols were unchanged by stanol ester feeding but became undetectable by feeding cholestyramine/lovastatin. Cholesterol and stanols accumulated in enterocytes of animals fed cholesterol and stanol esters, respectively. ABCG5 and ABCG8 mRNA levels were decreased by stanol esters and cholestyramine/lovastatin. Cholesterol feeding markedly increased ABCA1 and ABCG1 expression and modestly increased ABCG5/ABCG8. NPC1L1 mRNA was not significantly altered by any of the diets. ABCG1, ABCG5, ABCG8, and NPC1L1 mRNAs were highest in cells of the upper villus, whereas ABCA1 mRNA was highest in cells of the lower villus. The results suggest that cholesterol lowering effect of stanol esters is unrelated to changes in mRNA levels of intestinal ABC sterol transporters or NPC1L1. Cholesterol flux regulates ABC expression but not NPC1L1. The different localization of ABCA1 suggests a different function for this protein than for ABCG1, ABCG5, ABCG8, and NPC1L1.

LXR/RXR ligand activation enhances basolateral efflux of beta-sitosterol in CaCo-2 cells.

To examine whether intestinal ABCA1 was responsible for the differences observed between cholesterol and beta-sitosterol absorption, ABCA1-facilitated beta-sitosterol efflux was investigated in CaCo-2 cells following liver X receptor/retinoid X receptor (LXR/RXR) activation. Both the LXR agonist T0901317 and the natural RXR/LXR agonists 22-hydroxycholesterol and 9-cis retinoic acid enhanced the basolateral efflux of beta-sitosterol without altering apical efflux. LXR-mediated enhanced beta-sitosterol efflux occurred between 6 h and 12 h after activation, suggesting that transcription, protein synthesis, and trafficking was likely necessary prior to facilitating efflux. The transcription inhibitor actinomycin D prevented the increase in beta-sitosterol efflux by T0901317. Glybenclamide, an inhibitor of ABCA1 activity, and arachidonic acid, a fatty acid that interferes with LXR activation, also prevented beta-sitosterol efflux in response to the LXR ligand activation. Influx of beta-sitosterol mass did not alter the basolateral or apical efflux of the plant sterol, nor did it alter ABCA1, ABCG1, ABCG5, or ABCG8 gene expression or ABCA1 mass. Similar to results observed with intestinal ABCA1-facilitated cholesterol efflux, LXR/RXR ligand activation enhanced the basolateral efflux of beta-sitosterol without affecting apical efflux. The results suggest that ABCA1 does not differentiate between cholesterol and beta-sitosterol and thus is not responsible for the selectivity of sterol absorption by the intestine. ABCA1, however, may play a role in beta-sitosterol absorption.

Liver-X-receptor-mediated increase in ATP-binding cassette transporter A1 expression is attenuated by fatty acids in CaCo-2 cells: effect on cholesterol efflux to high-density lipoprotein.

The effect of fatty acids on LXR (liver X receptors)-mediated enhancement of ABCA1 (ATP-binding cassette transporter A1) expression and cholesterol efflux was investigated in human intestinal cells CaCo-2. LXR activation by T0901317 increased basolateral cholesterol efflux to lipoprotein particles isolated at a density of 1.21 g/ml or higher. Oleic and arachidonic acids attenuated the amount of cholesterol isolated from these particles. Stearic, linoleic and docosahexaenoic acids also decreased cholesterol efflux from basolateral membranes, with the polyunsaturated fatty acids being the most potent. Although oleic, arachidonic and docosahexaenoic acids modestly decreased ABCA1 mRNA levels in response to LXR activation, stearic and linoleic acids did not. Except for oleic acid, all fatty acids substantially attenuated an increase in ABCA1 mass secondary to LXR activation. Inhibiting acyl-CoA:cholesterol acyltransferase activity prevented the decrease in cholesterol efflux caused by oleic acid. Thus, in response to LXR activation, all fatty acids decreased the efflux of cholesterol from the basolateral membrane of CaCo-2 cells. Although modest suppression of ABCA1 gene expression by oleic, arachidonic and docosahexaenoic acids cannot be completely excluded as a mechanism, the predominant effect of fatty acids on ABCA1 expression and cholesterol efflux is at a post-transcriptional level.

Fatty acid flux suppresses fatty acid synthesis in hamster intestine independently of SREBP-1 expression.

Hamsters were fed a control diet or diets containing palm, olive, safflower, or fish oil for 2 weeks. In villus cell populations from duodenum, jejunum, and ileum, rates of intestinal fatty acid and cholesterol synthesis were estimated, as were sterol regulatory element-binding protein (SREBP)-1a, SREBP-1c, SREBP-2, HMG-CoA synthase, fatty acid synthase, ATP citrate lyase, acetyl-CoA carboxylase mRNA levels, and SREBP-1 and SREBP-2 mass. Plasma cholesterol and triacylglcerol levels were increased in animals ingesting palm oil and decreased in animals ingesting fish oil. Fatty acid synthesis and fatty acid synthase activity were decreased in the proximal intestine of animals ingesting all the fat-containing diets. Intestinal cholesterol synthesis was unaltered. In animals fed fat, SREBP-1c gene expression was modestly increased in the duodenum of hamsters fed palm oil or olive oil, and decreased in animals ingesting safflower oil or fish oil. Fatty acid synthase, acetyl-CoA carboxylase, ATP citrate lyase, SREBP-2, and HMG-CoA synthase mRNA levels were not altered, nor were SREBP-1 or SREBP-2 mass. In the intestine, dietary polyunsaturated fatty acids suppress SREBP-1c mRNA without altering expression of its target genes, fatty acid synthase, acetyl-CoA carboxylase, or ATP citrate lyase. Fatty acid influx decreases intestinal fatty acid synthesis by a posttranscriptional mechanism independent of the SREBP pathway.

Polyunsaturated fatty acids decrease the expression of sterol regulatory element-binding protein-1 in CaCo-2 cells: effect on fatty acid synthesis and triacylglycerol transport.

Regulation of sterol regulatory element-binding proteins (SREBPs) by fatty acid flux was investigated in CaCo-2 cells. Cells were incubated with 1 mM taurocholate with or without 250 microM 18:0, 18:1, 18:2, 20:4, 20:5 or 22:6 fatty acids. Fatty acid synthase (FAS) and acetyl-CoA carboxylase mRNA levels and gene and protein expression of SREBPs were estimated. 18:2, 20:4, 20:5 and 22:6 fatty acids decreased the amount of mature SREBP-1 and mRNA levels of SREBP-1c, SREBP-1a, FAS and acetyl-CoA carboxylase. SREBP-2 gene or mature protein expression was not altered. Liver X receptor (LXR) activation by T0901317 increased gene expression of SREBP-1c, SREBP-1a, FAS and acetyl-CoA carboxylase without altering SREBP-2. 20:5, but not 18:1, prevented the full expression of SREBP-1c mRNA by T0901317. T0901317 increased SREBP-1 mass without altering the mass of mature SREBP-2. Although only 18:2, 20:4, 20:5 and 22:6 suppressed SREBP-1, acetyl-CoA carboxylase and FAS expression, all fatty acids decreased the rate of fatty acid synthesis. T0901317 increased endogenous fatty acid synthesis yet did not increase secretion of triacylglycerol-rich lipoproteins. In CaCo-2 cells, polyunsaturated fatty acids decrease gene and protein expression of SREBP-1 and FAS mRNA, probably through interference with LXR activity. Since all fatty acids decreased fatty acid synthesis, mechanisms other than changes in SREBP-1c expression must be entertained. Increased endogenous fatty acid synthesis does not promote triacylglycerol-rich lipoprotein secretion.

LXR/RXR activation enhances basolateral efflux of cholesterol in CaCo-2 cells.

Regulation of gene expression of ATP-binding cassette transporter (ABC)A1 and ABCG1 by liver X receptor/retinoid X receptor (LXR/RXR) ligands was investigated in the human intestinal cell line CaCo-2. Neither the RXR ligand, 9-cis retinoic acid, nor the natural LXR ligand 22-hydroxycholesterol alone altered ABCA1 mRNA levels. When added together, ABCA1 and ABCG1 mRNA levels were increased 3- and 7-fold, respectively. T0901317, a synthetic non-sterol LXR agonist, increased ABCA1 and ABCG1 gene expression 11- and 6-fold, respectively. ABCA1 mass was increased by LXR/RXR activation. T0901317 or 9-cis retinoic acid and 22-hydroxycholesterol increased cholesterol efflux from basolateral but not apical membranes. Cholesterol efflux was increased by the LXR/RXR ligands to apolipoprotein (apo)A-I or HDL but not to taurocholate/phosphatidylcholine micelles. Actinomycin D prevented the increase in ABCA1 and ABCG1 mRNA levels and the increase in cholesterol efflux induced by the ligands. Glyburide, an inhibitor of ABCA1 activity, attenuated the increase in basolateral cholesterol efflux induced by T0901317. LXR/RXR activation decreased the esterification and secretion of cholesterol esters derived from plasma membranes. Thus, in CaCo-2 cells, LXR/RXR activation increases gene expression of ABCA1 and ABCG1 and the basolateral efflux of cholesterol, suggesting that ABCA1 plays an important role in intestinal HDL production and cholesterol absorption.