Comparative gene array analyses of severe elastic fiber defects in late embryonic and newborn mouse aorta.

Elastic fibers provide reversible elasticity to the large arteries and are assembled during development when hemodynamic forces are increasing. Mutations in elastic fiber genes are associated with cardiovascular disease. Mice lacking expression of the elastic fiber genes elastin ( Eln-/-), fibulin-4 ( Efemp2-/-), or lysyl oxidase ( Lox-/-) die at birth with severe cardiovascular malformations. All three genetic knockout models have elastic fiber defects, aortic wall thickening, and arterial tortuosity. However, Eln-/- mice develop arterial stenoses, while Efemp2-/- and Lox-/- mice develop ascending aortic aneurysms. We performed comparative gene array analyses of these three genetic models for two vascular locations and developmental stages to determine differentially expressed genes and pathways that may explain the common and divergent phenotypes. We first examined arterial morphology and wall structure in newborn mice to confirm that the lack of elastin, fibulin-4, or lysyl oxidase expression provided the expected phenotypes. We then compared gene expression levels for each genetic model by three-way ANOVA for genotype, vascular location, and developmental stage. We found three genes upregulated by genotype in all three models, Col8a1, Igfbp2, and Thbs1, indicative of a common response to severe elastic fiber defects in developing mouse aorta. Genes that are differentially regulated by vascular location or developmental stage in all three models suggest mechanisms for location or stage-specific disease pathology. Comparison of signaling pathways enriched in all three models shows upregulation of integrins and matrix proteins involved in early wound healing, but not of mature matrix molecules such as elastic fiber proteins or fibrillar collagens.

Effects of Increased Arterial Stiffness on Atherosclerotic Plaque Amounts.

Increased arterial stiffness is associated with atherosclerosis in humans, but there have been limited animal studies investigating the relationship between these factors. We bred elastin wildtype (Eln+/+) and heterozygous (Eln+/-) mice to apolipoprotein E wildtype (Apoe+/+) and knockout (Apoe-/-) mice and fed them normal diet (ND) or Western diet (WD) for 12 weeks. Eln+/- mice have increased arterial stiffness. Apoe-/- mice develop atherosclerosis on ND that is accelerated by WD. It has been reported that Apoe-/- mice have increased arterial stiffness and that the increased stiffness may play a role in atherosclerotic plaque progression. We found that Eln+/+Apoe-/- arterial stiffness is similar to Eln+/+Apoe+/+ mice at physiologic pressures, suggesting that changes in stiffness do not play a role in atherosclerotic plaque progression in Apoe-/- mice. We found that Eln+/-Apoe-/- mice have increased structural arterial stiffness compared to Eln+/+Apoe-/- mice, but they only have increased amounts of ascending aortic plaque on ND, not WD. The results suggest a change in atherosclerosis progression but not end stage disease in Eln+/-Apoe-/- mice due to increased arterial stiffness. Possible contributing factors include increased blood pressure and changes in circulating levels of interleukin-6 (IL6) and transforming growth factor beta 1 (TGF-ß1) that are also associated with Eln+/- genotype.

Hypertension and decreased aortic compliance due to reduced elastin amounts do not increase atherosclerotic plaque accumulation in Ldlr-/- mice.

BACKGROUND AND AIMS: High blood pressure and reduced aortic compliance are associated with increased atherosclerotic plaque accumulation in humans. Animal studies support these associations, but additional factors, such as fragmented elastic fibers, are present in most previous animal studies. Elastin heterozygous (Eln+/-) mice have high blood pressure and reduced aortic compliance, with no evidence of elastic fiber fragmentation and represent an appropriate model to directly investigate the effects of these factors on atherosclerosis. METHODS AND RESULTS: Eln+/- and Eln+/+ mice were crossed with low density lipoprotein receptor knockout (Ldlr-/-) and wild-type (Ldlr+/+) mice and fed normal or Western diet (WD) for 16 weeks. We hypothesized that on WD, Eln+/-Ldlr-/- mice with high blood pressure and reduced aortic compliance would have increased atherosclerotic plaque accumulation compared to Eln+/+Ldlr-/- mice. We measured serum cholesterol and cytokine levels, blood pressure, aortic compliance, and plaque accumulation. Contrary to our hypothesis, we found that on WD, Eln+/-Ldlr-/- mice do not have increased plaque accumulation compared to Eln+/+Ldlr-/- mice. At the aortic root, there are no significant differences in plaque area between Eln+/-Ldlr-/- and Eln+/+Ldlr-/- mice on WD (p = 0.89), while in the ascending aorta, Eln+/-Ldlr-/- mice on WD have 29% less normalized plaque area than Eln+/+Ldlr-/- mice on WD (p = 0.009). CONCLUSION: Using an atherogenic mouse model, we conclude that increased blood pressure and reduced aortic compliance are not direct causes of increased aortic plaque accumulation. We propose that additional insults, such as fragmentation of elastic fibers, are necessary to alter plaque accumulation.

Differences in genetic signaling, and not mechanical properties of the wall, are linked to ascending aortic aneurysms in fibulin-4 knockout mice.

Fibulin-4 is an extracellular matrix protein that is essential for proper assembly of arterial elastic fibers. Mutations in fibulin-4 cause cutis laxa with thoracic aortic aneurysms (TAAs). Sixty percent of TAAs occur in the ascending aorta (AA). Newborn mice lacking fibulin-4 (Fbln4(-/-)) have aneurysms in the AA, but narrowing in the descending aorta (DA), and are a unique model to investigate locational differences in aneurysm susceptibility. We measured mechanical behavior and gene expression of AA and DA segments in newborn Fbln4(-/-) and Fbln4(+/+) mice. Fbln4(-/-) AA has increased diameters compared with Fbln4(+/+) AA and Fbln4(-/-) DA at most applied pressures, confirming genotypic and locational specificity of the aneurysm phenotype. When diameter compliance and tangent modulus were calculated from the mechanical data, we found few significant differences between genotypes, suggesting that the mechanical response to incremental diameter changes is similar, despite the fragmented elastic fibers in Fbln4(-/-) aortas. Fbln4(-/-) aortas showed a trend toward increased circumferential stretch, which may be transmitted to smooth muscle cells (SMCs) in the wall. Gene expression data suggest activation of pathways for SMC proliferation and inflammation in Fbln4(-/-) aortas compared with Fbln4(+/+). Additional genes in both pathways, as well as matrix metalloprotease-8 (Mmp8), are upregulated specifically in Fbln4(-/-) AA compared with Fbln4(+/+) AA and Fbln4(-/-) DA. Mmp8 is a neutrophil collagenase that targets type 1 collagen, and upregulation may be necessary to allow diameter expansion in Fbln4(-/-) AA. Our results provide molecular and mechanical targets for further investigation in aneurysm pathogenesis.

Electrospray ionization tandem mass spectrometry of sodiated adducts of cholesteryl esters.

Cholesteryl esters (CE) are important lipid storage molecules. The present study demonstrates that sodiated adducts of CE molecular species form positive ions that can be detected in both survey scan mode as well as by exploiting class-specific fragmentation in MS/MS scan modes. A common neutral loss for CE is the loss of cholestane (NL 368.5), which can be used to specifically quantify tissue CE molecular species. Using this MS/MS technique, CE molecular species were quantified in mouse monocyte-derived macrophages (J774 cells) incubated with either linoleic (18:2) or arachidonic acid (20:4). These studies revealed that arachidonic acid was not only incorporated into the CE pool, but also was elongated resulting in the accumulation of 22:4 and 24:4 CE molecular species in macrophages. Additionally, this technique was used to quantify CE molecular species present in crude lipid extracts from plasma of female mice fed a Western diet, which led to an enrichment in CE molecular species containing monounsaturated fatty acids compared to female mice fed a normal chow diet. Last, NL 368.5 spectra revealed the oxidation of the aliphatic fatty acid residues of CE molecular species containing polyunsaturated fatty acids. Taken together, these studies demonstrate the utility of using sodiated adducts of CE in conjunction with direct infusion electrospray ionization tandem mass spectrometry to rapidly quantify CE molecular species in biological samples.

Cyclooxygenase (COX)-1 and COX-2 participate in 5,6-epoxyeicosatrienoic acid-induced contraction of rabbit intralobar pulmonary arteries.

Epoxyeicosatrienoic acids (EETs) have been reported to contract intralobar pulmonary arteries (PA) of the rabbit in a cyclooxygenase (COX)-dependent manner. In the present study, we observed that COX-1 and COX-2 isoforms were expressed in freshly isolated PA of healthy rabbits. We examined the hypothesis that both COX isoforms participate in 5,6-EET-induced contraction of rabbit intralobar PA. Selective inhibition of COX-1 with 300 nM 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole (SC-560) prevented 5,6-EET (1x10(-8)-1x10(-5) M)-induced contractions of isolated intralobar rabbit PA rings in a manner similar to that observed with the nonselective cyclooxygenase inhibitor indomethacin at 10 microM. Selective inhibition of COX-2 with either 100 nM 5-bromo-2-(4-fluorophenyl)-3-(4-methylsulfonyl) thiophene (DUP-697) or 3 microM N-(2-cyclohexyloxy-4-nitrophenyl) methanesulfonamide (NS-398) shifted the EC50 value of 5,6-EET-induced PA contraction to the right but with considerably lower efficacy than SC-560. In rabbit PA, 5,6-EET-induced contraction was primarily dependent on COX-1 activity. Differential metabolism of 5,6-EET by COX-1 and COX-2 does not explain the primary dependence of PA contraction on COX-1 activity because 5,6-EET was metabolized similarly by both COX isoforms. COX-1 and -2 were expressed primarily in PA endothelium where COX-1 expression was dense and uniform, whereas COX-2 expression was sparse and nonuniform. 5,6-EET-induced PA contraction was endothelium-dependent. These results suggest that 5,6-EET-induced contraction is primarily dependent on COX-1 activity.