Susanne Bro

Chronic Renal Failure Accelerates Atherogenesis in Apolipoprotein E–Deficient Mice

Cardiovascular mortality is 10 to 20 times increased in patients with chronic renal failure (CRF). Risk factors for atherosclerosis are abundant in patients with CRF. However, the pathogenesis of cardiovascular disease in CRF remains to be elucidated. The effect of CRF on the development of atherosclerosis in apolipoprotein E-deficient male mice was examined. Seven-week-old mice underwent 5/6 nephrectomy (CRF, n = 28), unilateral nephrectomy (UNX, n = 24), or no surgery (n = 23). Twenty-two weeks later, CRF mice showed increased aortic plaque area fraction (0.266 +/- 0.033 versus 0.045 +/- 0.006; P < 0.001), aortic cholesterol content (535 +/- 62 versus 100 +/- 9 nmol/cm(2) intimal surface area; P < 0.001), and aortic root plaque area (205,296 +/- 22,098 versus 143,662 +/- 13,302 micro m(2); P < 0.05) as compared with no-surgery mice; UNX mice showed intermediate values. The plaques from uremic mice contained CD11b-positive macrophages and showed strong staining for nitrotyrosine. Systolic BP and plasma homocysteine concentrations were similar in uremic and nonuremic mice. Plasma urea and cholesterol concentrations were elevated 2.6-fold (P < 0.001) and 1.5-fold (P < 0.001) in CRF compared with no-surgery mice. Both variables correlated with aortic plaque area fraction (r(2) = 0.5, P < 0.001 and r(2) = 0.3, P < 0.001, respectively) and with each other (r(2) = 0.5, P < 0.001). On multiple linear regression analysis, only plasma urea was a significant predictor of aortic plaque area fraction. In conclusion, the present findings suggest that uremia markedly accelerates atherogenesis in apolipoprotein E-deficient mice. This effect could not be fully explained by changes in BP, plasma homocysteine levels, or total plasma cholesterol concentrations. Thus, the CRF apolipoprotein E-deficient mouse is a new model for studying the pathogenesis of accelerated atherosclerosis in uremia.

Effects of excess PTH on nonclassical target organs

The classical target organs for parathyroid hormone (PTH) are the bone and kidneys. In uremia, however, numerous studies have shown that PTH may also affect the function of a number of nonclassical organs and tissues besides the bone and kidney, including the brain, heart, smooth muscles, lungs, erythrocytes, lymphocytes, pancreas, adrenal glands, and testes. Most of these effects do not apply to the generally accepted actions or normal regulatory mechanisms of PTH. Thus, the potential role of PTH as one of the possibly many toxins in uremia is of current interest. The molecular basis for the actions of elevated PTH levels on various nonrenal and nonskeletal organs or tissues might be mediated via the widespread distribution of the classical PTH/PTH-related peptide (PTHrP) receptors and via the novel PTH2 receptors. The present survey deals with an evaluation of the nonrenal and nonskeletal effects of excess PTH in uremia, taking into consideration the presently available information on the organ-specific expression of the classical and novel PTH receptors, and of the expression and function of PTHrP.

Increased Expression of Adhesion Molecules in Uremic Atherosclerosis in Apolipoprotein-E–Deficient Mice

Chronic renal failure markedly accelerates atherosclerosis in apolipoprotein-E-deficient mice, but the mechanism is unknown. The recruitment of inflammatory cells in the arterial wall by vascular adhesion molecules plays a key role in the formation of classical atherosclerosis. This study examines whether the expression of vascular adhesion molecules is increased in uremic atherosclerosis. Uremia was induced by 5/6 nephrectomy; control mice were sham-operated. After 2 wk of uremia, no lesion formation could be demonstrated in uremic or control mice. After 12 wk, aortas from uremic mice had a 9.8-fold increase of the aortic plaque area fraction compared with control mice (P < 0.0001). The aortic expression of intercellular adhesion molecule-1 (ICAM-1) mRNA in uremic mice was 215 +/- 31% (P < 0.05) and 243 +/- 55% (P < 0.05) of that in controls after 2 and 12 wk, respectively (n = 9 x 4). In contrast, aortic expression of vascular cell adhesion molecule-1 (VCAM-1) mRNA in uremic mice was unchanged after 2 wk but increased to 237 +/- 40% (P < 0.01) of that in control mice after 12 wk. On immunohistochemistry of aortas from uremic mice, ICAM-1 was predominantly present in endothelial cells both in nonlesioned and lesioned aortas, whereas VCAM-1 was predominantly present in the medial smooth muscle cell layer in lesioned aortas. The plasma concentration of soluble ICAM-1 (sICAM-1) (but not of sVCAM-1) was slightly elevated after 2 wk of uremia. In contrast, both sICAM-1 and sVCAM-1 plasma concentrations were markedly higher in uremic than control mice after 12 wk. These results suggest that uremic atherosclerosis is preceded by an upregulation of ICAM-1 expression in arterial endothelium and that formation of early uremic lesions is accompanied by upregulation of VCAM-1 expression in the medial smooth muscle cell layer.

A neutralizing antibody against receptor for advanced glycation end products (RAGE) reduces atherosclerosis in uremic mice

Chronic renal failure markedly accelerates atherogenesis in apolipoprotein E-deficient (apoE(-/-)) mice. To study the putative role of receptor for advanced glycation end products (RAGE) in development of uremic atherosclerosis, apoE(-/-) mice received intraperitoneal injections thrice weekly of a neutralizing murine RAGE-antibody (RAGE-ab) (n=21) or an isotype-matched control antibody (placebo-ab) (n=23). Treatment was started 4 weeks after surgical 5/6 nephrectomy in 16 weeks old mice and continued for 12 weeks. The RAGE-ab did not affect blood pressure, plasma cholesterol or measures of uremia. However, the aortic plaque area fraction was reduced by 59% in RAGE-ab compared with placebo-ab-treated mice (0.016 +/- 0.002 versus 0.039 +/- 0.005, P<0.001). In plasma, the RAGE-ab reduced concentrations of oxidized phospholipid neo-epitopes in plasma as detected by the specific monoclonal antibody EO6 (P<0.05) and titers of IgG antibodies against oxidized low-density lipoprotein (P<0.001). In the aorta of treated mice, the RAGE-ab did not affect the mRNA expression of eight selected genes associated with inflammation. The results suggest that blockade of RAGE reduces the proatherogenic effects of uremia, possibly through a systemic decrease in oxidative stress.

Inhibition of the renin-angiotensin system abolishes the proatherogenic effect of uremia in apolipoprotein E-deficient mice

Objective—Uremia accelerates atherosclerosis in apolipoprotein E-deficient (apoE−/−) mice. We examined whether this effect may be preventable by pharmacological blockade of the renin-angiotensin system (RAS). Methods and Results—Uremia was induced in apoE−/− mice by 5/6 nephrectomy (NX). Treatment with the angiotensin converting enzyme inhibitor enalapril (2 or 12 mg/kg/d) from week 4 to 36 after NX reduced the aortic plaque area fraction from 0.23±0.02 (n=20) in untreated mice to 0.11±0.01 (n=21) and 0.08±0.01 (n=23), respectively (P<0.0001); the aortic plaque area fraction was 0.09±0.01 (n=22) in sham-operated controls. Enalapril from week 20 to 44 after NX also retarded the progression of atherosclerosis. Plasma levels of soluble intercellular adhesion molecule-1 (sICAM-1) and vascular cell adhesion molecule-1 (sVCAM-1) and concentrations of IgM antibodies against oxidized low density lipoprotein (OxLDL) increased after NX (P<0.01). Enalapril (12 mg/kg/d) attenuated these increases (P<0.05) and reduced aortic expression of vascular cell adhesion molecule (VCAM)-1 mRNA (P<0.05). Atherosclerosis in NX mice was also reduced by losartan (an angiotensin II receptor-blocker), but not when blood pressure was lowered with hydralazine (a non-RAS-dependent vasodilator). Conclusion—The results suggest that inhibition of RAS abolishes the proatherogenic effect of uremia independent of its blood pressure-lowering effect, possibly because of antiinflammatory and antioxidative mechanisms.

Uremia-Specific Effects in the Arterial Media During Development of Uremic Atherosclerosis in Apolipoprotein E–Deficient Mice

Objective—Uremia accelerates formation of atherosclerosis-like lesions in apolipoprotein E–deficient (apoE−/−) mice. In this study, we compared gene expression patterns in classical and uremic atherosclerosis. Methods and Results—High-density oligonucleotide microarray analyses were performed with aortic RNA from 5/6 nephrectomized (NX) and sham-operated mice. After 12 weeks, NX apoE−/− mice had more atherosclerosis and 24 genes were differentially expressed as compared with sham apoE−/− mice. Nine genes expressed in muscle cells displayed reduced expression (3.3- to 142-fold, P<0.05), whereas osteopontin gene expression was increased 8.7-fold (P<0.05) in NX mice. Studies of NX wild-type mice suggested that the changes in NX apoE−/− mice were dependent on hypercholesterolemia. Nevertheless, lesioned versus nonlesioned areas of aortas from nonuremic apoE−/− mice with classical atherosclerosis displayed less pronounced reductions in expression of the muscle cell related genes than seen in NX apoE−/− mice even though the osteopontin gene expression was increased ≈15-fold. Electron microscopy showed more vacuolized and necrotic smooth muscle cells within the media underneath both nonlesioned and lesioned intima in NX than in sham apoE−/− mice. Conclusion—The results suggest that uremic vasculopathy in apoE−/− mice, in addition to intimal atherosclerosis, is characterized by a uremia-specific medial smooth muscle cell degeneration, which appears to be accentuated by hypercholesterolemia.

Effect of treatment with human apolipoprotein A-I on atherosclerosis in uremic apolipoprotein-E deficient mice.

OBJECTIVE Uremia markedly increases the risk of atherosclerosis. Thus, effective anti-atherogenic treatments are needed for uremic patients. This study examined effects of non-lipidated recombinant human apoA-I (h-apoA-I) and a recombinant trimeric apoA-I molecule (TripA-I) on lipid metabolism and atherosclerosis in uremic apoE-/- mice. METHODS AND RESULTS Upon intraperitoneal injection, h-apoA-I and TripA-I rapidly associated with plasma HDL and reduced mouse apoA-I plasma levels without affecting total or HDL cholesterol concentrations. The plasma half-life was approximately 36 h for TripA-I and approximately 16 h for h-apoA-I. Injection of h-apoA-I (100mg/kg) or TripA-I (100mg/kg) twice weekly for 7 weeks did not affect the cross-sectional area of atherosclerotic lesions in the aortic root, or the en face lesion area and cholesterol content in the thoracic aorta in uremic apoE-/- mice. Also, the treatments did not affect expression of selected inflammatory genes in the thoracic aorta or plasma concentrations of soluble ICAM-1 and VCAM-1. However, h-apoA-I-treated mice had larger smooth muscle cell-staining areas in aortic root plaques than PBS-treated mice (4.8+/-0.8% vs. 2.5+/-0.6%, P<0.05). CONCLUSIONS The data suggest that long-term treatment with non-lipidated h-apoA-I or TripA-I might affect plaque composition but does not reduce atherosclerotic lesion size in uremic apoE-/- mice.

ACE inhibition attenuates uremia-induced aortic valve thickening in a novel mouse model.

BackgroundWe examined whether impaired renal function causes thickening of the aortic valve leaflets in hyperlipidemic apoE-knockout (apoE-/-) mice, and whether the putative effect on the aortic valves could be prevented by inhibiting the angiotensin-converting enzyme (ACE) with enalapril.MethodsThickening of the aortic valve leaflets in apoE-/- mice was induced by producing mild or moderate chronic renal failure resulting from unilateral nephrectomy (1/2 NX, n = 18) or subtotal nephrectomy (5/6 NX, n = 22), respectively. Additionally, the 5/6 NX mice were randomized to no treatment (n = 8) or enalapril treatment (n = 13). The maximal thickness of each leaflet was measured from histological sections of the aortic roots.ResultsLeaflet thickness was significantly greater in the 5/6 NX mice than in the 1/2 NX mice (P = 0.030) or the unoperated mice (P = 0.003). The 5/6 NX mice treated with enalapril had significantly thinner leaflets than did the untreated 5/6 NX mice (P = 0.014).ConclusionModerate uremia causes thickening of the aortic valves in apoE-/- mice, which can be attenuated by ACE inhibition. The nephrectomized apoE-/- mouse constitutes a new model for investigating the mechanisms of uremia-induced aortic valve disease, and also provides an opportunity to study its pharmacologic prevention.

Lipoprotein(a) accelerates atherosclerosis in uremic mice

Uremic patients have increased plasma lipoprotein(a) [Lp(a)] levels and elevated risk of cardiovascular disease. Lp(a) is a subfraction of LDL, where apolipoprotein(a) [apo(a)] is disulfide bound to apolipoprotein B-100 (apoB). Lp(a) binds oxidized phospholipids (OxPL), and uremia increases lipoprotein-associated OxPL. Thus, Lp(a) may be particularly atherogenic in a uremic setting. We therefore investigated whether transgenic (Tg) expression of human Lp(a) increases atherosclerosis in uremic mice. Moderate uremia was induced by 5/6 nephrectomy (NX) in Tg mice with expression of human apo(a) (n = 19), human apoB-100 (n = 20), or human apo(a) + human apoB [Lp(a)] (n = 15), and in wild-type (WT) controls (n = 21). The uremic mice received a high-fat diet, and aortic atherosclerosis was examined 35 weeks later. LDL-cholesterol was increased in apoB-Tg and Lp(a)-Tg mice, but it was normal in apo(a)-Tg and WT mice. Uremia did not result in increased plasma apo(a) or Lp(a). Mean atherosclerotic plaque area in the aortic root was increased 1.8-fold in apo(a)-Tg (P = 0.025) and 3.3-fold (P = 0.0001) in Lp(a)-Tg mice compared with WT mice. Plasma OxPL, as detected with the E06 antibody, was associated with both apo(a) and Lp(a). In conclusion, expression of apo(a) or Lp(a) increased uremia-induced atherosclerosis. Binding of OxPL on apo(a) and Lp(a) may contribute to the atherogenicity of Lp(a) in uremia.

Cardiac structure and function in a mouse model of uraemia without hypertension

Kidney dysfunction is often associated with cardiac left ventricular hypertrophy and increased cardiovascular mortality. Objective. The aim of this study was to find out whether this reflects direct effects of uraemia on the heart or is dependent on accompanying hypertension. Material and methods. Apolipoprotein‐E (apoE)‐deficient C57BL/6 mice are resistant to development of hypertension after renal mass reduction. To evaluate the impact of uraemia without hypertension on the heart, apoE‐deficient mice underwent 5/6 nephrectomy (NX) or sham operation (Sh) and were randomized to treatment with the angiotensin converting enzyme inhibitor enalapril (12 mg kg−1 d−1) or no medication. Results. NX did not affect systolic blood pressure (BP), but reduced mean creatinine clearance, body weight and blood haemoglobin to 27 % (p<0.01), 82 % (p<0.0001) and 73 % (p<0.0001), respectively, of the values in Sh mice. Thirty‐six weeks after NX, heart wet weight, echocardiographic estimates of left ventricular mass and left ventricular diastolic and systolic functions were similar in NX and Sh mice. NX did not increase cardiac fibrosis or cardiac mRNA expression of biglycan, whereas it decreased the mRNA expression of procollagen (p<0.01). Enalapril reduced BP (p<0.001), heart wet weight and estimated left ventricular mass in both NX (p<0.01) and Sh mice (p<0.05), but did not affect cardiac diastolic or systolic function. Conclusions. The results suggest that uraemia does not impair cardiac structure or function in apoE‐deficient mice. Since NX has no effect on BP in apoE‐deficient mice, the results may indicate that hypertension is important for development of left ventricular disease in uraemia.