Microsatellite instability of selective target genes in HNPCC-associated colon adenomas.

Microsatellite instability (MSI) occurs in most hereditary nonpolyposis colorectal cancers (HNPCC) and less frequently in sporadic tumors as the result of DNA mismatch repair (MMR) deficiency. Instability at coding microsatellites (cMS) in specific target genes causes frameshift mutations and functional inactivation of affected proteins, thereby providing a selective growth advantage to MMR deficient cells. At present, little is known about Selective Target Gene frameshift mutations in preneoplastic lesions. In this study, we examined 30 HNPCC-associated MSI-H colorectal adenomas of different grades of dysplasia for frameshift mutations in 26 cMS-bearing genes, which, according to our previous model, represent Selective Target genes of MSI. About 30% (8/26) of these genes showed a high mutation frequency (> or =50%) in colorectal adenomas, similar to the frequencies reported for colorectal carcinomas. Mutations in one gene (PTHL3) occurred significantly less frequently in MSI adenomas compared to published mutation rates in MSI carcinomas (36.0 vs 85.7%, P=0.023). Biallelic inactivation was observed in nine genes, thus emphasizing the functional impact of cMS instability on MSI tumorigenesis. Some genes showed a high frequency of frameshift mutations already at early stages of MSI colorectal tumorigenesis that increased with grade of dysplasia and transition to carcinoma. These include known Target Genes like BAX and TGFBR2, as well as three novel candidates, MACS, NDUFC2, and TAF1B. Overall, we have identified genes of potential relevance for the initiation and progression of MSI tumorigenesis, thus representing promising candidates for novel diagnostic and therapeutic approaches directed towards MMR-deficient tumors.

No aggravation of renal injury in apolipoprotein E knockout mice (ApoE(-/-)) after subtotal nephrectomy.

BACKGROUND: There is substantial experimental evidence that various forms of dyslipidaemia aggravate the course of renal failure and that reversal of dyslipidaemia ameliorates progression of renal failure. The apolipoprotein E knockout mouse (ApoE(-/-)) is an established model of accelerated atherogenesis. We investigated whether the course of renal disease after uninephrectomy (UNX) and subtotal nephrectomy (SNX) is altered in ApoE(-/-) mice compared with their genetic controls. METHODS: Ten-week-old, male ApoE(-/-) mice (body weight 25+/-2 g) were subjected either to sham operation (sham), UNX or SNX. C57BL/6 sham, UNX and SNX mice served as controls (body weight 26+/-3 g). The food intake of ApoE(-/-) and C57BL/6 mice was kept identical by a pair-feeding protocol. After 12 weeks, mean arterial blood pressure and heart rate were measured in awake resting mice, the kidneys were perfusion fixed and analysed using quantitative histological methods, immunohistochemistry and RT-PCR. RESULTS: At baseline, the sham ApoE(-/-) mice had significantly higher (P<0.05) serum cholesterol and triglycerides than the controls. In parallel, mean arterial blood pressure was significantly elevated in sham ApoE(-/-) mice compared with controls (137+/-15 vs 116+/-4 mmHg; P<0.05). In the sham groups, the glomerulosclerosis index was significantly higher in the ApoE(-/-) mice (1.05+/-0.14 vs 0.57+/-0.07; P<0.05), whereas the tubulointerstitial damage score was comparable (0.06+/-0.04 vs 0.04+/-0.02; n.s.). After SNX there was a significant increase in glomerulosclerosis index, but no difference could be detected between ApoE(-/-) and controls (1.75+/-0.16 vs 1.61+/-0.01, n.s.). The same was true for the tubulointerstitial damage index. CONCLUSIONS: Despite some glomerulosclerosis and elevated mean arterial blood pressure at baseline, no acceleration of progression of renal disease was found in this genetic model of hyperlipoproteinaemia. This observation suggests that despite the known spontaneous histological changes in untouched kidneys, however, the presence of hyperlipidaemia in the ApoE(-/-) mouse does not cause more severe progression in the present models of moderate renal disease.

Myocyte loss in early left ventricular hypertrophy of experimental renovascular hypertension.

Left ventricular hypertrophy (LVH) develops very early in experimental renovascular hypertension after clipping of one renal artery and is accompanied by a remodeling of cardiac structure which has not yet been investigated in detail. It was the aim of the present study to analyze changes in cardiomyocyte number and volume in LVH after 2 weeks of renovascular hypertension. Sprague-Dawley rats were subjected to clipping of the left renal artery (2K1C) or sham operation (sham). One group of 2K1C rats received antihypertensive treatment with dihydralazine. The experiment was terminated after 2 weeks. Hearts were investigated using stereological methods, electron microscopy, immunohistology for the proliferation marker proliferating cell nuclear antigen, the pro- and anti-apoptotic proteins Bax and Bcl-2 as well as the TUNEL technique. After 2 weeks, systolic blood pressure and relative left ventricular weight were significantly higher in untreated 2K1C animals than in sham and dihydralazine-treated 2K1C rats. Volume fraction of interstitial tissue and capillary length density were not different, whereas wall thickness of intramyocardial arteries was significantly higher in untreated 2K1C (5.12+/-0.7 micro m) than in sham (3.92+/-0.6 micro m) and in dihydralazine-treated 2K1C (3.91+/-0.7 micro m) rats. Cardiomyocyte diameter and volume were significantly higher in untreated 2K1C than in sham animals. The number of cardiomyocytes per left ventricle was significantly lower in untreated 2K1C rats (5.5+/-1.6 vs 3.9+/-6.9 x10(7)). Using immunohistochemistry, no direct evidence of apoptosis was found, but a relative higher expression of the anti-apoptotic protein bcl-2 expression was seen in untreated 2K1C than in sham animals. This may reflect a protective mechanism as a consequence of earlier occurring apoptosis. These observations document that experimental renovascular hypertension induces a rapidly developing LVH characterized by marked cardiac remodeling and substantial loss of cadiomyocytes.

The apolipoprotein e knockout mouse: a model documenting accelerated atherogenesis in uremia.

Rodents do not develop spontaneous atherosclerosis. Currently, there is no good animal model to study the effect of uremia on atherosclerosis. This study evaluated whether apolipoprotein E knockout (Apoe-/-) mice are useful to study the effect of renal dysfunction on cardiovascular risk. Apoe-/- mice have decreased serum apolipoprotein E and exhibit lipid abnormalities and atherosclerosis even on a low-cholesterol diet. Ten-wk-old Apoe-/- mice were subtotally nephrectomised (SNX Apoe-/-; n = 8), uninephrectomised (UNX Apoe-/-; n = 5), or sham-operated (sham Apoe-/-; n = 5) and compared with their genetic controls (SNX C57/BL6; UNX C57/BL6; sham C57/BL6). After 12 wk, BP was measured intraarterially, blood samples were taken, and the experiment was terminated by perfusion fixation. The heart weight was determined, and quantitative morphologic analysis of intramyocardial arteries and aortic changes was performed. At the end of the experiment, heart weight and relative left ventricular weight were comparable in all groups. Intraarterial BP was somewhat higher in Apoe-/- mice compared with controls. Baseline serum cholesterol and triglyceride levels were higher in Apoe-/- mice than in C57/BL6. Atherosclerotic plaques were not present in sham or UNX C57/BL6, but minor plaque formation was noted in some SNX control animals. In contrast, beginning plaques were seen even in untouched Apoe-/- mice, and strikingly increased plaque formation was noted in UNX and SNX Apoe-/- mice. Maximal plaque diameter (cross-section) was 37 +/- 74 micro m in SNX C57/BL6, 191 +/- 90 micro m in sham Apoe-/-, 323 +/- 66 micro m in UNX Apoe-/-, and 457 +/- 17 micro m in SNX Apoe-/-. The plaque morphology corresponded with that of early plaques characterized by foam cells and virtual absence of lymphocytes or smooth muscle cell infiltration. In conclusion, even mild renal dysfunction, i.e., after uninephrectomy, causes a dramatic increase in plaque size and aggressive morphology (foam cell rich soft plaques) in the animal model of the Apoe-/- mouse.

Effect of antioxidant therapy with dl-alpha-tocopherol on cardiovascular structure in experimental renal failure.

BACKGROUND: Chronic renal failure is characterized by remodeling of the structure of the heart and the vasculature, for example, left ventricular hypertrophy, myocardial fibrosis, capillary/myocyte mismatch, as well as thickening of intramyocardial arteries and of peripheral arteries and veins. Furthermore, uremia is a state of increased oxygen stress. It was the purpose of this study to examine whether these findings are interrelated. METHODS: To investigate whether antioxidative therapy with dl-alpha-tocopherol (Toco; vitamin E) interferes with the development of abnormal cardiovascular structure in experimental renal failure, 28 male Sprague-Dawley rats were subjected to partial renal ablation (subtotal nephrectomy, SNX) or to sham operation (sham). SNX were either left untreated or received the antioxidant Toco (2 x 1500 IE/kg BW/week in the pellets). Blood pressure was measured using tail plethysmography. The experiment was terminated after 12 weeks. Heart and left ventricular weight were determined and the following parameters were measured using morphometry and stereology: volume densities of cardiomyocytes, capillaries and non-vascular interstitium; length density and total length of cardiac capillaries, wall thickness of intramyocardial arterioles and of the aorta. RESULTS: Systolic blood pressure and body weight were comparable in all groups. Treatment with Toco led to significantly increased plasma concentrations of Toco. Left ventricular weight and wall thickness of intramyocardial arteries were significantly higher in both SNX groups compared to sham controls. Volume density of the cardiac interstitial tissue was significantly higher in untreated SNX than in Toco treated SNX and sham control rats. Length density of capillaries was significantly lower in untreated SNX than in control rats; however, the values were significantly higher, and even higher than in sham controls, when SNX were treated with Toco. CONCLUSIONS: Treatment with the antioxidant dl-alpha-tocopherol prevented cardiomyocyte/capillary mismatch, and to some extent also myocardial fibrosis in rats with renal failure. The results point to a role of oxidative stress in the genesis of myocardial interstitial fibrosis and capillary deficit of the heart.