Beta2-adrenergic activity modulates vascular tone regulation in lecithin:cholesterol acyltransferase knockout mice.

Lecithin:cholesterol acyltransferase (LCAT) deficiency is associated with hypoalphalipoproteinemia, generally a predisposing factor for premature coronary heart disease. The evidence of accelerated atherosclerosis in LCAT-deficient subjects is however controversial. In this study, the effect of LCAT deficiency on vascular tone and endothelial function was investigated in LCAT knockout mice, which reproduce the human lipoprotein phenotype. Aortas from wild-type (Lcat(wt)) and LCAT knockout (Lcat(KO)) mice exposed to noradrenaline showed reduced contractility in Lcat(KO) mice (P<0.005), whereas acetylcholine exposure showed a lower NO-dependent relaxation in Lcat(KO) mice (P<0.05). Quantitative PCR and Western blotting analyses suggested an adequate eNOS expression in Lcat(KO) mouse aortas. Real-time PCR analysis indicated increased expression of ß2-adrenergic receptors vs wild-type mice. Aorta stimulation with noradrenaline in the presence of propranolol, to abolish the ß-mediated relaxation, showed the same contractile response in the two mouse lines. Furthermore, propranolol pretreatment of mouse aortas exposed to L-NAME prevented the difference in responses between Lcat(wt) and Lcat(KO) mice. The results indicate that LCAT deficiency leads to increased ß2-adrenergic relaxation and to a consequently decreased NO-mediated vasodilation that can be reversed to guarantee a correct vascular tone. The present study suggests that LCAT deficiency is not associated with an impaired vascular reactivity.

Cathepsin-k as a diagnostic marker in the identification of micro-granulomas in Crohn's disease.

Crohn's disease is a chronic inflammatory bowel disease, whose aetiology and pathogenesis are still unknown. The occurrence of epithelioid granulomas is one characteristic feature of the disease since these lesions are found in the bowel wall in 50-87% of colectomy specimens. Although granulomas are not pathognomonic, their identification is considered a relevant element for diagnosis. Cathepsin-k, a papain-like cysteine protease, is involved in bone remodelling, and has been widely used as a immunohistochemical marker for the in situ detection of osteoclasts. Interestingly, the expression of this potent protease is also significantly increased in stimulated tissue macrophages, epithelioid cells and granulomas, but is not expressed in resident tissue macrophages. In the present study, we evaluated Cathepsin-k expression as a diagnostic tool in the identification of small granulomas in Crohn's disease. Formalin-fixed and paraffin-embedded samples of 10 cases of Crohn's disease were collected from surgical ileo-colic resections followed by comparison of Cathepsin-k and CD68 immunoreactivity. Granulomas were identified in 4 of 10 cases examined in haematoxylin & eosin preparations. Cathepsin-k enabled the identification of small granulomas (with a diameter between 100 and 200 microm) in 6 of 10 cases, mainly localized within the submucosa and muscular layers. When compared to CD68, Cathepsin-k immunoreactivity was generally absent or only weakly expressed in resting tissue macrophages, thus allowing better identification of activated epithelioid cells. Based on these results, Cathepsin-k appears to be a reliable tool for the precise and rapid identification of small epithelioid granulomas in Crohn's disease.

Evaluation of a soft atherosclerotic lesion in the rabbit aorta by an invasive IVUS method versus a non-invasive MRI technology.

The intravascular ultrasound (IVUS) modality has rapidly gained acceptance for the measurement of arterial plaque thickness and for anatomical characterization. In view, however, of the growing interest in the direct assessment of plaque size after therapeutic modalities directly reducing plaque burden, a non-invasive method such as magnetic resonance imaging (MRI) may be of help for repeated evaluations. The two methods were compared directly on a focal plaque developed at the abdominal aortic level by a combination of local electric lesion followed by a hypercholesterolemic diet. The plaque was fully characterized histopathologically at intervals up to 120 days from lesion induction, and maximal plaque formation was detected at 90 days from electrical injury. Plaques could be well assessed by IVUS at each time point analyzed and data correlated very well to histopathologic findings (r = 0.969, P = 0.0014). The MRI technology provided reliable determinations only at 90 days after lesion induction, i.e. at maximal plaque formation, with excellent correspondence to IVUS determinations (r = 0.989, P = 0.0111). Altogether these findings indicate that the non-invasive MRI technology, when applied to the analysis of arterial plaques of adequate size, can be used successfully for plaque determination, with results comparable to the invasive IVUS technique.

Resistive index in chronic nephropathies: predictive value of renal outcome.

UNLABELLED: The study of renovascular resistances by color Doppler ultrasound has become a useful diagnostic resource for nephrologists. In recent nephrological literature, many papers deal with the correlations between resistive index, anatomo-pathological patterns and renal function. AIMS: In our study, we have tried to discover if resistive index represents a prognostic index of progressive renal failure. MATERIAL AND METHODS: To this purpose we compared renal resistive index and blood creatinine obtained from 28 nephropathic patients at their first control, with blood creatinine values after a 3-year follow-up period. Using a linear regression test, we found a strong correlation between the initial value of resistive index and the value of creatinine variation (p = 0.006). RESULTS: All of the patients with normal resistive index at the beginning maintained a stable renal function. Conversely, the patients with high resistive index at their first control showed a progressive renal failure. CONCLUSION: Our study shows the reliability of resistive index in the prognostic evaluation of renal outcome.

Development of a lipid-rich, soft plaque in rabbits, monitored by histology and intravascular ultrasound.

Lipid rich, soft plaques in the clinic are a common forerunner to occlusive thrombus formation, even with modest arterial stenosis. Animal models of atherosclerosis, obtained by various methods, do not generally allow direct in vivo evaluation of the lesion and, furthermore, cannot be examined more than once. The aim of the study was the generation of a rabbit model of atherosclerosis, with morphological characteristics similar to human lipid-rich, soft atheromatous plaques, and the evaluation of the reliability of intravascular ultrasound (IVUS) technology in the study of the development of atherosclerotic lesions in this model. Briefly, New Zealand white rabbits undergo perivascular electrical injury at both common carotid arteries, together with a 1.5% cholesterol diet for up to 90 days. The lesioned arterial segments show progressive changes, from diffuse cellular mortality, to macrophage infiltration in the media, up to the final migration of macrophages to the neointima, resulting in bulky, eccentric, macrophage and lipid-rich lesions. At IVUS, the produced lesions clearly resemble those described as 'soft plaques' in the clinical setting, with minimal calcification and reduced echo-reflectivity versus the adventitial layer. Quantitative and morphometric analysis of plaques shows a significant correlation between histological and IVUS measurements at each time point. In conclusion, vascular injury in the common carotids of rabbits generates atherosclerotic lipid-rich, soft plaques, that can be properly assessed by the IVUS methodology. The easy accessibility of the arterial lesion allows serial IVUS investigations and the direct evaluation of a number of locally or generally delivered therapeutic agents.

Increased cholesterol efflux potential of sera from ApoA-IMilano carriers and transgenic mice.

The ability of HDL to remove cholesterol from peripheral cells and drive it to the liver for excretion is believed to explain most of the strong inverse correlation between plasma HDL cholesterol levels and coronary heart disease. Carriers of the ApoA-IMilano (A-IM) mutant have a severe hypoalphalipoproteinemia but are not at increased risk for premature of coronary heart disease. To explain this apparent paradox, we compared the capacity of serum from A-IM and control subjects to extract cholesterol from Fu5AH cells. Because the A-IM carriers are all heterozygotes for the mutation, we also compared the cholesterol efflux capacity of serum from transgenic mice expressing A-IM or wild-type ApoA-I (A-IWT), in the absence of murine ApoA-I. In the whole series of human or mouse sera, cholesterol efflux was significantly correlated with several HDL-related parameters; after adjustment for concomitant variables, the only parameter that remained significantly correlated with cholesterol efflux was the serum ApoA-I concentration (r2=0.85 in humans and 0.84 in mice). The same was true when samples from control subjects, A-IM carriers, A-IWT or A-IM mice were analyzed separately. Cholesterol efflux to sera from the A-IM carriers was only reduced slightly compared with control sera (25.0+/-4.2% versus 30.4+/-3.3%), although there was a large reduction (-45%) in the serum ApoA-I concentration in the former. Cholesterol efflux was also lower to sera from A-IM than A-IWT mice (15.6+/-3.8% versus 30. 1+/-7.1%), but less than expected from the 70% reduction in serum ApoA-I concentration. A relative efflux potential of serum was calculated in each group as the slope of the regression line fitting cholesterol efflux to ApoA-I concentrations. Therefore, the relative efflux potential reflects the relative efficiency of ApoA-I in determining cell cholesterol efflux. The relative efflux potential of mouse and human sera was in the following order: A-IM mice>A-IM carriers>A-IWT mice=control subjects, suggesting a gene-dosage effect of the A-IM mutation on the efficiency of serum to extract cholesterol from cells. The high efficiency of A-IM-containing HDL for cell cholesterol uptake would result in an improved reverse cholesterol transport in the A-IM carriers, possibly explaining the low susceptibility to atherosclerosis development.

Human apolipoproteins A-I and A-II in cell cholesterol efflux: studies with transgenic mice.

The first step in reverse cholesterol transport is the movement of cholesterol out of cells onto lipoprotein acceptors in the interstitial fluid. The contribution of specific lipoprotein components to this process remains to be established. In this study, the role of human apolipoproteins (apo) A-I and A-II in the efflux of cellular cholesterol was investigated in transgenic mouse models in which the expression of murine apoA-I was abolished due to gene targeting (A-IKO). Serum from A-IKO mice and from mice expressing human apoA-I and/or human apoA-II was incubated with [3H]cholesterol-labeled Fu5AH rat hepatoma cells for 4 hours at 37 degrees C. The cholesterol efflux to the serum of A-IKO mice was markedly lower than that to the serum of mice transgenic for human apoA-I (5.0 +/- 1.5% versus 25.0 +/- 4.0%). Expression of human apoA-II alone did not modify the cholesterol efflux capacity of A-IKO mouse serum. Cholesterol efflux to serum of mice expressing human apoA-II together with human apoA-I was significantly lower than that to human apoA-I mouse serum (20.0 +/- 2.3% versus 25.0 +/- 4.0%). Regression analysis of cholesterol efflux versus the lipid/apolipoprotein concentrations of mouse serum suggested that 3 independent factors contribute to determine the cholesterol efflux potential of serum: the apolipoprotein composition of HDL, the serum concentration of HDL phospholipids, and the presence of a small fraction of particles containing apoA-I.

A transgenic mouse model for the detection of cellular stress induced by toxic inorganic compounds.

Transgenic mice for genotoxicity testing have been developed, although no such models have been produced for the evaluation of toxic, nongenotoxic chemical compounds. We have developed a transgenic mouse model for the analysis of toxic inorganic compounds. We engineered a mouse lineage with the human growth hormone (hGH) gene under the control of the human hsp70 promoter, in which a plasma-detectable hGH response can be elicited by exposure to heat shock. In primary cell cultures from these mice, hGH release was observed following treatment with several toxic inorganics. Transgenic mice injected intraperitoneally with sodium arsenite, cadmium chloride, copper sulphate, or methylmercurium chloride showed significant hGH levels in plasma.

Recombinant apolipoprotein A-IMilano dimer inhibits carotid intimal thickening induced by perivascular manipulation in rabbits.

Apolipoprotein A-IMilano (apoA-IM), a natural variant of apolipoprotein A-I (apoA-I), confers to the carriers a significant protection against vascular disease. The antiatherogenic activity of a recombinant disulfide-linked apoA-IM dimer (rA-IM/A-IM) was analyzed in vivo by evaluating its effect on neointimal formation induced by periarterial manipulation in 1% cholesterol-fed rabbits. A flexible collar was applied around the carotid artery 21 days after the beginning of the dietary regimen, and animals were killed 10 days later. Rabbits were injected five times with reconstituted high-density lipoprotein containing egg phosphatidylcholine (EPC) and rA-IM/A-IM (119 mg EPC + 40 mg protein per dose) or with EPC liposomes (119 mg EPC per dose) beginning either 5 days before or at the day of collar positioning. Neither treatment affected plasma cholesterol levels. A significant intimal thickening was observed in control animals; the intima-to-media (I/M) ratio was 0.63 +/- 0.11 versus 0.03 +/- 0.05 for the sham-operated contralateral arteries. Neointimal formation was markedly inhibited in animals pretreated with rA-IM/A-IM before lesion induction (I/M, 0.26 +/- 0.19) but not in those in which treatment began the day of collar insertion (I/M, 0.74 +/- 0.14). EPC liposomes did not affect neointimal formation (I/M, 0.50 +/- 0.14 and 0.51 +/- 0.07 in the two treatment groups). Proliferation of smooth muscle cells, assessed by direct incorporation of bromo-2'-deoxyuridine (BrdU) into replicating DNA, was reduced by approximately 30% and 75% in the intimal and medial tissues of rA-IM/A-IM-pretreated rabbits.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of isoprenoid biosynthesis and arterial smooth-muscle cell proliferation.

Recently, we provided in vitro and in vivo evidence that several vastatins with different potencies decrease arterial smooth-muscle cell (SMC) proliferation independently of their hypocholesterolemic properties. In this study, the in vivo dose-dependent antiproliferative activity of fluvastatin on neointimal formation induced by the insertion of a collar around one carotid artery was investigated in normocholesterolemic rabbits (five animals per treatment group). Intraperitoneal fluvastatin treatment progressively inhibited intimal to medial tissue ratios (I/M) by 5, 48, and 64% versus controls at doses of 3, 5, or 10 mg/kg/day, respectively. Local arterial delivery by an Alzet pump of mevalonate (8 mg/kg/day) at the site of collar placement fully prevented a fluvastatin (5 mg/kg/day) inhibitory effect on both I/M and SMC proliferation, as assessed by direct incorporation of bromodeoxyuridine (BrdU) into replicating DNA. The results suggest that vastatins exert a direct antiproliferative effect on intimal myocytes beyond their effects on plasma lipids, probably through local inhibition of isoprenoid biosynthesis.

Effect of lacidipine on the carotid intimal hyperplasia induced by cuff injury.

The in vivo antiatherogenic activity of the calcium antagonist lacidipine was investigated in arterial hyperplasia induced by perivascular manipulation of hypercholesterolemic carotid rabbits. This was accomplished by positioning a hollow silastic collar around one carotid, which within a few days induces an atherosclerotic lesion (proliferative lesion) showing biochemical and morphologic changes similar to those of early human atherosclerosis: the contralateral carotid, with no collar, served as control in the same animal. The effect of lacidipine was also investigated in aortic atherosclerotic lesions (fatty lesions) induced by hypercholesterolemia mixed with either cholesterol (1%) and lacidipine (3 mg/kg/day) or cholesterol (1%) alone for 8 weeks. Hypercholesterolemic New Zealand White rabbits were fed daily a standard diet. Intimal hyperplasia was mechanically induced in one carotid artery of each rabbit 6 weeks after dietary and drug treatment started. Neointimal formation was followed by measuring by light microscopy the cross-sectional thickness of intimal (I) and medial (M) tissue of fixed arteries. In positive control animals receiving dietary cholesterol only (n = 10), by 14 d after collar positioning the process of intimal hyperplasia was significantly pronounced. The control arteries showed an I:M tissue ratio of 0.03 +/- 0.02, whereas in the carotid with collar the ratio was 0.56 +/- 0.11. In the animals receiving lacidipine, neointimal formation was significantly lower [I:M tissue ratio 0.32 +/- 0.1 (n = 10), about 60% of positive controls]. Measurement of the percent area of the aortic intima covered by plaques did not show significant differences between control and lacidipine-treated animals. These results suggest a direct antiatherosclerotic effect of lacidipine on proliferative lesions.

HMG CoA reductase inhibitors. In vivo effects on carotid intimal thickening in normocholesterolemic rabbits.

The in vivo activity of different 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors (vastatins) on neointimal formation induced by insertion of a flexible collar around one carotid artery of normocholesterolemic rabbits was investigated. The contralateral carotid artery served as a sham control. Pravastatin, lovastatin, simvastatin, and fluvastatin were given mixed with food at daily doses of 20 mg/kg body wt for 2 weeks starting on the day of collar placement. The treatment with vastatins did not modify rabbit plasma cholesterol concentrations. The neointimal formation was assessed by measuring the cross-sectional thickness of intimal and medial tissues of fixed arteries with light microscopy. Fourteen days after collar placement, intimal hyperplasia (mostly cellular) was pronounced in treated carotid arteries. The intimal/medial (I/M) tissue ratio was 12-fold higher in treated arteries than in arteries without the collar (0.36 +/- 0.04 versus 0.03 +/- 0.02). Animals treated with lovastatin (n = 12), simvastatin (n = 12), and fluvastatin (n = 12) showed significantly less neointimal formation; I/M tissue ratios were 0.24 +/- 0.03, 0.20 +/- 0.03, and 0.17 +/- 0.03, respectively. The inhibition elicited by pravastatin (n = 12, 0.32 +/- 0.03) did not reach statistical significance. alpha-Actin antibody immunofluorescence analysis of serial sections revealed that cells present in the hyperplastic intima were mostly myocytes. Rates of intimal myocyte proliferation were also measured by incorporation of 5-bromo-2'-deoxyuridine, a thymidine analogue, into replicating DNA. Immunofluorescence analysis showed that 5-bromo-2'-deoxyuridine was actively incorporated into intimal myocytes after ++reinsertion of the collar, with a labeling index (percent of labeled myocytes) of 2.15 after 14 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Light and electron microscopic findings in five cases of cryoglobulinemic glomerulonephritis.

Renal tissue from five patients with cryoglobulinemia was studied by light and electron microscopy and immunofluorescence. None of the histologic features observed at the light microscopic level seems to be specific for cryoglobulinemia. Electron microscopic investigations have shown very large electron dense deposits in almost every examined lobule in all cases. The deposits displayed two main patterns; a homogeneous texture in two cases and tubular or annular structures in three cases. The patients with typically structured deposits had IgG-IgM cryoglobulinemia (2 cases) or monoclonal IgM cryoglobulinemia (1 case). The presence of IgM in cryoglobulinemia may be the cause of the peculiar structure of the deposits.