Marked induction of sterol 27-hydroxylase (CYP27) activity and MRNA levels during differentiation of human cultured monocytes into macrophages

Hasegawa expansion of the atherosclerotic plaque, its stability and the potential for smooth muscle cell proliferation. Several groups of proteases have been suggested to be involved in the remodelling of the extracellular matrix (ECM). Members of the serine protease family, such as plasmin and tissue-type and urokinase-type plasminogen activators, are able to degrade parts of the ECM. Cysteine proteases such as the cathepsins have the capacity to degrade elastin fibres. However, the most important family of matrix-degrading enzymes is probably the matrix metalloproteinases (MMPs) since these proteases can degrade all macromolecules present in the connective tissue matrix. Finally, the catalytic activity of the different families of proteases is regulated by groups of specific inhibitors, e.g. tissue inhibitors of metalloproteinases (TIMPs), plasminogen activator inhibitor (PAI) and cystatins, all of which inactivate the proteolytic activity by complex binding. Studies in our group over the past several years have shown that there is a genetic component in the regulation of MMP expression thats is associated with cardiovascular disease. Similarly, at least one functional polymorphism has been detected in the promoter region of the cysteine protease inhibitor cystatin C, the expression of which is reduced in atherosclerotic and aneurysmal lesions. Evidence has also been provided that insulin might be an important regulator of MMP action and that MMP activity might be particularly implicated in type-2 diabetes. ~-~THE CORONARY HEART DISEASE RISK IN NEWLY DIAGNOSED TYPE 2 DIABETIC PERSONS N.D. Hancu. Iuliu Hatieganu University, Cluj Napoca, Cluj, Romania Aim: To assess the coronary heart disease risk in newly diagnosed type 2 diabetic persons. Method: Clinical and biochemical data collected from 1034 newly diagnosed type 2 diabetic persons in Diabetes Center Cluj Napoca during 2001(EPIDIAB Programme). The coronary heart disease risks were estimated with UK 99 chart proposed by J.S. Yudkin and N. Chaturvedi. Of the 1034 persons, 428 fulfilled the criteria for assessment by UK 99 chart and thus further analysis. Results: The segregation in risk classes was as follows: very low risk 4.4%, low risk 10.5%, moderate risk 32.2%, high risk 44.9% and very high risk 7.9%. The mean coronary heart disease risk (%) was 15.94±9.35. Conclusions: Managing risk factors is mandatory in people with diabetes mellitus as they carry a high risk at diagnosis (52.28% of them have a cardiovascular risk higher than 20%). The management of risk factors in diabetes should precede the onset of heart disease and should be pursued as aggressively as it would be in individuals with established vascular disease. Keywords: coronary heart disease risk, diabetes mellitus, UK 99 ~-~

MARKED INDUCTION OF STEROL 27-HYDROXYLASE (CYP27) ACTIVITY AND MRNA LEVELS DURING DIFFERENTIATION OF HUMAN CULTURED MONOCYTES INTO MACROPHAGES

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~--f~ PPE-1-REGULATED ENDOTHELIAL-SPECIFIC GENE THERAPY USING VEGF & PDGF-B INDUCED ANGIOGENESIS AND VESSEL MATURATION IN ISCHEMIC LIMB MODEL A. Shaish 1, K. Levanon 1, N. Vardr-Bloom 1, S. Greenberger1, I. Barshak2, A. Orenstein3, E. Breitbart 4, D. Harats 1'4. J / h e Institute of Lipid &

Atherosclerosis Research," 2The Institute of Pathology," 3The Institute of Advanced Technologies," 4 Vascular Biogenics, Ltd., Israel Angiogenic therapy with VEGF is short lasting and is reversible when the expression of VEGF ceases, since the resulting vessels are immature. Objective: to induce mature vessels, by using systemic gene therapy with adenoviral vectors that express VEGF & PDGF-B specifically within angiogenic endothelial cells. Materials & Methods: We constructed adenovirus vectors with expression cassette that includes the modified promoter of the murine preproendothelin-1 (PPE-1 3x) and the cDNA of VEGF165 or PDGF-B. Adenoviruses with CMV promoter and VEGF or PDGF-B serve as non-tissue-specific controls. Mice with ischemic hind limb, were systemically administered one of the following vectors: 1. Ad5PPE-I~xVEGF; 2. Ad5 CMV.VEGF; 3. Ad5PPE-I~xPDGF-B; 4. Ad5CMV.PDGF-B; 5. combination of Ad5PPE-I~xVEGF and Ad5PPE-1 3xPDGF-B; 6. control, Ad5PPE-1 3xGFP or saline. The effect of the therapy was monitored by ultrasonic imaging of perfusion, and by histological analysis. Results: 70 days after ligation, there was increase of 57% and 117% in mean capillary density in the Ad5PPE-1 3xVEGF group compared to the Ad5CMV.VEGF and control groups, respectively (p<0.001). 90 days after ligation, combination treatment and treatment with Ad5PPE-1 3x-PDGFB yielded 6% and 13% higher capillary density than Ad5PPE-1 3xVEGF alone, respectively. Vessel maturation was detected by the presence of SMC in the angiogenic vessels in mice treated with Ad5PPE-1 3x-PDGF-B or combination therapy. To lesser degree there was also vessel maturation in Ad5PPE-I~xVEGF treated animals. Conclusions: Systemic gene therapy with adenoviral vectors and the PPE-1- promoter with either VEGF, PDGF-B or a combination of them, induced stable, mature angiogenesis in the mouse ischemic hind-limb model, with no systemic adverse effects.

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ALTERED EXPRESSION OF INTERMEDIATE CONDUCTANCE CALCIUM-ACTIVATED POTASSIUM CHANNELS INDUCED BY LONG-TERM BLOCKADE OF NITRIC OXIDE SYNTHESIS

H. Hase~awa 1, T. Saito 1, Y. Chiba 1, Y. Fujiwara 1, A. Shoji 1, H. Miura 2, M. Miura 1. 1Aldta University, Akita, Japan," 2Medical College of Wisconsin,

MI, USA

M.D. Hansson1, E. Ellis 2, M.C. Hunt 1, G. Schmitz 3, A. Babiker 1.

I Diuision of Clinical Chemistry, 2Division of Gastroenterology and Hepatology, Karolinska Institutet Huddinge University Hospital, Stockholm, Sweden," 3Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg, Germany Objective: The cytochrome P-450 sterol 27-hydroxylase (CYP27), is capable to oxidize cholesterol into 27-hydroxycholesterol and cholestenoic acid. The latter metabolites may be secreted from the cells, and CYP27 has therefore been suggested to be an antiatherogenic enzyme. Macrophages have been shown to contain relatively high amounts of CYP27. In the present work we investigated the possibility that there is an upregulation of CYP27 during differentiation of monocytes into macrophages. Methods: Human monocytes were differentiated to macrophages in vitro for one week using serum free medium. The 27-oxygenated products of cholesterol (27-hydroxycholesterol and cholestenoic acid) in the medium were determined by isotop dilution mass spectrometry. CYP27 mRNA was measured using RNA Protection Assay. Results: A marked increase of CYP27 oxygenated products in the medium with a concomitant increase in the mRNA in the cells was detected during the differentiation of monocytes into macrophages starting on day 4. Addition of M-CSF had no significant effect. Conclusion: CYP27 is induced during differentiation from monocytes to macrophages leading to an increased removal of cholesterol from the cells. These results support the contention that CYP27 is an antiatherogenic factor.

To test the hypothesis that expression of Ca-activated K channels (Kca) are up-regulated in the condition with impaired NO production, alteration in expression of Kca mRNA was examined in the model of long-term inhibition of NO synthesis with N-nitro-L-arginine methyl ester (LNAME). LNAME (30mg/kg/day) was administered with osmotic mini-pump for 4 weeks in Sprague-Dawley rats. Differential expression of Kca mRNA was examined by RT-PCR using specific primers of large conductance (BK), intermediate conductance (ImK) and small conductance (SK1, SK2 and SK3) Kca. Rat ImK was cloned from cDNA library of vascular smooth muscle cells in pulmonary artery. Results: long-term LNAME administration produced hypertension and resulted in cardiac hypertrophy with marked perivascular fibrosis of coronary arterioles. RT-PCR revealed significant and selective up-regulation of ImK mRNA after 2 weeks in LNAMEtreated rats. While no significant change was noticed in expression of BK, SK1, SK2 nor SK3 mRNA. Up-regulation of ImK expression was also confirmed by in situ hybridization and immnohistochemical staining using antibody to ImK protein. These changes were completely abrogated by co-treatments with quinapril or AT1 receptor antagonist, candesartan. ImK has tyrosine phosphorylation sequence on the C-termius and motifs for AP1 in the promotor. These findings indicate that chronic inhibition of NO synthesis promotes augmented expression of ImK via AT1 receptormediated AP1 signaling pathway and results in compensatory enhancement of Kca-mediated vasorelaxation.

73rd EAS Congress