Experimental hypercholesterolemia induces apoptosis in the aortic valve.

BACKGROUND AND AIM OF THE STUDY: Aortic valve disease is presently the number one indication for valve replacement in the United States, yet its molecular mechanisms remain unknown. As apoptosis (programmed cell death) occurs in degenerative disease states, it was postulated that experimental hypercholesterolemia is associated with apoptosis in rabbit aortic valves. METHODS: New Zealand White rabbits (n = 8) were fed a 1% cholesterol diet for 12 weeks; control rabbits (n = 8) were fed a normal diet. After sacrifice of the animals, the aortic valves were dissected. Apoptosis was identified in the valvular lesion by TdT-mediated dUTP-biotin nick end-labeling (TUNEL) technique, and confirmed with transmission electron microscopy. The number of apoptotic cells was measured by computed morphometry. RESULTS: Valves from hypercholesterolemic rabbits showed an increase in apoptosis. TUNEL staining was identified in the atherosclerotic layer of hypercholesterolemic valves (0.1% of cells), but not in the cells of controls (p <0.0001). CONCLUSION: Apoptosis is increased in rabbit aortic valves during experimental hypercholesterolemia. If fatal cellular degeneration occurs in hypercholesterolemic valve disease, these data suggest that apoptosis may play a role in the mechanism of valvular disease.

In vivo gene transfer of endothelial nitric oxide synthase to carotid arteries from hypercholesterolemic rabbits enhances endothelium-dependent relaxations.

BACKGROUND AND PURPOSE: Hypercholesterolemia is associated with abnormal endothelium-dependent vasorelaxation due to decreased nitric oxide bioavailability. Our aim was to examine the effect of adenovirus-mediated gene transfer of endothelial nitric oxide synthase (eNOS) to the hypercholesterolemic rabbit carotid artery in vivo. In addition, we examined whether adenovirus-mediated gene transfer was associated with vascular dysfunction. METHODS: Rabbits were fed a 1% cholesterol diet for 4 weeks followed by a 0.5% cholesterol diet for 6 weeks. Vascular reactivity was assessed in nontransduced carotid arteries from chow- and cholesterol-fed animals. In addition, carotid arteries were surgically isolated, and 2 separate doses of adenoviral vectors encoding eNOS or beta-galactosidase (AdbetaGal) on the contralateral side were delivered to the lumen (1x10(10) and 5x10(10) pfu/mL). RESULTS: Abnormal acetylcholine-mediated endothelium-dependent vasorelaxation was detected in the carotid artery from cholesterol-fed animals, whereas responses to calcium ionophore A23187 and diethylamine NONOate were normal. Vascular reactivity was similar in nontransduced and AdbetaGal-transduced hypercholesterolemic vessels. In vessels transduced with eNOS, transgene expression was demonstrated by immunostaining in both the endothelium and the adventitia and by Western blot analysis. High-dose but not low-dose eNOS gene transfer enhanced endothelium-dependent relaxation in vessels from cholesterol-fed rabbits. CONCLUSIONS: Adenovirus-mediated gene transfer of eNOS to carotid arteries of cholesterol-fed animals improves endothelium-dependent relaxation when an optimal viral titer is administered.

L-arginine availability is not limiting for nitric oxide generation from recombinant endothelial nitric oxide synthase.

L-arginine (L-Arg) may be limiting for inducible nitric oxide synthase (NOS) activity and under certain circumstances, such as increased concentrations of a NOS inhibitor, may also be limiting for endothelial NOS activity. It is unknown if L-Arg is limiting for recombinant eNOS activity in the vascular wall after adenoviral mediated gene transfer. Our aim was to examine, if L-Arg is limiting for recombinant eNOS activity in the normal or atherosclerotic vessel wall. Rings of rabbit aorta from chow or cholesterol fed animals were transduced with adenovirus vector encoding eNOS (AdeNOS) or beta-galactosidase (AdbetaGal). After 24 h, transgene expression was confirmed and vasomotor studies were performed in the absence or presence of L-Arg. During maximal contractions to phenylephrine (10(-5) M), L-Arg (3 mM) was added to the organ chamber for 30 min. Subsequently, relaxations to acetylcholine during half-maximal contractions were obtained. In the chow- and cholesterol-fed animals, relaxations were significantly enhanced in the NOS and NOS + L-Arg groups compared to the betaGal and betaGal + L-Arg groups. There was no difference between NOS and NOS + L-Arg or betaGal and betaGal + L-Arg rings from chow- or cholesterol-fed animals. While gene transfer of eNOS enhances endothelium-dependent vasorelaxation in the normal and atherosclerotic vessel wall, L-arginine is not limiting for recombinant eNOS activity.

Ex vivo gene transfer of endothelial nitric oxide synthase to atherosclerotic rabbit aortic rings improves relaxations to acetylcholine.

Cholesterol feeding results in impaired endothelium dependent vasorelaxation. The role of nitric oxide in this process is unclear. The aim of this study was to evaluate the role of nitric oxide in cholesterol-induced vasomotor dysfunction by examining the effect of overexpression of eNOS in the hypercholesterolemic rabbit aorta on vascular reactivity. Vascular rings from the thoracic aorta of hypercholesterolemic rabbits were exposed ex vivo either to an adenoviral vector encoding endothelial nitric oxide synthase (AdeNOS) or Escherichia coli beta Galactosidase (AdbetaGal). Transgene expression was examined by histochemistry for beta galactosidase, immunohistochemistry for eNOS and cyclic GMP measurements and vasomotor studies were performed. Transgene expression was found to localize to the endothelium and adventitia. cGMP levels were significantly greater in AdeNOS compared to AdbetaGal transduced rings. Acetylcholine mediated relaxation was significantly impaired in cholesterol fed rabbits and was markedly improved by overexpression of eNOS. These results suggest that reduced NO bioavailability observed in cholesterol-induced vascular dysfunction can be partially overcome by eNOS gene transfer.

Adenovirus-mediated gene transfer of macrophage colony stimulating factor to the arterial wall in vivo.

Macrophage colony stimulating factor (MCSF) is believed to play a key role in one of the earliest events in atherosclerosis, ie, monocyte to macrophage differentiation in the arterial intima. The aim of this study was to examine the biological effects of vascular wall expression of MCSF. A recombinant adenovirus vector encoding human MCSF (AdMCSF) was generated by standard techniques of homologous recombination in 293 cells. The rabbit carotid artery was transduced with AdMCSF. As negative controls, carotid arteries were transduced with either an adenoviral vector encoding beta-galactosidase, an adenoviral vector encoding apolipoprotein E, or diluent alone. Intima-media thickness ratio was calculated 5 and 21 days after transduction. The cell type present in intimal infiltrates was analyzed by immunohistochemistry. MCSF expression was demonstrated in the vessel wall of AdMCSF-transduced vessels by reverse transcription-polymerase chain reaction and immunofluorescence. In contrast to control vessels, adenovirus-mediated MCSF expression was associated with an intimal cellular infiltrate consisting of smooth muscle cells and small numbers of macrophages. Whereas the intima-media thickness ratio was greater in AdMCSF-transduced vessels at 5 days, this difference was no longer statistically significant at 21 days. These results suggest that MCSF may play a role in recruitment of monocytes and macrophages to the vessel wall and may contribute to smooth muscle cell proliferation and migration.

From methylglyoxal to new immunopotentiating ascorbic acid derivatives.

In Szent-Györgyi's search for alpha-dicarbonyl compounds that play an important role in cell regulation, 3-desoxyglucosulose was isolated first from liver but did not prove active. Methylglyoxal came next, however, its toxicity prompted Szent-Györgyi to suggest a combination with ascorbic acid which, indeed led to immunopotentiating enediol acetals although of low stability. Therefore the vinylogue of methylglyoxal, acetylacrolein was coupled with L-ascorbic acid carbanion. This second new reaction, of the aldol-type, led to the stable, potent immunoactive compound, 2-(5-methylfuryl)-3-ketogulonolactone cyclohemiketal that forms a completely surprising H-bond with succinic anhydride and succinimide based on an X-ray study. A third new reaction in which ascorbic acid plays the role of a Michael donor to alpha,beta-unsaturated aldehydes and ketones proved now to be of general validity; it is unexpectedly acid catalyzed and the adducts formed with aliphatic and alicyclic olefin ketones have definite immunopotentiating effect. A brief description of the biological effects of all types of new compounds is outlined.