Aaron Hoffman

Human Serum Paraoxonases (PON1) Q and R Selectively Decrease Lipid Peroxides in Human Coronary and Carotid Atherosclerotic Lesions PON1 Esterase and Peroxidase-Like Activities

BACKGROUND Human serum paraoxonase (PON1) exists in two polymorphic forms: one that differs in the amino acid at position 192 (glutamine and arginine, Q and R, respectively) and the second one that differs in the amino acid at position 55 (methionine and leucine, M and L, respectively). PON1 protects LDL from oxidation, and during LDL oxidation, PON1 is inactivated. METHODS AND RESULTS The present study compared PON1 isoforms Q and R for their effect on lipid peroxide content in human coronary and carotid lesions. After 24 hours of incubation with PON1Q or PON1R (10 arylesterase units/mL), lipid peroxides content in both coronary and carotid lesion homogenates (0.1 g/mL) was reduced up to 27% and 16%, respectively. The above incubation was associated with inactivation of PON1Q and PON1R by 15% and 45%, respectively. Lesion cholesteryl linoleate hydroperoxides and cholesteryl linoleate hydroxides were hydrolyzed by PON1 to yield linoleic acid hydroperoxides and linoleic acid hydroxides. Furthermore, lesion and pure linoleic acid hydroperoxides were reduced to yield linoleic acid hydroxides. These results thus indicate that PON1 demonstrates esterase-like and peroxidase-like activities. Recombinant PON1 mutants in which the PON1-free sulfhydryl group at cysteine-284 was replaced with either alanine or serine were no longer able to reduce lipid peroxide content in carotid lesions. CONCLUSIONS We conclude that PON1 may be antiatherogenic because it hydrolyzes lipid peroxides in human atherosclerotic lesions.

Prosthetic Vascular Graft Infection: The Role of 18F-FDG PET/CT

Graft infection after prosthetic vascular reconstruction is an uncommon but severe complication. The clinical presentation is often subtle and nonspecific and may occur long after surgery. Although defining a prosthetic vascular graft infection can be difficult, early diagnosis and treatment are important because of the relatively high rates of amputation and death. The present study assessed the role of PET/CT using 18F-FDG for the diagnosis of vascular graft infections. Methods: Thirty-nine patients (35 men and 4 women; age range, 44–82 y) with suspected vascular graft infection underwent 18F-FDG PET/CT. The performance of PET/CT for the diagnosis of an infectious process and its localization to the graft or soft tissues was assessed. The final diagnosis was based on histopathologic findings and microbiologic assays obtained at surgery or on clinical and imaging follow-up. Results: PET/CT detected foci of increased 18F-FDG uptake suspected as infection in 27 patients and localized these findings to the graft in 16 patients. Vascular graft infection was confirmed in 14 of these patients (88%). PET/CT excluded graft involvement in 11 patients, and in 10 (91%) of these 11, long-term follow-up further confirmed that the infectious process was limited to surrounding soft tissues only. No abnormal 18F-FDG uptake was found in any of the 12 patients with no further evidence of infection. PET/CT had a sensitivity of 93%, specificity of 91%, positive predictive value of 88%, and negative predictive value of 96% for the diagnosis of vascular graft infection. Conclusion: 18F-FDG PET/CT is a reliable noninvasive imaging modality for the diagnosis of vascular graft–related infection. The precise anatomic localization of increased 18F-FDG uptake provided by PET/CT enables accurate differentiation between graft and soft-tissue infection.

Selective distribution of oxysterols in atherosclerotic lesions and human plasma lipoproteins

The presence of oxidized sterols (oxysterols) in human serum and lesions has been linked to the initiation and progression of atherosclerosis. Data concerning the origin, identity and quantity of oxysterols in biological samples are controversial and inconsistent. This inconsistency may arise from different analytical methods or handling conditions used by different investigators. In the present study, oxysterol levels and distribution were analyzed by an optimized GC-MS method, in human atherosclerotic coronary and carotid lesions, in atherosclerotic apolipoprotein E deficient mice (E° mice) and in native and in vitro oxidized human low and high density lipoproteins. Oxysterol levels were analyzed with a limit of detection of 0.06 – 0.24 ng, with 25-hydroxycholesterol (25-OH) being the least sensitive. In human coronary and carotid lesions, obtained from endatherectomic samples, 27-hydroxycholesterol (27-OH) was the major oxysterol, with about 85% as sterols esterified to fatty acids. While total cholesterol and oxysterols levels were similar in both kinds of human lesions, oxysterol distribution was significantly different. In coronary lesions the mean levels of 27-OH and 7β-hydroxycholesterol (7β-OH) were 38% and 20% of total oxysterols, whereas in carotid lesions their mean levels were 66% and 5%, respectively. Unlike in human aortic lesions, 27-OH was entirely absent in E° mice, whereas the level of 7α-hydroxycholesterol (7α-OH) was 28% of the total oxysterols, vs. 5% in human coronary lesions. As 27-OH is an enzymatic product of cholesterol oxidation, this finding may indicate that such an enzymatic process does not take place in E° mice.

Carotid Plaque Vulnerability: Quantification of Neovascularization on Contrast-Enhanced Ultrasound With Histopathologic Correlation

OBJECTIVE The purpose of this research is to develop a computerized method to quantify carotid plaque neovascularization on contrast-enhanced ultrasound images and to compare the results with the histopathologic analysis of the plaque. SUBJECTS AND METHODS Twenty-seven patients (age range, 48-84 years; mean [± SD] age, 68.4 ± 9.72 years) were recruited before endarterectomy. Contrast-enhanced ultrasound examination of the carotid artery was performed by applying low mechanical index and harmonics with pulse inversion. An algorithm was developed that implemented several image processing methods to automatically quantify neovascularization and reconstruct the vascular tree in the atheromatous plaque. Neovascularization and the number of inflammatory cells seen on histopathologic analysis of the plaque after endarterectomy were compared with neovascularization determined by the computerized method. The mean (± SD) ratios of the ultrasound and histopathologic measurements were calculated. RESULTS In five patients, heavy calcification of the plaque prevented visualization of plaque texture. Intraplaque neovascularization on contrast-enhanced ultrasound images was significant in 19 patients and low in three patients. The ratio of the neovascularization area to the total plaque area on contrast-enhanced ultrasound images was well correlated with the same histopathologic ratio (R(2) = 0.7905) and with the number of inflammatory cells present in the plaque (R(2) = 0.6109). The histopathologic ratio and the number of intraplaque inflammatory cells also were well correlated (R(2) = 0.7034). CONCLUSION The newly developed method allowed quantification of the intraplaque neovascularization as a feature of vulnerability in the carotid plaque and proved to be highly correlated with histopathologic results.

Mechanisms of big endothelin-1-induced diuresis and natriuresis : role of ET(B) receptors.

Endothelin-1 (ET-1) at high concentrations has marked antidiuretic and antinatriuretic activities, whereas its precursor, big endothelin-1 (big ET-1), has surprisingly potent diuretic and natriuretic actions. The mechanisms underlying the excretory effects of big ET-1 have not been fully elucidated. To explore these mechanisms, we examined the effects of a highly selective ET(B) antagonist (A-192621.1), a calcium channel blocker (verapamil), a nitric oxide synthase inhibitor (N-nitro-L-arginine methyl ester [L-NAME]), and a cyclooxygenase inhibitor (indomethacin) on the systemic and renal actions of big ET-1 in anesthetized rats. An intravenous bolus injection of incremental doses of big ET-1 (0.3, 1. 0, and 3.0 nmol/kg) produced a significant hypertensive effect that was dose dependent and prolonged (from 113+/-7 mm Hg to a maximum of 148+/-6 mm Hg). The administration of big ET-1 induced marked diuretic and natriuretic responses (urinary flow rate increased from 8.5+/-1 to 110+/-14 microL/min, and fractional excretion of sodium increased from 0.38+/-0.13% to 7.51+/-1.24%). Glomerular filtration rate and renal plasma flow significantly decreased only at the highest dose of big ET-1. Pretreatment with A-192621.1 (3 mg/kg plus 3 mg. kg(-1). h(-1)) significantly abolished the diuretic (17+/-5 microL/min to a maximum of 19+/-3 microL/min) and natriuretic (0. 29+/-0.1% to a maximum of 1.93+/-0.37%) responses induced by big ET-1. Moreover, A-192621.1 potentiated the decline in glomerular filtration rate and renal plasma flow and the increase in mean arterial blood pressure produced by the low doses of big ET-1. Similar to A-192621.1, pretreatment with a nitric oxide synthase inhibitor (L-NAME, 10 mg/kg plus 5 mg. kg(-1). h(-1)) significantly and comparably reduced the diuretic and natriuretic actions of big ET-1 and augmented the hypoperfusion/hypofiltration and systemic vasoconstriction induced by high doses of the peptide. Pretreatment with verapamil (2 mg. kg(-1). h(-1)) slightly inhibited the diuretic/natriuretic effects of the high-dose big ET-1 and completely prevented the increase in mean arterial blood pressure provoked by the peptide. Unlike verapamil and L-NAME, only indomethacin administration was associated with significant natriuretic/diuretic responses and did not influence the pressor effect and renal actions of big ET-1. Taken together, these results suggest that big ET-1-induced diuretic and natriuretic responses are mediated mainly by stimulation of nitric oxide production coupled to ET(B) receptor subtype activation.

Impaired Nitric Oxide–Mediated Renal Vasodilation in Rats With Experimental Heart Failure Role of Angiotensin II

BACKGROUND Congestive heart failure (CHF) is associated with a decrease in renal perfusion. Because endothelium-derived NO is important in the regulation of renal blood flow (RBF), we tested the hypothesis that an impairment in the NO system may contribute to the decrease in RBF in rats with experimental CHF. METHODS AND RESULTS Studies were performed in rats with experimental high-output CHF induced by aortocaval (AV) fistula and sham-operated controls. In controls, incremental doses of acetylcholine (ACh, 1 to 100 microg x kg(-1) x min(-1)) increased RBF and caused a dose-related decrease in renal vascular resistance (RVR). However, the increase in RBF and decrease in RVR were markedly attenuated in rats with CHF. Likewise, the effects of ACh on urinary sodium and cGMP excretion were also diminished in CHF rats, as was the renal vasodilatory effect of the NO donor S-nitroso-N-acetylpenicillamine (SNAP). These attenuated responses to endothelium-dependent and -independent renal vasodilators in CHF rats occurred despite a normal baseline and stimulated NO2+NO3 excretion and normal expression of renal endothelial NO synthase (eNOS), as determined by eNOS mRNA levels and immunoreactive protein. Infusion of the NO precursor L-arginine did not affect baseline RBF or the response to ACh in rats with CHF. However, administration of the nonpeptide angiotensin II receptor antagonist A81988 before ACh completely restored the renal vasodilatory response to ACh in CHF rats. CONCLUSIONS This study demonstrates that despite a significant attenuation in the NO-related renal vasodilatory responses, the integrity of the renal NO system is preserved in rats with chronic AV fistula. This impairment in NO-mediated renal vasodilation in experimental CHF appears to be related to increased activity of the renin-angiotensin system and may contribute further to the decrease in renal perfusion seen in CHF.

Regulation of intrarenal blood flow in experimental heart failure: role of endothelin and nitric oxide.

Congestive heart failure(CHF) is associated with a marked decrease in cortical blood flow and preservation of medullary blood flow. In the present study we tested the hypothesis that changes in the endothelin (ET) and nitric oxide (NO) systems in the kidney may contribute to the altered intrarenal hemodynamics in rats with aortocaval fistula, an experimental model of CHF. Cortical and medullary blood flow were measured simultaneously by laser-Doppler flowmetry in controls and rats with compensated and decompensated CHF. As previously reported [K. Gurbanov, I. Rubinstein, A. Hoffman, Z. Abassi, O. S. Better, and J. Winaver. Am. J Physiol. 271 (Renal Fluid Electrolyte Physiol. 40): F1166-F1172, 1996], administration of ET-1 in control rats produced a sustained cortical vasoconstriction and a transient medullary vasodilatory response. In rats with decompensated CHF, cortical vasoconstriction was severely blunted, whereas ET-1-induced medullary vasodilation was significantly prolonged. This prolonged response was mimicked by IRL-1620, a specific ETB agonist, and partially abolished by NO synthase (NOS) blockade. In line with these findings, expression of ET-1, ETA and ETB receptors, and endothelial NOS (eNOS), assessed by RT-PCR, and eNOS immunoreactivity, assessed by Western blotting, was significantly higher in the medulla than in the cortex. Moreover, expression of ET-1 mRNA in the cortex and eNOS mRNA in the cortex and the medulla increased in proportion to the severity of heart failure. These findings indicate that CHF is associated with altered regulation of intrarenal blood flow, which reflects alterations in expression and activity of the ET and NO systems. It is further suggested that exaggerated NO activity in the medulla contributes to preservation of medullary blood flow in the face of cortical vasoconstriction in CHF.Congestive heart failure (CHF) is associated with a marked decrease in cortical blood flow and preservation of medullary blood flow. In the present study we tested the hypothesis that changes in the endothelin (ET) and nitric oxide (NO) systems in the kidney may contribute to the altered intrarenal hemodynamics in rats with aortocaval fistula, an experimental model of CHF. Cortical and medullary blood flow were measured simultaneously by laser-Doppler flowmetry in controls and rats with compensated and decompensated CHF. As previously reported [K. Gurbanov, I. Rubinstein, A. Hoffman, Z. Abassi, O. S. Better, and J. Winaver. Am. J. Physiol. 271 ( Renal Fluid Electrolyte Physiol. 40): F1166-F1172, 1996], administration of ET-1 in control rats produced a sustained cortical vasoconstriction and a transient medullary vasodilatory response. In rats with decompensated CHF, cortical vasoconstriction was severely blunted, whereas ET-1-induced medullary vasodilation was significantly prolonged. This prolonged response was mimicked by IRL-1620, a specific ETB agonist, and partially abolished by NO synthase (NOS) blockade. In line with these findings, expression of ET-1, ETA and ETB receptors, and endothelial NOS (eNOS), assessed by RT-PCR, and eNOS immunoreactivity, assessed by Western blotting, was significantly higher in the medulla than in the cortex. Moreover, expression of ET-1 mRNA in the cortex and eNOS mRNA in the cortex and the medulla increased in proportion to the severity of heart failure. These findings indicate that CHF is associated with altered regulation of intrarenal blood flow, which reflects alterations in expression and activity of the ET and NO systems. It is further suggested that exaggerated NO activity in the medulla contributes to preservation of medullary blood flow in the face of cortical vasoconstriction in CHF.

Human carotid atherosclerotic plaque increases oxidative state of macrophages and low-density lipoproteins, whereas paraoxonase 1 (PON1) decreases such atherogenic effects

Human atherosclerotic plaque contains a variety of oxidized lipids, which can facilitate further oxidation. Paraoxonase 1 (PON1) is a high-density lipoprotein (HDL)-associated esterase (lipolactonase), exhibiting antiatherogenic properties. The aims of the present study were to examine the oxidizing potency of the human carotid plaque lipid extract (LE), and the antiatherogenic role of PON1 on LE oxidation competence. Human carotid plaques were extracted by organic solvent, and the extract was incubated with lipoprotein particles, with macrophages, or with probes sensitive to oxidative stress, with or without preincubation with PON1, followed by oxidative-stress assessment. Our findings imply that the LE oxidized LDL, macrophages, and exogenous probes and decreases HDL-mediated cholesterol efflux from macrophages, in a dose-dependent manner. Incubation of PON1 with LE significantly affects LE composition, reduces LE atherogenic properties, and decreases the extracts total peroxide concentration by 44%, macrophage oxidation by 25%, and probe oxidation by up to 52%. We conclude that these results expand our understanding of how the plaque itself accelerates atherogenesis and provides an important mechanism for attenuation of atherosclerosis development by the antioxidant action of PON1 on the atherosclerotic plaque.

Endothelin induces an initial increase in cardiac output associated with selective vasodilation in rats

The systemic and regional hemodynamic effects of endothelin (ET), a novel endothelial derived vasoconstrictor peptide were studied in Wistar Kyoto rats. A bolus of 1 nmol/Kg ET intravenously induced a transient 43% decrease in blood pressure associated with a 57% decrease in systemic resistance and a 30% increase in cardiac output (p less than 0.01 for all parameters). This was followed by an increase of 20% in arterial pressure and of 71% in systemic resistance and a decrease of 30% in cardiac output at 10 minutes. The initial fall in blood pressure was not abolished by pretreatment with verapamil, captopril, indomethacin, ketanserin, atropine, methylene blue or ethanol. Verapamil abolished the hypertensive phase by markedly decreasing cardiac output. ET had selective effects on the arterial tree; during the hypotensive phase it caused a transient increase in blood flow in the carotid and femoral arteries (+41% and +83% respectively, p less than 0.01) but a decrease in flow in the renal and mesenteric arteries (-53% and -44% respectively, p less than 0.05). Accordingly, there was a decrease in resistance in the carotid and femoral beds (-55% and -67% respectively, p less than 0.01) and an increase in resistance in the renal and mesenteric beds (+102%; p less than 0.01 and +23%; p = N.S. respectively). Subsequently there was an increase in resistance in all vascular beds to variable degrees. The maximal increase in resistance was in the renal bed (+156%). Thus, ET causes initially a potent systemic vasorelaxation and an increase in cardiac output later progressing to systemic vasoconstriction and a decrease in cardiac output. The initial vasodilation is selective, appearing in musculocutaneous beds but not in visceral beds.

Possible role for endothelins in penile erection

The effects of endothelins (endothelin-1 and endothelin-3) on penile erection were studied in vivo in the pithed rat. Penile erectile responses were evaluated by measurements of intracorporal pressure via a needle inserted into one corpus cavernosum. Injection of endothelins (0.1-10 micrograms/kg i.v.) was followed by a rapid rise in corporal pressure. The responses to endothelin-1 and endothelin-3 were equivalent at the lower doses, but at 10 micrograms/kg the response to endothelin-3 was greater than that to endothelin-1. NG-Nitro-L-arginine-methyl ester (20 mg/kg) but not indomethacin (10 mg/kg) antagonized the rise in corporal pressure caused by submaximal doses of endothelins. Electrical nerve stimulation-induced increase in corporal pressure was reduced after 5 micrograms/kg endothelin-1 but not after the same dose of endothelin-3. The results indicate that in corporal vasculature endothelin-3 has a predominantly vasodilator effect, while endothelin-1 has a vasodilator effect at lower doses and a vasoconstrictor effect at higher doses. The vasodilator action of endothelins is probably mediated by activation of endothelin ETB receptors and local release of nitric oxide. Thus, endothelins may participate in initiation and/or maintenance of penile erection.