Richard I. Levin

Adenosine: an endogenous inhibitor of neutrophil-mediated injury to endothelial cells.

Since adenosine and its analogue 2-chloroadenosine prevent neutrophils from generating superoxide anion in response to chemoattractants, we sought to determine whether these agents could inhibit neutrophil-mediated injury of endothelial cells. The chemoattractant N-formyl-methionyl-leucyl-phenylalanine (FMLP, 0.1 microM) enhanced the adherence of neutrophils to endothelial cells twofold (18 +/- 2% vs. 39 +/- 3% adherence, P less than 0.001) and caused substantial neutrophil-mediated injury to endothelial cells (2 +/- 2% vs. 39 +/- 4% cytotoxicity, P less than 0.001). 2-Chloroadenosine (10 microM) not only inhibited the adherence of stimulated neutrophils by 60% (24 +/- 2% adherence, P less than 0.001) but also diminished the cytotoxicity by 51% (20 +/- 4% cytotoxicity, P less than 0.002). Furthermore, depletion of endogenously released adenosine from the medium by adenosine deaminase-enhanced injury to endothelial cells by stimulated neutrophils (from 39 +/- 4% to 69 +/- 3% cytotoxicity, P less than 0.001). Indeed, in the presence of adenosine deaminase, even unstimulated neutrophils injured endothelial cells (19 +/- 4% vs. 2 +/- 2% cytotoxicity, P less than 0.001). These data indicate that engagement of adenosine receptors prevents both the adhesion of neutrophils and the injury they cause to endothelial cells. Adenosine inhibits injury provoked not only by cells that have been stimulated by chemoattractants but also by unstimulated cells. Based on this model of acute vascular damage we suggest that adenosine is not only a potent vasodilator, but plays the additional role of protecting vascular endothelium from damage by neutrophils.

Natriuretic peptides: Physiology, therapeutic potential, and risk stratification in ischemic heart disease ☆ ☆☆ ★

BACKGROUND The natriuretic peptide family consists of four molecules that share significant amino acid sequence homologic characteristics and a looped motif. Atrial natriuretic peptide and brain natriuretic peptide are similar in their ability to promote natriuresis and diuresis, inhibit the renin-angiotensin-aldosterone axis, and act as vasodilators. Understanding of the actions of C-type natriuretic peptide and dendroaspis natriuretic peptide is incomplete, but these two new family members also act as vasodilators. Because of the rapid evolution of information about this peptide family, we reviewed the state of the art with respect to risk stratification and therapeutic ability. METHODS English-language papers were identified by a MEDLINE database search covering 1966 through 1997 and supplemented with bibliographic references and texts. CONCLUSIONS The natriuretic peptides are counterregulatory hormones with prognostically important levels. They are similarly upregulated in heart failure and counteract neurohormones that induce vasoconstriction and fluid retention. BNP may be the superior prognosticator for risk stratification after myocardial infarction and is independent of left ventricular ejection fraction. Lastly, experimental trials suggest that administration of exogenous natriuretic peptides or inhibitors of their catabolism to patients with ischemic heart disease may be clinically beneficial.

Nitroglycerin Stimulates Synthesis of Prostacyclin by Cultured Human Endothelial Cells

Nitroglycerin (NTG), the agent most commonly used to treat acute angina pectoris, is a vasodilator whose mechanism of action remains unknown. We hypothesized that NTG might induce endothelial cells to synthesize prostacyclin (PGI(2)), a known vasodilator and inhibitor of platelet aggregation. Therefore, cultured human endothelial cells were incubated with NTG at various concentrations for 1-3 min. PGI(2) biologic activity in the endothelial cell supernates was assayed by inhibition of platelet aggregation in vitro. The concentration of 6-keto-PGF(1alpha), the stable hydrolysis product of PGI(2), was measured by specific radioimmunoassay.NTG alone significantly inhibited platelet aggregation and thromboxane A(2) synthesis only at suprapharmacologic concentrations (>/=1 mug/ml). However, when NTG at clinically attainable concentrations (0.1-10 ng/ml) was incubated with endothelial cells, the endothelial cell supernates inhibited platelet aggregation in a dose-dependent manner. The inhibitor was heat labile. Radioimmunoassay of the endothelial cell supernates for 6-keto-PGF(1alpha) demonstrated that NTG elicited dose-dependent increments in the synthesis of PGI(2) by endothelial cells, ranging from 13% at NTG 10 pg/ml to 63% at NTG 10 ng/ml (P < 0.01, n = 10). Pretreatment of endothelial cells with either aspirin (50 muM for 120 min) or the prostacyclin synthetase inhibitor 15-hydroperoxyarachidonic acid (20 mug/ml for 15 min) abolished production of the platelet inhibitory substance. Synergy between NTG and PGI(2) in the inhibition of platelet aggregation was not present at clinically attainable concentrations of NTG.Thus, NTG at clinically attainable concentrations causes a dose-dependent increase in PGI(2) synthesis by endothelial cells. If this phenomenon occurs in vivo, the PGI(2) produced could ameliorate myocardial ischemia by causing peripheral vasodilation and decreasing cardiac work, inhibiting platelet aggregation and thromboxane A(2) synthesis, and possibly reversing coronary artery vasospasm.

Colchicine alters the quantitative and qualitative display of selectins on endothelial cells and neutrophils.

Since colchicine-sensitive microtubules regulate the expression and topography of surface glycoproteins on a variety of cells, we sought evidence that colchicine interferes with neutrophil-endothelial interactions by altering the number and/or distribution of selectins on endothelial cells and neutrophils. Extremely low, prophylactic, concentrations of colchicine (IC50 = 3 nM) eliminated the E-selectin-mediated increment in endothelial adhesiveness for neutrophils in response to IL-1 (P < 0.001) or TNF alpha (P < 0.001) by changing the distribution, but not the number, of E-selectin molecules on the surface of the endothelial cells. Colchicine inhibited stimulated endothelial adhesiveness via its effects on microtubules since vinblastine, an agent which perturbs microtubule function by other mechanisms, diminished adhesiveness whereas the photoinactivated colchicine derivative gamma-lumicolchicine was inactive. Colchicine had no effect on cell viability. At higher, therapeutic, concentrations colchicine (IC50 = 300 nM, P < 0.001) also diminished the expression of L-selectin on the surface of neutrophils (but not lymphocytes) without affecting expression of the beta 2-integrin CD11b/CD18. In confirmation, L-selectin expression was strikingly reduced (relative to CD11b/CD18 expression) on neutrophils from two individuals who had ingested therapeutic doses of colchicine. These results suggest that colchicine may exert its prophylactic effects on cytokine-provoked inflammation by diminishing the qualitative expression of E-selectin on endothelium, and its therapeutic effects by diminishing the quantitative expression of L-selectin on neutrophils.

Nonsteroidal antiinflammatory agents inhibit stimulated neutrophil adhesion to endothelium: Adenosine dependent and independent mechanisms

All nonsteroidal antiinflammatory drugs (NSAIDs) inhibit neutrophil aggregation (homotypic cell-cell adhesion) and do so without affecting expression of CD11b/CD18. Since the first step in acute inflammation is a critical interaction between neutrophils and the vascular endothelium (heterotypic cell-cell adhesion), we determined whether NSAIDs diminish the adherence of neutrophils to the endothelium. At antiinflammatory concentrations (0.5–5 mM) sodium salicylate, an NSAID that does not inhibit prostaglandin synthesis, inhibited stimulated but not unstimulated neutrophil adherence to endothelial cells (IC50 < 1 mM,P < 0.00001). Salicylates have previously been shown to inhibit oxidative phosphorylation and, predictably, sodium salicylate inhibited oxidative phosphorylation, as evidenced by depletion of ATP stores (875±75 pmol/106 PMN, [2.92±0.25 mM]) in stimulated (FMLP, 0.1μM) but not resting neutrophils treated with antiinflammatory doses of sodium salicylate (EC50=1 mM,P < 0.00001). Indomethacin and piroxicam (10 and 30μM) only minimally decreased ATP concentrations in stimulated and resting neutrophils. ATP is metabolized to adenosine, and we have previously demonstrated that both endogenously released (180–200 nM) and exogenous adenosine (IC50=250 nM) inhibit stimulated neutrophil adherence to endothelial cells. To determine whether the increased metabolism of ATP and the resultant increase in adenosine release were responsible for inhibition of neutrophil adhesion to endothelium, we determined whether addition of adenosine deaminase (ADA, 0.125 IU/ml), an enzyme that converts extracellular adenosine to its inactive metabolite, inosine, affected inhibition of neutrophil adhesion to endothelium by stimulated neutrophils. ADA significantly reversed inhibition of neutrophil adherence to endothelium by sodium salicylate (0.5–5 mM,P < 0.00001). This suggests that sodium salicylate inhibits neutrophil adherence by increasing adenosine release. Whereas indomethacin and piroxicam (10–50μM) also inhibited stimulated neutrophil adherence to endothelial cells, ADA did not affect their inhibition of adherence. These studies demonstrate a heretofore unexpected antiinflammatory mechanism for salicylates: salicylates increase ATP hydrolysis and thereby enhance release of adenosine. Moreover, these data are consistent with the hypothesis that NSAIDs differ from one another with respect to their mechanisms of action.

Effects of Amlodipine on Glomerular Filtration, Growth, and Injury in Experimental Hypertension

The objective of this study was to determine whether the calcium antagonist amlodipine could slow the progression of chronic renal disease. We examined the effects of amlodipine on kidney structure and function in two experimental models of hypertension. In the first study, adult, male Munich Wistar rats underwent uninephrectomy and were given weekly injections of desoxycorticosterone and 1% saline for drinking. Rats ingested normal chow or chow containing amlodipine for 8 weeks. The drug reduced systemic blood pressure, but glomerular filtration rate, kidney weight, proteinuria, and morphological evidence of glomerular injury were not affected. In the second study, male spontaneously hypertensive rats underwent uninephrectomy at 5 weeks of age and were followed for 6 months, during which they received no therapy or amlodipine. The drug dose was determined in preliminary studies to be the highest dose not associated with marked growth retardation. Again, although systemic blood pressure was significantly reduced by amlodipine, proteinuria and the prevalence of glomerulosclerosis were similar in amlodipine-treated and control spontaneously hypertensive rats. Micropuncture studies revealed that glomerular pressure remained elevated in amlodipine-treated spontaneously hypertensive rats. Kidney weight and glomerular volume were also similar in amlodipine-treated and control rats. Amlodipine also failed to inhibit platelet aggregation. Therefore, antihypertensive therapy with amlodipine fails to reduce glomerular pressure in spontaneously hypertensive rats as well as glomerular size and injury in spontaneously hypertension rats and desoxycorticosterone-salt hypertension. Although other dihydropyridine calcium antagonists have been found to reduce experimental glomerular injury, these data suggest that amlodipine may not prevent hypertensive nephrosclerosis.

Quantitation of transient myocardial ischemia by digital, ambulatory electrocardiography☆

Transient myocardial ischemia is more frequently silent than accompanied by angina. The frequency of ischemia varies markedly from day to day, so that in order to accurately define the total ischemic burden, it may be necessary to quantitate ischemic episodes for periods longer than 24 hours. Therefore, a programmable, digital device was developed for long-term, interactive, ambulatory monitoring of the electrocardiogram, which uses variations in a time-averaged ST level as an indicator of myocardial ischemia. The electrocardiographic signal is digitized at 256 Hz and analyzed by an algorithm. If ST depression is planar or downsloping and persists for more than 40 seconds, and if the ST depression is equal to or more than a user-programmed threshold, the device marks the onset of an ischemic event and times it. The algorithm has been validated by comparison of its analysis of the ST segment to human and computerized analyses of frequency-modulated Holter recordings and stress tests. To assess the feasibility and utility of long-term monitoring, patients with documented coronary artery disease were monitored continuously for 14-day periods. Of 26 patients enrolled, 8 completed a protocol for individualization of anti-ischemic therapy using transdermal nitroglycerin. Over 90% of ischemic episodes in this group of patients, all of whom had had a previous myocardial infarction, were silent. Treatment with 10 mg of transdermal nitroglycerin reduced the number of ischemic episodes by 59% and the duration of ischemia by 60% (p less than 0.001); there was no diminution in the effectiveness of treatment from week 1 to week 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Uremic levels of oxalic acid suppress replication and migration of human endothelial cells.

Patients with chronic renal failure who undergo hemodialysis experience accelerated atherosclerosis and premature death. Since the end-metabolite, oxalic acid, accumulates in plasma in proportion to the severity of renal failure, we studied whether sodium oxalate (0 to 300 microM) is an endothelial toxin and, therefore, might enhance atherogenesis. Exposure to uremic levels of oxalate (greater than 30 microM) for 9 to 28 days depressed endothelial cell replication by 33% to 84% (mean +/- SD, 54% +/- 15.7%, n = 17 experiments, p = 0.002). In contrast, replication of fibroblasts exposed to 200 microM oxalate for 45 days was not inhibited. The inhibitory effect of oxalate on endothelial cell replication was both dose- and time-dependent (both p less than 0.0001) and was first detected 3 to 7 days after the initial exposure to oxalate. Further, the inhibitory effect was fully reversible upon removal of oxalate, but only if exposure was limited to 5 days or less. Sodium salts of other carboxylic acids (citric, succinic, glyoxylic, and malonic; 200 microM) as well as HCl (200 microM) did not suppress endothelial cell replication. Oxalate also inhibited endothelial cell migration but had no effect on basal, thrombin-induced, or arachidonate-induced prostacyclin production by endothelial cells. Exposure of endothelial cells to sodium oxalate (200 microM) for as little as 24 hours-a time period sufficient to induce delayed, transient inhibition of replication not detectable until approximately 1 week after exposure-inhibited incorporation of 3H-leucine into protein by 40% (p = 0.009). We conclude that sodium oxalate acts as a uremic toxin, inhibiting endothelial cell replication and migration, functions which may be important for constitutive inhibition of atherosclerosis.

Physician choices in the treatment of angina pectoris

Uncertainty about optimal treatment for many diseases results in heterogeneous management by definition. It was hypothesized that identifiable characteristics in a physicians background would influence the management of any such condition and thereby explain some of this heterogeneity. A vignette describing a patient with new-onset angina and a questionnaire ascertaining individual physician characteristics and management preferences were sent to attending physicians and house staff in the Department of Medicine at New York University School of Medicine. Although physicians believed very strongly that the patient had angina on the basis of the history, there was no consensus about managing the hypothetic patient. The age of the physician was the single most important predictor of management, with the younger half of the sample more likely to hospitalize (p less than 0.001), less likely to prescribe nitroglycerin as a sole therapy (p less than 0.005), and more likely to prescribe beta blockers (p less than 0.005). The era in which a physician trains may determine practices that persist for a lifetime. These findings may have important implications for medical education and the quality and cost of medical care.

Myocardial preservation during ischemia and reperfusion

Protection of the myocardium during cardiac surgery is necessary because of two distinctly different forms of injury: ischemia during cross-clamping of the aorta and secondary destructive phenomena during reperfusion. The primary injury of ischemia is caused by a combination of inadequate or no delivery of substrate and inadequate or no flow, which prevents the removal of toxic intermediary and end products of anaerobic metabolism. The secondary injury, that due to myocardial reperfusion after prolonged ischemia, has been shown in experimental models to accentuate ischemic injury, hasten cell death, and thus may extend infarct size. Protection of the myocardium from the primary ischemic injury with chemical cardioplegia was first introduced by Melrose et al.’ in 1955. Since then extensive animal studies2e5 have provided not only a foundation for understanding the physiologic and metabolic derangements in myocardium during ischemia but also the basis for formulating the various cardioplegic solutions for clinical use. Roe et a1.6 were among the first to describe a large clinical experience with single-dose, crystalloid, hypothermic potassium cardioplegia, which yielded very low morbidity and mortality. Tylers et a1.,7 Conti et al.,s and Adams et al9 reported similar results when multidose cardioplegia was used. The use of oxygenated blood as a vehicle for cardioplegic arrest was advocated by Follette et al.l” in 1978. The superiority of sanguineous cardioplegia was then demonstrated in several clinical studies”-l3 both in terms of longer cross-clamping time and lower perioperative