Serum complement activation in congestive heart failure.

BACKGROUND: Although activation of the complement system in myocardial infarction and cardiopulmonary bypass has been shown to contribute to myocardial injury, its role in congestive heart failure (CHF) is unknown. The purpose of this study was to determine the presence of terminal complement activation and its relation to clinical outcomes in patients with CHF. METHODS: We measured serum levels of the terminal complement complex C5b-9 in 36 patients with symptomatic heart failure and left ventricular ejection fraction <40%. We compared the serum C5b-9 levels of these patients with CHF with a group of 12 age-matched control patients. Combined clinical outcomes (death, urgent heart transplantation, or hospitalization with worsening heart failure) at 6 months were determined. RESULTS: The serum C5b-9 [median (25th to 75th percentiles)] levels in 36 patients with CHF [101.5 ng/mL (40 to 164)] were significantly (P =.003) higher than in the 12 control patients [36.5 ng/mL (22 to 50)]. Significantly more of the patients with CHF with the highest levels of C5b-9 (highest 50th percentile) had New York Heart Association class IV symptoms (67% vs 33%; P =.04) and adverse clinical outcomes by 6 months (56% vs 17%; P =.02) compared with the patients with CHF with lower levels (lowest 50th percentile). CONCLUSIONS: We have described a significant elevation in circulating C5b-9, the terminal complement complex, in patients with symptomatic heart failure and have observed an association between high levels of C5b-9 and near-term adverse events.

Gender differences in referral to cardiac rehabilitation programs after revascularization.

BACKGROUND: This study examines the influence of gender on the healthcare provider's secondary prevention instruction and cardiac rehabilitation (CR) referral after coronary revascularization procedures such as balloon angioplasty/coronary stenting and coronary bypass surgery (CABG). Cardiac rehabilitation decreases mortality and morbidity, yet only a small percentage of women and men are referred to these programs. The patient population of our university-affiliated CR program consisted of 88% men and 12% women. METHODS: In a matched case observational study, 80 patients (40 men, 40 women) who had undergone coronary revascularization procedures between 1997 and 1998 completed a questionnaire on secondary prevention instruction and written referral to CR programs. Patients were matched for age and coronary revascularization procedure. RESULTS: Women were less likely to be instructed on secondary prevention strategies and CR or referred to CR as compared to men despite being matched for age and undergoing the same procedure. The data demonstrate a gender difference in hospital teaching and referral information for CR after revascularization (P < 0.001). Being a woman was associated with a decreased likelihood of receiving a physician referral to CR after revascularization (P < 0.001). CONCLUSION: The instruction of patients concerning secondary prevention and CR postrevascularization procedures is poor. Within that group, women were far less likely to have CR discussed or referrals made by healthcare professionals. This study demonstrates the need for education initiatives of all healthcare providers on the comprehensive nature and benefits of CR in the secondary prevention of cardiovascular disease, with a particular emphasis on women.

Myocardial blood-flow response during mental stress in patients with coronary artery disease.

Positron emission tomography was used to quantify changes in myocardial blood flow during mental stress in patients with and without coronary artery disease. Blunted augmentation of myocardial blood flow during mental stress was observed in regions without significant epicardial stenosis.

Effect of 6-wk estrogen withdrawal or replacement on myocardial ischemic tolerance in rats.

Menopausal status is a risk factor for coronary artery disease death, but the mechanism underlying this association is uncertain. To test whether estrogen ameliorates the effects of acute myocardial ischemia in ways likely to translate into a mortality difference, we compared the response to brief (6-min) and prolonged (45-min) coronary occlusion in vivo in five groups (each n = 16) of rats: ovariectomized females; ovariectomized females after 6 wk 17beta-estradiol replacement; male rats supplemented with estradiol for 6 wk; normal males; and normal females. Coronary occlusion produced a uniform ischemic risk area averaging 53 +/- 3% of left ventricular volume. After a brief occlusion, reperfusion ventricular tachycardia/fibrillation occurred with >85% frequency in all groups. During a prolonged occlusion, ischemic ventricular tachycardia occurred in 100% and sustained tachycardia requiring cardioversion in >75% of rats in all groups. Myocardial infarct size averaged 52 +/- 4% of the ischemic risk area and was similarly unaffected by gender or estrogen status. We conclude that neither short-term estrogen withdrawal, replacement, nor supplementation significantly affects the potentially lethal outcomes from acute coronary occlusion in this species.

Estrogen stimulates heat shock protein 90 binding to endothelial nitric oxide synthase in human vascular endothelial cells. Effects on calcium sensitivity and NO release.

Estradiol (E(2)) causes endothelium-dependent vasodilation, mediated, in part, by enhanced nitric oxide (NO) release. We have previously shown that E(2)-induced activation of endothelial nitric oxide synthase (eNOS) reduces its calcium dependence. This pathway of eNOS activation is unique to a limited number of stimuli, including shear stress, the response to which is herbimycin-inhibitable. Consistent with this, herbimycin and geldanamycin pretreatment of human umbilical vein endothelial cells (HUVEC) abrogated E(2)-stimulated NO release and cGMP production, respectively. These benzoquinone ansamycins are potent inhibitors of Hsp90 function, which has recently been shown to play a role in stimulus-dependent eNOS activation. As in response to shear, E(2) induced an Hsp90-eNOS association, peaking at 30 min and completely inhibited by the conventional estrogen receptor antagonist ICI 182,780. These findings suggest that Hsp90 plays an important role in the rapid, estrogen receptor-mediated modulation of eNOS activation by estrogen.

Modulation of circulating cellular adhesion molecules in postmenopausal women with coronary artery disease.

OBJECTIVES: The present study examined the association of estrogen (E2) and the inflammatory response of endothelium in coronary artery disease (CAD) by measuring circulating cellular adhesion molecules (cCAMs) in subjects with atherosclerosis. BACKGROUND: Atherosclerotic plaque demonstrates features similar to inflammation. Endothelial cell activation by inflammatory cytokines induces expression of cellular adhesion molecules (CAMs), thereby perhaps augmenting leukocyte adhesion and recruitment and subsequent development of atherosclerosis. The incidence of CAD is lower in women; this may be due to the cardioprotective effects of E2. METHODS: Consecutive eligible subjects with CAD admitted for cardiac catheterization were studied. The groups evaluated were men, postmenopausal women receiving E2 replacement therapy (ERT), postmenopausal women not receiving ERT and premenopausal women. Control groups included men and women without CAD. Preprocedural blood samples were drawn from all groups. Measurements of cCAMs, E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 were performed by enzyme-linked immunoabsorbant assay. E2 levels were assessed by radioimmunoassay. RESULTS: We observed a statistically significant increase in all cCAMs in men with CAD and postmenopausal women with CAD not receiving ERT compared with postmenopausal women with CAD receiving ERT. Premenopausal women with CAD and postmenopausal women with CAD receiving ERT had a significant increase in VCAM-1 alone compared with the female control group. CONCLUSIONS: A possible mechanism by which E2 exerts one of its cardioprotective effects is by limiting the inflammatory response to injury by modulating the expression of CAMs from the endothelium.

17 beta-estradiol regulation of human endothelial cell basal nitric oxide release, independent of cytosolic Ca2+ mobilization.

Estradiol retards the development of atherosclerosis. Animal models have suggested that NO may be a critical effector molecule in this cardiovascular protection. In this study, female human umbilical vein endothelial cells (HUVECs) were propagated in phenol red-free gonadal hormone-free medium and pretreated with 17 beta-estradiol (E2). Reduced NO2- and NO3- (NOx) concentration, determined by chemiluminescence, demonstrated a rapid increase in basal HUVEC NO release in response to physiological concentrations of E2. The estrogen receptor (ER) antagonist ICI 164,384 inhibited the augmented NO release, demonstrating an ER-mediated component of this response. Because endothelial NO synthase (eNOS) activity is largely regulated by cytosolic Ca2+, relative [Ca2+]i in response to E2 was determined in a fluorometric assay. E2 did not promote HUVEC Ca2+ fluxes. Furthermore, eNOS activity in E2-pretreated endothelial whole-cell lysates was not dependent on additional Ca2+. Despite involving the ER, this is a nongenomic effect E2, as demonstrated by maintained responses in transcriptionally inhibited cells and by the rapidly (10 minutes) of cGMP formation in an NO bioassay. We demonstrate, for the first time, that independent of cytosolic Ca2+ mobilization, there is augmentation of eNOS activity with a resultant increase in HUVEC basal NO release in response to short-term estradiol exposure. Implications for the cardiovascular protective role of estrogen are discussed.

Effects of 17beta-estradiol on cytokine-induced endothelial cell adhesion molecule expression.

One of the earliest events in atherosclerosis is interaction of circulating mononuclear leukocytes and the endothelium. Endothelial cell (EC) activation by cytokines results in expression of adhesion molecules and production of chemotactic factors, augmenting leukocyte adhesion and recruitment, respectively. The incidence of atherosclerosis in premenopausal women is significantly less than that observed in age-matched males with similar risk profiles. Because estrogen has gene regulatory effects, we investigated whether 17beta-estradiol (E2) can inhibit cytokine-mediated EC adhesion molecule transcriptional activation. Cultured human umbilical vein EC (estrogen receptor-positive) were propagated in gonadal hormone-free medium and were E2-pretreated for 48 h before IL-1 activation. Detected by FACS analysis, E2 strongly (60-80%) inhibited IL-1-mediated membrane E-selectin and vascular cell adhesion molecule-1 induction, and intercellular adhesion molecule-1 hyperinduction. 17alpha-estradiol (an inactive E2 stereoisomer) had no effect. This inhibition correlated with similar reductions in steady state-induced E-selectin mRNA levels, and was abrogated by the E2 antagonist ICI 164,384, demonstrating a specific, estrogen receptor-mediated effect. Nuclear run-offs confirmed suppression at the transcriptional level. The implications of these results for the cardiovascular protective role of estrogen are discussed.

Interaction of immune complexes with glomerular heparan sulfate-proteoglycans.

The binding characteristics of cationic and more neutral immune complexes with heparan sulfate-proteoglycan enriched anionic sites of glomerular basement membrane and mesangial matrix were studied. Rat kidneys were treated either with buffers alone or buffers containing heparitinase or chondroitinase-ABC followed by perfusion with cationic or native immune complexes. Tissues were processed for immunofluorescence and transmission electron microscopy after fixation with glutaraldehyde or tannic acid glutaraldehyde. Kidneys perfused with radioiodinated immune complexes were processed for light and electron microscopic autoradiography. In addition, glomeruli from kidneys perfused with radioiodinated immune complexes were isolated and counted for radioactivity. By immunofluorescence the cationic immune complexes deposited linearly along the glomerular basement membrane. By electron microscopy, the cationic complexes localized mainly in the inner and outer layers of the glomerular basement membrane and to a certain extent in the mesangial matrix in a distribution that corresponded to previously documented anionic sites. Whereas heparitinase treatment abrogated the binding of cationic immune complexes in both glomerular basement membrane and mesangial matrix, chondroitinase-ABC treatment did not cause any decrease in binding. In contrast, more neutral immune complexes appeared to be nonspecifically trapped in the mesangium, and their distribution was unaffected by both enzymatic treatments. Light and electron microscopic autoradiography and counts of isolated glomeruli confirmed these findings. The results overall indicate that cationic immune complexes bind electrostatically to the heparan sulfate-proteoglycan enriched anionic sites of the glomerular basement membrane and mesangial matrix, while more neutral immune complexes are nonspecifically trapped in the mesangium of the renal glomerulus.

Charge-related deposition of immune complexes in the glomerular basement membrane is independent of Fc effector function.

Differently charged immune complexes composed of fragments of bovine gamma globulin (BGG) antigen and fragments of rabbit anti-BGG antibody lacking FC portions were used for a study of the role of charge in deposition and localization within the mouse glomerulus. Preformed soluble complexes were made from native heterogeneous, cationic, or anionic F(ab')2 fragments of BGG and of rabbit antibody to BGG, and were injected intravenously. The distribution in the kidney was studied by immunofluorescence and electron microscopy. Cationic but not anionic or heterogeneous complexes diffusely localized in the glomerular basement membrane in both subepithelial and subendothelial sites. In contrast, more neutral or anionic complexes localized less prominently and were limited to the mesangium. The findings suggest that electrostatic interactions independent of Fc effector functions of immunoglobulins can be responsible for glomerular immune complex localization.

Nondissociating cationic immune complexes can deposit in glomerular basement membrane.

The mechanisms by which immune complexes deposit in the glomerular basement membrane have been the subject of much debate, with the relative importance of direct deposition of circulating immune complexes (IC) vs. formation of IC in situ from the binding of circulating antibody to structural or exogenous planted antigen being at issue. In order to determine whether intact IC can deposit as such, covalently linked IC were prepared by a two-step reaction involving the bifunctional reagent toluene-2,4-diisocyanate (TDI), the antigen bovine gamma globulin (BGG), and rabbit anti-BGG antibody. Antigen and antibody were covalently cross-linked, with little self-linkage of antigen or antibody, and IC were purified by gel filtration. The net charge of the complexes was varied by chemical means, either before or after IC formation. When cationic IC were injected intravenously into mice, there was codeposition of antigen and antibody diffusely in the glomerular basement membrane (GBM), and deposits were observed ultrastructurally in the laminae rarae, interna and externa, and the lamina densa. Thus, under conditions of restricted appropriate charge, intact IC can cross the glomerular basement membrane and deposit in subepithelial sites without being excluded by size alone.

Nephritogenicity and differential distribution of glomerular immune complexes related to immunogen charge.

Nephritogenicity and the differential distribution of glomerular deposits as a function of immunogen charge were examined in a murine model of chronic active serum sickness. A range of differentially charged immunogens was used, i.e., chemically modified highly cationic or anionic bovine gamma-globulin (BGG), native unmodified isoelectrically focused slightly cationic or anionic BGG, and heterogeneous native BGG. The amounts and distribution of immune complexes in glomeruli were compared by immunofluorescence and electron microscopy 3 to 10 weeks after the initiation of 15 intravenous injections of the five immunogens and 13 days after intraperitoneal continuous release of the native unmodified BGGs by osmotic pumps. The results demonstrate that the more cationic the immunogen, the more nephritogenic and the greater the tendency to form subepithelial deposits in the glomerular basement membrane. The observed differences in nephritogenicity and localization induced by focused unmodified cationic and anionic fractions of BGG particularly emphasize immunogen charge as a major factor influencing glomerular distribution of immune complexes in this model. The findings indicate that several degrees of nephritogenicity and the differential distribution of immune complexes can be related to immunogen charge and strongly suggest that charge interactions may be of fundamental importance in the pathogenesis of immune complex glomerulonephritis.

Structural studies on an IgM-lambda pyroglobulin.

An IgM-lambda pyroglobulin from a patient with Waldenström's syndrome was studied. Heavy and light chains were separated and their N-terminal amino acid sequence determined. The heavy chain was unblocked and belonged to the VHIII subclass, and the light chain belonged to the lambda I subclass. Factors influencing pyroprecipitability were examined through experiments designed to study some of the physical and chemical properties of an IgM-lambda pyroglobulin. Pyroprecipitability was affected by pH, ionic strength, urea, and reducing agents, suggesting an involvement of noncovalent electrostatic interactions. It was also demonstrated through recombinant experiments that it is necessary to have covalently joined homologous heavy and light chains in pentameric form for pyroprecipitation to occur. Since neither heavy nor light chains had any unique structural features, the reasons for this property remain obscure but may reflect the result of conformational factors.

Charge of circulating immune complexes as a factor in glomerular basement membrane localization in mice.

The effect of the charge of circulating immune complexes on glomerular localization was studied in a model of passive serum sickness. Preformed immune complexes of heterogeneous or restricted charge, prepared in vitro from isoelectrically focused or chemically modified proteins, were injected intravenously into mice. The distribution of immune complexes in the kidney was compared by immunofluorescence and electron microscopy. Cationic but not anionic or electrophoretically heterogeneous immune complexes gave rise to diffuse localization in the glomerular basement membrane. The binding in subepithelial and subendothelial sites correlated with the known distribution of structural anionic sites. The observations suggest that electrostatic interactions between fixed anionic sites and immune complexes may be an important factor in glomerular trapping. Alternative mechanisms based on initial localization of excess free cationic antigen cannot be completely excluded and are also considered.