Lipoprotein-heparin-fibronectin-denatured collagen complexes enhance cholesteryl ester accumulation in macrophages.

The sequestration of low-density lipoprotein (LDL) by components of the vascular extracellular matrix has long been recognized as a contributing factor to lipid accumulation during atherogenesis. The effects, however, that components of the extracellular matrix might have on LDL catabolism by scavenger cells have been little investigated. For these purposes we have prepared insoluble complexes of LDL, heparin, fibronectin, and denatured collagen (gelatin) and examined their effects on lipid accumulation, LDL uptake and degradation, and cholesteryl ester synthesis in mouse peritoneal macrophages. The results of these experiments have demonstrated that the cholesteryl ester content of macrophages incubated with a particular suspension of LDL, heparin, fibronectin, and collagen complexes is four- to fivefold that of cells incubated with LDL alone. The uptake of complexes containing 125I-LDL is rapid; however, in contrast to either endocytosed 125I-LDL or 125I-acetyl LDL, the degradation of complex-derived LDL is impaired. In addition, the uptake of complex-derived LDL stimulates the incorporation of [14C]oleic acid into cholesteryl oleate, however, the stimulation was a small fraction of that observed in cells incubated with acetyl LDL. Ultrastructurally, macrophages incubated with LDL, heparin, fibronectin, and collagen complexes did not contain many lipid droplets, but rather their cytoplasm is filled with phagosomes containing material similar in appearance to LDL-matrix complexes. These results indicate that components of the extracellular matrix can alter the catabolism of LDL by scavenger cells, suggesting that they may play a role in cellular lipid accumulation in the atherosclerotic lesion.

Transforming growth factor-beta activity is potentiated by heparin via dissociation of the transforming growth factor-beta/alpha 2-macroglobulin inactive complex.

The control of smooth muscle cell (SMC) proliferation is determined by the combined actions of mitogens, such as platelet-derived growth factor, and the opposing action of growth inhibitory agents, such as heparin and transforming growth factor-beta (TGF-beta). The present studies identify an interaction between heparin and TGF-beta in which heparin potentiates the biological action of TGF-beta. Using a neutralizing antibody to TGF-beta, we observed that the short term antiproliferative effect of heparin depended upon the presence of biologically active TGF-beta. This effect was observed in rat and bovine aortic SMC and in CCL64 cells, but not in human saphenous vein SMC. Binding studies demonstrated that the addition of heparin (100 micrograms/ml) to medium containing 10% plasma-derived serum resulted in a 45% increase in the specific binding of 125I-TGF-beta to cells. Likewise, heparin induced a twofold increase in the growth inhibitory action of TGF-beta at concentrations of TGF-beta near its apparent dissociation constant. Using 125I-labeled TGF-beta, we demonstrated that TGF-beta complexes with the plasma component alpha 2-macroglobulin, but not with fibronectin. Heparin increases the electrophoretic mobility of TGF-beta apparently by freeing TGF-beta from its complex with alpha 2-macroglobulin. Dextran sulfate, another highly charged antiproliferative molecule, but not chondroitin sulfate or dermatan sulfate, similarly modified TGF-beta's mobility. Relatively high, antiproliferative concentrations of heparin (1-100 micrograms/ml) were required to dissociate the TGF-beta/alpha 2-macroglobulin complex. Thus, it appears that the antiproliferative effect of heparin may be partially attributed to its ability to potentiate the biological activity of TGF-beta by dissociating it from alpha 2-macroglobulin, which normally renders it inactive. We suggest that heparin-like agents may be important regulators of TGF-beta's biological activity.

Herpes simplex virus infection in human arterial cells. Implications in arteriosclerosis.

Herpesviruses have been implicated as etiologic factors in the pathogenesis of human arteriosclerosis. We have examined the pathobiological effects of human herpes simplex virus (HSV-1) infection in influencing lipid accumulation and metabolism in human and bovine arterial smooth muscle cells (SMC). Significantly greater amounts of saturated cholesteryl esters (CE) and triacylglycerols (TG) accumulate in HSV-1-infected human and bovine arterial SMC than uninfected cells. This CE accumulation results, in part, from decreased CE hydrolysis. Furthermore, arachidonate-stimulated, HSV-1-infected arterial SMC have a reduced capacity to produce prostacyclin (an agonist of intracellular CE hydrolytic activity) than uninfected, stimulated SMC. It appears that HSV-1 may induce lipid accumulation in arterial SMC similar, in part, to the lipid accumulation observed in vivo during human atherogenesis. Thus, herpesviruses may contribute to lipid accumulation, which is a characteristic feature of atherosclerosis.

Decreased type II/type I TGF-beta receptor ratio in cells derived from human atherosclerotic lesions. Conversion from an antiproliferative to profibrotic response to TGF-beta1.

Atherosclerosis and postangioplasty restenosis may result from abnormal wound healing. The present studies report that normal human smooth muscle cells are growth inhibited by TGF-beta1, a potent wound healing agent, and show little induction of collagen synthesis to TGF-beta1, yet cells grown from human vascular lesions are growth stimulated by TGF-beta1 and markedly increase collagen synthesis. Both cell types increase plasminogen activator inhibitor-1 production, switch actin phenotypes in response to TGF-beta1, and produce similar levels of TGF-beta activity. Membrane cross-linking of 125I-TGF-beta1 indicates that normal human smooth muscle cells express type I, II, and III receptors. The type II receptor is strikingly decreased in lesion cells, with little change in the type I or III receptors. RT-PCR confirmed that the type II TGF-beta1 receptor mRNA is reduced in lesion cells. Transfection of the type II receptor into lesion cells restores the growth inhibitory response to TGF-beta1, implying that signaling remains responsive. Because TGF-beta1 is overexpressed in fibroproliferative vascular lesions, receptor-variant cells would be allowed to grow in a slow, but uncontrolled fashion, while overproducing extracellular matrix components. This TGF-beta1 receptor dysfunction may be relevant for atherosclerosis, restenosis and related fibroproliferative diseases.

Recovery of prostacyclin production by de-endothelialized rabbit aorta. Critical role of neointimal smooth muscle cells.

Prostacyclin (PGI2) synthetic capacity was assayed at the surface of aortas at various intervals after removal of endothelium with a balloon catheter. Results were correlated with morphologic changes in the vessel wall seen by light microscopy, scanning and transmission electron microscopy. To assay PGI2 synthetic capacity, we applied an incubation chamber to the luminal surface of the aortas; after arachidonic acid stimulation we assayed the PGI2 synthesized with a bioassay and radioimmunoassay. PGI2 synthesis in de-endothelialized aortas was determined immediately after balloon-catheter injury and at intervals of 1 h and 2, 4, 15, 35, and 70 d. PGI2 synthesis was low at 1 h and increased over time with levels at 35 and 70 d reaching that of normal artery. Scanning and transmission electron microscopy of de-endothelialized areas showed persistent absence of endothelium with formation of a neointima composed of smooth muscle cells. De-endothelialized aorta was covered with adherent platelets shortly after injury, however several days later only a few platelets adhered to the denuded surface. Results indicated that (a) endothelium is responsible for nearly all PGI2 production at the luminal surface of the normal aorta, (b) de-endothelialized muscular neointima synthesized increasing quantities of PGI2 with time after injury, and (c) increase of PGI2 production at the luminal surface of de-endothelialized aorta correlates with formation of a neointima and with the acquired thromboresistance of the aorta.

Prostacyclin modulates cholesteryl ester hydrolytic activity by its effect on cyclic adenosine monophosphate in rabbit aortic smooth muscle cells.

We tested the hypothesis that prostacyclin (PGI2), 6-keto-prostaglandinF1 alpha(6-keto-PGF1 alpha), and several E series prostaglandins (PG) may affect the activity of cholesteryl ester (CE) hydrolase since our previous experiments indicated that smooth muscle cells (SMC) in neointima of injured rabbit aorta (a) acquire the capacity to produce PGI2 and (b) have increased lysosomal CE hydrolytic (acid cholesteryl ester hydrolase [ACEH])activity. Using cultured SMC from rabbit thoracic aorta, we demonstrated that PGI2, 6-keto-PGF1 alpha, and 6-keto-PGE1 enhanced ACEH activity fourfold. No significant effects on ACEH activity were observed with PGE1 or PGE2. Preincubation of SMC with an inhibitor of adenylate cyclase activity (dideoxyadenosine) abolished the effect of these PG on CE hydrolytic activity. Addition of dibutyryl cAMP to these SMC significantly increased ACEH activity. Although concentrations of PGI2 used significantly increased cAMP levels, proliferation of these SMC was not observed. In related experiments, we determined if the addition of PGI2, 6-keto-PGF1 alpha, or 6-keto-PGE1 to cultured aortic SMC would enhance the egress of unesterified cholesterol and CE from these SMC. A significant loss of total cholesterol from PG-treated SMC was observed at the end of 14 d. Results suggest that increased synthesis of PGI2 by neointimal SMC in the injured aortic wall may, at least in part, explain the changes in CE catabolism and accumulation following injury. These PG may also be important in CE metabolism and accumulation in human arteries.

Specific inhibition of eIF-5A and collagen hydroxylation by a single agent. Antiproliferative and fibrosuppressive effects on smooth muscle cells from human coronary arteries.

Restenosis occurs in 35% of patients within months after balloon angioplasty, due to a fibroproliferative response to vascular injury. These studies describe a combined fibrosuppressive/antiproliferative strategy on smooth muscle cells cultured from human primary atherosclerotic and restenotic coronary arteries and from normal rat aortas. L-Mimosine suppressed the posttranslational hydroxylation of the precursors for collagen and for eukaryotic initiation factor-5A (eIF-5A) by directly inhibiting the specific protein hydroxylases involved, prolyl 4-hydroxylase (E.C. 1.14.11.2) and deoxyhypusyl hydroxylase (E.C. 1.14.99.29), respectively. Inhibition of deoxyhypusyl hydroxylation correlated with a dose-dependent inhibition of DNA synthesis. Inhibition of prolyl hydroxylation caused a dose-dependent reduction in the secretion of hydroxyproline-containing protein and decreased the formation of procollagen types I and III. The antifibroproliferative action could not be attributed to nonspecific or toxic effects of mimosine, appeared to be selective for the hydroxylation step in the biosynthesis of the procollagens and of eIF-5A, and was reversible upon removal of the compound. The strategy of targeting these two protein hydroxylases has important implications for the pathophysiology of restenosis and for the development of agents to control fibroproliferative diseases.

Evidence for an age-related dysfunction in the antiproliferative response to transforming growth factor-beta in vascular smooth muscle cells.

Previous studies have indicated that aged animals show an increased intimal hyperplasia after arterial injury. The present studies examined the hypothesis that the increased serum-free proliferation of aged smooth muscle cells (SMC), in vitro, was due to a loss of an antiproliferative signal, such as transforming growth factor-beta 1 (TGF-beta 1). Northern blot analysis of the mRNA derived from old (> 19 mo) or young (3-4 mo) rat aortic SMC indicated that both groups had an equivalent level of the 2.5 kB TGF-beta 1 message. Metabolic labeling with 35S-methionine and immunoprecipitation for TGF-beta 1 confirmed the de novo synthesis of TGF-beta 1 in rat SMC. Old and young SMC supernatants showed equal levels of active or latent (acid-activated) TGF-beta activity. Despite the similarities in the production of TGF-beta 1, old SMC were refractory to inhibition by TGF-beta 1, whereas young SMC were markedly inhibited (80%) by low levels of TGF-beta 1 (IC50 < 5 pg/ml). Binding studies at 4 degrees C indicated that old SMC exhibited reduced binding capacity for 125I-TGF-beta 1. Cross-linking studies confirmed that old SMC showed reduced binding of 125I-TGF-beta 1 to membrane sites corresponding to the high molecular weight type III receptor, as well as the 85-kDa type II and 65-kDa type I receptor. However, at 37 degrees C, old SMC degraded 125I-TGF-beta 1 more rapidly than young SMC. Combined, this data suggests that SMC derived from older animals are capable of normal production of TGF-beta 1 but fail to respond to the autocrine growth inhibitory effects of this agent, thereby leading to enhanced proliferation.

Periprostatic adipose inflammation is associated with high-grade prostate cancer.

BACKGROUND: Obesity, a cause of subclinical inflammation, is associated with increased risk of high-grade prostate cancer (PC) and poor outcomes. Whether inflammation occurs in periprostatic white adipose tissue (WAT), and contributes to the negative impact of obesity on PC aggressiveness, is unknown. METHODS: In a single-center, cross-sectional design, men with newly diagnosed PC undergoing radical prostatectomy were eligible for study participation. The primary objective was to examine the prevalence of periprostatic WAT inflammation defined by the presence of crown-like structures (CLS-P) as detected by CD68 immunohistochemistry. Secondary objectives were to explore the clinical and systemic correlates of periprostatic WAT inflammation. Tumor characteristics and host factors including BMI, adipocyte diameter, and circulating levels of lipids, adipokines, and other metabolic factors were measured. Wilcoxon rank-sum, Chi-square, or Fisher's exact tests, and generalized linear regression were used to examine the association between WAT inflammation and tumor and host characteristics. RESULTS: Periprostatic fat was collected from 169 men (median age 62 years; median BMI 28.3). Periprostatic WAT inflammation was identified in 49.7% of patients and associated with higher BMI (P=0.02), larger adipocyte size (P=0.004) and Gleason grade groups IV/V tumors (P=0.02). The relationship between WAT inflammation and high Gleason grade remained significant after adjusting for BMI (P=0.04). WAT inflammation correlated with higher circulating levels of insulin, triglycerides, and leptin/adiponectin ratio, and lower high density lipoprotein cholesterol, compared to those without WAT inflammation (P's <0.05). CONCLUSION: Periprostatic WAT inflammation is common in this cohort of men with PC and is associated with high-grade PC.

Fluorescent opsonization assay: binding of plasma fibronectin to fibrin-derivatized fluorescent particles does not enhance their uptake by macrophages.

A simple method for assessing the opsonic properties of plasma fibronectin is described. The method is based on the uptake of protein-derivatized fluorescent particles by mouse peritoneal macrophages. With this system, fibronectin stimulated the uptake of gelatin conjugated to carboxylated fluorescent particles. In contrast to the gelatinized particles, neither the covalent nor noncovalent interaction of fibronectin with fibrin particles stimulated their uptake by macrophages. These results provide additional evidence that fibronectin is not a conventional opsonin and that enhanced phagocytosis is not an obligatory response by macrophages to fibronectin-coated particles.

A mediator of cell surface-specific plasmin generation.

It has become increasingly evident that the generation of cell surface proteases including plasmin is fundamental to a wide variety of in vivo biological processes. Cell surface receptors allow for specific controlled proteolysis, provide protection from inhibitors, and enhance catalytic efficiency. Here we describe one such receptor, annexin II, which serves as a coreceptor for tissue plasminogen activator and plasminogen and is found on a wide variety of cell types including endothelial cells, some tumor cells, monocytes and macrophages, and neuronal cells. Evidence indicates that annexin II may be crucial to the efficient generation of cell surface plasmin, endothelial cell formation of new blood vessels, and maintenance of vascular patency. Additionally, it has been shown that annexin II expression in acute promyelocytic leukemia contributes to the bleeding diathesis seen in this disease and that inhibition of annexin II may be an important mechanism in the formation of atherosclerotic plaque. Furthermore, emerging evidence reveals the importance of annexin II on the surface of monocytes and macrophages, where it may contribute to the cells' ability to degrade extracellular matrix proteins and migrate to sites of injury or inflammation.

Local factors affecting the tendency to bypass local hospitals for inpatient mental health care: an exploratory analysis.

Using the records of 2,171 rural residents of Illinois who received inpatient treatment for mental illness or substance abuse, this paper examines factors that influence the tendency to seek service from a distant rather than a local hospital. Results indicate that the age and insurance coverage of the individual, the per capita income of the community area, surrogates for the service orientation of the local hospital and the proximity of the patient's residence to an urban center are significant influences. With the exceptions of drug abuse requiring detoxification or other symptomatic treatment, drug abuse accompanied by comorbidity and psychosocial disorders, psychosis, and childhood disorders, the primary diagnosis of the individual failed to have a significant effect on the propensity to bypass local sources of inpatient treatment.

Laterality and field dependence.

The purposes of this study were (1) to replicate a right visual-field advantage for a version of the Embedded Figures Test modified for tachistoscopic presentation and (2) to test for a relationship between field independence and performance on modified Embedded Figures Test items presented to the right visual-field. 34 right-handed subjects were presented Embedded Figures Test items to the left, center, and right visual-fields for 150 msec. The Group Embedded Figures Test was also given. Results showed a right visual-field advantage on the modified embedded-figures test and (2) a negative relationship between Group Embedded Figures Test performance and right visual-field scores. These findings were discussed in terms of the differences between the two versions of the embedded-figures test, memory for simple figures, specificity of judgment, and sustained versus tachistoscopic perception. A tentative conclusion suggests that a better internal representation of the briefly presented complex figures places field-independent subjects at a disadvantage on the modified version of the test.

A modified lateral eye-movement measure, the right hemisphere and creativity.

The relationship between direction of gaze and creativity was studied using a modified measure of lateral eye movement which recorded the total time of gaze rather than initial direction of gaze only. Twelve subjects were presented the Uses Test and the Remote Associates Test orally while direction of gaze was monitored. Analysis yielded a high positive correlation between left gaze and upward gaze for the Uses Test scores while directional gaze did not correlate with Remote Associates Test performance. These findings would have been obscured if the conventional method of recording lateral eye movements had been used.

Progress toward Healthy People 2000: a preliminary look at Oklahoma.

Over 10,000 Oklahomans die each year from coronary artery disease or stroke. This study examined the behavioral risk factors for these illnesses present in Oklahomans. Oklahomans are at considerably higher risk than the desirable national goals for such risk factors. For example, too many Oklahomans smoke and too few exercise. The data to support these findings are included herein, and some steps to reverse these trends are recommended.

Health care reform? An American obsession with prescriptive incrementalism.

A rounded evaluation of the national health insurance proposals that now seem to be taken seriously by political elites requires conceptual organization. This article adopts a typology that describes each major proposal as a social, mixed or a private insurance scheme depending on the source(s) of funding, method of compensating hospitals and physicians, the unit of payment, and mechanism for financing capital. Not surprisingly, the analysis suggests that the social insurance model, closely resembling the Canadian system, is more likely to control inflation and redress distributional inequities than are other approaches. Why, then, has this approach not been adopted? The answer may be found in the widespread acceptance of disjointed incrementalism as a valid description of the policy process which yields an ideological orientation that can be termed "prescriptive incrementalism." This orientation is closely related to a belief in an "American exceptionalism," a belief that is not warranted by a cross-sectional examination of the political culture infusing issues about the proper role of government in health care financing and delivery. Unfortunately for advocates, the truly exceptional factor restricting the United States' ability to effect national health reform is a quite delberately obstruction-oriented political structure.

Heparin stimulation of plasminogen activator secretion by macrophage-like cell line RAW264.7: role of the scavenger receptor.

Secretion of urokinase-type plasminogen activator (uPA) by RAW264.7 cells was stimulated by heparin in a dose- and time-dependent manner. Secretion of uPA was not detected when cells were exposed to heparin at 4 degrees C, indicating that heparin was not simply releasing receptor-bound uPA. Furthermore, prior removal of membrane-associated uPA did not influence heparin's ability to stimulate the release of uPA from the macrophage-like line. Low molecular weight and weakly anticoagulant heparins stimulated uPA secretion but less effectively than other heparin fractions. The observed stimulation in macrophage uPA secretion by heparin is similar to that previously reported for polyanions recognized by the scavenger receptor including fucoidan, polyinosinic acid, dextran sulfate, and acetyl-LDL (Falcone and Ferenc: J. Cell. Physiol., 135:387-396, 1988). Evidence that heparin's binding to RAW264.7 cells is mediated by the scavenger receptor is derived from experiments in which fucoidan blocked the specific binding of [3H]-heparin to RAW264.7 cells. However, heparin partially inhibited the stimulation of cholesteryl [3H]-oleate synthesis observed in these cells upon incubation with acetyl-LDL and weakly inhibited cellular binding of 125I-acetyl-LDL at 4 degrees C. These data indicate that heparin's binding to RAW264.7 cells is mediated, only in part, by the scavenger receptor. Nonetheless, neither heparin nor fucoidan was able to stimulate the release of plasminogen activator activity from monocyte-like U937 cells which are devoid of scavenger receptor activity.

The challenge of comparative health policy for political science.

After a brief review of the literature and some reflection on the state of the art, this paper argues that more analyses of health policy in an explicitly comparative perspective are needed. This conclusion is based on a discussion of the largely unexploited benefits that could accrue from a welding of political science and health services research and a consideration of a research design illustrating the compatibility of these two endeavors.

Acetyl-LDL stimulates macrophage-dependent plasminogen activation and degradation of extracellular matrix.

The ability of acetyl-LDL to stimulate macrophage-dependent plasminogen activation and degradation of extracellular matrix was examined. We have found that expression of plasminogen activator activity in response to the scavenger receptor ligand varied among cell populations. Exposure to acetyl-LDL stimulated plasminogen activator expression by cells which constitutively released low levels of activator. These include a virally transformed macrophage-like cell line (RAW246.7), concanavalin A and C. parvum-activated macrophages. The stimulation of plasminogen activator activity was independent of cellular lipid accumulation since nonlipoprotein inhibitors of acetyl-LDL binding to the scavenger receptor stimulated activator expression in great excess to that observed with acetyl-LDL. In contrast, acetyl-LDL was unable to induce soluble plasminogen activator activity in cells which normally do not express it. These include a macrophage-like cell line (J774A.1) and resident peritoneal macrophages. Furthermore, acetyl-LDL was unable to modulate the copious secretion of activator by inflammatory macrophages elicited with thioglycolate. When macrophages were tested for their ability to degrade smooth muscle cell derived matrix, solubilization by resident, elicited, and activated cells was variously increased in the presence of plasminogen. Furthermore, exposure to acetyl-LDL enhanced plasmin-dependent degradation by resident cells and activated cells, whereas matrix degradation by elicited cells was unaffected.