High sensitivity C-reactive protein in cardiovascular risk assessment. CRP mania or useful screening?

Elevated level of the acute phase reactant C-reactive protein (CRP) is a very sensitive marker of acute inflammatory reactions. Using high sensitivity assays for CRP, recent observations indicate that slightly elevated CRP levels which would be in the normal range of conventional assays are a novel marker for an increased risk for cardiovascular events, especially coronary artery disease and myocardial infarction. Various large scale prospective trials including the Physicians' Health Study and the Women's Health Study revealed that slightly increased hsCRP levels at base line in apparently healthy persons are associated with a 2-fold increase in the risk of a future myocardial infarction. The predictive value of hsCRP was found to be independent from classic risk factors, in particular from elevated serum cholesterol. An increase in hsCRP levels was also associated with a higher risk to develop peripheral artery disease and with a faster progression of carotid artery disease. Until now, treatment with HMG-CoA reductase inhibitors (statins) has been found to be the only medication to consistently decrease hsCRP levels, although by about 15% only. Despite the association of elevated hsCRP levels with future cardiovascular events, routine measurement of hsCRP for cardiovascular risk assessment is currently not recommended because of its low accuracy, the lack of a clear cut-off point for elevated hsCRP levels and the lack of an absolute predictive value. So far, hsCRP remains an interesting potential risk marker for cardiovascular disease whose definite relevance remains to be established.

Polyunsaturated fatty acids and cardiovascular risk: interference at the level of gene expression.

Long-chain polyunsaturated fatty acids (PUFA) are important structural components of the cell membrane as well as precursors of highly active lipid mediators, the eicosanoids. In addition, they have been identified as novel intracellular signaling molecules which either directly or indirectly through the modulation of other signaling pathways activate transcription factors and gene expression. With regard to cardiovascular risk and diseases, effects of PUFA have been observed on genes whose products regulate either metabolic processes, especially fatty acid metabolism, or are involved in inflammatory and mitogenic processes of vascular cells. Often, the effects of n-3 and n-6 PUFA on the expression of both types of genes are antagonistic indicating a balancing role of n-3 and n-6 PUFA in cell metabolism and function already at the level of gene expression.

Hyaluronan fragments induce the synthesis of MCP-1 and IL-8 in cultured human peritoneal mesothelial cells.

Human peritoneal mesothelial cells (HMC) play an important role in inflammatory processes by their ability to produce various cytokines and chemokines, such as monocyte chemoattractant protein 1 (MCP-1) and interleukin 8 (IL-8). In this study we investigated the effect of experimentally generated hyaluronan (HA) fragments, degradation products of the extracellular matrix component hyaluronan, which accumulate at inflammatory sites, on the expression of MCP-1 and IL-8 in cultured HMC. MCP-1 and IL-8 mRNA expression was determined by RNase protection assays, and protein levels in the supernatants were measured by enzyme-linked immunosorbent assays. HA fragments with a molecular mass of approximately 1-7x10(5) daltons upregulate MCP-1 and IL-8 synthesis in HMC dose and time dependently. The effect of HA fragments could be blocked by Ro31-8220, a specific protein kinase C inhibitor, and by PD98059, an inhibitor of the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway. Upregulation of chemokine synthesis was preceded by an increase in NF-kappaB and AP-1 DNA-binding activity, suggesting that these transcription factors are activated to increase MCP-1 and IL-8 expression by HA fragments. These data demonstrate that HA fragments markedly enhance the mRNA expression and protein synthesis of MCP-1 and IL-8 in HMC. In concert with previous findings, our observations indicate that enhanced levels of HA, which are present in the peritoneal cavity of peritoneal dialysis patients, may account for a locally increased chemokine production.

Effect of high glucose concentration on the synthesis of monocyte chemoattractant protein-1 in human peritoneal mesothelial cells: involvement of protein kinase C.

Human peritoneal mesothelial cells (HMC) contribute to the activation and control of inflammatory processes in the peritoneum by their potential to produce various inflammatory mediators. The present study was designed to assess the effect of glucose, the osmotic active compound in most commercially available peritoneal dialysis fluids, on the synthesis of the C-C chemokine monocyte chemoattractant protein-1 (MCP-1) in cultured HMC. The MCP-1 concentration in the cell supernatants was determined by enzyme-linked immunosorbent assay and the MCP-1 mRNA expression was examined using Northern blot analysis. Incubation of HMC with glucose (30-120 mM) resulted in a time- and concentration-dependent increase in MCP-1 protein secretion and mRNA expression. After 24 h the MCP-1 synthesis was increased from 2.8 +/- 0.46 to 4.2 +/- 0.32 ng/10(5) cells (n = 5, p < 0.05) in HMC treated with 60 mM glucose. In contrast, osmotic control media containing either the metabolically inert monosaccharide mannitol or NaCl did not influence MCP-1 production. The stimulating effect of high glucose on MCP-1 expression in HMC was mimicked by activation of protein kinase C (PKC) with the phorbol ester PMA (20 nM). Coincubation of the cells with glucose and the specific PKC inhibitor Ro 31-8220 completely blunted glucose-mediated MCP-1 expression. In summary, our results indicate that glucose induces MCP-1 synthesis by a PKC-dependent pathway. Since osmotic control media did not increase MCP-1 release, it is suggested that the effect of glucose is mainly related to metabolism and not to hyperosmolarity. These data may in part explain elevated steady-state levels of MCP-1 found in the dialysis effluent of continuous ambulatory peritoneal dialysis patients.

Lipids in vascular function.

Physiological and pathological vascular responses depend on the action of numerous intercellular mediators, ranging from hormones to gases like nitric oxide, proteins, and lipids. The last group consists not only of the different types of lipoproteins, but also includes a broad array of other lipophilic signaling molecules such as fatty acids, eicosanoids, phospholipids and their derivatives, sphingolipids and isoprenoids. Due to space limitations, it is impossible to discuss all the vascular effects of lipophilic mediators or compounds. Therefore, we will focus on one of the most important lipid-mediated diseases, atherosclerosis. Lipoproteins and especially their native or oxidized lipid compounds affect vascular function in many different ways, and these effects do not only modulate atherogenesis but are of paramount physiological and pathophysiological importance in other diseases, such as inflammation, tumor metastasis, or normal wound healing.

Long-chain polyunsaturated fatty acids and eicosanoids in infants--physiological and pathophysiological aspects and open questions.

Eicosanoids are highly active lipid mediators in physiologic and pathologic processes, with their effects ranging from cytoprotection and vasoactivity to modulation of inflammatory and proliferative reactions. Generation of eicosanoids can be affected by changes in the pools of their precursors, the long-chain polyunsaturated fatty acids (LCPUFA). Thus, dietary interventions such as supplementation of infant formula with specific n-3 and n-6 LCPUFA will alter formation as well as activity of the eicosanoids produced. This report summarizes the results and discussion of the workshop on "Eicosanoids and Polyunsaturated Fatty Acids in Infants." The intention of the workshop organizers was to give an overview of the role of eicosanoids in physiological and pathophysiological processes in infants, to discuss the implications that an increased n-3 and n-6 LCPUFA intake may have on eicosanoid generation, and to point out open questions and controversies for future research.

High glucose increases prostaglandin E2 synthesis in human peritoneal mesothelial cells: role of hyperosmolarity.

Peritoneal mesothelial cells are considered the predominant source of peritoneal prostanoid formation because they represent the largest resident cell population in the peritoneal cavity. The present study was designed to evaluate the effect of D-glucose, which is widely used in commercially available peritoneal dialysis fluids as an osmotic compound, on the synthesis of prostaglandins in cultured human mesothelial cells (HMC). Analysis of eicosanoid synthesis in HMC by reversed-phase HPLC revealed that 6-keto-PGF1alpha, the spontaneous hydrolysis product of prostacyclin (PGI2), and prostaglandin E2 (PGE2) were the main eicosanoids produced. Addition of D-glucose resulted in a time- and concentration-dependent (30 to 120 mM) increase in PGE2 production in HMC (24 h, 90 mM: 3.9+/-0.5 ng/10(5) cells versus 2.3+/-0.3 in untreated cells; P < 0.05). Mannitol (90 mM) or L-glucose (90 mM). nonmetabolizable osmotic compounds, also led to a significant (P < 0.05) but less intense increase in PGE2 synthesis (3.3+/-0.4 and 3.2+/-0.5 ng/10(5) cells, respectively). Increased PGE2 synthesis was completely blunted by coincubation with the specific protein kinase C (PKC) inhibitor Ro 31-8220 or downregulation of PKC activity by preincubation with phorbol myristate acetate for 16 h. Furthermore, coincubation with PD 98059, an inhibitor of the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway, also inhibited increased PGE2 synthesis by D-glucose or mannitol. In contrast, the iso-osmolar glucose polymer icodextrin, which is used as an alternative to D-glucose in peritoneal dialysis solutions, had no effect on PGE2 synthesis. These data indicate that D-glucose and metabolically inert sugars increase PGE2 synthesis in HMC at least in part by hyperosmolarity and that this effect requires activation of PKC and the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway of intracellular signaling.

Hemodialysis-induced upregulation of granulocyte adhesion molecules CD11b/CD18 is independent from 5-lipoxygenase activity.

Hemodialysis with new cuprophane membranes upregulates expression of granulocyte adhesion molecules and activates 5-lipoxygenase as reflected by the enhanced generation of leukotriene B4 (LTB4). We assessed the role of 5-lipoxygenase activity in hemodialysis-induced upregulation of the granulocyte adhesion molecules, CD11b and CD18, and granulocyte adhesion to human umbilical vein endothelial cells in an ex vivo dialysis model. 5-Lipoxygenase was effectively inhibited by preincubation of human whole blood with the specific inhibitor, BAY X1005. Dialysis with new cuprophane but not with new acrylonitrile membranes significantly upregulated expression of CD11b and CD18 by 6-fold and 4-fold, respectively. Inhibition of 5-lipoxygenase did not affect the expression of CD11b and CD18 during dialysis with either of the two membranes. In contrast to the enhanced expression of CD11b and CD18, adhesion of granulocytes to human umbilical vein endothelial cells did not increase during dialysis, nor was it affected by BAY X1005. These data indicate that ex vivo dialysis with cuprophane membranes upregulates expression of CD11b and CD18 on granulocytes independent of the activation of 5-lipoxygenase.

Intrinsic cyclooxygenase activity is not required for monocytic differentiation of U937 cells.

Metabolism of arachidonic acid has been found to modulate cell growth and differentiation. Since differentiation of premonocytic U937 cells is paralleled by increase expression of cyclooxygenase and prostanoid synthesis, we assessed the role of cyclooxygenase in the differentiation of U937 cells. Treatment with retinoic acid (RA, 1 microM) or 1,250(OH)2-vitamin D3 (1,25-D3, 10 nM) increased the expression of the monocytic surface antigens CD11b plus CD11c, and CD11b, CD11c, CD14 plus CD18, respectively. Addition of the cyclooxygenase inhibitors indomethacin (10 microM) or piroxicam (20 microM) slightly increased expression of CD11b and CD18 in cells differentiated with RA, but did not alter expression of surface antigens in cells treated with 1,25-D3. Stimulus-dependent rises in the cytosolic-free calcium concentrations remained unchanged by the inhibitors, as was superoxide anion generation in cells stimulated with phorbol myristate acetate. Effective inhibition of cyclooxygenase over the 72 h of differentiation was proven by the marked decrease in A23187-stimulated prostanoid formation in differentiated cells. To assess whether prostaglandins negatively control monocytic differentiation, as suggested by the stimulatory effects of cyclooxygenase inhibitors on CD11b/CD18 expression in RA-treated cells, prostaglandin E2 (PGE2; 100 nM) was added to the differentiation with RA or 1,25-D3. However, the addition of PGE2 increased expression of CD11b and CD11b plus CD14, as well as superoxide anion generation. These data indicate that intrinsic cyclooxygenase activity is not required for monocytic differentiation of U937 cells. In addition, basal prostanoid secretion does not measurably contribute to monocytic differentiation.

Modulation of the expression of early genes by polyunsaturated fatty acids.

Expression of early genes is a characteristic immediate cellular response to mitogenic or inflammatory stimulation. Various second messenger systems have been found to transduce the signal from the plasma membrane to the nucleus. Recent observations indicate that in addition to well characterized second messenger systems, polyunsaturated fatty acids, especially the n-6 fatty acid arachidonic acid and its endogenously produced metabolites affect the expression of early genes in different cell types. At least in fibroblasts, the stimulatory effect of arachidonic acid can be antagonized by n-3 polyunsaturated fatty acids. Further identification of the mechanisms through which polyunsaturated fatty acids modulate early gene expression and regulate subsequent cellular responses, like cell growth, may help to define novel concepts in the management of cardiovascular diseases.

Differential effects of n-6 and n-3 polyunsaturated fatty acids on cell growth and early gene expression in Swiss 3T3 fibroblasts.

Dietary n-3 polyunsaturated fatty acids (PUFAs) have been found to reduce accelerated cell growth. To study the underlying molecular mechanisms, we evaluated the effects of the n-3 PUFAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) compared with the n-6 PUFA arachidonic acid (AA) on cell growth and early gene mRNA accumulation in Swiss 3T3 fibroblasts. AA significantly increased cell numbers and incorporation of [3H]-thymidine compared with cells treated with EPA and DHA, which did not stimulate cell growth. In contrast to AA and parallel to its effect on cell growth, EPA and DHA did not lead to a pronounced increase in Egr-1 and c-fosmRNA levels. When they were incubated together with AA, both DHA and EPA reduced AA-induced Egr-1 and c-fosmRNA accumulation and incorporation of [3H]-thymidine. We have recently shown that AA strongly increases Egr-1 and c-fosmRNA accumulation 3T3 fibroblasts through its metabolism to prostaglandin E2 (PGE2) and its subsequent activation of protein kinase C (Danesch et al., 1994, J. Biol. Chem., 269:27258-27263). Consistent with the notion that increased PGE2 formation is required for the AA-induced early gene mRNA accumulation, EPA and DHA reduced PGE2 formation from exogenous [14C]-AA by more than 60%, but they did not decrease mRNA levels following stimulation with PGE2. We suggest that, in 3T3 fibroblasts, EPA and DHA antagonize AA-induced early gene mRNA accumulation and cell growth by reducing PGE2 formation.

Linoleic acid esterified in low density lipoprotein serves as substrate for increased arachidonic acid synthesis in differentiating monocytic cells.

The cellular metabolism of albumin- and lipoprotein-bound 18:2(n - 6) during monocytic differentiation was examined in the human premonocytic U937 and Mono Mac 6 cells. Differentiation for 72 h of U937 cells with retinoic acid (RA, 1 microM) or 1,25-(OH)2-vitamin D3 (1,25-D3, 10 nM) and of Mono Mac 6 cells with RA (1 microM) or lipopolysaccharide (LPS, 10 ng/ml) increased the desaturation and elongation of [1-14C]18:2(n - 6) to [1-14C]20:4(n - 6). In undifferentiated U937 and Mono Mac 6 cells, incubations with human LDL (100 micrograms/ml, 18 h) resulted in a 2.5-fold increase in 18:2(n - 6) levels in the cellular phospholipids. Differentiation of U937 cells with RA or or of Mono Mac 6 cells with LPS prior to LDL addition. Significantly reduced 18:2(n - 6) and elevated 20:4(n - 6) levels in cellular phospholipids. This increase in 20:4(n - 6) was likely not due to an increased incorporation of preformed 20:4(n - 6) esterified in LDL, as the receptor-specific degradation of [125I]LDL was reduced in both the RA-treated U937 and LPS-treated Mono Mac 6 cells. In U937 cells incubated with [1-14C]18:2(n - 6), the synthesis of TXB2, PGE2 and HHT could be detected after differentiation with RA. suggesting the availability of [1-14C]20:4(n - 6), derived from [1-14C]18:2(n - 6), for cyclooxygenase metabolism. Our results show that the conversion of 18:2(n - 6) to 20:4(n - 6) increases during monocyte differentiation. The 18:2(n - 6) supplied to the cells via the receptor-mediated uptake of LDL was utilized as substrate for the increased 20:4(n - 6) synthesis.

Arachidonic acid increases activation of NADPH oxidase in monocytic U937 cells by accelerated translocation of p47-phox and co-stimulation of protein kinase C.

Arachidonic acid (AA) has been implicated as an important amphiphilic co-factor in the activation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in neutrophils and reconstituted cell-free systems. To assess the role of AA in the activation of O2- generation in monocytic cells, we studied pre-monocytic U937 cells differentiated with 1,25-(OH)2-vitamin D3 plus interferon-gamma (IFN-gamma). AA dose-dependently enhanced phorbol myristate acetate (PMA)-stimulated O2- generation, with a maximum increase of 4,5-fold, through: (1) a more than 50% reduction of the lag-phase, defined as the time between addition of PMA and detection of O2-; and (2) a more than 60% increase in the constant rate of O2- generation. Reduction of the lag phase was associated with increased protein kinase C (PKC)-independent translocation of the cytosolic subunit of NADPH oxidase p47-phox to the cell membrane, whereas increased generation of O2- correlated with enhanced activation of PKC. The data indicate that AA increases activation of NADPH oxidase by accelerating its assembly and by co-stimulating PKC in monocytic U937 cells.

Effects of different polyunsaturated fatty acids on growth-related early gene expression and cell growth.

Effects of polyunsaturated fatty acids on expression of early-response genes c-fos and Egr-1 and induction of cell growth were assessed in Swiss 3T3 fibroblasts. Stimulation with arachidonic acid increased mRNA levels of c-fos and Egr-1. This effect was inhibited by preincubation with cyclooxygenase inhibitors and restored by addition of prostaglandin E2 (PGE2), the predominant eicosanoid produced in Swiss 3T3 fibroblasts. Further signaling of PGE2, was mediated by a protein kinase C-dependent pathway, since downregulation, or inhibition, of protein kinase C reduced increases in mRNA levels. Parallel to the stimulatory effects on mRNA levels, AA and PGE2 also increased cell growth, as determined by uptake of [3H]-thymidine. In contrast to arachidonic acid, n-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) did not increase c-fos and Egr-1 mRNA levels or cell growth. Furthermore, preliminary data indicate that EPA and DHA even reduce the stimulatory effect of AA, which is associated with reduced formation of PGE2. In conclusion, our data indicate that AA increases expression of growth-related early genes c-fos and Egr-1 Swiss 3T3 fibroblasts by its conversion to PGE2 and subsequent activation of protein kinase C, whereas n-3 fatty acids do not activate this signaling cascade.

[Nutrition in prevention of coronary heart disease]. / Ernährung in der Prävention der koronaren Herzkrankheit.

Clinical as well as basic research in the field of atherogenesis indicates that the progression of this disease process can be slowed down or even reversed. It is well established that nutrition plays an important role in the prevention and treatment of the classical atherogenic risk factors such as obesity, diabetes mellitus and hyperlipidemia. In addition, some nutrients such as the polyunsaturated n-3-fatty-acids or antioxidative vitamins can intervene directly by influencing one or more steps of the atherogenetic and/or thrombogenetic process. A comprehensive understanding of the pathogenesis of this disease as well as of the mechanisms of nutrient action are essential to the planning of successful nutritional prevention strategies. Because most nutrients influence mainly the slow and long-standing development of the atherosclerotic lesion, their inclusion in primary nutritional prevention should be started at an early age. Few nutrients such as the n-3 fatty acids, which also reduce the thrombogenetic risk factors, have demonstrated some success in the secondary prevention of CHD. Given the complexity with which nutrients intervene in the atherosclerotic process and their interactions with each other, nutritional prevention strategies should be based on well-grounded dietary modifications rather than supplementation with individual nutrients.

Effects of dietary fish oil on ventricular premature complexes.

For ethical and practical reasons, in this study the antiarrhythmic potential of fish oil was evaluated in patients free from complex ventricular arrhythmias and severe heart failure. Although subjects without overt structural heart disease had ventricular arrhythmias that were not associated with an increased risk for sudden cardiac or coronary death, recent data suggest that frequent VPCs in patients similar to our study population may reflect subclinical cardiac disease amenable to the multiple beneficial actions of n-3 fatty acids. The potential and safety of fish oil as a treatment for more complex cardiac arrhythmias or arrhythmias in higher risk patients with more severe heart disease deserve further study.

Cooperative effects of interferon-gamma on the induction of NADPH oxidase by retinoic acid or 1,25(OH)2-vitamin D3 in monocytic U937 cells.

The effect of retinoic acid (RA), 1,25-dihydroxyvitamin D3 (1,25-D3) or human recombinant interferon-gamma (IFN-gamma) on the induction of NADPH oxidase was studied in premonocytic U937 cells. Differentiation with the combination of either RA (1 microM) or 1,25-D3 (10 nM) with IFN-gamma (100 IU/ml) induced NADPH oxidase activity as demonstrated by increased superoxide anion (O2-) generation in response to stimulation with phorbol myristate acetate (PMA, 100 nM). Induction of NADPH oxidase activity was preceded by increases in mRNA levels of p47-phox, p67-phox and gp91-phox, which encode three subunits of the enzyme, and immunoblot analysis of the p47-phox and p67-phox proteins revealed that the increases in mRNA levels were equally reflected by increases in protein levels. In contrast, RA, 1,25-D3 or IFN-gamma alone did not induce NADPH oxidase activity which correlated with their failure to increase p67-phox and gp91-phox mRNA levels. The mRNA of p21 rac1, a GTP-binding protein that regulates NADPH oxidase activity in macrophages, was constitutively expressed in undifferentiated cells and was not affected by differentiation. These data indicate that induction of a functional NADPH oxidase in premonocytic U937 cells requires the cooperative actions of IFN-gamma plus RA or 1,25-D3 and is reflected in the increased expression of p67-phox and gp91-phox.

Differential effects of polyunsaturated fatty acids on cell growth and differentiation of premonocytic U937 cells.

The effect of long chain polyunsaturated fatty acids (PUFA) on cell growth and differentiation was assessed in human premonocytic U937 cells. Addition of either 10 microM arachidonic acid (AA, 20:4n-6), eicosapentaenoic acid (EPA, 20:5n-3) or docosahexaenoic acid (DHA, 22:6n-3) resulted in the rapid incorporation of these fatty acids into cellular phospholipids. Their uptake was greatest in the first 2 h. AA and EPA reached steady-state levels after 8 h, while levels of DHA increased steadily over 72 h. In parallel, fatty acid metabolites derived from AA and EPA, 22:4n-6, 22:5n-6 and 22:5n-3, 22:6n-3, respectively, increased continuously indicating an active fatty acid elongation and desaturation. The effects of PUFA on monocytic differentiation were examined in cells which had been enriched with AA, EPA or DHA for 8 h and subsequently treated with retinoic acid (RA), 1,25-(OH)2-vitamin D3 (1,25-D3), interferon-gamma (IFN-gamma) or their combinations for 72 h. Growth of differentiating or non-differentiating U937 cells was not affected by enrichment with PUFA. However, in cells differentiated with 1,25-D3 plus IFN-gamma, prior enrichment with all three PUFA slightly but significantly (P < 0.05) increased the expression of the monocytic surface antigens CD11b and CD14 and generation of superoxide anion. The data indicate that although n-6 and n-3 PUFA are rapidly incorporated into phospholipids, they do not affect cell growth. However, enrichment with PUFA increases monocytic differentiation of U937 cells when induced most effectively with 1,25-D3 plus IFN-gamma.