Herman J. M. van Rijn

Nitric Oxide Production Is Reduced in Patients With Chronic Renal Failure

In patients with chronic renal failure (CRF), atherosclerosis is a major cause of cardiovascular morbidity and mortality. Generally, atherosclerosis has been associated with a reduced bioavailability of nitric oxide (NO). Experimental studies have indicated the presence of enhanced NO degradation by reactive oxygen species as well as decreased NO production as possible causes for this reduced NO bioavailability. So far, the question whether or not NO production is impaired in patients with CRF has never been investigated. Therefore, we measured whole body NO production in 7 patients with CRF, and in 7 matched healthy subjects. To assess the relative importance of a dysfunction of NO synthase (NOS), we compared the NO production of these patients to that of 2 other groups known to have endothelial dysfunction, ie, 7 patients with familial hypercholesterolemia (FH) who did not yet have signs of clinical cardiovascular disease (all nonsmokers), and 5 cigarette smokers. These groups were also compared with 7 nonsmoking, age-matched healthy subjects. Whole body NO production, determined as in vivo arginine-to-citrulline conversion, was assessed by giving an intravenous infusion of [15N2]-arginine as a substrate for NOS and measuring isotopic plasma enrichment of [15N]-citrulline by LC-MS. NO production in the CRF patients (0.13+/-0.02 micromol. kg-1. h-1) was significantly lower (P<0.05) than in the corresponding control group (0.23+/-0.09 micromol. kg-1. h-1). NO production also tended to be lower in the FH patients (0.16+/-0.04 micromol. kg-1. h-1), but the difference with the corresponding control group did not reach significance (0.22+/-0.06 micromol. kg-1. h-1). In the group of smokers, NO production was similar to that in nonsmokers (0. 22+/-0.09 micromol. kg-1. h-1). In conclusion, it is demonstrated for the first time that basal whole body NO production is reduced in patients with CRF. This finding implies that therapeutic interventions to endothelial dysfunction in these patients should be primarily directed toward improvement of NO production. The finding of only a tendency toward reduction of NO production in patients with FH and the absence of a reduction in cigarette smokers suggests that other mechanisms such as enhanced NO degradation may be involved in the decrease of NO bioavailability in these groups.

Low-Density Lipoprotein Enhances Platelet Secretion Via Integrin-αIIbβ3–Mediated Signaling

Abstract —LDL is known to increase the sensitivity of human platelets for agonists and to induce aggregation and secretion independently at high concentrations, but its mechanism of action is largely obscure. To clarify how LDL increases platelet sensitivity, cells were incubated in lipoprotein-poor plasma and treated with collagen at a concentration that induced ≈20% secretion of 14C-serotonin. Preincubation with LDL (30 minutes at 37°C) enhanced secretion in a dose-dependent manner to 60±14% at a concentration of 2 g LDL protein/L. Similar stimulation by LDL was seen when secretion was induced by the thrombin receptor–activating peptide. This enhancement was strongly reduced (1) in the presence of monoclonal antibody PAC1 against activated αIIbβ3, a polyclonal antibody against αIIb, and in the presence of the fibrinogen peptides GRGDS and HHLGGAKQAGDV; (2) in αIIbβ3-deficient platelets; and (3) after dissociation of αIIbβ3. In contrast, binding of 125I-LDL to normal platelets in the presence of PAC1, anti-αIIb, GRGDS, and HHLGGAKQAGDV, and to αIIbβ3-deficient platelets was normal. LDL increased the surface expression of fibrinogen in lipoprotein-poor plasma and fibrinogen-free medium, suggesting that extracellular and granular fibrinogen bind to αIIbβ3 after platelet-LDL interaction. Platelets deficient in fibrinogen (<0.5% of normal) or von Willebrand Factor (<1% of normal) but containing normal amounts of other ligands for αIIbβ3 preserved responsiveness to LDL, indicating that occupancy of αIIbβ3 was not restricted to fibrinogen. Inhibition of protein kinase C (bisindolylmaleimide) diminished fibrinogen binding and sensitization by LDL; inhibition of tyrosine kinases (herbimycin A) left fibrinogen binding unchanged but diminished sensitization by LDL. We conclude that an increased concentration of LDL, such as observed in homozygous familial hypercholesterolemia, sensitizes platelets to stimulation by collagen and thrombin receptor–activating peptide via ligand-induced outside-in signaling through integrin-αIIbβ3.

Ischemia modified albumin in normal pregnancy and preeclampsia.

Objective. Ischemia-modified albumin (IMA) has emerged as a new biomarker of myocardial ischemia. Currently, no information is available on maternal IMA levels during normal and complicated pregnancy. Preeclampsia is associated with ischemia and increased formation of free radicals in the placenta. We therefore hypothesized that production of IMA may occur in women with preeclampsia. Methods. Serum IMA and albumin concentrations were assessed in 12 patients with preeclampsia, 12 normal pregnant controls, and 12 nonpregnant controls. IMA levels were compared between groups and corrected for albumin by multivariate regression analysis. Results. Mean IMA levels were elevated in normal pregnant controls (107.3 U/mL; 95% CI, 102.5 to 112.01), compared with nonpregnant controls (94.5 U/mL; CI, 89.4 to 99.6; p = 0.015). In patients with preeclampsia, IMA levels were similar to those in normal pregnant controls (109.7 U/mL; CI, 102.2 to 117.2; p = 0.65). Also, no difference in IMA levels was observed between women with preeclampsia who delivered small-for-gestational-age (SGA) infants (99.0 U/mL; CI, 87.9 to 110.1; p = 0.13) and women with preeclampsia but without SGA. Conclusion. Serum IMA, which has been advocated as a clinical marker of cardiac ischemia, appears to be elevated during normal pregnancy. We found no significant relationship between IMA levels and preeclampsia, in women with or without SGA infants.

NO activity in familial combined hyperlipidemia: potential role of cholesterol remnants

OBJECTIVE Patients with familial combined hyperlipidemia (FCH) have an increased cardiovascular mortality despite only moderate elevations of LDL-cholesterol. Since endothelial NO release is intimately involved in the anti-atherosclerotic effects of the endothelium, we studied the effect of short-term lipid-lowering therapy on NO-mediated vasodilatation in patients with FCH. In view of only moderate LDL elevations, we evaluated whether alterations in other lipid fractions upon therapy correlated to changes in NO-mediated vasodilatation. METHODS NO activity was assessed by serotonin-induced, nitric oxide-mediated increase in forearm blood flow (FBF). Measurements were performed 2 weeks off and 4 weeks on lipid-lowering therapy in 12 FCH patients using forearm venous occlusion plethysmography. Control experiments were performed in 12 healthy subjects. RESULTS Serotonin-induced vasodilatation was impaired in FCH patients (FBF (unit ml/100 ml forearm tissue/min) from 3.0 (0.3) to 4.8 (0.4)) compared to controls (FBF from 2.9 (0.3) to 6.5 (0.6); p < 0.05 vs. FCH). FBF response to serotonin improved significantly upon lipid-lowering therapy (from 3.0 (0.3) to 5.7 (0.5); p < 0.05 treated vs. untreated). The level of improvement in endothelial function was significantly correlated to the absolute reduction of intermediate density lipoproteins upon lipid-lowering therapy (r = -0.64; p < 0.05), whereas it did not correlate to changes in VLDL- or LDL-cholesterol, nor to Lp(a). CONCLUSION Patients with familial combined hyperlipidemia have impaired NO-mediated vasodilatation, that responds rapidly to lipid lowering medication, and may be related to changes in intermediate density lipoproteins.

Effect of Oxidation on the Platelet-Activating Properties of Low-Density Lipoprotein

Objective—Because of the large variation in oxidizing procedures and susceptibility to oxidation of low-density lipoprotein (LDL) and the lack in quantification of LDL oxidation, the role of oxidation in LDL–platelet contact has remained elusive. This study aims to compare platelet activation by native LDL (nLDL) and oxidized LDL (oxLDL). Methods and Results—After isolation, nLDL was dialyzed against FeSO4 to obtain LDL oxidized to well-defined extents varying between 0% and >60%. The oxLDL preparations were characterized with respect to their platelet-activating properties. An increase in LDL oxidation enhances platelet activation via 2 independent pathways, 1 signaling via p38MAPK phosphorylation and 1 via Ca2+ mobilization. Between 0% and 15% oxidation, the p38MAPK route enhances fibrinogen binding induced by thrombin receptor (PAR-1)-activating peptide (TRAP), and signaling via Ca2+ is absent. At >30% oxidation, p38MAPK signaling increases further and is accompanied by Ca2+ mobilization and platelet aggregation in the absence of a second agonist. Despite the increase in p38MAPK signaling, synergism with TRAP disappears and oxLDL becomes an inhibitor of fibrinogen binding. Inhibition is accompanied by binding of oxLDL to the scavenger receptor CD36, which is associated with the fibrinogen receptor, &agr;IIb&bgr;3. Conclusion—At >30% oxidation, LDL interferes with ligand binding to integrin &agr;IIb&bgr;3, thereby attenuating platelet functions.

Platelet endothelial cell adhesion molecule-1 (PECAM-1) inhibits low density lipoprotein-induced signaling in platelets

At physiological concentrations, low density lipoprotein (LDL) increases the sensitivity of platelets to aggregation- and secretion-inducing agents without acting as an independent activator of platelet functions. LDL sensitizes platelets by inducing a transient activation of p38MAPK, a Ser/Thr kinase that is activated by the simultaneous phosphorylation of Thr180 and Tyr182 and is an upstream regulator of cytosolic phospholipase A2 (cPLA2). A similar transient phosphorylation of p38MAPK is induced by a peptide mimicking amino acids 3359–3369 in apoB100 called the B-site. Here we report that the transient nature of p38MAPK activation is caused by platelet endothelial cell adhesion molecule 1 (PECAM-1), a receptor with an immunoreceptor tyrosine-based inhibitory motif. PECAM-1 activation by cross-linking induces tyrosine phosphorylation of PECAM-1 and a fall in phosphorylated p38MAPK and cPLA2. Interestingly, LDL and the B-site peptide also induce tyrosine phosphorylation of PECAM-1, and studies with immunoprecipitates indicate the involvement of c-Src. Inhibition of the Ser/Thr phosphatases PP1/PP2A (okadaic acid) makes the transient p38MAPK activation by LDL and the B-site peptide persistent. Inhibition of Tyr-phosphatases (vanadate) increases Tyr-phosphorylated PECAM-1 and blocks the activation of p38MAPK. Together, these findings suggest that, following a first phase in which LDL, through its B-site, phosphorylates and thereby activates p38MAPK, a second phase is initiated in which LDL activates PECAM-1 and induces dephosphorylation of p38MAPK via activation of the Ser/Thr phosphatases PP1/PP2A.

Low Density Lipoprotein Phosphorylates the Focal Adhesion-associated Kinase p125FAK in Human Platelets Independent of Integrin αIIbβ3

Low density lipoprotein (LDL) is known to sensitize platelets to agonists via integrin mediated outside-in signaling (Hackeng, C. M., Huigsloot, M., Pladet, M. W., Nieuwenhuis, H. K., Rijn, H. J. M. v., and Akkerman, J. W. N. (1999) Arterioscler. Thromb. Vasc. Biol., in press). As outside in signaling is associated with phosphorylation of p125FAK, the effect of LDL on p125FAK phosphorylation in platelets was investigated. LDL induced p125FAK phosphorylation in a dose- and time- dependent manner. The phosphorylation was independent of ligand binding to integrin αIIbβ3 and aggregation, such in contrast to α-thrombin-induced p125FAK phosphorylation, that critically depended on platelet aggregation. Platelets from patients with Glanzmann’s thrombastenia showed the same LDL- induced phos- phorylation of p125FAK as control platelets, whereas α-thrombin completely failed to phosphorylate the kinase in the patients platelets. LDL signaling to p125FAK was independent of integrin α2β1, the FcγRII receptor, and the lysophosphatidic acid receptor and not affected by inhibitors of cyclooxygenase, protein kinase C, ERK1/2 or p38MAPK. Phosphorylation of p125FAK by LDL was strongly inhibited by cyclic AMP. These observations indicate that LDL is a unique platelet agonist, as it phosphorylates p125FAKin platelet suspensions, under unstirred conditions and independent of integrin αIIbβ3.

Site-specific phosphorylation of platelet focal adhesion kinase by low-density lipoprotein.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase implicated in signalling pathways mediated by integrins and G-protein-coupled receptors (GPCRs). Upon stimulation FAK is phosphorylated on six tyrosine residues. Here we report the site-specific phosphorylation by low-density lipoprotein (LDL), which is known to induce integrin-independent FAK phosphorylation, and compare this with the effect of thrombin, which phosphorylates FAK via integrin alphaIIbbeta3. Stimulation with LDL reveals (i) a major role for Tyr-925 phosphorylation which surpasses the phosphorylation of the other residues, including Tyr-397, in rate and extent, (ii) alphaIIbbeta3-independent phosphorylation of Tyr-925 and Tyr-397, and (iii) complex formation between FAK and the Src-kinase Fgr but not with c-Src. These patterns differ profoundly from those induced by thrombin. LDL-induced phosphorylation of Tyr-925 and Tyr-397 was inhibited by 60-75% by receptor-associated protein, an inhibitor of members of the LDL receptor family. Thus these findings reveal a novel mechanism of FAK phosphorylation by signalling cascades involving a member of the LDL receptor family.

Lysophosphatidic acid-independent platelet activation by low-density lipoprotein

Mildly oxidized low‐density lipoprotein activates platelets through lysophosphatidic acid (LPA). Hence, the platelet‐activating properties attributed to native low‐density lipoprotein (nLDL) might be caused by LPA contamination. We show that nLDL enhances thrombin receptor‐activating peptide (TRAP)‐induced fibrinogen binding to αIIbβ3. The LPA receptor blocker N‐palmitoyl‐L‐serine‐phosphoric acid did not affect nLDL‐enhanced fibrinogen binding induced by TRAP, but reduced TRAP‐induced binding. cAMP and inhibitors of protein kinase C and Ca2+ rises completely blocked ligand binding by TRAP and nLDL/TRAP. Inhibitors of p38MAPK and ADP secretion interfered only partially. Blockade of Rho‐kinase increased ligand binding 2–3‐fold. We conclude that nLDL enhances TRAP‐induced fibrinogen binding independent of LPA.

The functional and clinical significance of the Met-->Thr substitution in Kringle IV type 10 of apolipoprotein(a).

Lipoprotein(a) [Lp(a)], an independent risk factor for the development of atherosclerosis, contains an apolipoprotein(a) [apo(a)] moiety covalently linked to a LDL moiety. Apo(a) is a glycoprotein homologous to plasminogen as it contains multiple repeats of a lysine binding domain resembling plasminogen kringle IV (K.IV). The multiple K.IV repeats can be differentiated in ten types that show a variation in their lysine binding capacity. Since K.IV type 10 shows the highest conservation of the amino acids postulated to form the lysine binding pocket, this kringle is suggested to be the main lysine binding site of apo(a). Recently, a T-->C polymorphism in the apo(a)-gene was reported, leading to a Met-->Thr substitution at amino acid position 66 of K.IV type 10, in the vicinity of the postulated lysine binding pocket. To investigate the significance of this substitution on some in vitro characteristics of Lp(a), the affinity for lysine-Sepharose and the binding affinity for limited plasmin digested des AA fibrin (Desafib-X) of the two subtypes was determined using plasma of donors homozygous for the polymorphism. These studies revealed a large heterogeneity in the binding characteristics, irrespective of the subtype. The comparison of the allele frequencies of this polymorphism in 155 patients having symptomatic atherosclerosis versus 153 normolipidemic controls revealed no significant differences. In conclusion, this study suggests that the presence of either a Met66 or a Thr66 residue in K.IV type 10 of apo(a) has no consequences for the binding characteristics of Lp(a) toward lysine-Sepharose or Desafib-X, nor is it associated with the presence of symptomatic atherosclerosis.