Frans A. Kuypers

Mammalian Long-Chain Acyl-CoA Synthetases

Acyl-CoA synthetase enzymes are essential for de novo lipid synthesis, fatty acid catabolism, and remodeling of membranes. Activation of fatty acids requires a two-step reaction catalyzed by these enzymes. In the first step, an acyl-AMP intermediate is formed from ATP. AMP is then exchanged with CoA to produce the activated acyl-CoA. The release of AMP in this reaction defines the superfamily of AMP-forming enzymes. The length of the carbon chain of the fatty acid species defines the substrate specificity for the different acyl-CoA synthetases (ACS). On this basis, five sub-families of ACS have been characterized. The purpose of this review is to report on the large family of mammalian long-chain acyl-CoA synthetases (ACSL), which activate fatty acids with chain lengths of 12 to 20 carbon atoms. Five genes and several isoforms generated by alternative splicing have been identified and limited information is available on their localization. The structure of these membrane proteins has not been solved for the mammalian ACSLs but homology to a bacterial form, whose structure has been determined, points at specific structural features that are important for these enzymes across species. The bacterial form acts as a dimer and has a conserved short motif, called the fatty acid Gate domain, that seems to determine substrate specificity. We will discuss the characterization and identification of the different spliced isoforms, draw attention to the inconsistencies and errors in their annotations, and their cellular localizations. These membrane proteins act on membrane-bound substrates probably as homo- and as heterodimer complexes but have often been expressed as single recombinant isoforms, apparently purified as monomers and tested in Triton X-100 micelles. We will argue that such studies have failed to provide an accurate assessment of the activity and of the distinct function of these enzymes in mammalian cells.

Chlorohydrin formation from unsaturated fatty acids reacted with hypochlorous acid

Stimulated neutrophils produce hypochlorous acid (HOCl) via the myeloperoxidase-catalyzed reaction of hydrogen peroxide with chloride. The reactions of HOCl with oleic, linoleic, and arachidonic acids both as free fatty acids or bound in phosphatidylcholine have been studied. The products were identified by gas chromatography-mass spectrometry of the methylated and trimethylsilylated derivatives. Oleic acid was converted to the two 9,10-chlorohydrin isomers in near stoichiometric yield. Linoleic acid, at low HOCl:fatty acid ratios, yielded predominantly a mixture of the four possible monochlorohydrin isomers. Bischlorohydrins were also formed, in increasing amounts at higher HOCl concentrations. Arachidonic acid gave a complex mixture of mono- and bischlorohydrins, the relative proportions depending on the amount of HOCl added. Linoleic acid appears to be slightly more reactive than oleic acid with HOCl. Reactions of oleic and linoleic acids with myeloperoxidase, hydrogen peroxide, and chloride gave chlorohydrin products identical to those with HOCl. Lipid chlorohydrins have received little attention as products of reactions of neutrophil oxidants. They are more polar than the parent fatty acids, and if formed in cell membranes could cause disruption to membrane structure. Since cellular targets for HOCl appear to be membrane constituents, chlorohydrin formation from unsaturated lipids could be significant in neutrophil-mediated cytotoxicity.

Patterns of arginine and nitric oxide in patients with sickle cell disease with vaso-occlusive crisis and acute chest syndrome.

Purpose Our objective was to evaluate l-arginine and nitric oxide metabolite (NOx) levels in children with sickle cell disease (SCD) at steady-state and during vaso-occlusive crisis (VOC). Because alterations in nitric oxide production may have an important role in the pathophysiology of SCD, our second aim was to determine if a relationship exists between these levels and vaso-occlusive crisis (VOC). Patients and Methods Plasma l-arginine and serum NOx levels were examined in 36 patients with SCD with 39 episodes of VOC and 10 children with SCD at steady-state. Daily levels were obtained in children requiring hospitalization. Results Steady-state l-arginine levels were normal in children with SCD. l-arginine levels were low, however, in children with VOC (37.4 ± 2.7 vs. 53.6 ± 4.6 &mgr;mol/L;P = 0.008) but returned to baseline during hospitalization. In contrast, NOx levels were normal at presentation but decreased during hospitalization for both patients with VOC and patients with acute chest syndrome (ACS) (21.1 ± 2.0, 17.4 ± 2.4, and 12.3 ± 1.6 &mgr;mol/L, respectively;P < 0.05). In the patients with VOC who had ACS develop, l-arginine decreased to the lowest levels at the time of the ACS diagnosis, correlating with decreasing NOx levels. Conclusion These data suggest that there may be a relationship between the l-arginine–nitric oxide pathway and vaso-occlusion in SCD. Low arginine levels during VOC could reflect a state of acute substrate depletion that results in a decrease in nitric oxide production.

VEGF modulates erythropoiesis through regulation of adult hepatic erythropoietin synthesis

Vascular endothelial growth factor (VEGF) exerts crucial functions during pathological angiogenesis and normal physiology. We observed increased hematocrit (60–75%) after high-grade inhibition of VEGF by diverse methods, including adenoviral expression of soluble VEGF receptor (VEGFR) ectodomains, recombinant VEGF Trap protein and the VEGFR2-selective antibody DC101. Increased production of red blood cells (erythrocytosis) occurred in both mouse and primate models, and was associated with near-complete neutralization of VEGF corneal micropocket angiogenesis. High-grade inhibition of VEGF induced hepatic synthesis of erythropoietin (Epo, encoded by Epo) >40-fold through a HIF-1α–independent mechanism, in parallel with suppression of renal Epo mRNA. Studies using hepatocyte-specific deletion of the Vegfa gene and hepatocyte–endothelial cell cocultures indicated that blockade of VEGF induced hepatic Epo by interfering with homeostatic VEGFR2-dependent paracrine signaling involving interactions between hepatocytes and endothelial cells. These data indicate that VEGF is a previously unsuspected negative regulator of hepatic Epo synthesis and erythropoiesis and suggest that levels of Epo and erythrocytosis could represent noninvasive surrogate markers for stringent blockade of VEGF in vivo.NOTE: In the version of this article initially published, the name of one of the authors, Nihar R. Nayak, was misspelled as Nihar R. Niyak. The error has been corrected in the HTML and PDF versions of the article.

Kinetics and site specificity of hydroperoxide-induced oxidative damage in red blood cells

To provide a detailed description of the time course and the site specificity of hydroperoxide-induced oxidative stress in red blood cells (RBCs), we have characterized the action of a membrane-soluble (cumene hydroperoxide [cumOOH]) and a water-soluble (hydrogen peroxide [H2O2]) oxidant. The fluorescent polyunsaturated fatty acid (PUFA) parinaric acid (PnA) was used to probe peroxidation processes in the membrane, and oxidation of hemoglobin (Hb) was measured spectrophotometrically as an indicator of cytosolic oxidative stress. The observed degradation patterns of PnA and Hb were clearly distinct for each oxidant. At comparable oxidant concentrations, the cumulative oxidative stress on the RBC membrane was always much higher with cumOOH, whereas much more Hb oxidation was measured with H2O2. The kinetics of Hb oxidation as well as the nature of the products formed were different for each oxidant. The main Hb oxidation product generated gradually by cumOOH was metHb, whereas H2O2 caused the rapid formation of ferrylHb. CumOOH caused more oxidation of endogenous PUFAs and of vitamin E, while the degradation pattern of vitamin E closely resembled that of PnA. At high oxidant concentrations, extensive cell lysis was observed after prolonged incubation. Butylated hydroxytoluene (BHT) completely prevented oxidation of endogenous PUFAs but did not completely prevent hemolysis, indicating that factors other than lipid peroxidation are also important in causing lysis of RBCs. The action of cumOOH is characterized by a gradual reaction with Hb, generating radicals that produce an oxidative stress primarily directed at the membrane, which increases in time to a maximum and then gradually decreases. In contrast, H2O2 crosses the RBC membrane and reacts rapidly with Hb, generating a very reactive radical species that has Hb, not the membrane, as a prime target. H2O2-induced oxidative stress is at a maximum immediately after addition of this oxidant and decreases rapidly to zero in a short time. These findings provide further insight into the mode of action of hydroperoxides and the mechanism of compartmentalization of RBC oxidative damage.

Atherosclerosis and plasma and liver lipids in nine inbred strains of mice.

Nine inbred strains of mice, which are progenitors of recombinant inbred sets, were evaluated for aortic lesion formation and plasma and liver lipid levels. This survey was done to determine if a semi-synthetic high-fat diet could elicit the same extent of diet-induced atherosclerosis as that observed in mice fed a natural ingredient highfat diet and to discover strain-specific plasma and liver lipid variants for future genetic characterization. Evaluation of aortic lesions after 18 wk of diet consumption showed that strains C57BL/6J, C57L/J, SWR/J and SM/J were susceptible to atherosclerosis and that A/J, AKR/J, C3H/HeJ, DBA/2J and SJL/J were relatively resistant. High-density lipoprotein cholesterol (HDL-C) levels were negatively correlated to lesion formation. Susceptible strains had decreased HDL-C levels when switched from chow to the semi-synthetic high-fat, high cholesterol diet, whereas resistant strains either showed no change or a slight increase in HDL-C levels. The exception to this pattern was found in SM mice, which were susceptible to aortic lesion formation but maintained the same HDL-C level on both chow and high-fat diets. HDL size differed among the strains, and levels of plasma apolipoprotein A-I and A-II correlated with HDL-C levels. Liver damage was not correlated to HDL-C levels or to susceptibility to atherosclerosis. Mice from strain A, which are resistant to atherosclerosis, had evidence of liver damage as observed by elevated levels of plasma alanine aminotransferase activity, by liver histology, by increased liver weight and by exceptionally high hepatic cholesterol content. For all strains, the levels of liver cholesterol and triglycerides were inversely correlated with each other; phospholipids did not vary greatly among strains. No remarkable differences in hepatic fatty acid profile were noted among the strains fed the atherogenic diet, but the fatty acid profile did differ considerably from that found in the diet itself.

Parinaric acid as a sensitive fluorescent probe for the determination of lipid peroxidation

The decrease in fluorescence of conjugated polyenic acyl chains is used as a sensitive assay for lipid peroxidation. The fatty acid cis-trans-trans-cis-9,11,13, 15-octadecatetraenoic acid (cis-parinaric acid) is introduced into liposomal membranes as free fatty acid or, by using the PC specific transfer protein from bovine liver, as 1-palmitoyl-2-cis-parinaroyl-sn-glycero-3-phosphocholine. The peroxidation process as monitored by the decrease in fluorescence intensity is compared with other peroxidation assay systems. Applications of the new assay system are discussed.

Hemolysis-associated pulmonary hypertension in thalassemia.

Abstract: Accumulating evidence supports the existence of a condition involving hemolysis‐associated pulmonary hypertension (PHT). Hemolysis‐induced release of cell‐free hemoglobin and red blood cell arginase, resulting in impaired nitric oxide bioavailability, endothelial dysfunction, and PHT, has been reported in sickle cell disease. Since thalassemia is also a condition of chronic hemolysis, these patients are at risk. The data demonstrate that hemolysis‐induced dysregulation of arginine metabolism and PHT also occurs in thalassemia. Erythrocyte release of arginase during hemolysis contributes to the development of PHT. Therapies that maximize arginine and nitric oxide bioavailability may benefit patients with thalassemia.

Hydroxyurea and Arginine Therapy: Impact on Nitric Oxide Production in Sickle Cell Disease

Purpose: Recent data suggest that hydroxyurea (HU) increases the production of nitric oxide (NO), a potent vasodilator. NO is normally metabolized from l‐Arginine (Arg). However, in vitro and animal experiments suggest that HU is the NO donor itself. In contrast, a recent study indicates that nitric oxide synthase (NOS) may play a role. Since adults with sickle cell disease (SCD) are Arg‐deficient, Arg availability may limit the ability of HU to maximally impact NO production if an NOS mechanism is involved. The authors have previously shown that Arg supplementation alone induces a paradoxical decrease in NO metabolite (NOx) production. Methods: The authors studied the effects of HU and Arg supplementation on NOx production. HU alone or HU + Arg was administered to patients with SCD at steady state, and sequential levels of Arg, serum NOx and exhaled NO were followed over 4 hours. Results: After HU + Arg, all patients demonstrated a significant increase in serum NOx production within 2 hours. When the same patients were treated with HU alone (5.1 ± 2 &mgr;mol/L), a mixed response occurred. NOx levels increased in four patients and decreased in one patient (‐23.3 &mgr;mol/L). Conclusions: While Arg alone does not increase serum NOx production in SCD patients at steady state, it does when given together with HU. Hence, co‐administration of Arg with HU may augment the NOx response in SCD and improve utilization of Arg in patients at steady state.

Hydrolysis of Phosphatidylserine-exposing Red Blood Cells by Secretory Phospholipase A2 Generates Lysophosphatidic Acid and Results in Vascular Dysfunction

Secretory phospholipase A2 (sPLA2) type IIa, elevated in inflammation, breaks down membrane phospholipids and generates arachidonic acid. We hypothesized that sPLA2 will hydrolyze red blood cells that expose phosphatidylserine (PS) and generate lysophosphatidic acid (LPA) from phosphatidic acid that is elevated in PS-exposing red blood cells. In turn, LPA, a powerful lipid mediator, could affect vascular endothelial cell function. Although normal red blood cells were not affected by sPLA2, at levels of sPLA2 observed under inflammatory conditions (100 ng/ml) PS-exposing red blood cells hemolyzed and generated LPA (1.2 nm/108 RBC). When endothelial cell monolayers were incubated in vitro with LPA, a loss of confluence was noted. Moreover, a dose-dependent increase in hydraulic conductivity was identified in rat mesenteric venules in vivo with 5 μm LPA, and the combination of PS-exposing red blood cells with PLA2 caused a similar increase in permeability. In the presence of N-palmitoyl l-serine phosphoric acid, a competitive inhibitor for the endothelial LPA receptor, loss of confluence in vitro and the hydraulic permeability caused by 5 μm LPA in vivo were abolished. The present study demonstrates that increased sPLA2 activity in inflammation in the presence of cells that have lost their membrane phospholipid asymmetry can lead to LPA-mediated endothelial dysfunction and loss of vascular integrity.