[Association between blood pressure during 12-28 weeks gestation and pre-eclampsia: predictive value of blood pressure trajectories constructed by latent class growth modeling].

Objective: To explore the associations between blood pressure trajectories during pregnancy and risk of future pre-eclampsia in a large cohort enrolling pregnant women at gestational age of ~12 weeks from community hospitals in Tianjin. Latent class growth modeling (LCGM) was used to model the blood pressure trajectories. Methods: This was a large prospective cohort study. The study enrolled pregnant women of ~12 weeks of gestation in 19 community hospitals in Tianjin from November 1, 2016 to May 30, 2018. We obtained related information during 5 antepartum examinations before gestational week 28, i.e., week 12, week 16, week 20, week 24 and week 28. LCGM was used to model longitudinal systolic (SBP) and diastolic blood pressure (DBP) trajectories. For the association study, the predictors were set as SBP and DBP trajectory membership (built separately), the outcome was defined as the occurrence of preeclampsia after 28 weeks of gestation. Results: A total of 5 809 cases with known pregnant outcomes were documented. After excluding 249 cases per exclusion criteria, 5 560 cases with singleton pregnancy were included for final analysis. There were 128 cases preeclampsia and 106 cases gestational hypertension in this cohort. Univariate logistic regression and multivariate logistic regression showed the higher baseline SBP level and DBP level were related with increased risk of preeclampsia. Four distinctive SBP trajectories and DBP trajectories from 12 weeks to 28 weeks of gestation were identified by LCGM. After controlling for potential confounders (baseline BMI, being primipara or not, white blood cell counts, hemoglobin level, platelet counts and alanine aminotransferase level), the OR for SBP latent classification trajectory_ 4 was 4.023 (95%CI: 2.368 to 6.835, P<0.001), and the OR for SBP latent classification trajectory_3 was 1.854 (95%CI: 1.223 to 2.811, P=0.004). Logistic regression showed that: using the DBP latent classification trajectory_1 as the reference group, the OR for DBP latent classification trajectory_4 was 4.100 (95%CI: 2.571 to 6.538, P<0.001), and 2.632 (95%CI: 1.570 to 4.414, P<0.001) for DBP latent classification trajectory_2. After controlling for potential confounders (baseline BMI, being primipara or not, white blood cell counts, hemoglobin level, platelet counts and alanine aminotransferase level), the OR for DBP_traj_4 was 2.527 (95%CI: 1.534 to 4.162, P<0.001), and the OR for DBP_traj_3 was 1.297 (95%CI: 0.790 to 2.128, P=0.303), and 2.238 (95%CI: 1.328 to 3.772, P=0.002) for DBP_traj_2. Therefore, BP trajectories from 12 weeks to 28 weeks identified by LCGM served as novel risk factors that independently associated with the occurrence of preeclampsia. Receiver operating characteristic (ROC) curve analysis showed incremental diagnostic performance by combing baseline blood pressure levels with blood pressure trajectories. Conclusion: By applying LCGM, we for the first time identified distinctive BP trajectories from gestational week 12 to 28, which can independently predict the development of preeclampsia after 28 weeks of gestation.

[Association between weight gain during the first half of pregnancy and the risk of hypertension disorder of pregnancy: a prospective cohort study].

Objective: To explore the association between weight gain during the first half of pregnancy and the risk of hypertension disorder of pregnancy (HDP). Methods: This prospective cohort study recruited singleton pregnant women in the first trimester from November 2016 to March 2019 at 19 community hospitals in Tianjin. According to pre-pregnancy body mass index (BMI), the cohort was divided into 3 groups: underweight(BMI<18.5 kg/m2), normal-weight(18.5-24.9 kg/m2), and overweight/obese(≥25.0 kg/m2). The basic information of the participants was gathered through questionnaires, and the height, weight, and blood pressure of the participants were measured along with routine pregnancy examinations. The rate of gestational weight gain (rGWG) in the 3 periods (0-13+6, 14+0-20+6, and 0-20+6 weeks) of the participants was calculated. To observe the occurrence of HDP, the participants were followed up to 42 days postpartum. Using a generalized linear model, the association between rGWG at the 3 periods during the first half of pregnancy and HDP after 20 weeks of gestation was evaluated. Results: A total of 9 805 pregnant women were finally included, with the age of (30.6±3.8) years old, 9 418 (96.1%) Han ethnicity, and 6 845 (69.8%) primipara. There were 1 184 (12.1%), 6 831 (69.7%) and 1 790 (18.3%) participants in the underweight, normal-weight, and overweight/obese groups. Five hundreds and eight pregnant women were diagnosed with HDP (5.2%). The incidences of HDP were 1.8% (21/1 184), 3.9% (269/6 831), and 12.2% (218/1 790), respectively, in underweight, normal-weight, and overweight/obese groups. Adjusted for age, pre-pregnancy BMI, primipara, and family history of hypertension, women in the entire cohort with rGWG ≥ 0.18 kg/week before 13+6 weeks of pregnancy had a 28% higher HDP risk than those with rGWG ≤ 0.00 kg/week (RR=1.28, 95%CI 1.04-1.55, P=0.015), and the risk of HDP was increased by 39% in the overweight/obese group (RR=1.39, 95%CI 1.04-1.85, P=0.026), while no correlation was found between rGWG and HDP in underweight and normal-weight pregnant women (P>0.05). Weight gain during 14+0-20+6 weeks of pregnancy in any group was not related to the risk of HDP (P>0.05).In the entire cohort, compared to rGWG ≤0.14 kg/week, rGWG≥0.28 kg/week prior to 20+6 weeks increased HDP risk by 36% (RR=1.36, 95%CI 1.11-1.67, P=0.003). Normal-weight pregnant women with rGWG≥0.29 kg/week faced a 46% higher risk of HDP than those with rGWG≤0.15 kg/week (RR=1.46, 95%CI 1.11-1.93, P=0.008).In the overweight/obese group, excessive weight gain before 20+6 weeks seemed to increased risk of HDP, but the difference was not statistically significant (RR=1.35,95%CI 0.99-1.85, P=0.059), while the connection was nonexistent in underweight women. Conclusions: Except for pre-pregnancy underweight women, excessive weight gain during the first half of pregnancy is associated with increased risk of HDP among pregnant women.

[Clinical phenotype analysis of 6 cases of TTC21B gene related nephronophthisis].

Objective: To analyze the clinical characteristics of 6 children with TTC21B-related nephronophthisis to provide reference for early clinical diagnosis. Methods: The general condition, clinical manifestations, laboratory tests and other clinical data of 6 children from 4 families diagnosed with nephronophthisis by genetic testing in Shanghai Children's Hospital from January 2015 to December 2020 were analyzed retrospectively. Results: A total of 6 children (3 males and 3 females) developed proteinuria and progressive renal dysfunction in early infancy. The onset age of proteinuria was 18 (6, 25) months. The age at the onset of renal impairment was 22 (10, 36) months. All 6 children progressed to end-stage renal disease (ESRD) within 10 (4, 65) months of onset. Five children had hypertension, 3 children with abnormal liver function, 2 children with visceral translocation and 1 child with growth retardation. The genetic results suggested that all children carried variations TTC21B gene p.C518R. Conclusions: Children with TTC21B gene p.C518R nephronophthisis had proteinuria and progressed to ESRD at the early stage of life. These nephronophthisis patients commonly presented with liver and renal dysfunction.

[Clinical features and expression of PLA(2)R in renal tissue with idiopathic membranous nephropathy in children].

Objective: To explore the clinical features and expression of PLA(2)R in renal tissue of children with idiopathic membranous nephropathy. Methods: Retrospective study was performed in patients with membranous nephropathy diagnosed through renal biopsy and the follow-up time was at least half a year in Shanghai Children's Hospital from January 2010 to February 2017. We compared their clinicopathological and pathological findings of IMN. Indirect immunofluorescence assay was used to detect glomerular PLA(2)R expression. We analyzed the differences of clinical features between the PLA(2)R negative and positive groups. T test, rank-sum test and Fisher exact test were used. Results: Eleven cases had hematuria and proteinuria, 9 cases presented with nephrotic syndrome, and 2 cases showed isolated proteinuria. Of the 22 cases of children with IMN, 16 patients had complete remission (complete remission rate was 72.8%), and 22 patients had partial remission. The renal function of all cases was normal and in all cases the estimated glomerular filtration rate was > 90 ml/(min·1.73m(2)). Of 22 cases with IMN, 7 cases were PLA(2)R-positive in renal tissue and 15 cases were PLA(2)R-negative. The age of positive group (10 years old) was older than the negative group (6 years old)(Z=-2.483, P<0.05) and the time of positive group (6 months) for urine protein to return to negative was longer than the negative group (2.5 months) through treatment. These differences were significantly different (Z=-2.072, P<0.05). Conclusions: Hematuria and proteinuria can be found in most children with idiopathic primary membranous nephropathy. Prednisone combined with immunosuppressant was effective. The positive rate of PLA(2)R in renal tissue of children with IMN was about 32%. The age of PLA(2)R positive group was older than the negative group. And the time of urine protein turning to negative in positive group was longer than that in the negative group.

Effects of MK-801 concentration on cell proliferation in rats with focal cerebral ischemia-reperfusion.

We explored the relationship between MK-801 concentration and neural stem cell proliferation in rats with focal cerebral ischemia-reperfusion (FCIR). A total of 60 male Sprague Dawley rats were randomized into control (six rats), sham-operation (six rats), operation (12 rats), and MK-801 groups. The MK-801 group comprised 36 rats that were subjected to different doses of MK-801 (0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mg/kg). Suture occlusion was used to establish an ischemia reperfusion model of middle cerebral artery occlusion (MCAO); 30 min before establishing the FCIR model, the MK-801 group rats were intraperitoneally injected with different doses of MK-801, while the sham-operation and control groups were injected with normal saline. Seven days after model establishment, bromodeoxyuridine-positive cerebral cortex cells adjacent to the focus of infarction were labeled for immunohistochemistry. MK-801 at a concentration of 0.4 mg/kg prevented endogenous neural stem cell proliferation, and this inhibitory effect was strengthened with increasing MK-801 concentration, especially at concentrations greater than 0.8 mg/kg. MK-801 inhibits endogenous neural stem cell proliferation in rats with FCIR, and the inhibitory effect is strengthened with increasing MK-801 concentration.

Activation and blockade of serotonin7 receptors in the prelimbic cortex regulate depressive-like behaviors in a 6-hydroxydopamine-induced Parkinson's disease rat model.

The role of serotonin7 (5-HT7) receptors in the regulation of depression is poorly understood, particularly in Parkinson's disease-associated depression. Here we examined whether 5-HT7 receptors in the prelimbic (PrL) sub-region of the ventral medial prefrontal cortex (mPFC) involve in the regulation of depressive-like behaviors in sham-operated rats and rats with unilateral 6-hydroxydopamine lesions of the medial forebrain bundle. The lesion induced depressive-like responses as measured by the sucrose preference and forced swim tests when compared to sham-operated rats. Intra-PrL injection of 5-HT7 receptor agonist AS19 (0.5, 1 and 2 µg/rat) increased sucrose consumption, and decreased immobility time in sham-operated and the lesioned rats, indicating the induction of antidepressant-like effects. Further, intra-PrL injection of 5-HT7 receptor antagonist SB269970 (1.5, 3 and 6 µg/rat) decreased sucrose consumption, and increased immobility time, indicating the induction of depressive-like responses. However, the doses producing these effects in the lesioned rats were higher than those in sham-operated rats. Neurochemical results showed that intra-PrL injection of AS19 (2 µg/rat) increased dopamine, 5-hydroxytryptamine (5-HT) and noradrenaline (NA) levels in the mPFC, habenula and ventral hippocampus (vHip) in sham-operated and the lesioned rats; whereas SB269970 (6 µg/rat) decreased 5-HT levels in the habenula and vHip, and the levels of NA in the mPFC, habenula and vHip in the two groups of rats. The results suggest that 5-HT7 receptors in the PrL play an important role in the regulation of these behaviors, which attribute to changes in monoamine levels in the limbic and limbic-related brain regions after activation and blockade of 5-HT7 receptors.

Agonists at PPAR-gamma suppress angiotensin II-induced production of plasminogen activator inhibitor-1 and extracellular matrix in rat cardiac fibroblasts.

BACKGROUND AND PURPOSE: Peroxisome proliferator-activated receptor (PPAR)-gamma ligands have been shown to inhibit cardiac fibrosis. However, the underlying mechanisms are poorly understood. We investigated the regulation by PPAR-gamma ligands of angiotensin (Ang) II-induced plasminogen activator inhibitor (PAI)-1, extracellular matrix (ECM) production and cell growth in cardiac fibroblasts. EXPERIMENTAL APPROACH: The effects of PPAR-gamma ligands on Ang II-induced PAI-1, ECM expression and cell growth were assessed in primary-cultured rat cardiac fibroblasts; cardiac PAI-1 and ECM production was examined in Ang II-infused rats. KEY RESULTS: In growth-arrested cardiac fibroblasts, PPAR-gamma ligands rosiglitazone and 15-deoxy-Delta(12,14)-prostaglandin J2 (15d-PGJ2) dose-dependently attenuated Ang II-induced cell proliferation and expression of PAI-1, collagen type-I, collagen type-III and fibronectin. An accompanying increase in PPAR-gamma expression and activation was also observed. These suppressive effects were attenuated by the PPAR-gamma antagonists GW9662 and bisphenol A diglycidyl ether (BADGE). Moreover, rosiglitazone and 15d-PGJ2 inhibited in part the expression and phosphorylation of Ang II-induced transforming growth factor (TGF)-beta1, Smad2/3 and c-Jun NH(2)-terminal kinase (JNK). Ang II infusion in rats markedly increased left ventricular production of PAI-1, collagen and fibronectin, with a concurrent increase in the ratios of heart weight/body weight and left ventricle weight/body weight. Co-treatment with rosiglitazone significantly decreased these levels and upregulated PPAR-gamma expression. CONCLUSIONS AND IMPLICATIONS: Rosiglitazone and 15d-PGJ2 suppress Ang II-induced production of PAI-1 and ECM probably via interactions between PPAR-gamma and TGF-beta1/Smad2/3 and JNK signalling pathways. It is suggested that PPAR-gamma and its ligands may have potential applications in preventing cardiac fibrosis.

[Preparation and characterization of the monoclonal antibodies against Japanese encephalitis virus.].

BACKGROUND: To prepare mouse monoclonal antibodies (McAbs) against Japanese encephalitis virus (JEV)and evaluate their biological characteristics. METHODS: McAbs against JEV were prepared by immunizing, fusing, cloning and screening. Their sensitivity, specificity, universality and neutralizing function were analyzed with ELISA, IFA, NT and Western blot. RESULTS: Titers of three McAbs against JEV were higher than 106. Three McAbs only reacted with JEV and not with other nine arboviruses. F12.37 could react with ten strains of JEV and sensitively detected ten replicating strains of viruses in BHK cell. The strains P3 and SH03-103 of JEV were neutralized by F12.37, its titers of protecting 50% cell were 3.2x105 and 105. Western blot showed that F12.37 reacted with envelop(E)protein of JEV. CONCLUSION: Three McAbs against JEV had high titer and good specificity. And F12.37 was very sensitive and universal in reacting with JEV, and neutralized JEV of Genotype I and Genotype .The binding site of F12.37 lays in E protein of JEV.

Inducible nitric oxide synthase deficiency does not affect the susceptibility of mice to atherosclerosis but increases collagen content in lesions.

BACKGROUND: Although endothelial nitric oxide synthase (NOS) is antiatherogenic, the role of inducible NOS (iNOS) in the development of atherosclerosis is not established. METHODS AND RESULTS: We compared the susceptibility of iNOS knockout (iNOS(-/-)) and wild-type (iNOS(+/+)) mice to the development of atherosclerosis induced by feeding an atherogenic diet for 15 weeks. Plasma lipid level, atherosclerotic lesion size, and cellular density in the lesions were all similar in the 2 strains (lesion size: iNOS(+/+) 285+/-73x10(3) microm(2), iNOS(-/-) 293+/-82x10(3) microm(2), n=10). iNOS mRNA was detected in the lesions of iNOS(+/+) but not iNOS(-/-) mice through RT-PCR. Immunohistochemically, iNOS(+/+) mice showed iNOS staining in macrophages and medial smooth muscle cells in the lesions. Nitrotyrosine staining showed a similar distribution, whereas it was absent in iNOS(-/-) mice. There was no apparent difference in the intensity or distribution of vascular cell adhesion molecule-1 staining in the lesions of the 2 strains. However, the lesions of iNOS(+/+) mice showed a markedly decreased extracellular collagen content compared with those of iNOS(-/-) mice CONCLUSIONS: iNOS induction does not affect the development of atherosclerosis in mice fed an atherogenic diet, but the resulting lesions show decreased levels of extracellular collagen and may be more fragile.

Tanshinone II-A inhibits low density lipoprotein oxidation in vitro.

Tanshinone II-A (TSII-A) is a major component of Salvia miltorrhiza Bunge which has long been used for preventing and ameliorating anginal pain in China. However the effect of TSII-A on low density lipoprotein (LDL) oxidation has not been studied. The present study was performed to investigate the effects of TSII-A on LDL oxidation using four oxidizing systems, including copper-, peroxyl radical- and peroxynitrite-initiated and macrophage-mediated LDL oxidation. LDL oxidation was measured in terms of formation of thiobarbituric acid-reactive substances (TBARS), relative electrophoretic mobility (REM) on agarose gel and lag time. In all four systems, TSII-A has apparent antioxidative effects against LDL oxidation, as evidenced by its dose-dependent inhibition of TBARS formation, prolongation of lag time and suppression of increased REM. Regarding the mechanism underlying its antioxidative effect, TSII-A neither scavenged superoxide nor peroxynitrite. It also did not chelate copper. But it has mild peroxyl radical scavenging activity. The direct binding to LDL particles and conformational change of LDL structure by TSII-A were suggested, because it increased negative charge of LDL which was shown by increased REM on agarose gel. In conclusion, TSII-A is an effective antioxidant against LDL oxidation in vitro. The underlying mechanism appears to be related to its peroxyl radical scavenging and LDL binding activity.

Inducible nitric oxide synthase knockout mouse macrophages disclose prooxidant effect of interferon-gamma on low-density lipoprotein oxidation.

To test our hypothesis that interferon-gamma (IFN-gamma) has a direct prooxidant effect on macrophage-mediated LDL oxidation behind its antioxidant effect via induction of inducible nitric oxide synthase (iNOS), we incubated LDL with wild-type (iNOS(+/+)) or iNOS knockout mouse (iNOS(-/-)) macrophages preincubated with IFN-gamma or IFN-gamma plus lipopolysaccharide (IFN-gamma/LPS) for 24 h. LDL oxidation was measured in terms of formation of thiobarbituric acid reactive substances (TBARS) and electrophoretic mobility. Thiol production, nitrite production, and superoxide production from macrophages were measured by using Ellman's assay, the Griess reagent, and the SOD-inhibitable cytochrome c reduction method, respectively. IFN-gamma alone or combined with LPS induced iNOS expression and increased nitrite production in iNOS(+/+) macrophages, but not in iNOS(-/-) macrophages. TBARS formation from LDL was suppressed in IFN-gamma- and IFN-gamma/LPS-treated iNOS(+/+) macrophages but was increased in IFN-gamma-treated iNOS(-/-) macrophages. In the presence of N(G)-monomethyl-l-arginine (l-NMMA), a NOS inhibitor, the suppressive effect of IFN-gamma and IFN-gamma/LPS was abolished and TBARS formation was even increased to a level above that of untreated iNOS(+/+) macrophage. NOC 18, an NO donor, dose dependently inhibited macrophage-mediated LDL oxidation. IFN-gamma increased superoxide and thiol productions in both types of macrophages. We conclude that IFN-gamma promotes macrophage-mediated LDL oxidation by stimulating superoxide and thiol production under conditions where iNOS-catalyzed NO release is restricted.

Estrogens induce apoptosis in mouse peritoneal macrophages.

AIM: To study whether estrogen might induce apoptosis in mouse peritoneal macrophages (MPM). METHOD: The MPM were isolated and incubated in culture medium containing 17-beta-estradiol, estrone, or equal volume of 100% ethanol as control. DNA fragmentation was visualized by agarose gel electrophoresis. RESULTS: 17-beta-Estradiol 0.01-1 mumol.L-1 or estrone 10-20 mumol.L-1 elicited typical morphological apoptosis and DNA fragmentation in a concentration-dependent manner in MPM. Staurosporine (Sta) 0.01 mumol.L-1, cycloheximide (Cyc) 1 mg.L-1, and tamoxifen (Tam) 10 mumol.L-1 inhibited the DNA fragmentation induced by 17-beta-estradiol 1 mumol.L-1 or estrone 20 mumol.L-1. CONCLUSION: Estradiol and estrone induced apoptosis in MPM.

Oxidized low-density lipoproteins stimulate adhesion of monocytes to endothelial cells.

AIM: To study the effects of oxidized low-density lipoproteins (ox-LDL) on the adhesiveness of monocytes to endothelial cells. METHODS: LDL was obtained from healthy human plasma by ultracentrifugation, and oxidized by CuSO4 10 mumol.L-1. The assay of adhesion was performed using cultured bovine aortic endothelial cells (BAEC) and human peripheral blood monocytes. RESULTS: Pretreatment BAEC with ox-LDL enhanced monocyte adhesion to BAEC in time- and dose-dependent manner. ox-LDL as little as 10 mg.L-1 and 30 min of preincubation stimulated monocyte adhesion. Cycloheximide (Cyc, a protein synthesis inhibitor) 1 mg.L-1 and staurosporine (Sta, a PKC inhibitor) 20 nmol.L-1 abolished the effect of ox-LDL (60 mg.L-1), but dextran sulfate 20 mg.L-1 had no effect on monocyte adhesion. Phorbol 12-myristate 13-acetate (PMA) 1 nmol.L-1 and lysophosphatidylcholine (Lys) 6 mumol.L-1 mimicked the effects of ox-LDL and potentiated monocyte adhesion. Sta also suppressed the augmentative effects of Lys and PMA. CONCLUSION: ox-LDL enhances the adhesion of monocytes to BAEC through the activation of PKC.

Oxidized low-density lipoproteins induce apoptosis in aortic and endocardial endothelial cells.

AIM: To examine whether oxidized low-density lipoproteins (ox-LDL) might induce apoptosis in bovine aortic and endocardial endothelial cells (BAEC and BEEC). METHODS: Low-density lipoproteins (LDL) were isolated from healthy human plasma by ultracentrifugation and oxidized by CuSO4 10 mumol.L-1. BAEC and BEEC were incubated in a medium containing ox-LDL, LDL, or phosphate-buffer solution (PBS) as control. DNA fragmentation was visualized by agarose gel electrophoresis and determined quantitatively using Hoechst-33258 fluorochrome. RESULTS: Ox-LDL, not LDL, elicited typical apoptotic changes and DNA fragmentation in BAEC and BEEC. In BAEC, dextran sulfate, and cicloheximide (Cic) exhibited no effect on DNA fragmentation induced by ox-LDL. Butylated hydroxytoluene (BHT) 20 mumol.L-1 completely inhibited Cu(2+)-mediated oxidation of LDL as well as the apoptosis-inducing effect of Cu(2+)-exposed LDL. Lysophosphatidylcholine (LPC) did not elicit DNA fragmentation in BAEC and in BEEC. DNA fragmentation induced by ox-LDL in BAEC and in BEEC was blocked by chelating the calcium of the culture medium by egtazic acid. CONCLUSION: Ox-LDL induces apoptosis in BAEC and BEEC without involving the LPC.

High density lipoproteins increase cytoplasmic free calcium in bovine aortic endothelial cells.

This study examined the influence of human high density lipoproteins (HDL) on the intracellular free calcium of cultured bovine aortic endothelial cells (BAECs). Intracellular Ca2+ concentration ([Ca2+]i) was determined by a fluorescent calcium indicator, Fura-2. It was found that, in the presence of 1 mmol/L extracellular calcium, HDL resulted in a biphasic elevation of [Ca2+]i in BAECs, consisting of an initial, transient component followed by a lower, but more sustained component. Doses of HDL from 25 to 200 micrograms protein/ml induced marked concentration-dependent elevations of [Ca2+]i in BAECs. The sustained component was abolished by deprivation of extracellular calcium or by pretreatment of endothelial cells with a calcium influx blocker, NiCl2, HDL-induced elevation of [Ca2+]i was attenuated in a concentration-dependent way by an inhibitor of calcium release, tetracaine. Repeated applications of HDL (100 micrograms protein/ml) markedly blunted the initial peak component of the calcium transient of BAECs. These results demonstrate that both intracellular and extracellular calcium pools are responsible for the biphasic elevation of [Ca2+]i induced by HDL in cultured BAECs.

Oxidized low-density lipoproteins induce apoptosis in macrophages.

AIM: To examine whether oxidized low density lipoproteins (ox-LDL) might induce apoptosis in mouse peritoneal macrophages (MPM). METHODS: Low density lipoproteins (LDL) were isolated from healthy human plasma by ultracentrifugation and oxidized by CuSO4 10 mumol.L-1. MPM were incubated in a medium containing ox-LDL, LDL, or phosphate-buffer solution (PBS) as control. DNA fragmentation was visualized by agarose gel electrophoresis and determined quantitatively using Hoechst 33,258 fluorochrome. RESULTS: Ox-LDL, not LDL, elicited typical apoptotic morphological changes (shrinkage of cytoplasm and condensation of chromatin) and DNA fragmentation in a time- and dose-dependent manner. Incubation for 24 h was necessary for ox-LDL 200 mg protein.L-1 to induce DNA fragmentation, and the maximal effect reached at 72 h. The DNA fragmentation after 24 h incubation with ox-LDL at concentrations of 100, 200, and 400 mg protein.L-1 amounted to 6.0% (P > 0.05), 9.3% (P < 0.05), and 30.9% (P < 0.01), respectively vs PBS control. Dextran sulfate, a scavenger receptor blocker and cycloheximide, a protein synthesis inhibitor, exhibited no effect on DNA fragmentation. However, antioxidant butylated hydroxytoluene (BHT) 20 mumol.L-1 completely inhibited Cu(2+)-mediated oxidation of LDL as well as the apoptosis-inducing effect of Cu(2+)-exposed LDL. Lysophosphatidylcholine (LPC), an active component in ox-LDL, at concentration up to 60 mumol.L-1, did not elicit DNA fragmentation in MPM. CONCLUSION: Ox-LDL induces apoptosis in MPM without involving LPC.

Some similarities in vascular effects of oleic acid and oxidized low-density lipoproteins on rabbit aorta.

In the present study oxidized low-density lipoproteins (ox-LDL) was prepared by a new simple method: oxidizing LDL by electrolysis-generated free radicals. In endothelium-intact norepinephrine(NE)-precontracted rabbit aortic rings, ox-LDL (2 mg protein/ml)-incubation for 30 min or 3 mM oleic acid for 10 min, significantly attenuated the acetylcholine (ACh)-induced endothelium-dependent relaxation (EDR) (both P < 0.01 v control). Such attenuated EDR were sustained after washout. The oleic acid-induced endothelial dysfunction was associated with concomitant reduction of cGMP level in aortic rings. Preincubation of aortic rings with 500 microM L-arginine or 100 u/ml superoxide dismutase for 10 min partly prevented the oleic acid-induced attenuation of EDR and reduction of cGMP, indicating that oleic acid may impair the L-arginine-nitric acid pathway and/or inactivate the nitric oxide. Both ox-LDL and oleic acid potentiated NE-induced aortic ring contraction (both P < 0.01 v control). Such potentiating effects were abolished by preincubation with 1 microM verapamil, indicating the possible involvement of calcium influx in vascular smooth muscle cells during the enhanced contraction. Gas-chromatographic analysis showed that oleic acid content is the highest among all free fatty acids in ox-LDL. In conclusion, we found that oleic acid possesses certain similar vascular effects as ox-LDL in inducing endothelial dysfunction and in enhancing NE-induced vasocontraction in rabbit aortic ring. We proposed that the vasoactive effects of ox-LDL may be resulted partly from the activation or release of active oleic acid molecule during oxidative modification of LDL.