Highly Atherogenic Lipid Particles are Associated with Preeclampsia After Successful Fertility Treatment for Obese Women who have Unexplained Infertility.

Atherogenic dyslipidemia-before or during pregnancy-may contribute to preeclampsia and subsequent cardiovascular disease risk. We performed a nested case-control study to further understand dyslipidemia associated with preeclampsia. The cohort consisted of participants in the randomized clinical trial "Improving Reproductive Fitness Through Pretreatment with Lifestyle Modification in Obese Women with Unexplained Infertility" (FIT-PLESE). FIT-PLESE was designed to study the effect of a pre-fertility treatment 16-week randomized lifestyle intervention program (Nutrisystem diet + exercise + orlistat vs. training alone) on improvement in live birth rate among obese women with unexplained infertility. Of the 279 patients in FIT-PLESE, 80 delivered a viable infant. Maternal serum was analyzed across five visits: before and after lifestyle interventions and also at three pregnancy visits (16, 24, and 32 weeks gestation). Apolipoprotein lipids were measured in a blinded fashion using ion mobility. Cases were those who developed preeclampsia. Controls also had a live birth but did not develop preeclampsia. Generalized linear and mixed models with repeated measures were used to compare the mean lipoprotein lipid levels of the two groups across all visits. Complete data were available for 75 pregnancies, and preeclampsia developed in 14.5% of the pregnancies. Cholesterol/high-density lipoprotein (HDL) ratios (p < 0.003), triglycerides (p = 0.012), and triglyceride/HDL ratios, all adjusted for BMI, were worse in patients with preeclampsia (p < 0.001). Subclasses a, b, and c of highly atherogenic, very small, low-density lipoprotein (LDL) particles were higher during pregnancy for the preeclamptic women (p < 0.05). Very small LDL particle subclass d levels were significantly greater only at 24 weeks (p = 0.012). The role of highly atherogenic, very small LDL particle excess in the pathophysiology of preeclampsia awaits further investigation.

Proprotein Convertase Subtilisin Kexin 9 (PCSK9) and nonHDL particles rise during normal pregnancy and differ by BMI.

BACKGROUND: Serum lipids, including total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-c), increase during pregnancy. Serum Proprotein Convertase Subtilisin Kexin 9 (PCSK9) is a vital regulator in lipoprotein metabolism. Circulating PCSK9 downregulates the LDL receptor on the surface of liver cells inhibiting clearance of LDL-c. OBJECTIVE: To determine the influence of weeks of pregnancy and obesity on circulating levels of essential lipid lipoproteins and PCSK9 in women with normal, uncomplicated pregnancies and deliveries. METHODS: We performed a comprehensive lipid and lipoprotein profile during each trimester of pregnancy in 70 mostly Caucasian women with uncomplicated normal pregnancies and deliveries. Based on their first trimester BMI, we placed them into one of three categories: (<25 kg/m2 n=23, 25-30 kg/m2 n=25, or >30 n=22) kg/m2. Cholesterol, triglycerides, LDL cholesterol (LDL-c), non-HDL particles, and lipoprotein(a) were measured by spectrophotometry, ion mobility, and immunoturbidimetric assays. Elisa assay determined PCSK9 (active and total). Homeostatic Model Assessment (HOMA-IR) assessed insulin resistance in the second and third trimesters of pregnancy. RESULTS: Total and active PCSK9, LDL-c, and nonHDL particle concentrations were higher than reported for non-pregnant normal values, increased after the first trimester of pregnancy, and were highest from mid-gestation to the last trimester of pregnancy in the overweight and the obese. CONCLUSION: PCSK9 levels rise as normal pregnancy progresses. Levels are higher in persons who are obese, even after adjustment for insulin resistance. Defining normal PCSK9 levels during pregnancy must adjust for gestational age and BMI.

Atrial natriuretic peptide expression in rats with different pulmonary hypertensive responses to hypoxia.

Mechanisms that regulate atrial natriuretic peptide (ANP) expression during hypoxia are not well defined. We hypothesized that plasma immunoreactive ANP (irANP) and right heart irANP and ANP mRNA levels would be greater in a strain of Sprague-Dawley rats that develops more severe hypoxic pulmonary hypertension (H rats) than another strain (M rats). After 3 wk of hypoxia (0.5 atm), right ventricular systolic pressure (RVSP) and the right ventricle (RV) weight-to-left ventricle plus septum (LV (+) S) weight ratio [RV/(LV+S)] were greater in H rats than in M rats (70 +/- 4 vs. 40 +/- 2 mmHg and 0.59 +/- 0.02 vs. 0.50 +/- 0.02, respectively; P < 0.05 for both), but plasma ANP increased twofold and RV irANP and ANP mRNA increased fivefold in both rat strains. After 3 days of normoxic recovery from chronic hypoxia, RVSP, RV/(LV+S), and RV irANP and ANP mRNA levels decreased in M rats but not in H rats. Plasma irANP decreased to baseline levels in both rat strains. We conclude that, in addition to changes in RV pressure and hypertrophy, hypoxia acts through other mechanisms to modulate RV ANP synthesis and circulating ANP levels in hypoxia-adapted rats.

Effects of natriuretic peptides on vascular smooth-muscle cells derived from different vascular beds.

1. This study explored the hypothesis that atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-natriuretic peptide (CNP) have differing antiproliferative and antihypertrophic effects on pulmonary artery (PA) and thoracic aorta (TA) smooth-muscle cells (SMCs). 2. Cultured cells were exposed to 5% fetal calf serum (FCS) and angiotensin II (A-II) to induce DNA and protein synthesis, respectively. 3. ANP (10(-7) M) significantly reduced thymidine uptake in TA by 31% +/- 2% (P < or = 0.01) but not in PA (P > or = 0.05). 4. In parallel experiments, BNP (10(-7) M) significantly reduced thymidine uptake in TA (-22% +/- 5%, P < or = 0.01), but not in PA cells (P > or = 0.05). 5. CNP (10(-7) M) did not significantly alter thymidine uptake in TA cells exposed to FCS, but it did significantly reduce uptake in PA (-28.5% +/- 4%) 2(P < or = 0.05). 6. Blunting by ANP (10(-7) M) of the A-II (10(-8) M)-induced increase in protein synthesis was significantly greater in PA than in TA cells. 7. However, BNP and CNP (10(-7) M) exerted similar antihypertrophic effects on TA and PA cells exposed to A-II. 8. The antiproliferative effects of BNP and ANP exceed those of CNP in TA SMCs, but CNP appears to be the most effective antiproliferative agent in PA SMCs. In addition, PA-derived SMCs are more sensitive to the antihypertrophic effects of ANP than TA-derived cells, suggesting phenotypic differences. The findings indicate that the natriuretic peptides may play complementary roles in modulating SMC proliferation and protein synthesis.

Brain natriuretic peptide: possible role in the modulation of hypoxic pulmonary hypertension.

To test the hypothesis that brain natriuretic peptide (BNP) plays a role similar to that of atrial natriuretic peptide (ANP) in modulating pulmonary vascular responses to hypoxia, we measured the vasodilator potency of ANP and BNP in rat pulmonary artery (PA) and thoracic aorta (TA) rings and in isolated rat lungs. We also measured the effect of chronic hypoxia on plasma levels and cardiac gene expression of both peptides. BNP had a vasorelaxant effect equipotent to that of ANP on preconstricted TA and PA rings, but was less potent than ANP in relaxing the vasoconstrictor response to hypoxia in isolated lungs [mean 50% inhibitory concentration (IC50) 10(-7) vs. 10(-6) M for ANP and BNP, respectively]. Plasma BNP levels were 30-fold lower than ANP, but both peptides increased approximately 70% during chronic hypoxia. In the right atrium, hypoxia lowered BNP mRNA slightly, but had no effect on ANP mRNA or tissue levels of either peptide. However, hypoxia increased right ventricular content and mRNA levels of both peptides by three- to fourfold. We conclude that BNP and ANP have similar pulmonary vasodilator effects and are upregulated proportionally during chronic hypoxia. These results support a role for BNP in modulating the pulmonary hypertensive response to chronic hypoxia.

Investigation of a unique time-space cluster of sarcoidosis in firefighters.

A unique cluster of three cases of sarcoidosis developed recently among 10 white firefighters who trained together as apprentices in 1979. This led us to hypothesize that firefighters are at increased risk of this condition because of the combined effect of smoke exposure and infection with a communicable agent, such as Chlamydia pneumoniae, a recently proposed cause of sarcoidosis. We conducted a case-finding questionnaire survey of 1,282 active and retired male Providence firefighters and police officers and then evaluated both the index apprenticeship class and two control cohorts by chest radiography, seromarkers of T lymphocyte activation (neopterin and sIL-2R), and chlamydial serology. One additional case of sarcoidosis was identified among the 990 (77%) survey respondents. No new cases were detected in the subsequent laboratory investigation of 46 (87%) firefighters from the index 1979 apprenticeship class, 53 (75%) firefighter controls from the 1974 and 1980 classes, or 50 (30%) police officer controls from 1973-1981 classes. The cohorts did not differ with regard to either C. pneumoniae antibody titers or sIL-2R levels, but serum neopterin was elevated (> 9.0 nmol/L) in 20% (eight of 41) of the index cohort, 22% (11 of 51) of firefighter controls, and 4% (two of 48) of police officers. Logistic regression found firefighting to be the only significant predictor of neopterin elevation (odds ratio 5.8; 95% CI, 1.3 to 26.9). Our results suggest that firefighters may be at risk of T lymphocyte activation. Determining whether this reflects an enhanced risk of lymphocytic alveolitis and whether firefighters are more likely to develop sarcoidosis requires further study.

Neutral endopeptidase inhibition attenuates development of hypoxic pulmonary hypertension in rats.

Neutral endopeptidase (NEP) inhibition is thought to blunt hypoxic pulmonary hypertension by reducing atrial natriuretic peptide (ANP) metabolism, but this hypothesis has not been confirmed. We measured NEP activity, guanosine 3',5'-cyclic monophosphate (cGMP) production, plasma ANP levels, and cardiac ANP synthesis in rats given an orally active NEP inhibitor (SCH-34826) during 3 wk of hypoxia. Under normoxic conditions, SCH-34826 had no effect on plasma ANP levels but reduced pulmonary and renal NEP activity by 50% and increased urinary cGMP levels (60 +/- 6 vs. 22 +/- 4 pg/mg creatinine; P < 0.05). Under hypoxic conditions, SCH-34826-treated rats had lower plasma ANP levels (1,259 +/- 361 vs. 2,101 +/- 278 pg/ml; P < 0.05), lower right ventricular systolic pressure (53 +/- 5 vs. 73 +/- 2 mmHg; P < 0.05), lower right ventricle weight-to-left ventricle+septum weight ratio (0.47 +/- 0.04 vs. 0.53 +/- 0.03; P < 0.05), and less muscularization and percent medial wall thickness of peripheral pulmonary arteries (22 +/- 5 vs. 45 +/- 8% and 17 +/- 1 vs. 25 +/- 1%, respectively; P < 0.05 for all values) than did rats treated with vehicle alone. These values were not affected by SCH-34826 under normoxic conditions. SCH-34826 decreased right ventricular ANP tissue levels in hypoxic rats (27 +/- 10 vs. 8 +/- 1 ng/mg protein; P < 0.05) but did not affect steady-state ANP mRNA levels. We conclude that NEP inhibition blunts pulmonary hypertension without increasing plasma ANP levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiopulmonary responses to chronic hypoxia in transgenic mice that overexpress ANP.

Elevated plasma atrial natriuretic peptide (ANP) levels have been shown to blunt pulmonary hemodynamic responses to chronic hypoxia, but whether elevated circulating ANP levels negatively feedback on cardiac expression of the ANP gene is unknown. Using a recently developed strain of transgenic mouse (TTR-ANF) that expresses a transthyretin promoter-ANP fusion gene in the liver, we studied the effect of chronically elevated plasma ANP levels on cardiac hypertrophic and pulmonary hemodynamic responses and expression of the endogenous cardiac ANP gene during chronic hypoxia. Plasma ANP levels were 10-fold higher in TTR-ANF mice than in their non-transgenic littermates. After 3 wk of hypobaric hypoxia (0.5 atm), right ventricular hypertrophy and pulmonary hypertension had developed in both groups of mice, but TTR-ANF mice had lower right ventricle-to-left ventricle plus septum weight ratios (0.39 +/- 0.01 vs. 0.45 +/- 0.02), right ventricular systolic pressures (25 +/- 2 vs. 29 +/- 2 mmHg), and lung dry weight-to-body weight ratios (0.48 +/- 0.03 vs. 0.57 +/- 0.01 mg/g) and less muscularization of peripheral pulmonary vessels (8.3 +/- 1.4 vs. 17.4 +/- 2.5%) than nontransgenic controls. Right atrial and ventricular steady-state ANP mRNA levels were the same in both groups of mice under normoxic and hypoxic conditions despite much higher plasma ANP levels and less pulmonary hypertension in TTR-ANF mice. We conclude that chronically elevated plasma ANP levels attenuate the development of hypoxic pulmonary hypertension in mice but do not suppress cardiac expression of the endogenous ANP gene under normoxic conditions nor blunt the upregulation of right ventricular ANP expression during chronic hypoxia.

C-receptor ligand blocks pulmonary clearance of atrial natriuretic peptide in isolated rat lungs.

Pulmonary clearance of atrial natriuretic peptide (ANP) was measured by indicator dilution technique in isolated perfused rat lungs with and without ANP clearance receptor (C-receptor) blockade. Approximately 50% of a bolus injection of 125I-ANP was removed during a single pass through the lungs compared with the intravascular marker 14C-dextran. Pulmonary clearance of 125I-ANP was suppressed in a dose-dependent fashion by unlabeled ANP. C-receptor blockade suppressed pulmonary clearance of 125I-ANP to the same degree as unlabeled ANP. High-performance liquid chromatography analysis of the pulmonary venous effluent from lungs treated with C-receptor ligand demonstrated intact 125I-ANP. We conclude that virtually all of the pulmonary vascular uptake of 125I-ANP during a single pass through isolated lungs is secondary to removal by ANP C-receptors.

Elastin binds to a multifunctional 67-kilodalton peripheral membrane protein.

Elastin binding proteins from plasma membranes of elastin-producing cells were isolated by affinity chromatography on immobilized elastin peptides. Three proteins of 67, 61, and 55 kDa were released from the elastin resin by guanidine/detergent, soluble elastin peptides, synthetic peptide VGVAPG, or galactoside sugars, but not by synthetic RGD-containing peptide or sugars not related to galactose. All three proteins incorporated radiolabel upon extracellular iodination and contained [3H]leucine following metabolic labeling, confirming that each is a synthetic product of the cell. The 67-kDa protein could be released from the cell surface with lactose-containing buffers, whereas solubilization of the 61- and 55-kDa components required the presence of detergent. Although all three proteins were retained on elastin affinity columns, the 61- and 55-kDa components were retained only in the presence of 67-kDa protein, suggesting that the 67-kDa protein binds elastin and the 61- and 55-kDa proteins bind to the 67-kDa protein. We propose that the 67-, 61-, and 55-kDa proteins constitute an elastin-receptor complex that forms a transmembrane link between the extracellular matrix and the intracellular compartment.

Characterization of elastin protein and mRNA from salmonid fish (Oncorhynchus kisutch).

1. Elastin was isolated from the bulbus arteriosus of a salmonid fish. Monoclonal and polyclonal antibodies, elicited against a CNBr digest of this protein, immunoprecipitated a polypeptide of Mr 43,000 from fish cell culture medium. 2. Cell-free translation of salmon poly A+ RNA produced a protein of approximately 43 kD that was immunoprecipitated with anti-elastin antibodies. The corresponding mRNA had an approximate Mr of 2 kb. 3. Despite similarities in amino acid composition, the differences in Mr between mammalian and salmon mRNA and protein suggest a divergence of fish and higher vertebrate elastins from an earlier ancestral gene.

The elastin receptor: a galactoside-binding protein.

The elastin receptor complex contains a component of 67 kilodaltons that binds to a glycoconjugate affinity column containing beta-galactoside residues and is eluted from this column with lactose. This protein component is also released from the surface of cultured chondroblasts by incubation with lactose, and its association with immobilized elastin is inhibited by lactose. Since lactose also blocks elastic fiber formation by cultured chondroblasts, the galactoside-binding property of the elastin receptor is implicated in this process.

Kinetics of receptor-mediated binding of tropoelastin to ligament fibroblasts.

Soluble 125I-labeled tropoelastin bound to confluent cultures of bovine ligamentum nuchae fibroblasts and to fibroblast plasma membrane preparations in a time-dependent, saturable, and reversible manner. Scatchard analysis indicates that there are approximately 2 X 10(6) binding sites/cell with a binding efficiency (Kd) of 8 X 10(-9) M. Binding of tropoelastin to cells and membranes reached equilibrium by 90 min and was reversible with 50% of specifically bound material released by 40 min. Specific binding of tropoelastin to cells pre-treated with dilute trypsin solutions was reduced significantly when compared with controls. Four polypeptides of estimated molecular masses of 67, 61, 55, and 43 kDa were obtained from detergent extracts of plasma membranes by elution affinity chromatography on elastin-Affi-Gel. Our findings establish that elastin-specific binding proteins displaying characteristics of a true receptor are present on the surface of elastin-producing cells.

Smooth muscle-mediated connective tissue remodeling in pulmonary hypertension.

Abnormal accumulation of connective tissue in blood vessels contributes to alterations in vascular physiology associated with disease states such as hypertension and atherosclerosis. Elastin synthesis was studied in blood vessels from newborn calves with severe pulmonary hypertension induced by alveolar hypoxia in order to investigate the cellular stimuli that elicit changes in pulmonary arterial connective tissue production. A two- to fourfold increase in elastin production was observed in pulmonary artery tissue and medial smooth muscle cells from hypertensive calves. This stimulation of elastin production was accompanied by a corresponding increase in elastin messenger RNA consistent with regulation at the transcriptional level. Conditioned serum harvested from cultures of pulmonary artery smooth muscle cells isolated from hypertensive animals contained one or more low molecular weight elastogenic factors that stimulated the production of elastin in both fibroblasts and smooth muscle cells and altered the chemotactic responsiveness of fibroblasts to elastin peptides. These results suggest that connective tissue changes in the pulmonary vasculature in response to pulmonary hypertension are orchestrated by the medial smooth muscle cell through the generation of specific differentiation factors that alter both the secretory phenotype and responsive properties of surrounding cells.

Identification of multiple tropoelastins secreted by bovine cells.

High resolution gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis, cell-free translation, and elastin-specific antibodies were used to identify three tropoelastin isoforms secreted by bovine tissue and cells. Tropoelastin isolated from nuchal ligament and from conditioned culture medium or cell-matrix extracts of ligament fibroblasts and auricular chondrocytes resolved as three distinct bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with molecular weights of approximately 67,500 (tropoelastin I), 65,000 (tropoelastin II), and 62,000 (tropoelastin III). Three tropoelastin polypeptides with molecular mass 2-3 kDa higher than their corresponding tissue forms were also evident in cell-free translation products of ligamentum nuchae RNA, suggesting that each tropoelastin species is encoded by a unique mRNA. The presence of cysteine in all three tropoelastin isoforms was demonstrated by the incorporation of [35S]cysteine into newly synthesized tropoelastin polypeptides and by immunoreactivity with an antibody raised against a synthetic peptide that defines the cysteine-containing carboxyl-terminal region of tropoelastin. Immunological co-localization of the carboxyl-terminal antibody with insoluble elastin in lung vasculature and parenchyma suggests that intact tropoelastin and not a processed form is incorporated into the elastin fiber.

Characterization of biologically active domains on elastin: identification of a monoclonal antibody to a cell recognition site.

Monoclonal antibodies to bovine alpha-elastin were characterized with solid-phase ELISA, Western blot, immunoprecipitation, and immunoaffinity chromatography. One monoclonal antibody, BA-4, bound to insoluble elastin, alpha-elastin, and tropoelastin and to peptide fragments generated by proteolytic digestion of insoluble elastin. Immunoaffinity chromatography of elastin fragments released from insoluble elastin with pancreatic elastase demonstrated that BA-4 was specific for a chemotactically active epitope composed of valine, glycine, alanine, and proline in a molar ratio of approximately 2:2:1:1. This composition matches the Val-Gly-Val-Ala-Pro-Gly repeating sequence in elastin that has been shown to be a chemoattractant for fibroblasts and monocytes. Specific ablation of the chemotactic activity of synthetic Val-Gly-Val-Ala-Pro-Gly by BA-4 IgG confirmed the identity of the epitope recognized by the monoclonal antibody and suggests that, despite its hydrophobic nature, this cell recognition domain is accessible on the surface of elastin and is strongly immunogenic. BA-4 should prove useful for investigating cell surface receptors for elastin.

Increased cyclic GMP levels lead to a stimulation of elastin production in ligament fibroblasts that is reversed by cyclic AMP.

The effects of cyclic nucleotides on elastin synthesis were studied in ligamentum nuchae fibroblasts by adding exogenous cyclic nucleotide derivatives or beta-adrenergic agents to cell culture medium. Elastin synthesis was enhanced (approximately 80%) by dibutyryl cGMP (Bt2cGMP) in concentrations ranging from 0.01 to 100 nM. Two other cGMP derivatives, 8-bromoguanosine 3':5'-cyclic monophosphate (8-Br-cGMP) and 2'-deoxy-cGMP, were also potent stimulators of elastin synthesis. In the absence of calcium, basal elastin production was substantially decreased (40% of control) and cGMP analogs no longer stimulated elastin synthesis, suggesting a role for calcium in the cGMP response. Bt2cAMP had no demonstrable effect on elastin production except at high concentrations which produced a nonspecific decrease equivalent to the decrease in total protein synthesis. Similarly, elevation of endogenous cellular cAMP levels by beta-adrenergic stimulation produced no change in elastin production. When 8-Br-cGMP was added to cells together with Bt2cAMP, cGMP-dependent stimulation of elastin production was abolished by cAMP in a dose-dependent fashion. These results suggest a coordinated means by which elastin production is controlled in ligament cells, i.e. increased cGMP levels lead to a stimulation of elastin production that is reversed by cAMP.

Differential sensitivity of chicken MM-creatine kinase to trypsin and proteinase-K.

Under several conditions of SDS-PAGE, the chicken MM-creatine kinase (MM-CK) monomer migrated as a approximately 50,000 dalton polypeptide, approx 25% larger than usually reported. Characterization by sedimentation equilibrium indicated that the anomalous molecular weight was an artifact of electrophoresis. Digestion with trypsin caused only moderate reductions in CK activity, despite extensive degradation of the denatured enzyme revealed by SDS-PAGE. Characterization of trypsinized MM-CK under non-denaturing conditions of electrophoresis and HPLC revealed no fragmentation of the native enzyme, suggesting that MM-CK quaternary structure was maintained despite extensive tryptic nicking. In contrast, much lower concentrations of proteinase-K generated only a single fragment in SDS-PAGE while causing a nearly total loss of enzyme activity.

Glucocorticoids stimulate elastin production in differentiated bovine ligament fibroblasts but do not induce elastin synthesis in undifferentiated cells.

Glucocorticoid treatment of fibroblasts from late gestation fetal bovine ligamentum nuchae resulted in a time- and dose-dependent selective increase in elastin production. Tropoelastin levels increased 2-3-fold in the presence of 10 nM dexamethasone while total protein synthesis and the rate of cell division decreased with glucocorticoid exposure. Two tropoelastin bands of molecular weights 64,500 and 61,000 were identified by immunoprecipitation and sodium dodecyl sulfate gradient-gel electrophoresis and both bands increased to an equal extent in the presence of dexamethasone. Undifferentiated cells from early-gestation animals did not synthesize elastin after hormone exposure, even though glucocorticoid receptors were demonstrated by nuclear-translocation experiments. These results indicate that glucocorticoids stimulate elastin production in elastin-producing ligament cells but do not induce elastin synthesis (differentiation) in undifferentiated cells.