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To evaluate the role of vascular endothelial growth factor (VEGF) in the pathogenesis of preeclampsia, we measured total VEGF, free VEGF and soluble Flt-1 (sFlt-1) concentrations and determined their relationships. Maternal serum samples were collected from 20 patients with preeclampsia and 20 normotensive women with uncomplicated pregnancies matched with the patients with preeclampsia for gestational age and parity. The serum concentrations of total VEGF (2.39±0.75 vs. 0.28±0.14) and sFlt-1 (934.5±235.5 vs. 298.0±161.2) were significantly increased in the patients with preeclampsia compared to the women with uncomplicated pregnancies. However the serum concentration of free VEGF (21.5±6.3 vs. 134.0±16.3) was lower in patients with preeclampsia. There was a positive correlation between the serum concentrations of total VEGF and sFlt-1 with systolic and diastolic blood pressure, respectively. There was a negative correlation between the serum concentration of free VEGF and systolic and diastolic blood pressure. There was a strong negative correlation between free VEGF and sFlt-1 concentrations. In conclusion, we found VEGF and sFlt-1 were related to the pathogenesis of preeclampsia. Although reduced concentrations of free VEGF might interfere with endothelial cell function and survival, further studies are required to clarify its specific role in the pathogenesis of preeclampsia.

The lutropin/choriogonadotropin receptor is a sevenhelix transmembrane (TM) receptor. A unique feature of TM helices is the content of Pro, which generally is absent in ␣ helices of globular proteins. Because Pro disrupts helices and introduces a ϳ26°kink, it has been speculated that Pro plays a crucial role in the structure of TM helices, exoloops, and cytoloops of TM receptors. To examine the roles of the five TM Pros of the lutropin/ choriogonadotropin receptor, these residues were individually substituted. Mutant receptors were examined for surface expression, hormone binding, and cAMP induction. Surface expression was monitored after introducing the flag epitope into the receptors. The LH/CG 1 receptor belongs to a subfamily of glycoprotein hormone receptors within the seven-transmembrane receptor family. Unlike most seven-TM receptors, it is comprised of a long extracellular N-terminal exodomain and a membraneassociated C-terminal endodomain containing 7 TM helices (1, 2). The exodomain is capable of high affinity hormone binding (3-5) without hormone action (5, 6). In contrast, the endodomain is capable of low affinity hormone binding (5, 7) and receptor activation (6, 8 -10). Low affinity hormone binding and initial receptor activation are likely to involve the exoloops and TM helices of the endodomain. The structure of these domains probably is affected by the organization of TM helices.A unique feature of TM helices is the presence of Pro (11), in contrast to the general lack of Pro in helices of globular proteins (12). Pro disrupts helices (13) and introduces a ϳ26°kink (14) in the helix backbone (15). The kink in TM helices is thought to orient so that the Pro-containing convex side is adjacent to neighboring TM helices, whereas the concave side faces the lipid phase (12). As a result, TM Pros are thought to influence the packing and organization of TM helices and thus, the mechanism of receptor activation (16). Despite their potential significance, the five TM Pros of the LH/CG receptor have not been examined together. Herein, we report the importance of two of the Pros in hormone binding and targeting of the receptor to the surface membrane. EXPERIMENTAL PROCEDURES Mutagenesis and Functional Expression of LH/CG Receptors-Mu-tant LH/CG receptor cDNAs were prepared in the pSELECT vector using the Altered Sites Mutagenesis System (Promega) and subcloned into pcDNA3 (Invitrogen) as described previously (9). Mutated LH/CG receptor plasmids were transfected into human embryonic kidney 293 cells by the calcium phosphate method. Stable cell lines were established in minimum essential medium containing 10% horse serum and 500 g/ml of G418. They were used for hormone binding, cAMP production, antibody binding, and fluorescence microscopy. I-hCG Binding and Intracellular cAMP Assay-Stable cells were assayed for125 I-hCG binding in the presence of 150,000 cpm of 125 I-hCG (17) and increasing concentrations of unlabeled hCG. The K d values were determined by Scatchard plots. hCG, batch CR 127, was supplied by t...

Background and Purpose— The role of circulating neutrophil extracellular traps (NETs) in cancer-related stroke is unknown. Methods— We conducted a prospective cohort study to test whether NETs are increased in cancer-related stroke and whether elevated NETs levels are associated with coagulopathy, assessed using D-dimer levels (≥2 μg/mL). Plasma DNA and nucleosome were assessed as NET-specific biomarkers. Results— In total, 138 patients were recruited; 38 patients had cancer-related stroke (active cancer and acute cryptogenic embolic stroke), 33 patients were healthy-controls, 27 patients were cancer-controls (active cancer but no stroke), and 40 patients were stroke-controls (acute ischemic stroke but no cancer). Plasma DNA and nucleosome levels were significantly elevated in cancer-related stroke patients than in healthy-controls ( P <0.05). These levels were correlated with the D-dimer levels ( P <0.01). In multiple regression analyses, increased plasma DNA levels were associated with cancer-related stroke (odds ratio=11.65 for highest quartile; 95% CI, 3.199–42.46) and D-dimer levels of ≥2 μg/mL (odds ratio=19.09 for highest quartile; 95% CI, 4.143–87.95) after adjusting for possible confounders. Conclusions— Increased circulating DNA levels were associated with cancer-related stroke, suggesting that NETosis is one of the molecular mechanisms of cancer-related stroke. Further long-term follow-up studies in large cohorts are needed to confirm the role of NET-specific biomarkers.