PlGF Levels Research Articles

Emerging Roles of Antiangiogenic and Angiogenic Proteins in Pathogenesis and Prediction of Preeclampsia

Hypertensive disorders are a major cause of maternal and fetal death, especially in developing nations, with the pregnancy-specific disease preeclampsia-eclampsia responsible for most of this mortality. Preeclampsia, recognized by ancient Greeks, was only sporadically investigated until late last century, current literature still labeling this disorder a “disease of theories.” Thus, not surprisingly, we have started the new millennium unable to predict or prevent preeclampsia, and management strategies remain but supportive, with pregnancy termination still the only definitive “therapy.” This may be about to change, as focused research, mainly in the past decade, has resulted in dramatic progress regarding pathogenesis of disease manifestations and risk assessment, while investigators have begun designing and testing definitive therapy.1 There are still a myriad of uncertainties and unanswered questions to address, but here we summarize the extent of 1 new and exciting chapter in preeclampsia research: the roles of angiogenic and antiangiogenic factors, to which an article in this issue by Rana and colleagues,2 contributes. A landmark report published in 2003 set the stage for all that followed.3 Dr Karumanchi’s laboratory, previously involved in renal cancer research, changed its focus to the kidney in preeclampsia. Using microarray chip technology, they probed gene expression profiles in placentas of women with preeclampsia and those with uncomplicated pregnancies. Of course many differences were noted, but one that caught the investigators’ attention was upregulation of the mRNA for the antiangiogenic protein soluble fms-like tyrosine kinase 1 (abbreviated as sFlt-1 or sVEGFR-1) in placentas from women with preeclampsia. Aware that de novo hypertension and proteinuria were 2 disturbing side effects of certain antiangiogenic compounds tested in cancer patients, these …

Maternal plasma and amniotic fluid SFLT-1 and PLGF levels at the time of mid-trimester amniocentesis in women who subsequently develop preeclampsia

Maternal plasma and amniotic fluid SFLT-1 and PLGF levels at the time of mid-trimester amniocentesis in women who subsequently develop preeclampsia

Association of maternal serum levels of soluble endoglin with small-for-gestational-age and preterm births

SMALL-FOR-GESTATIONAL-AGE AND PRETERM BIRTHS JILLIAN TSAI, KAI YU, CONG QIAN, CHUN LAM, ANANTH KARUMANCHI, RICHARD LEVINE, Vanderbilt University School of Medicine, Nashville, Tennessee, Department of Health and Human Services, National Institute of Child Health and Human Development, Division of Epidemiology, Statistics, and Prevention Research, Bethesda, Maryland, Allied TechnologyGroup, Rockville,Maryland, Center for Vascular Biology, Division of Nephrology, Department ofMedicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts OBJECTIVE: An imbalance of circulating angiogenic factors may contribute to the pathogenesis of small-for-gestational-age (SGA) births and spontaneous preterm delivery. We hypothesized that maternal levels of circulating soluble endoglin (sEng), an anti-angiogenic factor which binds TGF, are associated with SGA and preterm births. STUDY DESIGN: Random sample of 2200 women from the Calcium for Preeclampsia Prevention trial in healthy nulliparas. SGA was !10th percentile birthweight for GA, parity, infant sex, and race. After excluding women with gestational hypertension or preeclampsia and those without spontaneous onset of labor, 1206 subjects remained of whom 89 delivered SGAand 111 preterm infants. Odds ratios (ORs) and 95% confidence intervals (CIs) relating the 2d, 3d, or 4th quartilesofsEngto the1stquartile (lowest)were computedusing logistic regression, controlling formaternal bodymass index, age, gestational age at sample collection, smoking, race (for preterm births only), and PlGF and sFlt1 levels. RESULTS: In samples obtained at 21-32 (P=0.04) and 33-42 (P=0.09) weeks women who delivered SGA infants had higher sEng levels than women with infants of appropriate size. Similarly, compared to subjects with full-term deliveries, those with preterm deliveries had higher sEng levels at 21-32 (P=0.01) and 33-42 (P=0.01) weeks. In multivariate analyses sEng was positively associated with SGA at 33-42 weeks: ORs (95% CIs) by sEng quartiles: 1.00, 1.29 (0.59-2.84), 2.52 (1.16-5.47), and 2.49 (1.05-5.89). sEng also showed marginal significant associations with preterm delivery at 21-32 weeks: ORs (95% CIs) by sEng quartiles: 1.00, 0.80 (0.42-1.52), 1.63 (0.88-3.03), and 1.11 (0.98-1.25). CONCLUSION: Higher levels of sEng at 33-42 weeks were associated with SGA and at 21-32 weeks were marginally associated with preterm delivery. These associations may indicate a role for sEng in the pathogenesis of intrauterine growth restriction and spontaneous preterm delivery through blockade of TGF signaling, reducing vasodilation and placental perfusion.

1141584386 Cell type specific regulation of human placenta growth factor

Background: Maternal serum levels of PlGF are significantly lower in preeclamptic patients than in normal pregnant women. PlGF mRNA expression is cell type specific with high levels in trophoblast but comparatively low levels in most nontrophoblast. Despite two consensus hypoxia response elements (HREs), PlGF expression is down regulated by hypoxia in trophoblast, but increased in other cell types. These observations suggest unique regulatory mechanisms control PlGF expression. We sought to characterize PlGF promoter regions which function to regulate transcription rates in different cell types and under hypoxia.Methods: Regions of human PlGF gene (−1521/+34), (−828/+34), (−698/+34) were cloned into a reporter vector and (−844/−677) was cloned into a minimal TK promoter containing expression vectors. Trophoblast (JEG3 and JAR), and nontrophoblast (HeLa and hEK‐293) were transiently transfected to determine functional responsiveness of the regions. A PlGF gene region containing two HREs was cloned upstream of a TK containing vector to determine hypoxia (1% O2) responsiveness.Results: PlGF (−1521/+34), (−828/+34) regions showed high promoter activity in trophoblast cells, but little activity in nontrophoblast cells. PlGF (−844/−677) cloned downstream of the TK reporter, augmented promoter activity in trophoblast but not nontrophoblast cells. When cloned upstream to the TK promoter, this region significantly decreased activity in nontrophoblast cells. Surprisingly, the PlGF region containing HREs produced little increase in minimal promoter activity during hypoxia in any of the cells.Conclusions: PlGF transcription is cell type specific and a region of PlGF (−828/−698) may act as an enhancer in trophoblast yet mediates repressor activity in nontrophoblast. The putative HREs in PlGF 5’UTR do not seem to increase PlGF transcription under hypoxia suggesting that they are not essential for governing PlGF expression. Further understanding of transcriptional regulation of PlGF in trophoblast may facilitate approaches to increase expression during preeclampsia.

Placenta growth factor expression is regulated by stretch and correlates with microvascular dysfunction and plasma LDL

Enlargement of preexisting collateral arteries (arteriogenesis) is regulated by hemodynamic forces (shear, stretch). We thus hypothesized that hemodynamic forces regulate placenta growth factor (PlGF, a VEGF-related arteriogenic factor). We assessed PlGF expression in cultured human smooth muscle cells in response to 0–48 h. of cyclic stretch (N=6). Stretch mildly increased PlGF mRNA (~1.4 fold at 30 h). We further investigated hemodynamic regulation of PlGF expression in vivo by correlating coronary artery PlGF expression with microvascular function in Yucatan and Ossabaw swine. Microvascular function was assessed by administering bradykinin (BK, 4 ng/kg iv) and adenosine (ADO, 3 μg/kg iv) to anesthetized swine. Coronary vascular resistance (CVR) was calculated from measures of flow (Doppler Flowire) and vessel size (intravascular ultrasound). A higher CVR indicates microvascular dysfunction. A strong inverse correlation between PlGF levels and CVR was found in Ossabaws (N=8) for both BK (r2=0.51; p=0.02) and ADO (r2=0.30; p=0.05), with a similar but nonsignificant trend in Yucatans. PlGF mRNA was also inversely correlated with plasma total cholesterol (r2=0.28; p=0.01) and LDL (r2=0.27; p=0.01) in Yucatans and Ossabaws (N=22), but not HDL. These data suggest that LDL-mediated microvascular dysfunction and altered sensing of hemodynamic forces might reduce PlGF expression and blunt arteriogenesis. Support: HL62552, RR13223, ADA (MS).

Circulating Angiogenic Factors and Abnormal Uterine Artery Doppler Velocimetry in the Second Trimester

Objective: Circulating angiogenic growth factors (such as vascular endothelial growth factor [VEGF] and placental growth factor [PlGF]) and their interaction may be associated with vascular remodeling of spiral arteries in normal pregnancy. Soluble Flt-1, an antagonist of both VEGF and PlGF, has been shown to be increased, while PlGF is decreased in women prior to the onset of preeclampsia. The purpose of this study was to compare maternal soluble Flt-1 and PlGF levels in the second trimester with a marker of abnormal placentation, abnormal uterine artery Doppler (UAD). Method: A prospective cohort of women, 16 to 24 weeks estimated gestational age (EGA), with singleton pregnancies, underwent UAD and phlebotomy. Maternal soluble Flt-1 and free PlGF were measured by ELISA in samples from women with abnormal UAD with a group, controlled for EGA, with normal UAD. Mann-Whitney Rank-Sum test was used to compare maternal serum levels of both soluble Flt-1 and PlGF between women with abnormal uterine artery Doppler versus women with normal uterine artery Doppler. Results: Of the 222 study subjects enrolled, 34 (15%) had abnormal UAD. The mean EGA at enrollment of subjects in each group was 18 weeks. There was no difference in PlGF between subjects with abnormal UAD (median, 191 pg/mL; range, 187 to 337 pg/mL) versus controls (median, 171 pg/mL; range, 169 to 289 pg/mL) (p = 0.59) or soluble Flt-1 (median, 780 pg/mL; range, 280 to 3200 pg/mL) or between subjects with abnormal UAD versus controls (median, 720 pg/mL; range, 220 to 1980 pg/mL) (p = 0.36). Conclusion: Concentrations of maternal soluble Flt-1 and free PlGF in the second trimester do not appear to be altered in women with abnormal UAD. This suggests that these biochemical markers are independent of the increased placental resistance seen with abnormal uterine artery Doppler.

Insulin-like growth factor binding protein-4 (IGFBP-4) is a novel anti-angiogenic and anti-tumorigenic mediator secreted by dibutyryl cyclic AMP (dB-cAMP)-differentiated glioblastoma cells

cAMP has been shown to reverse the transformed phenotype of various cancer cells. Human glioblastoma U87MG cells exposed to 500 microM dB-cAMP for 6 days showed reduced proliferation, attenuated invasiveness, and inability to induce angiogenic responses in human brain endothelial cells (HBECs) grown in Matrigeltrade mark. VEGF was the principal mediator of angiogenic actions of U87MG conditioned media (CM), since VEGF neutralizing antibody completely inhibited U87MG-induced angiogenic responses and no detectable levels of IGF, bFGF, and PlGF were found in U87MG CM. VEGF release was induced ( approximately 20%) in dB-cAMP-treated U87MG cells, suggesting a simultaneous induction of anti-angiogenic mediators. Down-stream effectors of dB-cAMP actions in U87MG were investigated by microarray gene expression analysis. Detected increases in differentiation genes, staniocalcin-1 and Wnt-5a, and angiogenesis-related genes, PAI-1, SPARC, IGFBP-4, IGFBP-7, PAPP-A, and PRSS-11 in dB-cAMP-treated U87MG cells were validated by real-time PCR, Western blot, and/or ELISA. A subsequent series of experiments identified IGFBP-4 as the principal anti-angiogenic mediator secreted by glioblastoma cells in response to dB-cAMP. Human recombinant IGFBP-4 inhibited the angiogenic response of HBEC induced by U87MG CM, whereas anti-human IGFBP-4 antibody restored the pro-angiogenic activity of dB-cAMP-treated U87MG CM. Since neither U87MG nor HBEC cells secreted detectable levels of IGF-I, and there are no known cellular IGFBP-4 receptors, the anti-angiogenic effect of IGFBP-4 was likely IGF-I-independent and indirect. IGFBP-4 also antagonized angiogenic effects of VEGF(165), PlGF, and bFGF, and reduced U87MG colony formation in soft-agar. IGFBP-4 is a novel dB-cAMP-induced anti-angiogenic and anti-tumorigenic mediator that may be a promising candidate for glioblastoma therapy.

Protein microarray analysis of disease activity in pediatric inflammatory bowel disease demonstrates elevated serum PLGF, IL-7, TGF-beta1, and IL-12p40 levels in Crohn's disease and ulcerative colitis patients in remission versus active disease.

Cytokines and growth factors play a major role in the dysregulated immune response in inflammatory bowel disease (IBD). We hypothesized that significant differences exist between the serum cytokine and growth factor profiles of pediatric IBD patients with active disease (AD) and those in remission, and that levels of some of these soluble mediators may be used to define regulators in IBD and determine disease activity. Eighty-eight consecutive patients with confirmed Crohn's disease (CD) and ulcerative colitis (UC) seen at the Duke Children's Hospital were prospectively enrolled and a serum sample was obtained. Data were recorded at the time of serum collection to calculate disease activity indices. The relative expression of 78 cytokines, growth factors, and soluble receptors was determined using proprietary antibody-based protein microarrays amplified by rolling circle amplification. SPSS 8 (SPSS Inc., Chicago, IL) was used to compare protein profiles for CD and UC patients in clinical remission (CR) versus AD. Sixty-five CD patients and 23 UC patients were enrolled. Forty-one CD patients had available samples and PCDAI results. Twenty-two patients were in remission PCDAI < or = 12.5 (median 5), 19 patients had disease activity >15 (median 30). Univariate analysis revealed that PLGF, IL-7, IL-12p40, and TGF-beta1 cytokine levels were significantly elevated for patients in CR versus AD (p < 0.01). Twelve UC serum samples had Seo/Truelove Witt AI for analysis. Five patients were in remission by TW AI and Seo AI < or =110 and 7 patients had active mild-to-severe disease by TW and Seo AI >110. Only one cytokine, IL12p40, showed significance between CR versus AD (p < 0.02). Surprisingly, we found no differences in circulating levels of proinflammatory cytokines but found that pediatric IBD patients in remission compared to those with AD had higher levels of specific circulating cytokines, including the regulatory cytokines IL-12p40 and TGF-beta1. It may be that these cytokines directly regulate intestinal inflammation in IBD or reflect the activity of T regulatory cells in negatively regulating the inflammatory response. Further studies will be needed to validate our results to define the molecular pathways involved in the intestinal immune response in man.

Maternal plasma VEGF, sVEGF-R1, and PlGF concentrations in preeclamptic and normotensive pregnant Zimbabwean women.

Vascular endothelial growth factor (VEGF), a disulphide-linked homodimeric glycoprotein that is selectively mitogenic for endothelial cells, plays an important role in vasculogenesis and angiogenesis. Preeclampsia, a relatively common complication of pregnancy that is characterized by diffuse endothelial dysfunction possibly secondary to impaired trophoblast invasion of the spiral arteries during implantation, has recently been associated with alterations in maternal serum/plasma concentrations of VEGF, and other related growth factors and their receptors. We examined the relationship of maternal plasma VEGF, sVEGF-R1 and PlGF levels to the risk of preeclampsia among women delivering at Harare Maternity Hospital, Zimbabwe. 131 pregnant women with preeclampsia and 175 controls were included in a case-control study. Maternal plasma concentrations of each biomarker were measured using enzymatic methods. We used logistic regression to calculate odds ratios (OR) and 95 % confidence intervals (CI). Preeclampsia risk was inversely related with quartiles of plasma VEGF (OR: 1.0, 1.0, 0.7, and 0.5, with the lowest quartile as reference; p for trend = 0.06). We noted a strong positive association between preeclampsia risk and sVEGF-R1 concentrations (OR: 1.0, 6.5, 9.7, 31.6, with the first quartile as the referent group; p for trend &lt; 0.001). After adjusting for confounders, we noted that women with sVEGF-R1 concentrations in the highest quartile (≥ 496 pg/ml), as compared with those in the lowest quartile (&lt; 62 pg/ml) had a 31.6-fold increased risk of preeclampsia (OR = 31.6, 95 % CI 7.7-128.9). There was no clear evidence of a linear relation in risk of preeclampsia with PlGF concentrations. In conclusion, plasma VEGF, sVEGF-R1 and PlGF concentrations (measured at delivery) were altered among Zimbabwean women with preeclampsia as compared with normotensive women. Our results are consistent with some, though not all, previous reports. Prospective studies are needed to: 1) identify modifiable determinants of maternal plasma concentrations VEGF, sVEGF-R1, and PlGF; and 2) evaluate the temporal relationship between observed alterations of these biological markers in preeclamptic pregnancies.

SFlt-1, VEGF, and PLGF levels in the cerebrospinal fluid of women with preeclampsia

sFlt-1, VEGF, and PLGF levels in the cerebrospinal fluid of women with preeclampsia

Insulin resistance and alterations in angiogenesis: additive insults that may lead to preeclampsia.

Altered angiogenesis and insulin resistance, which are intimately related at a molecular level, characterize preeclampsia. To test if an epidemiological interaction exists between these two alterations, we performed a nested case-control study of 28 women who developed preeclampsia and 57 contemporaneous controls. Serum samples at 12 weeks of gestation were measured for sex hormone binding globulin (SHBG; low levels correlate with insulin resistance) and placental growth factor (PlGF; a proangiogenic molecule). Compared with controls, women who developed preeclampsia had lower serum levels of SHBG (208+/-116 versus 256+/-101 nmol/L, P=0.05) and PlGF (16+/-14 versus 67+/-150 pg/mL, P<0.001), and in multivariable analysis, women with serum levels of PlGF < or =20 pg/mL had an increased risk of developing preeclampsia (odds ratio [OR] 7.6, 95% CI 1.4 to 38.4). Stratified by levels of serum SHBG (< or =175 versus >175 mg/dL), women with low levels of SHBG and PlGF had a 25.5-fold increased risk of developing preeclampsia (P=0.10), compared with 1.8 (P=0.38) among women with high levels of SHBG and low levels of PlGF. Formal testing for interaction (PlGFxSHBG) was significant (P=0.02). In a model with 3 (n-1) interaction terms (high PlGF and high SHBG, reference), the risk for developing preeclampsia was as follows: low PlGF and low SHBG, OR 15.1, 95% CI 1.7 to 134.9; high PlGF and low SHBG, OR 4.1, 95% CI 0.45 to 38.2; low PlGF and high SHBG, OR 8.7, 95% CI 1.2 to 60.3. Altered angiogenesis and insulin resistance are additive insults that lead to preeclampsia.

Angiogenesis and villous development in human placenta

Fetal well being requires adequate growth and maturation of placental villous trees. During gestation new phenotypes and relationships appear: precursor villi produce stem villi (the tree trunks), terminal villi (the finest branches) and intermediate forms called immature and mature intermediate villi (IIV and MIV). Before about 20 week, the emphasis is on producing IIV and stem villi. Thereafter production switches from IIV to MIV and from these abundant terminal villi are generated. The switch depends on fetal vascular development influenced by local O2 tensions, growth factor receptors and their ligands (notably vascular endothelial growth factor, VEGF, and placental growth factor, PlGF). From 9 to 25 week (low O2 tensions, high VEGF levels) fetal capillaries grow by branching angiogenesis. Between 15 and 32 week (high O2 tensions, low VEGF, high PlGF levels) they grow via nonbranching angiogenesis (linear growth of existing capillaries). Quantitative measures of these differences have been made using indices of villous and vessel growth (volumes, surfaces, lengths; numbers of endothelial cells) coupled with estimators of vessel calibre (mean cross sectional area) and endothelial cell size (squame surface area). Estimates were obtained stereologically using randomly sampled histological wax sections of placentas from 10 to 41 week. Here the following definitions apply: the term TV embraces true terminal villi plus some of the thinner MIV, intermediate villus (IV) includes IIV and MIV, whilst stem villus includes stem villi and larger IIV. Results were analysed by regression analysis and analysis of variance on untransformed or log transformed data. From 10 week, all villous compartments grew but the volume of capillaries expanded disproportionately to stroma and trophoblast. Capillary growth was proliferative and essentially linear (i.e. not due to increases in calibre). The volume of TV altered dramatically but the increase in IV volume was modest and the volume of stem villi did not alter significantly. During villous growth TV and IV became longer and thinner with larger surface areas. For most variables gestational changes showed inflection points at 20–25 week and growth was faster after the inflection point. Exceptionally, IV volume increased up to 24 week but not thereafter. In addition capillary to villus length ratios displayed 2 growth phases: the first (peaking before 20 week) was followed by a sharp drop preceding gradual restoration of peak values by term. In the first phase the mean area of an endothelial squame was maintained but squame size increased during the second phase. These findings confirm and extend the spatiotemporal patterns of change identified by qualitative studies and are consistent with branching and nonbranching angiogenesis being important phases in villous development.

Human Herpesvirus-8-Transformed Endothelial Cells Have Functionally Activated Vascular Endothelial Growth Factor/Vascular Endothelial Growth Factor Receptor

Kaposi's sarcoma is a vascular tumor commonly associated with human immunodeficiency virus (HIV)-1 and human herpesvirus (HHV-8) also known as Kaposi's sarcoma-associated herpesvirus. The principal features of this tumor are abnormal proliferation of vascular structures lined with spindle-shaped endothelial cells. HHV-8 may transform a subpopulation of endothelial cells in vitro via viral and cellular gene expression. We hypothesized that among the cellular genes, vascular endothelial growth factors (VEGFs) and their cognate receptors may be involved in viral-mediated transformation. We have shown that HHV-8-transformed endothelial cells (EC-HHV-8) express higher levels of VEGF, VEGF-C, VEGF-D, and PlGF in addition to VEGF receptors-1, -2, and -3. Furthermore, antibodies to VEGF receptor-2 inhibited cell proliferation and viability. Similarly, inhibition of VEGF gene expression with antisense oligonucleotides inhibited EC-HHV-8 cell proliferation/viability. The growth and viability of primary endothelial cells and a fibroblast cell line however were unaffected by either the VEGF receptor-2 antibody or the VEGF antisense oligodeoxynucleotides. VEGF and VEGF receptors are thus induced in EC-HHV-8 and participate in the transformation. Inhibitors of VEGF may thus modulate the disease process during development and progression.

Isolation by immobilized artificial membrane chromatography (IAM-PC) and partial identification of potent biliary cholesterol crystallization inhibitor proteins

Intense inflammatory response and an anti-angiogenic state have been implicated in the pathogenesis of preeclampsia. Here, we investigated this hypothesis evaluating the serum concentrations of CXCL10/IP-10, sFlt-1, and PlGF in women with early-onset preeclampsia. CXCL10/IP-10 was measured by Cytometric Bead Array. sFlt-1 and PlGF were measured by automated electrochemiluminescence immunoassay. The median serum concentration of CXCL10/IP-10 was 109.5 pg/mL in preeclamptic women, as compared with 52.28 pg/mL in the controls (P = 0.0028). The mean serum level of sFlt-1 was 13,636 pg/mL in preeclamptic women, as compared with 2020 pg/mL in the controls (P < 0.0001). PlGF levels were significantly lower in women with preeclampsia.