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1.  Low-Molecular Weight Heparin Increases Circulating sFlt-1 Levels and Enhances Urinary Elimination 
PLoS ONE  2014;9(1):e85258.
Preeclampsia is a devastating medical complication of pregnancy which leads to maternal and fetal morbidity and mortality. While the etiology of preeclampsia is unclear, human and animal studies suggest that excessive circulating levels of soluble fms-like tyrosine-kinase-1 (sFlt-1), an alternatively spliced variant of VEGF-receptor1, contribute to the signs and symptoms of preeclampsia. Since sFlt-1 binds to heparin and heparan sulfate proteoglycans, we hypothesized that the anticoagulant heparin, which is often used in pregnancy, may interfere with the levels, distribution and elimination of sFlt-1 in vivo.
We systematically determined serum and urine levels of angiogenic factors in preeclamptic women before and after administration of low molecular weight heparin and further characterized the interaction with heparin in biochemical studies.
Methods and Results
Serum and urine samples were used to measure sFlt-1 levels before and after heparin administration. Serum levels of sFlt-1 increased by 25% after heparin administration in pregnant women. The magnitude of the increase in circulating sFlt-1 correlated with initial sFlt-1 serum levels. Urinary sFlt-1 levels were also elevated following heparin administration and levels of elimination were dependent on the underlying integrity of the glomerular filtration barrier. Biochemical binding studies employing cation exchange chromatography revealed that heparin bound sFlt-1 had decreased affinity to negatively charged surfaces when compared to sFlt-1 alone.
Low molecular weight heparin administration increased circulating sFlt1 levels and enhanced renal elimination. We provide evidence that both effects may be due to heparin binding to sFlt1 and masking the positive charges on sFlt1 protein.
PMCID: PMC3897409  PMID: 24465515
2.  Preeclampsia, gestational hypertension and subsequent hypothyroidism 
Pregnancy hypertension  2012;3(1):21-27.
To evaluate the effect of preeclampsia (PE) and gestational hypertension (GH) on subsequent hypothyroidism. Recent studies suggest that women with PE have increased risk for reduced thyroid function, but the association between PE and GH with overt hypothyroidism has not been examined.
Study design
Two prospective population-based cohort studies, the Northern Finland Birth Cohorts 1966 and 1986, followed women who had PE (N=955), GH (N=1449) or were normotensive (N=13531) during pregnancy. Finnish national registers were used to confirm subsequent hypothyroidism. Adjusted hazard ratios (aHRs) with 95% confidence intervals (CIs) estimated hypothyroidism risk when comparing women with PE or GH with normotensive women.
Main outcome measures
Primary hypothyroidism during follow-up of 20–40 years.
The subsequent prevalence of hypothyroidism was higher among women with PE (4.0%) and GH (4.5%) compared with normotensive women (3.5%), but the risk increase was not significant (aHR for PE 1.13, 95%CI 0.80–1.59 and aHR for GH 1.11, 95%CI 0.85–1.45).
Subgroup analysis among nulliparous women revealed a significant association between late PE and subsequent hypothyroidism (aHR 1.82, 95%CI 1.04–3.19).
Early or recurrent PE were not associated with hypothyroidism (aHR 0.93, 95%CI 0.46–1.81 and aHR 1.35, 95%CI 0.63–2.88, respectively).
Overall, PE or GH during pregnancy was not significantly associated with subsequent hypothyroidism in Finnish women after 20–40 years of follow-up. However, late PE in nulliparous women was associated with a 1.8-fold increased risk of subsequent hypothyroidism, a finding that merits further study in other populations.
PMCID: PMC3578699  PMID: 23439671
preeclampsia; gestational hypertension; thyroid; hypothyroidism
3.  VEGF-C, VEGF-A and related angiogenesis factors as biomarkers of allograft vasculopathy in cardiac transplant recipients 
Cardiac allograft vasculopathy (CAV), the major cause of late allograft loss after cardiac transplantation, results from donor-directed cellular and humoral alloimmune responses. Graft vascular endothelial cells (EC) are primary targets of these destructive responses, suggesting that factors associated with endothelial injury and repair could serve as biomarkers of CAV.
Using a protein profiler array platform, we measured the levels of 55 angiogenesis-related proteins in sera from 33 adult heart transplant recipients, including 17 with angiographically documented CAV and 16 age- and gender-matched controls without CAV. All patients were >2 years after heart transplant.
The study population was 75% male with a mean age of 62 ± 11 years. On average, patients were 12 ± 5 years after heart transplantation. We found that vascular endothelial growth factor (VEGF)-C, VEGF-A, angiopoietin-2, artemin, urokinase-type plasminogen activator and vasohibin were strongly associated with established CAV (all p < 0.01). Multivariable modeling identified VEGF-C, VEGF-A and platelet factor-4 (PF-4) as significant independent biomarkers of CAV. Furthermore, receiver-operating characteristic curve analysis demonstrated that the combination of all 3 molecules provided outstanding performance for the diagnosis of CAV (area under the curve [AUC] = 0.98; p < 0.001).
Serum levels of VEGF-C, VEGF-A and PF-4 demonstrate strong associations with established CAV and, together with related angiogenesis factors, may serve as a reliable, non-invasive diagnostic test for CAV in cardiac transplant recipients.
PMCID: PMC3597743  PMID: 23260712
heart transplantation; coronary artery disease; angiogenic proteins; biologic markers
4.  Angiopoietin-2 may contribute to multi-organ dysfunction and death in sepsis 
Critical care medicine  2012;40(11):3034-3041.
In sepsis, quiescent blood vessels become leaky and inflamed by mechanisms that are incompletely understood. We hypothesized that Angiopoietin-2 (Angpt-2), a partial antagonist of the endothelium-stabilizing receptor Tie-2 secreted by endothelium, contributes to adverse outcomes in this disease.
Laboratory and animal research
Research laboratories and Emergency Department of Beth Israel Deaconess Medical Center, Boston, MA
Angpt-2 heterozygous mice, Emergency Department patients
Mice with one functional Angpt-2 allele developed milder kidney and lung injury, less tissue inflammation, and less vascular leakage compared to wildtype counterparts. Heterozygotes experienced >40% absolute survival advantage following two different models of sepsis (p = 0.004 and 0.018). In human subjects presenting to our emergency department with suspected infection (n = 270 combined), circulating Angpt-2 was markedly elevated within the first hour of clinical care. First-hour Angpt-2 concentrations were proportional to current disease severity (p < 0.0001), rose further over time in eventual non-survivors (p < 0.0001), and predicted the future occurrence of shock (p < 0.0001) or death (p < 0.0001) in the original cohort and an independent validation group. Finally, septic human serum disrupted the barrier function of microvascular endothelial cells, an effect fully neutralized by an Angpt-2 monoclonal antibody.
We conclude that Angpt-2 induction precedes and contributes to the adverse outcomes in sepsis, opening a new avenue for therapeutic investigation.
PMCID: PMC3705559  PMID: 22890252
sepsis; multi-organ dysfunction; angiopoietin-2; Tie2; endothelium; vasculature
5.  Clinical Characterization and Outcomes of Preeclampsia with Normal Angiogenic Profile 
The objective of this study was to compare the clinical characteristics and outcomes of preeclamptic women presenting with a normal plasma angiogenic profile with those subjects that are characterized by an abnormal angiogenic profile.
This was a secondary analysis of a prospective cohort study in women presenting to obstetrical triage at <37 weeks of gestation and diagnosed with preeclampsia within 2 weeks of enrollment and in whom angiogenic factors (sFlt1 and PlGF) measurements were available. Patients were divided into two groups based on their circulating levels of these factors described as a ratio the sFlt1/PlGF ratio, non-angiogenic preeclampsia (sFlt1/PlGF ratio <85) and angiogenic preeclampsia (sFlt1/PlGF ratio ≥85). The data are presented by sFlt1/PlGF category using median and quartile 1-quartile 3 for continuous variables and by frequency and sample sizes for categorical variables.
In our cohort the patients with non-angiogenic preeclampsia (N=46) were more obese {BMI - 35.2 (31.6,38.7) vs. 31.1(28.0,39.0), p=0.04}, more likely to have preexisting diabetes (21.7% vs. 2.0%, p=0.002) and presented at a later gestational age {35 (32,37) vs. 32 (29,34) weeks, p<0.0001} as compared to women with angiogenic preeclampsia (N=51). Women with non-angiogenic preeclampsia had no serious adverse outcomes {Elevated LFT's/Low platelets: 0% vs. 23.5%, abruption: 0% vs. 9.8%, pulmonary edema: 0% vs. 3.9%, eclampsia: 0% vs. 2.0 %, small for gestational age: 0% vs. 17.7% and fetal/neonatal death: 0% vs. 5.9%} as compared to women with angiogenic preeclampsia. The rate of preterm delivery <34 weeks was 8.7% in non-angiogenic preeclampsia compared to 64.7% in angiogenic preeclampsia (p<0.0001). Interestingly, delivery between 34-37 weeks and resource utilization (hospital admission days) were similar in the two groups.
In contrast to the angiogenic form, the non-angiogenic form of preeclampsia is characterized by little to no risk of preeclampsia related adverse outcomes, other than iatrogenic prematurity. Incorporation of angiogenic biomarkers in the evaluation of preeclampsia may allow accurate and early identification of severe disease.
PMCID: PMC3744824  PMID: 23725084
non-angiogenic preeclampsia; angiogenic factors; adverse outcomes
6.  Conversion of peripheral blood NK cells to a decidual NK-like phenotype by a cocktail of defined factors 
Natural killer (NK) cells that populate the decidua are important regulators of normal placentation. In contrast to peripheral blood NK (pNK) cells, decidual NK cells (dNK) lack cytotoxicity, secrete pro-angiogenic factors and regulate trophoblast invasion. Here we show that exposure to a combination of hypoxia, transforming growth factor beta 1, and a demethylating agent, results in NK cells that express Killer cell Immunoglobulin like Receptors, the dNK cell markers CD9 and CD49a, and dNK pattern of chemokine receptors. These cells secrete vascular endothelial growth factor, a potent pro-angiogenic molecule, display reduced cytotoxicity and promote invasion of human trophoblast cell lines. These findings have potential therapeutic applications for placental disorders associated with altered NK cell biology.
PMCID: PMC3742368  PMID: 23487420
8.  First Trimester Vitamin D, Vitamin D Binding Protein, and Subsequent Preeclampsia 
Hypertension  2010;56(4):758-763.
Prior studies report an association between vitamin D deficiency and hypertension, including the pregnancy-specific disorder, preeclampsia. Circulating vitamin D is almost entirely bound to vitamin D binding protein, which increases 2-fold during pregnancy, but previous studies have not examined vitamin D binding protein or free vitamin D levels. We performed a nested case-control study within the Massachusetts General Hospital Obstetrical Maternal Study (MOMS), measuring first trimester total 25-hydroxyvitamin D and vitamin D binding protein, and calculating free 25-hydroxyvitamin D levels. We compared these levels from pregnancies complicated by subsequent preeclampsia (cases, n=39) with those from normotensive pregnancies (controls, n=131). First trimester total 25-hydroxyvitamin D levels were similar in cases and controls (27.4 ±1.9 ng/ml vs. 28.8±0.80 ng/ml, p=0.435). Despite an association between higher first trimester blood pressures and subsequent preeclampsia, first trimester total 25-hydroxyvitamin D was not associated with first trimester systolic (r=0.11, p=0.16) or diastolic blood pressures (r=0.03, p=0.72). Although there was a trend toward increased risk of preeclampsia with 25-hydroxyvitamin D levels less than 15 ng/ml (OR 2.5, 95% CI 0.89-6.9), this was attenuated after adjustment for body mass index and other covariates (OR 1.35 95% CI 0.40-4.5). First trimester vitamin D binding protein and free 25-hydroxyvitamin D levels were similar in cases and controls and were not associated with first trimester blood pressures. These data suggest that first trimester total and free 25-hydroxyvitamin D levels are not independently associated with first trimester blood pressure or subsequent preeclampsia.
PMCID: PMC3775612  PMID: 20733087
vitamin D; vitamin D Binding Protein; preeclampsia; pregnancy
9.  Carbamylation of Serum Albumin as a Risk Factor for Mortality in Patients with Kidney Failure 
Science translational medicine  2013;5(175):175ra29.
Urea, the toxic end-product of protein catabolism, is elevated in end-stage renal disease (ESRD), although it is unclear whether or how it contributes to disease. Urea can promote the carbamylation of proteins on multiple lysine side chains, including human albumin, which has a predominant carbamylation site on lysine 549. The proportion of serum albumin carbamylated on Lys-549 (%C-Alb) correlated with time-averaged blood urea concentrations and was twice as high in ESRD patients than in non-uremic subjects (0.90% vs. 0.42%, P<0.0001). Baseline %C-Alb was higher in ESRD subjects who died within 1-year than in those who survived longer than 1 year (1.01% vs. 0.77%, P<0.001) and was associated with an increased risk of death within 1 year (HR of 3.76, 95% CI: 2.20–6.43, P<0.0001). These findings were validated in an independent cohort of diabetic ESRD subjects (HR 3.73, 95% CI: 2.00–6.96, P<0.001). Decreased concentrations of serum amino acids correlated with higher %C-Alb in ESRD patients, and mice with diet-induced amino acid deficiencies exhibited greater susceptibility to albumin carbamylation than did chow-fed mice. In vitro studies showed that amino acids such as cysteine, histidine, arginine, lysine, as well as other nucleophiles such as taurine, inhibited cyanate-induced C-Alb formation at physiologic pH and temperature. Together, these results suggest that chronically elevated urea promotes carbamylation of proteins in ESRD, and that serum amino acid concentrations may modulate this protein modification. In summary, we have identified serum %C-Alb as a risk factor for mortality in patients with ESRD and propose that this risk factor may be modifiable with supplemental amino acid therapy.
PMCID: PMC3697767  PMID: 23467560
10.  Two Variants of the C-Reactive Protein Gene Are Associated with Risk of Pre-Eclampsia in an American Indian Population 
PLoS ONE  2013;8(8):e71231.
The etiology of pre-eclampsia (PE) is unknown; but it is accepted that normal pregnancy represents a distinctive challenge to the maternal immune system. C-reactive protein is a prominent component of the innate immune system; and we previously reported an association between PE and the CRP polymorphism, rs1205. Our aim was to explore the effects of additional CRP variants. The IBC (Cardiochip) genotyping microarray focuses on candidate genes and pathways related to the pathophysiology of cardiovascular disease.
This study recruited 140 cases of PE and 270 matched controls, of which 95 cases met criteria as severe PE, from an American Indian community. IBC array genotypes from 10 suitable CRP SNPs were analyzed. A replication sample of 178 cases and 427 controls of European ancestry was also genotyped.
A nominally significant difference (p value <0.05) was seen in the distribution of discordant matched pairs for rs3093068; and Bonferroni corrected differences (P<0.005) were seen for rs876538, rs2794521, and rs3091244. Univariate conditional logistic regression odds ratios (OR) were nominally significant for rs3093068 and rs876538 models only. Multivariate logistic models with adjustment for mother's age, nulliparity and BMI attenuated the effect (OR 1.58, P = 0.066, 95% CI 0.97–2.58) for rs876538 and (OR 2.59, P = 0.050, 95% CI 1.00–6.68) for rs3093068. An additive risk score of the above two risk genotypes shows a multivariate adjusted OR of 2.04 (P = 0.013, 95% CI 1.16–3.56). The replication sample also demonstrated significant association between PE and the rs876538 allele (OR = 1.55, P = 0.01, 95% CI 2.16–1.10). We also show putative functionality for the rs876538 and rs3093068 CRP variants.
The CRP variants, rs876538 and rs3093068, previously associated with other cardiovascular disease phenotypes, show suggestive association with PE in this American Indian population, further supporting a possible role for CRP in PE.
PMCID: PMC3733916  PMID: 23940726
11.  Circulating Lymphangiogenic Factors in Preeclampsia 
Preeclampsia, a human pregnancy specific disorder is characterized by an anti-angiogenic state due to high levels of circulating soluble vascular endothelial growth factor 1 (sVEGFR-1). However, the role of lymphangiogenesis in preeclampsia has not been investigated. Recently, impaired VEGF-C (factor that regulates lymphangiogenesis) signalling has been implicated in the pathogenesis of interstitial edema and salt-sensitive hypertension. Therefore, we hypothesized that circulating VEGF-C and its circulating receptors (sVEGFR-2 and sVEGFR-3) may also be altered in preeclampsia and correlate with the severity of the phenotype.
We analyzed plasma levels of VEGF-C, sVEGFR-1, sVEGFR-2 and sVEGFR-3 in women with gestational hypertension (GHTN, n=20), preeclampsia (PE, n=20) and normotensive pregnancies (NP, n=20) in the third trimester and values reported as mean ± SD in pg/ml.
As previously reported, sVEGFR-1 levels were significantly higher in subjects with PE (19938 ± 12973) than in GHTN (7156 ± 5432), p<0.01 or NP (7760 ± 6018), p<0.01. VEGF-C levels were lower in subjects with GHTN (676 ± 323) than in PE (1335 ± 625), p<0.01, but not statistically different than in NP (971 ± 556), p=0.11. There was a trend towards lower sVEGFR-2 in PE as compared to GHTN or NP. Interestingly sVEGFR-3 was significantly lower in PE (54371 ± 21107) as compared to NP (83709 ± 24983), p<0.01, but not different as compared to GHTN (54642 ± 26947). The ratio of sVEGFR-2+sVEGFR-3/VEGF-C was dramatically lower during PE (57 ± 38) as compared to GHTN (113 ± 72), p<0.01 or NP (133 ± 91), p<0.01.
Preeclampsia is characterized by circulating pro-lymphangiogenic state as evidenced by decreased sVEGFR-3, slightly decreased sVEGFR-2, increased VEGF-C and a dramatically lower ratio of sVEGFR-2+sVEGFR-3/VEGF-C. Our data suggests that the circulating pro-lymphoangiogenic state during preeclampsia may be a compensatory response to edema and hypertension. Additional studies are needed to evaluate the clinical relevance of the altered lymphangiogenic signalling pathway during preeclampsia.
PMCID: PMC3570685  PMID: 22957504
Preeclampsia; VEGF-C; sVEGFR-2; sVEGFR-3; Lymphangiogenesis
12.  First trimester PAPP-A levels correlate with sFlt-1 levels longitudinally in pregnant women with and without preeclampsia 
First trimester Pregnancy Associated Plasma Protein A (PAPP-A) levels, routinely measured for aneuploidy screening, may predict development of preeclampsia. This study tests the hypothesis that first trimester PAPP-A levels correlate with soluble fms-like tyrosine kinase-1 (sFlt-1) levels, an angiogenic marker associated with preeclampsia, throughout pregnancy.
sFlt-1 levels were measured longitudinally in 427 women with singleton pregnancies in all three trimesters. First trimester PAPP-A and PAPP-A Multiples of Median (MOM) were measured. Student’s T and Wilcoxon tests compared preeclamptic and normal pregnancies. A linear mixed model assessed the relationship between log PAPP-A and serial log sFlt-1 levels.
PAPP-A and PAPP-A MOM levels were significantly lower in preeclamptic (n = 19), versus normal pregnancies (p = 0.02). Although mean third trimester sFlt-1 levels were significantly higher in preeclampsia (p = 0.002), first trimester sFlt-1 levels were lower in women who developed preeclampsia, compared with normal pregnancies (p = 0.03). PAPP-A levels correlated significantly with serial sFlt-1 levels. Importantly, low first trimester PAPP-A MOM predicted decreased odds of normal pregnancy (OR 0.2, p = 0.002).
Low first trimester PAPP-A levels suggests increased future risk of preeclampsia and correlate with serial sFlt-1 levels throughout pregnancy. Furthermore, low first trimester PAPP-A status significantly predicted decreased odds of normal pregnancy.
PMCID: PMC3623663  PMID: 23557166
13.  Second trimester anti-angiogenic proteins and preeclampsia 
Pregnancy hypertension  2012;2(2):158-163.
To measure the relationships between soluble fms-like tyrosine kinase-1 (sFlt1), soluble endoglin (sEng) and preeclampsia.
Study design
We utilized a nested case-control study comprised of 211 preeclamptic women and 213 normotensive women with primiparous singleton pregnancies enrolled from ≥13 and <27 gestational weeks among the Danish National Birth Cohort of 100,000 women. Relationships between sFlt1, sEng and preeclampsia were estimated using smoothing splines in generalized linear models, adjusting for maternal age, body mass index, pre-existing hypertension, smoking, and gestational age.
Main outcome measures
Preeclampsia was confirmed by an International Classification of Diseases (ICD) discharge diagnosis of 637.03, 637.04 637.09, 637.19 (ICD-8) or DO14 to DO15 (ICD-10) in the National Hospital Discharge Registry. In this sample, few cases delivered small for gestational age infants (8.1%) and the mean gestational age at delivery was term (38.2 ± 2.3 weeks).
Doublings in the expressions of sFlt1 and sEng were associated with 39% (95% CI = 3%, 86%) and 74% (95% CI = 1%, 198%) increased risks of preeclampsia respectively.
We conclude that second trimester high sFlt1 and sEng levels were possibly associated with an increased risk of preeclampsia after adjustment for maternal factors traditionally associated with the syndrome.
PMCID: PMC3375839  PMID: 22712058
14.  Transcriptional Patterns in Peritoneal Tissue of Encapsulating Peritoneal Sclerosis, a Complication of Chronic Peritoneal Dialysis 
PLoS ONE  2013;8(2):e56389.
Encapsulating peritoneal sclerosis (EPS) is a devastating complication of peritoneal dialysis (PD), characterized by marked inflammation and severe fibrosis of the peritoneum, and associated with high morbidity and mortality. EPS can occur years after termination of PD and, in severe cases, leads to intestinal obstruction and ileus requiring surgical intervention. Despite ongoing research, the pathogenesis of EPS remains unclear. We performed a global transcriptome analysis of peritoneal tissue specimens from EPS patients, PD patients without EPS, and uremic patients without history of PD or EPS (Uremic). Unsupervised and supervised bioinformatics analysis revealed distinct transcriptional patterns that discriminated these three clinical groups. The analysis identified a signature of 219 genes expressed differentially in EPS as compared to PD and Uremic groups. Canonical pathway analysis of differentially expressed genes showed enrichment in several pathways, including antigen presentation, dendritic cell maturation, B cell development, chemokine signaling and humoral and cellular immunity (P value<0.05). Further interactive network analysis depicted effects of EPS-associated genes on networks linked to inflammation, immunological response, and cell proliferation. Gene expression changes were confirmed by qRT-PCR for a subset of the differentially expressed genes. EPS patient tissues exhibited elevated expression of genes encoding sulfatase1, thrombospondin 1, fibronectin 1 and alpha smooth muscle actin, among many others, while in EPS and PD tissues mRNAs encoding leptin and retinol-binding protein 4 were markedly down-regulated, compared to Uremic group patients. Immunolocalization of Collagen 1 alpha 1 revealed that Col1a1 protein was predominantly expressed in the submesothelial compact zone of EPS patient peritoneal samples, whereas PD patient peritoneal samples exhibited homogenous Col1a1 staining throughout the tissue samples. The results are compatible with the hypothesis that encapsulating peritoneal sclerosis is a distinct pathological process from the simple peritoneal fibrosis that accompanies all PD treatment.
PMCID: PMC3572070  PMID: 23418565
15.  Hemodynamic, Vascular, and Reproductive Impact of FMS-Like Tyrosine Kinase 1 (FLT1) Blockade on the Uteroplacental Circulation During Normal Mouse Pregnancy1 
Biology of Reproduction  2011;86(2):57.
To investigate the role of FMS-like tyrosine kinase 1 (FLT1, also known as VEGFR1) signaling during pregnancy, mice were injected with anti-FLT1 neutralizing antibody (Ab) beginning on Gestational Day 8 or 12 and every other day thereafter until Day 18; vehicle-only injected mice served as controls. Uterine artery blood flow was measured with ultrasound on Days 13 and 18, and morphometric measurements of the uterine arcade were carried out on Day 19 to provide a measure of gestational vascular remodeling; reproductive performance was evaluated by determining litter size, resorption rates, and pup and placental weights. Ab injections beginning on Day 8 or Day 12 resulted in significant reductions of uterine artery peak systolic and diastolic flows at Days 13 and 18. In addition, normal reproductive function was compromised, as evidenced by a significant reduction in average number of viable pups along with enhanced resorption rates. Reproductive performance was also significantly compromised in this group, although less severely. There was no evidence of a reduction in main uterine artery diameters, though arterial distensibility was reduced, and the diameter of the main uterine vein was significantly smaller in the Ab-injected mice. Significant reductions in main uterine artery and segmental artery length were also noted. Placental and pup weights were similar in all the groups. FLT1 inhibition during murine pregnancy impaired blood flow to the fetal-placental unit, compromised several indices of vascular remodeling, reduced fecundity, and increased fetal reabsorptions. The effects of FLT1 inhibition are most pronounced when targeted during early pregnancy.
Systemic inhibition of FLT1 signaling during normal murine pregnancy is associated with significant fetal loss, abnormal uteroplacental blood flow, and altered remodeling and biomechanical properties of the uterine circulation.
PMCID: PMC3290673  PMID: 22075472
angiogenesis; FLT1; pregnancy; sFLT1; ultrasound; uterine artery; uterine vein; vascular remodeling; VEGF
16.  Circulating angiogenic factors and risk of adverse maternal and perinatal outcomes in twin pregnancies with suspected preeclampsia 
Hypertension  2012;60(2):10.1161/HYPERTENSIONAHA.112.195065.
To evaluate whether angiogenic factor levels correlate with preeclampsia-related adverse maternal and perinatal outcomes in women with twin pregnancy, we studied 79 women with suspected preeclampsia in the 3rd trimester. Anti-angiogenic soluble fms-like tyrosine kinase 1 (sFlt1) and pro-angiogenic placental growth factor (PlGF) were measured at presentation on an automated platform. An adverse outcome was defined as hemolysis elevated liver enzymes and low platelets syndrome; disseminated intravascular coagulation; abruption; pulmonary edema; cerebral hemorrhage; maternal, fetal and neonatal death; eclampsia; acute renal failure; small for gestational age; and indicated delivery. All outcomes were ascertained 2 weeks after initial evaluation. Comparing the 52 (65.8%) women who experienced an adverse outcome to the 27 (34.2%) women without an adverse outcome, the median sFlt1 was elevated [11461.5 pg/ml (8794.0–14847.5) versus 7495.0 pg/ml (3498.0–10482.0, P=0.0004], PlGF was reduced [162.5 pg/ml (98.0–226.5) versus 224.0 pg/ml (156.0–449.0), P=0.005] and sFlt1/PlGF ratio was elevated [74.2 (43.5–110.5) versus 36.2 (7.1–71.3), P=0.0005]. Among those presenting <34 weeks (n=40), the difference in sFlt1/PlGF ratio was more striking [97.7 (76.6–178.1) versus 31.7 (6.5–48.7); P=0.001]. Addition of sFlt1/PlGF to the highest systolic blood pressure and proteinuria improved prediction of adverse outcomes. We conclude that in women with twin pregnancy and suspected preeclampsia, the sFlt1/PlGF ratio at the time of initial evaluation is associated with subsequent adverse maternal and perinatal outcomes. These findings are similar to singleton pregnancies and may implicate common pathogenic pathways.
PMCID: PMC3432569  PMID: 22753210
angiogenic factors; twin pregnancy; preeclampsia; adverse outcomes
17.  Correlation of Cystatin-C with Glomerular Filtration Rate by Inulin Clearance in Pregnancy 
To test utility of cystatin-C as a marker of glomerular filtration rate during pregnancy, we performed serial correlations with inulin clearance during pregnancy and postpartum.
Twelve subjects received inulin infusions and serum cystatin-C at three time points. Pearson's correlation coefficient was calculated.
Cystatin-C levels ranged 0.66 to 1.48 mg/L during pregnancy, and 0.72 to 1.26 mg/L postpartum. Inulin clearance ranged 130 to 188 ml/min during pregnancy, and 110 to 167 ml/min postpartum. Cystatin-C did not correlate with inulin clearance at any time point.
Serum cystatin-C did not correlate with inulin clearance during pregnancy or postpartum.
PMCID: PMC3536826  PMID: 22008011
cystatin-C; glomerular filtration rate; inulin; kidney function test; pregnancy
18.  The Impact of Magnesium Sulfate Therapy on Angiogenic Factors in Preeclampsia 
Pregnancy hypertension  2012;2(1):16-21.
The objective was to evaluate whether intravenous magnesium sulfate (magnesium) alters levels of angiogenic factors in women with preeclampsia.
Study Design
This was a prospective cohort study comparing women with preeclampsia treated with magnesium for seizure prophylaxis to those who were not. Serum levels of angiogenic factors, soluble fms-like tyrosine kinase 1, soluble endoglin and placental growth factor, were measured at the time of diagnosis and approximately 24 hours later. Secondary analysis compared women receiving magnesium for preeclampsia to women receiving magnesium for preterm labor. Analysis of covariance was used to compare levels at 24 hours, adjusting for levels at enrollment and potential confounders.
Angiogenic factor levels did not differ between preeclampsia groups with and without magnesium or between preeclampsia and preterm labor groups treated with magnesium (all P > 0.05).
Magnesium likely decreases seizure risk in preeclampsia by a mechanism other than altering angiogenic factor levels.
PMCID: PMC3254114  PMID: 22247820
angiogenic factors; magnesium sulfate; preeclampsia; pregnancy; toxemia
19.  Bioavailable vitamin D is more tightly linked to mineral metabolism than total vitamin D in incident hemodialysis patients 
Kidney International  2012;82(1):84-89.
Prior studies showed conflicting results regarding the association between 25-hydroxyvitamin D (25(OH)D) levels and mineral metabolism in end-stage renal disease. In order to determine whether the bioavailable vitamin D (that fraction not bound to vitamin D binding protein) associates more strongly with measures of mineral metabolism than total levels, we identified 94 patients with previously measured 25(OH)D and 1,25-dihydroxyvitamin D (1,25(OH)2D) from a cohort of incident hemodialysis patients. Vitamin D binding protein was measured from stored serum samples. Bioavailable 25(OH)D and 1,25(OH)2D were determined using previously validated formulae. Associations with demographic factors and measures of mineral metabolism were examined. When compared with whites, black patients had lower levels of total, but not bioavailable, 25(OH)D. Bioavailable, but not total, 25(OH)D and 1,25(OH)2D were each significantly correlated with serum calcium. In univariate and multivariate regression analysis, only bioavailable 25(OH)D was significantly associated with parathyroid hormone levels. Hence, bioavailable vitamin D levels are better correlated with measures of mineral metabolism than total levels in patients on hemodialysis.
PMCID: PMC3376220  PMID: 22398410
vitamin D; vitamin D binding protein; mineral bone disease; free hormone hypothesis; vitamin D deficiency; chronic kidney disease; dialysis
20.  Gestational Age, Infant Birth Weight, and Subsequent Risk of Type 2 Diabetes in Mothers: Nurses’ Health Study II 
Women with a history of gestational diabetes mellitus (GDM) are at higher risk of developing type 2 diabetes (T2DM); however, little is known about the association between other common pregnancy complications (eg, preterm birth, macrosomia) and T2DM risk. We examined the associations between first-pregnancy preterm, postterm birth, low birth weight, and macrosomia with subsequent risk of T2DM.
We conducted a prospective cohort study of Nurses’ Health Study II (NHSII) participants; 51,728 women in the study had a single live birth and complete pregnancy history. NHSII confirmed incident diabetes mellitus through supplemental questionnaires. Participants were followed from year of first birth until 2005. We defined gestational age as very preterm (20 to ≤32 weeks), moderate preterm (33 to ≤37 weeks), term (38 to ≤42 weeks), and postterm (≥43 weeks). We defined low birth weight as an infant born at term weighing less than 5.5 pounds, and we defined macrosomia as an infant born at term weighing 10 pounds or more. We used Cox proportional hazards models, adjusting for potential confounders.
Women with a very preterm birth (2%) had an increased T2DM risk (adjusted hazard ratio, 1.34; 95% confidence interval [CI], 1.05–1.71). This increased risk emerged in the decade following pregnancy. Macrosomia (1.5%) was associated with a 1.61 increased T2DM risk, after adjusting for risk factors, including GDM (95% CI, 1.24–2.08). This association was apparent within the first 5 years after pregnancy. Moderate preterm and term low birth weight did not significantly increase the risk of T2DM over the 35-year follow-up time.
Women who experienced a very preterm birth or had an infant that weighed 10 pounds or more may benefit from lifestyle intervention to reduce T2DM risk. If replicated, these findings could lead to a reduced risk of T2DM through improved primary care for women experiencing a preterm birth or an infant of nonnormal birth weight.
PMCID: PMC3780709  PMID: 24050526
21.  Cardiac Angiogenic Imbalance Leads to Peri-partum Cardiomyopathy 
Nature  2012;485(7398):333-338.
Peri-partum cardiomyopathy (PPCM) is a frequently fatal disease that affects women near delivery, and occurs more frequently in women with pre-eclampsia and/or multiple gestation. The etiology of PPCM, or why it associates with pre-eclampsia, remains unknown. We show here that PPCM is associated with a systemic angiogenic imbalance, accentuated by pre-eclampsia. Mice that lack cardiac PGC-1α, a powerful regulator of angiogenesis, develop profound PPCM. Importantly, the PPCM is entirely rescued by pro-angiogenic therapies. In humans, the placenta in late gestation secretes VEGF inhibitors like soluble Flt1 (sFlt1), and this is accentuated by multiple gestation and pre-eclampsia. This anti-angiogenic environment is accompanied by sub-clinical cardiac dysfunction, the extent of which correlates with circulating levels of sFlt1. Exogenous sFlt1 alone caused diastolic dysfunction in wildtype mice, and profound systolic dysfunction in mice lacking cardiac PGC-1α. Finally, plasma samples from women with PPCM contained abnormally high levels of sFlt1. These data strongly suggest that PPCM is in large part a vascular disease, caused by excess anti-angiogenic signaling in the peri-partum period. The data also explain how late pregnancy poses a threat to cardiac homeostasis, and why pre-eclampsia and multiple gestation are important risk factors for the development of PPCM.
PMCID: PMC3356917  PMID: 22596155
22.  Associations of Pregnancy Characteristics with Maternal and Cord Steroid Hormones, Angiogenic Factors, and Insulin-like Growth Factor Axis 
Cancer causes & control : CCC  2011;22(11):1587-1595.
The objective of this study was to comprehensively profile biological factors in pregnancy that have been postulated to be important components of the in utero environment and may also have relevance to later susceptibility to cancer and other chronic diseases.
Steroid sex hormones, IGFs, and angiogenic factors were measured in maternal and cord serum from term, normotensive pregnancies. Spearman correlations and linear regression estimated relationships among the biological factors and clinical characteristics.
The analytes were generally not correlated between maternal and fetal circulations. However, significant correlations were demonstrated among several analytes within maternal or cord samples. A few analytes were associated with clinical characteristics (e.g., maternal IGF-1and IGFBP-3 were inversely correlated with offspring birth weight, while maternal leptin and cord testosterone were positively correlated with this characteristic). Maternal androgens were higher in African-Americans than whites and maternal PlGF and soluble fms-like tyrosine kinase-1 (sFlt-1) were higher in male than female offspring.
There were significant correlations among analytes but the patterns differed depending on whether they were measured in the maternal or fetal circulation. The number and magnitude of correlations among analytes, however, should affect the design and interpretation of future studies.
PMCID: PMC3321929  PMID: 21947778
African-American; angiogenic factors; IGF; leptin; prolactin
23.  Plasma Concentrations of Soluble Endoglin versus Standard Evaluation in Patients with Suspected Preeclampsia 
PLoS ONE  2012;7(10):e48259.
The purpose of this study was to compare plasma soluble endoglin (sEng) levels with standard clinical evaluation or plasma levels of other angiogenic proteins [soluble fms-like tyrosine kinase 1 (sFlt1) and placental growth factor (PlGF)] in predicting short-term adverse maternal and perinatal outcomes in women with suspected preeclampsia presenting prior to 34 weeks.
Methods and Findings
Data from all women presenting at <34 weeks for evaluation of preeclampsia with singleton pregnancies (July 2009−October 2010) were included in this analysis and sEng levels were measured at presentation. Data was analyzed for 170 triage encounters and presented as median {25−75th centile}. Thirty-three percent of patients (56 of 170) experienced an adverse outcome. sEng levels (ng/ml) were significantly elevated in patients who subsequently experienced adverse outcomes compared to those who did not (32.3 {18.1, 55.8} vs 4.8 {3.2, 8.6}, p<0.0001). At a 10% false positive rate, sEng had higher detection rates of adverse outcomes than the combination of highest systolic blood pressure, proteinuria and abnormal laboratory tests (80.4 {70.0, 90.8} vs 63.8 {51.4, 76.2}, respectively). Subjects in the highest quartile of sEng were more likely to deliver early compared to those in the lowest quartile (HR: 14.96 95% CI: 8.73−25.62, p<0.0001). Natural log transformed sEng correlated positively with log sFlt1 levels (r = 0.87) and inversely with log PlGF levels (r = −0.79) (p<0.0001 for both). Plasma sEng had comparable area under the curve for prediction of adverse outcomes as measurement of sFlt1/PlGF ratio (0.88 {0.81, 0.95} for sEng versus 0.89 {0.83, 0.95} for sFlt1/PlGF ratio, p = 0.74).
In women with suspected preeclampsia presenting prior to 34 weeks of gestation, sEng performs better than standard clinical evaluation in detecting adverse maternal and fetal outcomes occurring within two weeks of presentation. Soluble endoglin was strongly correlated with sFlt1 and PlGF levels, suggesting common pathogenic pathways leading to preeclampsia.
PMCID: PMC3482204  PMID: 23110221
24.  Circulating anti-angiogenic factors during hypertensive pregnancy and increased risk of respiratory distress syndrome in preterm neonates 
To test the hypothesis that high circulating concentrations of maternal anti-angiogenic factors are associated with increased risk of respiratory distress syndrome (RDS).
This is a nested case-control study of nulliparous women who delivered less than 37 weeks of gestation within the Calcium for Preeclampsia Prevention (CPEP) trial. The study included 116 women with preeclampsia or gestational hypertension and 323 normotensive controls. Soluble fms-like tyrosine kinase 1 (sFlt1), placental growth factor [PlGF] and soluble endoglin [sEng] in maternal serum were measured at 21–32 weeks of gestation.
Preterm infants born to hypertensive mothers were more likely to develop RDS (22.5 % versus 20.9%, P=0.03). After adjustment for gestational age at delivery, the odds ratio for the relationship between hypertension in pregnancy and RDS was 2.18 (95% C.I. 1.08, 4.39). In hypertensive pregnancies women whose infants developed RDS had significantly higher circulating mean sFlt1 levels during mid-pregnancy (21–32 weeks of gestation) even after adjustment for gestational age at delivery (21,516 pg/mL versus 7,000 pg/mL, P = 0.01).
Preterm preeclampsia and gestational hypertension, characterized by high circulating levels of sFlt1, are associated with a twofold increased risk of RDS in infants delivered before 37 weeks. Among women with these hypertensive pregnancies circulating sFlt1 concentrations during mid-pregnancy were substantially higher in women whose infants developed RDS.
PMCID: PMC3414194  PMID: 22097923
anti-angiogenic; soluble fms-like tyrosine kinase 1; sVEGF R1; sFlt1; placental growth factor; PlGF; soluble endoglin; sEng; respiratory distress syndrome; RDS; neonate; preterm; preeclampsia; gestational hypertension
25.  Intravital high-frequency ultrasonography to evaluate cardiovascular and uteroplacental blood flow in mouse pregnancy 
Pregnancy hypertension  2011;2(2):84-92.
The objective of this study is to define the ultrasonographic changes in the cardiovascular and uteroplacental circulation of normal pregnant mice compared to non-pregnant mice using high-frequency, high-resolution ultrasonography.
Ten to twelve-week-old CD-1 mice (six non-pregnant and six pregnant animals) were used for all experiments. Vevo® 2100 (VisualSonics) was used to evaluate the cardiovascular and uteroplacental circulation physiology. Cardiac echocardiogram and uterine artery Doppler studies were performed on all animals. Pregnant animals were evaluated on embryonic day seven (E7), thirteen (E13) and eighteen (E18). Fetal heart rate and umbilical artery Doppler flows were obtained on pregnant animals. Three-dimensional ultrasonography imaging was utilized for quantification of placental volumes. All data are presented as median {10th–90th percentiles}.
In pregnant mice on E7 compared to non-pregnant mice, there was an increase in cardiac output (p=0.008), stroke volume (p=0.002), ejection fraction, (p=0.02) and fractional shortening (p=0.02). The maternal heart rate increased throughout gestation (p= 0.009). During pregnancy, a gestational sac was clearly visible on E7. Between E13 and E18, the fetal size and fetal heart rate increased (p=0.001) and the umbilical artery peak systolic velocity increased (p <0.001). Minimal diastolic blood flow was observed in the umbilical artery on E13, which increased slightly on day E18 (p=0.01). There was also no change in the uterine artery resistance index between non-pregnant and pregnant mice. The placental volume increased between E13 and E18 (p=0.03).
Several changes noted in cardiovascular and uteroplacental systems occurring during normal murine pregnancy have striking similarities to humans and can be accurately measured using newer ultrasonographic techniques. Further studies are needed to evaluate changes in these vascular beds in mouse models of diseases such as preeclampsia and intrauterine growth restriction.
PMCID: PMC3337859  PMID: 22544045
high-frequency ultrasonography; mouse pregnancy; echocardiography; uteroplacental Doppler flow

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