Bleeding patterns in patients before and after diagnosis of von Willebrand disease: Analysis of a US medical claims database.

INTRODUCTION: Von Willebrand disease (VWD) is the most common inherited bleeding disorder. The bleeding phenotype is variable, and some individuals have persistent symptoms post-diagnosis. AIM: To characterize bleeding patterns in patients with VWD before and after diagnosis. METHODS: De-identified claims data for commercially insured patients in the IQVIA PharMetrics® Plus US database (Jan-2006 to Jun-2015) were extracted. Eligible patients had ≥2 claims for VWD (ICD-9 code 286.4), and continuous health-plan enrolment for ≥2 years before and after diagnosis. Bleeding event, treatment and treating-physician type were analysed for 18 months before and 7-24 months after diagnosis, according to pre-diagnosis bleeding phenotype (claims from one vs multiple bleed sites) and post-diagnosis bleeding status (resolved [no post-diagnosis bleed claims] vs continued [≥1 claim]). RESULTS: Data for 3756 eligible patients (72.6% female; 71.0% aged ≥18 years at diagnosis) were analysed. Overall, 642 (17.1%) and 805 (21.4%) patients had single- and multiple-site bleed claims pre-diagnosis, respectively, and 1263 (33.6%) patients (38.5% of women, 20.8% of men) continued to bleed post-diagnosis. Multiple-site bleeding was associated with pre-diagnosis heavy menstrual bleeding (HMB), oral contraceptive (OC) use and nasal cauterization. Continued bleeding post-diagnosis was associated with pre-diagnosis gastrointestinal bleeding, HMB and epistaxis; pre-diagnosis use of OCs, aminocaproic acid and nasal cauterization; and younger age at diagnosis. Few patients consulted a haematologist for bleed management. CONCLUSION: Many patients with VWD have persistent bleeding from multiple sites and continue to bleed post-diagnosis. Our findings suggest a need to optimize management to reduce the symptomatic burden of VWD following diagnosis.

Pregnancy induces persistent changes in vascular compliance in primiparous women.

OBJECTIVE: Pregnancy induces rapid, progressive, and substantial changes to the cardiovascular system. The low recurrence risk of preeclampsia, despite familial predisposition, suggests an adaptation associated with pregnancy that attenuates the risk for subsequent preeclampsia. We aimed to evaluate the persistent effect of pregnancy on maternal cardiovascular physiology. STUDY DESIGN: Forty-five healthy nulliparous women underwent baseline cardiovascular assessment before conception and repeated an average of 30 months later. After baseline evaluation, 17 women conceived singleton pregnancies and all delivered at term. The remaining 28 women comprised the nonpregnant control group. We measured mean arterial blood pressure, cardiac output, plasma volume, pulse wave velocity, uterine blood flow, and flow-mediated vasodilation at each visit. RESULTS: There was a significant decrease in mean arterial pressure from the prepregnancy visit to postpartum in women with an interval pregnancy (prepregnancy, 85.3±1.8; postpartum, 80.5±1.8 mm Hg), with no change in nonpregnant control subjects (visit 1, 80.3±1.4; visit 2, 82.8±1.4 mm Hg) (P=.002). Pulse wave velocity was significantly decreased in women with an interval pregnancy (prepregnancy, 2.73±0.05; postpartum, 2.49±0.05 m/s), as compared with those without an interval pregnancy (visit 1, 2.56±0.04; visit 2, 2.50±0.04 m/s) (P=.005). We did not observe a residual effect of pregnancy on cardiac output, plasma volume, uterine blood flow, or flow-mediated vasodilation. CONCLUSION: Our observations of decreased mean arterial pressure and reduced arterial stiffness following pregnancy suggest a significant favorable effect of pregnancy on maternal cardiovascular remodeling. These findings may represent a mechanism by which preeclampsia risk is reduced in subsequent pregnancies.

Negative regulation of HIF-1α by an FBW7-mediated degradation pathway during hypoxia.

Hypoxia inducible factor-1α (HIF-1α) stimulates expression of genes associated with angiogenesis and is associated with poor outcomes in ovarian and other cancers. In normoxia, HIF-1α is ubiquitinated and degraded through the E3 ubiquitin ligase, von Hippel-Lindau; however, little is known about the regulation of HIF-1α in hypoxic conditions. FBW7 is an E3 ubiquitin ligase that recognizes proteins phosphorylated by glycogen synthase kinase 3ß (GSK3ß) and targets them for destruction. This study used an ovarian cancer cell model to test the hypothesis that HIF-1α phosphorylation by GSK3ß in hypoxia leads to interaction with FBW7 and ubiquitin-dependent degradation. Expression of constitutively active GSK3ß reduced HIF-1α protein and transcriptional activity and increased ubiquitination of HIF-1α in hypoxia, whereas pharmacologic inhibition of GSK3 or expression of siGSK3ß promoted HIF-1α stabilization and activity. A mechanism through FBW7 was supported by the observed decrease in HIF-1α stabilization when FBW7 was overexpressed and both the elevation of HIF-1α levels and decrease in ubiquitinated HIF-1α when FBW7 was suppressed. Furthermore, HIF-1α associated with FBW7γ by co-immunoprecipitation, and the interaction was weakened by inhibition of GSK3 or mutation of GSK3ß phosphorylation sites. The relevance of this pathway to angiogenic signaling was supported by the finding that endothelial cell tube maturation was increased by conditioned media from hypoxic SK-OV-3 cell lines expressing suppressed GSK3ß or FBW7. These data introduce a new mechanism for regulation of HIF-1α during hypoxia that utilizes phosphorylation to target HIF-1α for ubiquitin-dependent degradation through FBW7 and may identify new targets in the regulation of angiogenesis.

Reduced NO signaling during pregnancy attenuates outward uterine artery remodeling by altering MMP expression and collagen and elastin deposition.

Recent findings indicate that endothelial nitric oxide (NO) plays a key role in uterine artery outward circumferential remodeling during pregnancy. Although the underlying mechanisms are not known, they likely involve matrix metalloproteinases (MMPs). The goal of this study was to examine the linkage among NO inhibition, expansive remodeling, and MMP expression within the uterine vascular wall. Adult female rats were treated with N(G)-nitro-L-arginine methyl ester [L-NAME (LPLN)] beginning on day 10 of pregnancy and until death at day 20 and compared with age-matched controls [late pregnant (LP)]. Mean arterial pressure of LPLN rats was significantly higher than controls. LPLN fetal and placental weights were significantly reduced compared with controls. Main uterine arteries (mUA) were collected to determine dimensional properties (lumen area and wall thickness), collagen and elastin content, and levels of endothelial nitric oxide synthase (eNOS) and MMP expression. Circumferential remodeling was attenuated, as evidenced by significantly smaller lumen diameters. eNOS RNA and protein were significantly (>90%) decreased in the LPLN mUA compared with LP. Collagen and elastin contents were significantly increased in LPLN rats by ∼10 and 25%, respectively, compared with LP (P < 0.05). Both MMP-2 and tissue inhibitors of metalloproteinase-2 as assessed by immunofluorescence were lower in the endothelium (reduction of 60%) and adventitia (reduction of 50%) of LPLN compared with LP mUA. Membrane bound MMP-1 (MT1-MMP) as assessed by immunoblot was significantly decreased in LPLN. These data suggest a novel contribution of MMPs to gestational uterine vascular remodeling and substantiate the linkage between NO signaling and gestational remodeling of the uterine circulation via altered MMP, TIMP-2, and MT1-MMP expression and activity.

Examination of Prepregnancy and Pregnancy Urinary Protein Levels in Healthy Nulliparous Women.

During pregnancy, abnormal proteinuria is defined as urine protein excretion greater than 300 mg/24 h. Although widely accepted, this definition is not based on clinical outcomes. Our study aimed to longitudinally examine proteinuria in healthy women prior to, and in late pregnancy and to compare inpatient and outpatient 24-hour urine collections. Nulliparous women planning to conceive were recruited and completed a 24-hour urinary collection. Those who subsequently conceived completed a second 24-hour urinary collection in late pregnancy. In the first 5 years of the study, urinary collections were completed during an inpatient admission; all collections during the latter part of the study were performed as outpatients. Urine protein was measured using the VITROS UPRO Slide kit. Wilcoxon signed rank tests were used for paired comparisons of prepregnancy and late pregnancy proteinuria and Wilcoxon rank sum tests were used to compare inpatient and outpatient collections. Among 134 women completing a prepregnancy collection, median urinary protein excretion was 188 mg/24 h (IQR 103-280). Sixty-five women subsequently conceived and completed a late pregnancy collection. In healthy women, urinary protein increased to 254 mg/24 h during pregnancy (IQR 166-396). Forty-five percent of women exceeded the defined normal threshold of proteinuria in 24 hours in the absence of disease. Inpatient collections resulted in higher levels of urinary protein than outpatient at both time points. Our data suggest that significant proteinuria is present in healthy nonpregnant women. Even in the absence of disease, proteinuria increases during pregnancy. Outpatient collections may underestimate proteinuria, especially in late pregnancy.

Differences in cardiovascular function comparing prior preeclamptics with nulliparous controls.

OBJECTIVE: The objective of the current study was to evaluate cardiovascular function; including blood pressure, cardiac output, pulse wave velocity and vascular compliance in nonpregnant nulliparous women compared to women with a history of preterm preeclampsia. STUDY DESIGN: This was a case control study. Blood pressure was measured using the Finapres Pro. Baseline cardiac output was determined by echocardiography. Pulse wave velocity was estimated using simultaneous electrocardiographic tracings and ultrasound determined arterial flow waveforms and calculated as estimated distance divided by the time interval between EKG r-wave peak and ultrasound derived peak popliteal artery flow. During volume challenge, 500mL of lactated Ringers solution was infused through an indwelling antecubital catheter over 10min. Cardiac output and blood pressure during and 15min after the infusion were estimated using the Finapres Pro. MAIN OUTCOME MEASURES: Indices of arterial stiffness and vascular compliance. RESULTS: Previous preeclamptics exhibited a significant increase in pulse pressure and cardiac output in response to volume challenge when compared with nulliparous controls. Prior preeclamptics had a strong positive correlation between blood pressure indices (r=0.50-0.68, p⩽0.01) and pulse pressure (r=0.58, P=0.008) with pulse wave velocity that was not evident in control women. CONCLUSIONS: In women with prior preterm preeclampsia a relationship between blood pressure, intravascular volume and arterial stiffness, is evident in the nonpregnant state and in the absence of hypertension or overt cardiovascular disease. This supports an overarching hypothesis that nonpregnant physiology is an important contributor to pregnancy adaptations.

The relationship of a family history for hypertension, myocardial infarction, or stroke with cardiovascular physiology in young women.

Cardiovascular disease (CVD) and preeclampsia share several pathophysiologic risk factors. We examined family history (FH) and physiologic status in 60 healthy, nulliparous women to determine the relationship between FH and known risk factors for CVD. Data are presented as mean ± standard error (SE). Decreased uterine blood flow was observed in women with FH of hypertension (+FH: 21.5 ± 1.7, no FH: 33.3 ± 9.0 mL/min; P = .04). Women reporting an FH of stroke showed increased alpha- and beta-adrenergic response, as measured by Valsalva maneuver (α: FH: 24.7 ± 1.9, -FH: 18.9 ± 1.1 mm Hg, P = .02; ß: FH: 22.0 ± 2.1, -FH: 16.9 ± 1.4 mm Hg; P = .04), and increased cardiac output (4.83 ± 0.22 vs 4.31 ± 0.12 L/min; P = .01). We identified no significant physiologic associations linked to an FH of myocardial infarction. Our observations show significant differences in physiologic characteristics in women with specific CVD family histories. These data, coupled with known heritable contributions to CVD and preeclampsia, suggest a distinct physiologic phenotype that may link preeclampsia risk with FH of CVD, independent of pregnancy.

Prepregnancy Vascular Dysfunction in Women who Subsequently Develop Hypertension During Pregnancy.

OBJECTIVE: To evaluate vascular dysfunction using both physiologic measures and biochemical markers, longitudinally, prior to and during pregnancy, in nulliparous women who had uncomplicated pregnancies compared to those who developed complicated hypertension during pregnancy. METHODS: Twenty healthy nulliparous women were studied during the follicular phase and in early (EP) and late (LP) pregnancy. All had singleton conceptions and delivered at term, seventeen with uncomplicated pregnancies (NP) and three who developed complicated hypertension (HP) after the LP evaluation. We compared prepregnancy, EP and LP pulse wave velocity (PWV) and soluble vascular cell adhesion molecule (sVCAM-1) between the NP and HP groups. PWV was measured using ultrasound and simultaneous echocardiogram tracing then calculated as the estimated distance divided by interval between EKG r-wave peak and peak brachial artery flow. SVCAM-1 was measured using a commercially available kit. Data are means ± SE, significance accepted as p < 0.05. RESULTS: The NP group had significantly lower prepregnant PWV (NP: 2.66 ± 0.06 m/s, HP: 3.00 ± 0.04, p=.02), but PWV was not different at the EP or LP time points. SVCAM-1 was significantly lower prior to pregnancy and during EP and LP in the NP group (Prepregnancy: NP: 712 ± 32 ng/mL, HP: 1058 ± 107, p < .001; EP: NP: 695 ± 31 ng/mL, HP: 924 ± 52, p = .004; LP: NP: 663 ± 25 ng/mL, HP: 946 ± 36, p < .001). CONCLUSIONS: PWV and sVCAM-1 may be important prepregnancy discriminators useful in assessing risk for preeclampsia prior to pregnancy.

Coagulation and Fibrinolytic System Protein Profiles in Women with Normal Pregnancies and Pregnancies Complicated by Hypertension.

OBJECTIVE: The current study longitudinally evaluated concentrations of fibrinogen (Fib), D-Dimer, plasminogen activator type-1 (PAI-1) and tissue type plasminogen activator (T-Pa) before pregnancy and in the first and third trimesters of pregnancy with a focus on the pregnancy transition. STUDY DESIGN: Twenty healthy, nonsmoking, nulliparous women, aged 29.8 ± 3.0 years, BMI 23.3 ± 3.2 kg/m(2) were studied during menstrual cycle day 8 ± 4 and again in early (11 - 15 wks) and late (31 - 34 wks) pregnancy. Seventeen women had singleton conceptions and delivered at term with uncomplicated pregnancies (CTL) and three women developed complicated hypertension (CH) during pregnancy after the third trimester (late pregnancy) evaluation. Data are means ± SEM, Significance was based on p < 0.05. RESULTS: Fib was the only protein evaluated that increased in early pregnancy relative to the prepregnancy assessment. D-dimer, PAI-1 and T-Pa increased in the third trimester compared with prepregnant and early pregnant values (p < .001). T-PA was significantly higher during late pregnancy in CH subjects compared with CTL (8.1 ± 0.7 ng/ml vs. 5.0 ± 0.2 ng/ml, p = .02). There were no other differences between groups. CONCLUSIONS: Increases in fibrinogen are evident in early pregnancy whereas fibrinolysis, perhaps in response to the procoagulant environment of pregnancy, is increased during late pregnancy. Before development of clinically overt hypertension, T-Pa is increased without concomitant changes in other proteins assessed. This is consistent with altered endothelial function with preeclampsia that may contribute to, or reflect, the vasculopathy accompanying this disorder.

Sildenafil increases uterine blood flow in nonpregnant nulliparous women.

This study investigated the effect of sildenafil on uterine volumetric blood flow (UVF) and vascular impedance in nonpregnant, nulliparous women. Fifteen women were randomized in a double-blind fashion to receive either placebo or sildenafil (25 or 100 mg) during the luteal phase of the menstrual cycle. Color Doppler ultrasound of both uterine arteries was performed at baseline and at 1 and 3 hours postdosing to calculate resistance index (RI) and UVF. Those who received sildenafil significantly increased UVF and decreased RI over the 3-hour monitoring period. When UVF responses to sildenafil were examined as a function of baseline UVF, a significant increase in UVF was observed in only those participants with higher baseline UVF. Overall, women in the luteal phase demonstrated a significant increase in UVF in response to sildenafil. However, this increase appears to be directly associated with basal UVF.

Relationship between prepregnancy and early pregnancy uterine blood flow and resistance index.

We evaluated the relationship between prepregnancy and early pregnancy uterine blood flow (UBF) and resistance index (RI). Nineteen nulliparous participants were studied during cycle day 8 + 4, and early pregnancy (13.4 + 1.6 weeks). Color Doppler ultrasound of both uterine arteries and maternal heart was performed to calculate uterine RI, volumetric UBF, and cardiac output (CO), respectively. We observed a strong negative association of uterine RI with prepregnancy UBF (r = -.82, P < .001) that weakened, but remained significant in early pregnancy (r =-.48, P = .04). Prepregnancy uterine index (UBF/CO) was significantly associated with early pregnancy uterine index; r = .48, P = .04). There was also a trend associating prepregnancy and early pregnancy volumetric UBF (r = .44, P = .068). Prepregnancy UBF may be a determinant of early pregnancy UBF and UBF may have independent value as a predictor of adverse pregnancy outcome.

Erythropoietin disrupts hypoxia-inducible factor signaling in ovarian cancer cells.

OBJECTIVE: The objective of this study was to evaluate the effects of recombinant erythropoietin (EPO) on HIF-1alpha induced angiogenic pathways in ovarian cancer cells. METHODS: Using Western blots and both quantitative and non-quantitative RT-PCR, HIF-1alpha protein and VEGF transcription levels were assessed. Cell growth was measured using flow cytometry. RESULTS: EPO treatment decreased hypoxia-induced HIF-1alpha protein levels and VEGF transcription, with no effect on cell growth. Inhibition of HIF-1alpha signaling by EPO was also observed in MCF-7 breast cancer cells. CONCLUSION: These novel findings suggest that EPO may exhibit anti-angiogenic properties, thus encouraging further exploration of signaling pathways between EPO and HIF-1alpha.