Dysregulation of Oxygen Sensing/Response Pathways in Pregnancies Complicated by Idiopathic Intrauterine Growth Restriction and Early-Onset Preeclampsia.

Preeclampsia (PE) and intrauterine growth restriction (IUGR) are the leading causes of maternal and fetal morbidity/mortality. The central deficit in both conditions is impaired placentation due to poor trophoblast invasion, resulting in a hypoxic milieu in which oxidative stress contributes to the pathology. We examine the factors driving the hypoxic response in severely preterm PE (n = 19) and IUGR (n = 16) placentae compared to the spontaneous preterm (SPT) controls (n = 13) using immunoblotting, RT-PCR, immunohistochemistry, proximity ligation assays, and Co-IP. Both hypoxia-inducible factor (HIF)-1α and HIF-2α are increased at the protein level and functional in pathological placentae, as target genes prolyl hydroxylase domain (PHD)2, PHD3, and soluble fms-like tyrosine kinase-1 (sFlt-1) are increased. Accumulation of HIF-α-subunits occurs in the presence of accessory molecules required for their degradation (PHD1, PHD2, and PHD3 and the E3 ligase von Hippel-Lindau (VHL)), which were equally expressed or elevated in the placental lysates of PE and IUGR. However, complex formation between VHL and HIF-α-subunits is defective. This is associated with enhanced VHL/DJ1 complex formation in both PE and IUGR. In conclusion, we establish a significant mechanism driving the maladaptive responses to hypoxia in the placentae from severe PE and IUGR, which is central to the pathogenesis of both diseases.

Leukocyte matrix metalloproteinase-9 is elevated and contributes to lymphocyte activation in type I diabetes.

Degradation of extracellular matrix proteins by matrix metalloproteinases (MMPs) is integral to cell migration and tissue remodeling in diabetes mellitus and its complications. MMPs also regulate the function of leukocytes via proteolytic processing of cytokines/chemokines. In this study, we measured the production of MMP-9 and its natural tissue inhibitor (TIMP)-1 by leukocytes isolated from human type I diabetic patients. MMP-9 was also detected in serum and splenocytes from non-obese diabetic (NOD) and BALB/c mice. MMP-9 was markedly elevated in leukocytes from diabetics compared to non-diabetic controls. TIMP-1 production was also enhanced in leukocytes from diabetics, but substantially less than MMP-9, with the MMP-9/TIMP-1 ratio being 1.6-fold higher in neutrophils and 3-fold higher in monocytes than controls. Interleukin (IL)-2 or lipopolysaccharide (LPS) treatment increased MMP-9 production in leukocytes from both diabetics and normal controls, whereas insulin decreased MMP-9 expression. Recombinant MMP-9 stimulated the proliferation of mouse splenocytes from NOD or BALB/c and a MMP-9 inhibitor dose-dependently inhibited splenocyte proliferation. In conclusion, our results demonstrate firstly that MMP-9 expression is elevated in leukocytes from type I diabetic patients and NOD mice and secondly, that MMP-9 elevates proliferation of mouse splenocytes. These data suggest that elevated leukocyte MMP-9 may contribute to the pathogenesis of type I diabetes and its associated complications.

Isolating pure populations of monocytes from the blood of pregnant women: comparison of flotation in iodixanol with elutriation.

Observations that the innate arm of the immune system is upregulated in pregnancy have highlighted the need for methods of isolating pure populations of monocytes for studies into pregnancy and pre-eclampsia without activating them during the isolation process. Density gradient centrifugation using iodixanol is a useful method for isolating relatively pure populations of unactivated monocytes from human blood but has not been validated in pregnant subjects. We compared the ability of monocytes isolated from pregnant women by density gradient centrifugation using iodixanol (n=6) with monocytes isolated by countercurrent centrifugal elutriation (n=6) in terms of their ability to produce interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) under basal conditions and after stimulation with bacterial lipopolysaccharide (LPS). Under basal conditions, monocytes isolated by density gradient centrifugation produced low amounts of IL-6 and MCP-1. Production of IL-6 and MCP-1 after stimulation of the monocytes with LPS was much greater (p<0.01). There was no statistically significant difference between the two methods in terms of stimulated levels of either cytokine.

Nitric oxide production by decidual endothelial cells is not reduced in preeclampsia.

OBJECTIVE: To investigate whether decidual endothelial cells (DEC) contribute to the pathogenesis of preeclampsia through abnormal nitric oxide production. Decidual endothelial cells from normal (NDEC) and preeclamptic (PEDEC) pregnancies, and also human umbilical vein endothelial cells (HUVEC), were examined. METHODS: HUVEC, NDEC, and PEDEC were incubated for 45 min in serum-free media with the addition of potential stimulators [calcium ionophore (A23187), sepiapterin, and a combination of cytokines (TNF-alpha, gamma-IFN and LPS)], and the competitive inhibitor, NG-monomethyl-L-arginine (L-NMMA). These were added alone or in combination. Supernatants were measured for nitrate/nitrite (NOx) levels and the cells acid-extracted for measurement of cyclic guanosine monophosphate (cGMP). The effect of 30 min of shear stress (approximately 20 dynes/cm2) on NO and cGMP production by NDEC and PEDEC and on production of prostacyclin and thromboxane A2, was assessed. RESULTS: PEDEC and HUVEC both produced more NO than NDEC under all conditions examined. Cell-associated cGMP levels, however, were not different among the cell groups but were increased by A23187 and inhibited by L-NMMA. In control conditions, shear stress stimulated cGMP levels 5-fold (p<0.01) in both NDEC and PEDEC, and PGI2 production 2-fold (p<0.05). CONCLUSIONS: DEC from preeclamptic women do not have reduced NO production and respond normally to shear stress by increasing cGMP and PGI2 production. Our results are consistent with other reports of equal or higher NO levels in preeclampsia and indicate that reduced NO production by endothelial cells is not the explanation for the vasoconstriction of uterine vessels.

Excretion of antihypertensive medication into human breast milk: a systematic review.

OBJECTIVE: To establish which antihypertensive medications are safe for use while breastfeeding, by reviewing the available evidence. METHODS: Reports of studies examining the transfer of antihypertensive medications to breastmilk were identified from multiple MEDLINE and EMBASE searches, manual review of bibliographies of articles and textbooks on drug use during lactation. The reports were reviewed and the results were compiled. RESULTS: Prospective cohort studies and case reports constituted the only available evidence. Compilation of these results found that the milk to plasma (M/P) ratios varied widely across the beta-blocker family, the beta-blockers with low protein binding having the highest M/P ratios. The angiotensin-converting enzyme (ACE) inhibitors, methyldopa, and some calcium channel blockers had low M/P ratios. CONCLUSION: The available data to date indicate that ACE inhibitors, methyldopa, beta-blockers with high protein binding, and some calcium channel blockers all appear to be safe treatments of hypertension in a nursing mother. The data suggest that drugs to be avoided are beta-blockers with low protein binding. However, the available evidence is limited and further studies are needed to confirm these findings.

The long-term effects of prematurity and intrauterine growth restriction on cardiovascular, renal, and metabolic function.

Objective. To determine relative influences of intrauterine growth restriction (IUGR) and preterm birth on risks of cardiovascular, renal, or metabolic dysfunction in adolescent children. Study Design. Retrospective cohort study. 71 periadolescent children were classified into four groups: premature small for gestational age (SGA), premature appropriate for gestational age (AGA), term SGA, and term AGA. Outcome Measures. Systolic blood pressure (SBP), augmentation index (Al), glomerular filtration rate (GFR) following protein load; plasma glucose and serum insulin levels. Results. SGA had higher SBP (average 4.6 mmHg) and lower GFR following protein load (average 28.5 mL/min/1.73 m(2)) than AGA. There was no effect of prematurity on SBP (P = .4) or GFR (P = .9). Both prematurity and SGA were associated with higher AI (average 9.7%) and higher serum insulin levels 2 hr after glucose load (average 15.5 mIU/L) than all other groups. Conclusion. IUGR is a more significant risk factor than preterm birth for later systolic hypertension and renal dysfunction. Among children born preterm, those who are also SGA are at increased risk of arterial stiffness and metabolic dysfunction.

Intercellular adhesion molecule-1 (ICAM-1) expression is necessary for monocyte adhesion to the placental bed endothelium and is increased in type 1 diabetic human pregnancy.

BACKGROUND: That adhesion molecule expression is upregulated in endothelial cells of the placental bed in pregnancies complicated by type 1 diabetes mellitus, and that this is associated with increased adherence of peripheral blood monocytes, which can be reversed by reduction in activity or expression of relevant adhesion molecules. Specific aims were to compare the adherence of monocytes from normal pregnancies to decidual endothelial cells from both normal and diabetic pregnancies, and to examine the involvement of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in regulation of such adhesion. METHODS: We examined adhesion of peripheral blood monocytes (isolated by density gradient centrifugation) of normal third trimester pregnant women, to cultured endothelial cells (isolated from decidual biopsies collected at elective caesarean section) from both normal women and those with type 1 diabetes. Adhesion molecule expression was determined by flow cytometry. The role of ICAM-1 was further investigated by monoclonal antibody-blocking experiments and gene-silencing methodology. RESULTS: There was a significant increase in monocyte adhesion to decidual endothelial cells from diabetic pregnancies, associated with increased endothelial cell expression of ICAM-1, but not VCAM-1. ICAM-1 expression in normal decidual endothelial cells was stimulated by pro-atherogenic and pro-inflammatory stimuli. Following ICAM-1 antibody blockade, monocyte adhesion was decreased by > 70%. ICAM-1 silencing by small interfering RNAs also inhibited monocyte adhesion and ICAM-1 expression. CONCLUSIONS: These findings implicate upregulation of ICAM-1 in decidual endothelial cells in the development of placental bed vascular pathology in diabetic pregnancy.

Peripheral arterial pulse wave analysis in women with pre-eclampsia and gestational hypertension.

OBJECTIVE: To compare peripheral pulse pressure waveforms in normal pregnancy, gestational hypertension (GH) and pre-eclampsia (PE). DESIGN: Comparative study. SETTING: Tertiary referral teaching hospital. SAMPLE: Women with hypertensive disease in pregnancy and matched normal pregnant controls. METHODS: Women in the third trimester of pregnancy with newly developed PE (n= 27) or GH (n= 33) were studied by radial artery applanation tonometry. Values were compared with those for gestational age matched normal pregnant women (n= 39). MAIN OUTCOME MEASURES: Augmentation Index. RESULTS: Hypertension was of equal severity in PE and GH. Central augmentation pressure and augmentation index, indicating vasoconstriction, were mildly elevated in GH, but markedly elevated in PE. CONCLUSION: Non-invasive applanation tonometry discriminates among normal pregnancy, GH and PE, and may be useful to predict PE.

A method for the isolation of glomerular and tubulointerstitial endothelial cells and a comparison of characteristics with the human umbilical vein endothelial cell model.

BACKGROUND: Abnormalities in the structure and function of glomerular endothelial cells play a pivotal role in the development of progressive renal disease. The vascular abnormalities observed in the renal tubulointerstitium, however, correlate more strongly with progressive renal failure. Therefore, the successful isolation and culture of human renal microvascular endothelial cells from both the glomerulus and tubulointerstitium are paramount in studying renal disease models. METHODS AND RESULTS: This study describes a simple and reproducible method for the isolation of human tubulointerstitial and glomerular endothelial cells by using immunomagnetic separation with anti-platelet endothelial-cell adhesion (anti-PECAM-1) Dyna beads, followed by manual weeding of mesangial and fibroblast contamination. No significant changes in morphological or immunohistochemical characteristics were observed up to passage two of culture. The in vitro characteristics of the endothelial cells were compared to the renal cortical endothelial cells in vivo and the standard human umbilical vein endothelial cell model (HUVECs). Similar to HUVECs, both populations of renal microvascular endothelial cells had a classical cobblestone appearance, stained positively for von Willebrand Factor and PECAM-1 and negatively for antifibroblast surface antigen and anticytokeratin. Differences in the expression of von Willebrand Factor, Wiebel Palade bodies and Flk-1 staining were observed between glomerular and tubulointerstitial endothelial cells. These immunohistochemical characteristics suggested that tubulointerstital endothelial cells were more closely aligned to HUVECS than to the glomerular endothelial cells. This observation indicated that HUVECs may be a suitable model for determining the tubulointerstitial endothelial response to systemic injury. CONCLUSION: In conclusion, a unique and novel method for the differential isolation of both glomerular and tubulointerstitial endothelial cells has been developed. Significantly, characterization of these populations suggests a role for HUVECS in the study of renal tubulointerstitial disease.

Methods of assessment of the arterial pulse wave in normal human pregnancy.

OBJECTIVES: The study was undertaken to obtain normal values for characteristics of the peripheral arterial pulse wave, in nonpregnant women and in pregnant women at three different stages of gestation, with two devices, to describe cardiovascular hemodynamic variables induced by pregnancy. STUDY DESIGN: Sixty pregnant women were enrolled in the study at three stages of pregnancy, 17 to 20 weeks, 25 to 28 weeks, and 33 to 36 weeks gestation. Results are presented for 53 of these subjects who remained normotensive throughout pregnancy. The values measured were compared with those from 10 nonpregnant women. The two devices used were the SphygmoCor and a monitor invented and developed by Professor Edward Hon, not yet commercially available, referred to in this article as the "Hon" monitor. RESULTS: An increase in heart rate was observed in all pregnant women. Blood pressure values were lower in pregnancy than in normal nonpregnant women. With respect to the SphygmoCor, the time from the start of the arterial waveform to the second peak/shoulder, was significantly shorter, and augmentation pressure and augmentation index were significantly lower in pregnancy. Data obtained from the "Hon" monitor showed no significant differences in pulse wave arrival time, but rapid ejection time was significantly shortened in pregnancy. CONCLUSION: The clinical findings confirm the known cardiovascular changes of pregnancy associated with vasodilatation of peripheral vessels and expansion of blood volume. The fall in augmentation pressure and index are consistent with these changes. Rapid ejection time and the time from the start of the arterial waveform to the second peak/shoulder, which should reflect similar cardiovascular physiologic events, were weakly correlated.