Superior, WI, United States
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Ramadoss J.,University of Texas Medical Branch | Magness R.R.,Perinatal Research Laboratories
Physiological Genomics | Year: 2012

Genomic studies on fetal alcohol spectrum disorders (FASD) have utilized either genome-wide microarrays/bioinformatics or targeted real-time PCR (RT-PCR). We utilized herein for the first time a novel digital approach with high throughput as well as the capability to focus on one physiological system. The aim of the present study was to investigate alcohol-induced alterations in uterine angiogenesisrelated mRNA abundance using digital mRNA technology. Four biological and three technical replicates of uterine arterial endothelial cells from third-trimester ewes were fluorescence-activated cell sorted, validated, and treated without or with binge-like alcohol. A capture probe covalently bound to an oligonucleotide containing biotin and a color-coded reporter probe were designed for 85 angiogenesis-related genes and analyzed with the Nanostring nCounter system. Twenty genes were downregulated (↓) and two upregulated (↑), including angiogenic growth factors/receptors (↓ placental growth factor), adhesion molecules (↓ angiopoietin-like-3; ↓ collagen-18A1; ↓ endoglin), proteases/matrix proteins/inhibitors (↓ alanyl aminopeptidase; ↓ collagen-4A3; ↓ heparanase; ↓ plasminogen, ↑ plasminogen activator urokinase; ↓ platelet factor-4; ↓ plexin domain containing-1; ↓ tissue inhibitor of metalloproteinases-3), transcription/signaling molecules (↓ heart and neural crest derivatives-2; ↓ DNA-binding protein inhibitor; ↓ NOTCH-4; ↓ ribosomal protein L13a1; ↓ ribosomal protein large-P1), cytokines/chemokines (↓ interleukin-1B), and miscellaneous growth factors (↓ leptin; ↓ plateletderived growth factor-α); ↓ transforming growth factor (TGF-α; ↑ TGF-β receptor-1). These novel data show significant detrimental alcohol effects on genes controlling angiogenesis supporting a mechanistic role for abnormal uteroplacental vascular development in FASD. The tripartite digital gene expression system is therefore a valuable tool to answer many additional questions about FASD from both mechanistic as well as ameliorative perspectives. © 2012 the American Physiological Society.

Sprague B.,University of Wisconsin - Madison | Sprague B.,Perinatal Research Laboratories | Chesler N.C.,University of Wisconsin - Madison | Magness R.R.,Perinatal Research Laboratories
International Journal of Developmental Biology | Year: 2010

Hemodynamic shear stress is the most powerful physiological regulator of endothelial Nitric Oxide Synthase (eNOS), leading to rapid rises in nitric oxide (NO). The substantial increases in uterine and placental blood flows throughout gestation rely heavily on the action of NO. We and others have investigated endothelial function in response to shear stress with cell culture models of shear stress. In order to apply the results of these studies more effectively, we need a more complete understanding of the origin and coupling of the hemodynamic forces and vascular tissue behavior. For example, equations commonly used to calculate in vivo shear stress incorporate assumptions of steady (non-pulsatile) blood flow and constant viscosity of blood (Newtonian fluid). Using computational models, we can estimate a waveform of shear stress over a cardiac cycle and the change in blood viscosity with shear rate and hematocrit levels, two variables that often change with size of vessel and location within a vascular tree. This review discusses hemodynamics as they apply to blood flow in vessels, in the hope that an integration of these fields can lead to improved in vitro shear stress experiments and understanding of NO production in uterine and placental vascular physiology during gestation. © 2009 UBC Press.

Koch J.M.,Perinatal Research Laboratories | Ramadoss J.,University of Wisconsin - Madison | Magness R.R.,University of Wisconsin - Madison
Journal of Proteome Research | Year: 2010

Embryonic development is a time-sensitive period that requires a synchronized uterine environment, which is created by the secretion of proteins from both the embryo and uterus. Numerous studies have identified uterine luminal proteins and related these to specific adaptations during early pregnancy (EP). However, no study has yet utilized LC-MS/MS to identify the signature profile of proteins in the uterine lumen during EP. In this study, uterine luminal fluid from nonpregnant (NP; n = 3) and EP (n = 3; gestational day 16) ewes were analyzed by LC-MS/MS and validated by Western immunoblotting. We identified a unique signature profile for EP luminal fluid; 15 proteins related to specific aspects of embryonic development including growth and remodeling, immune system regulation, oxidative stress balance, and nutrition were significantly altered (up to 65-fold of NP) in EP profile. Specific uterine remodeling proteins such as transgelin (P = 0.008) and placental proteins like PP9 (P = 0.02) were present in EP luminal fluid but were barely detectable in the NP flushings. Direct correlations (R2 = 0.84, P = 0.01) were observed between proteomics and immunoblotting. These data provide information on dynamic physiological processes associated with EP at the level of the uterus and conceptus and may potentially demonstrate a signature profile associated with embryonic well-being. © 2010 American Chemical Society.

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