The over-arching hypothesis to be tested in this research is that vascular inflammation due to neutrophil infiltration is critical to the pathophysiology of preeclampsia. Neutrophils infiltrating the vasculature could cause inflammation and vasoconstriction by release of toxic compounds, such as reactive oxygen species (ROS), TNF??and thromboxane.
In Specific Aim 1, we will determine if maternal vascular infiltration by neutrophils is a hallmark of preeclampsia, and associated with vascular inflammation. Subcutaneous fat, omental fat and placental tissue from pregnant patients will be collected and evaluated by immunohisto chemistry to assess vascular phenotypes of normal nonpregnant, normal pregnant and preeclamptic women, and to determine if neutrophil infiltration is restricted to the maternal compartment. We will stain tissues for CD66b (a neutrophil marker), intercellular adhesion molecule-1 (an endothelial adhesion molecule for neutrophils), interleukin-8 (IL-8, a potent neutrophil chemokine), NF-?B and COX-2 (hallmarks of inflammation), myeloperoxidase (a potent oxidizing enzyme), and matrix metalloproteinases 1 and 8. The in situ findings of vascular pathology observed in this Aim will drive the mechanistic studies of Aims 2-4 which will examine mechanisms for neutrophil induced vascular inflammation and vasoconstriction.
In Specific Aim 2, we will evaluate whether neutrophils that have infiltrated into the intimal space could be responsible for transmitting inflammation to the vascular smooth muscle by co-culturing neutrophils with human vascular smooth cells. We will use transfection and cell culture to determine if neutrophils can activate NF-?B and induce expression of COX-2, IL-8 and thromboxane in human vascular smooth muscle cells (VSMC).
In Specific Aim 3, we will use a myograph system with isolated omental vessels and cell culture of VSMC to examine cellular mechanisms whereby neutrophils could cause vasoconstriction and/or enhance vessel reactivity to vasoconstrictors.
In Specific Aim 4, we will determine if neutrophils or neutrophil products induce epigenetic changes in vascular smooth muscle genes related to inflammation, and thus, may program adverse long-term effects on maternal cardiovascular function. Methylation patterns of candidate genes will be evaluated in vessels from women with normal pregnancies and women with preeclampsia by bisulfite DNA sequence analysis and COBRA (Combined Bisulfite Restriction Analysis). We will also determine if neutrophils or neutrophil products (ROS, TNF?) alter the methylation status of these genes in a similar pattern in cultured VSMC. This novel hypothesis may explain future health issues, including recurrent preeclampsia and future risk of cardiovascular disease.

Public Health Relevance

Preeclampsia is a leading cause of maternal and fetal morbidity and mortality. This research will test the hypothesis that vascular inflammation due to neutrophil infiltration is critical to the pathophysiology of preeclampsia. This hypothesis encompasses molecular explanations for a vicious cycle that reinforces vascular inflammation in preeclampsia, and creates an epigenetic imprint in the maternal vascular system that may predispose women to chronic cardiovascular disease.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL069851-09
Application #
8431376
Study Section
Pregnancy and Neonatology Study Section (PN)
Program Officer
Mitchell, Megan S
Project Start
2002-04-01
Project End
2014-12-31
Budget Start
2013-01-01
Budget End
2014-12-31
Support Year
9
Fiscal Year
2013
Total Cost
$345,180
Indirect Cost
$109,560
Name
Virginia Commonwealth University
Department
Obstetrics & Gynecology
Type
Schools of Medicine
DUNS #
105300446
City
Richmond
State
VA
Country
United States
Zip Code
23298
Vaughan, John E; Walsh, Scott W (2012) Activation of NF-?B in placentas of women with preeclampsia. Hypertens Pregnancy 31:243-51
Mousa, Ahmad A; Strauss 3rd, Jerome F; Walsh, Scott W (2012) Reduced methylation of the thromboxane synthase gene is correlated with its increased vascular expression in preeclampsia. Hypertension 59:1249-55
Estrada-Gutierrez, Guadalupe; Cappello, Renato E; Mishra, Nikita et al. (2011) Increased expression of matrix metalloproteinase-1 in systemic vessels of preeclamptic women: a critical mediator of vascular dysfunction. Am J Pathol 178:451-60
Mishra, Nikita; Nugent, William H; Mahavadi, Sunila et al. (2011) Mechanisms of enhanced vascular reactivity in preeclampsia. Hypertension 58:867-73
Bachawaty, Teddi; Washington, Sonya L; Walsh, Scott W (2010) Neutrophil expression of cyclooxygenase 2 in preeclampsia. Reprod Sci 17:465-70
Shah, Tanvi J; Leik, Courtney E; Walsh, Scott W (2010) Neutrophil infiltration and systemic vascular inflammation in obese women. Reprod Sci 17:116-24
Walsh, Scott W (2009) Plasma from preeclamptic women stimulates transendothelial migration of neutrophils. Reprod Sci 16:320-5
Cadden, Kristen A; Walsh, Scott W (2008) Neutrophils, but not lymphocytes or monocytes, infiltrate maternal systemic vasculature in women with preeclampsia. Hypertens Pregnancy 27:396-405
Shah, Tanvi J; Walsh, Scott W (2007) Activation of NF-kappaB and expression of COX-2 in association with neutrophil infiltration in systemic vascular tissue of women with preeclampsia. Am J Obstet Gynecol 196:48.e1-8
Vaughan, John E; Walsh, Scott W; Ford, George D (2006) Thromboxane mediates neutrophil superoxide production in pregnancy. Am J Obstet Gynecol 195:1415-20

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