C. Kevin Huls, MD
Campus Address: 6 Center, Meriter Hospital, 202 South Park St, Madison, WI 53715
Major Professor: Dr. Ron Magness
Email: huls@wisc.edu
Telephone: (608) 267-6618
Degree Objective: Post-doctoral fellow, MS in Endocrinology-Reproductive Physiology
Background: University of Iowa, M.D. 5/2001
Residency: Bayfront Medical Center
Research Project:
My research project focuses on endothelial nitric oxide synthase (eNOS) phosphorylation at the serine 635 position in response to shear stress, ATP and ionomycin. The effect of ATP and ionomycin on the activation of eNOS with no shear stress has been completed. The current work is focusing on the effect when shear stress is added to the model.
The project tests the hypothesis that increased shear stress will result in activation of signaling within the cell. Since it is the endothelial cell that encounters shear stress because of its proximity to blood flow within the vessellurnen, our focus is on the signaling that occurs within the cell. Endothelial cells are known to produce NO which will result in vasodilation of vascular smooth muscle in a paracrine manner.
It is unclear how eNOS is regulated by phosphorylation and which signaling pathways may be involved in controlling the phosphorylation of eNOS. Two potential pathways are the Akt and ERK Y2 pathways. The phosphorylation of both of these pathways is assessed to determine if there is an association between eNOS-Ser-635 phosphorylation and either of these pathways.
The model used for this project uses ovine endothelial cells from either Luteal, follicular or pregnant sheep that were previously harvested and frozen in liquid nitrogen. Those cells are then grown to passage 4-5 and are cultured on fibronectin-coated microscope slides. After 80~90% confluence is reached, a small chamber is applied that then allows media to pass over the slide at either 3 or 15 dynes/cm2 and thjs is maintajned for 48 hours.
At the end of 48 hours the cells ar~ serum starved for the last 4 hours. The slides are removed, treated with A TP or ionomycin for 10 minutes, then lysed and a protein assay is done. From here a standard amount of protein is added from each sample and western analysis is done to measure the amount of phosphorylated protein. The optical density of phosphorylated protein to total protein is compared between the different treatment groups.
One additional step added to this arrangement is to identify whether estrogen has an effect. All of these steps are repeated with a second comparison of these cell lines when they are cultured in the presence of 10nM estradiol.
Presentations:
"Estrogen Modulation of Endothelial Nitric Oxide Synthase (eNOS) Phosphorylation Responses in Follicular, Luteal and Pregnant Derived Ovine Uterine Artery Endothelial Cells (UAEC)". Annual Society of Gynecologic Investigation Meeting, Reno, NV. March 15, 2007.
"Antihypertensive Medications in Pregnancy". Annual Wisconsin Medical Society Meeting. July 15, 2006.
"Duffy Antibody, Intrauterine Transfusion and Postnatal Care". UW/Meriter Perinatal Conference. June 27, 2006
"Duffy Antibody, Intrauterine Transfusion". UW/Meriter Conference. April 18, 2006.
"Fetal Supravetricular Tachycardia". UW/Meriter Hospital Perinatal Conference. March 21, 2006.