1. Introduction
There is an abundance of molecular, cellular, biochemical, animal model and human patient literature to support the concept that estrogen (E2) impacts the cardiovascular system (CVS) in significant ways that may involve both genomic and non-genomic mechanisms

2. Intro (cont.)
Many prominent CVS physiological effects have also been attributed to the actions of E2 including vasodilation, anti-oxidant properties, decreased post-ischemic inflammation, and anti-atherogenesis effects
CVS disease is the leading cause of death among post-menopausal women in developed countries

3. Intro (cont.)
Recent studies have looked more critically at the original WHI trial design and have concluded that although the data was informative, patient age, dose levels, and treatment combinations need to be reconsidered in future clinical studies
In the blood vasculature, non-nuclear ERa stimulates endothelial cell proliferation and migration through regulation of Nitric Oxide Synthase (NOS) in those cells

4. Intro (cont.)
The mechanism(s) by which E2 exerts its effects on the developing CVS is little understood. Therefore, there is a need to establish an in vivo developmental model in which to study the role of E2 in these CVS maturation and protective phenomena

5. Intro (cont.)
Zebrafish contain two genes that code for aromatase, which convert the androgens, testosterone and androstenedione, into E2
Aromatase inhibitors (AIs) compromise aromatase enzyme activity, which is responsible for the synthesis of E2 from androgens

6. Zebrafish Maintenance
Adult zebrafish (Danio rerio) were maintained at 28.5 C in aquarium tanks. Fish were fed Aqueon goldfish flakes twice a day by automatic fish feeders, and freeze-dried brine shrimp at intervals throughout the week. The day-night cycle was controlled by an automatic timer, and was maintained at 14 h light-10 h dark.

7. Embryo Collection and Treatment
Wildtype embryos (strain AB) were obtained from the Zebrafish International Resource Center (University of Oregon, Eugene), the breeding facilities at Virginia Military Institute, or Roanoke College.
AI and E2 treatments consisted of 50 lM aromatase inhibitor (AI, 4-OH-A, Sigma) or 10 nM E2 (17b-Estradiol, Sigma)

8. Testing for the ‘listless’ condition
All fish were treated at 48 h post fertilization (hpf) with either ERS, AI, NOSI, AI+E2, AI+NO, AI+NOSI+E2, nNOSI, iNOSI, or eNOSI solutions for five additional days

9. Ventricle Size
Heart ventricles were visualized by whole mount in situ hybridization using cardiac myosin light chain-2
Prehybridization was carried out for at least 3 h at 65 C. Hybridization was carried out overnight with the probe at a concentration of 1.5 lg/ml. Embryos were blocked for 1 h at room temperature and incubated with antidigoxigenin alkaline phosphatase conjugated antibody (Roche) at 4 C overnight. A

10. Cardiac Sac Puncture
AI-treated ‘listless’ fish were immobilized in a 2% agarose gel (Sigma) containing 0.4% tricaine (Sigma) as an anesthetic. Embryos were punctured with a glass needle attached to a micromanipulator. At daily intervals, heart rates and percent survival of the control, AI-non-punctures (NP) and AI-Punctures (P) groups was noted over a 5 day period while still embedded in the gel

11. Blood Vessel Integrity
In order to further quantify vessel integrity, three additional abnormalities were analyzed: (1) the number of SE bifurcations, (2) the number of VA/SE misconnections, and (3) the number of gaps in the VA.

12. Blood Flow
Blood flow was measured from two locations in the trunk: just above the anal pore and 875 lm anterior to the end of the vessel bed

13. Data Analysis
Chi-Square, t-tests, and ANOVA were performed dependent on the data being analyzed to determine statistical significance.
Sample sizes for all experiments including heart rate, cardiac sac size, blood vessel structure and integrity, blood flow and NO were n = 30; ventricle measurement had a sample size of n = 50. All experiments were repeated in triplicate.