In 1993, the NHLBI’s Women's Health Initiative (WHI) began enrolling the first participants in what would become one of the most definitive, far-reaching clinical trials of post-menopausal women's health ever undertaken in the U.S. During its more than 20 years of work, this long-term national health study of 161,808 women aged 50-79 has focused on strategies for preventing heart disease, breast and colorectal cancer, and osteoporotic fractures in postmenopausal women. The study has changed public health practices and generated more than 1,000 publications.
Recently, three teams of researchers received contracts from NHLBI to leverage the wealth of clinical and biomarker data collected by WHI and combine it with new -omics technologies in the hopes of gleaning new insights into the role that genomics, gene biology and regulation, and metabolomics may play in the risk for or development of coronary heart disease (CHD).
“We funded these contracts with two aims,” said Jacques Rossouw, chief of the Women’s Health Initiative (WHI) Branch, Division of Cardiovascular Sciences, NHLBI. “First, we want a better understanding of the etiology of coronary heart disease in women to help instruct future prevention strategies.
“Second, we want to better understand the effect of hormone therapy on coronary heart disease,” Dr. Rossouw said. “We know what the clinical effects are, but we don’t know why hormone therapy has these effects, and we’re hoping these studies can help shed light on the why.”
“The Women’s Health Initiative was so important because its key finding that transformed care for women was that women on combined estrogen plus progestin therapy had high higher rates of coronary heart disease than women on placebo,” said Dr. Kathy Rexrode from Brigham and Women’s Hospital, a principal investigator for one of the studies. “In contrast, women on estrogen-alone did not have different rates than women on placebo. This went against all the evidence we had up to that point and was the opposite of what we expected.”
Each of the projects will use different techniques to assess the study’s genomic data, and will examine different aspects of the biology involved. Results from the studies could help improve current risk prediction algorithms and help identify the women at highest risk for heart disease.
Looking Beyond the Genome
In one study, led by Dr. Themistocles "Tim" Assimes and Dr. Phil Tsao from the Stanford University School of Medicine, researchers will identify genomic markers that may cause CHD in women and then examine miRNA and methylation in relation to those genomic markers to see whether those genes are expressed or repressed and, if they are expressed, the degree of expression. In contrast, most studies instead look at variations in the DNA that may be related to disease risk. Researchers also will look to determine if there is a relationship between that expression or repression and traditional risk factors.
miRNAs, or microRNAs, are tiny pieces of RNA (about 20-30 nucleotides long) that circulate in a cell and attach to mRNA (or messenger RNA) to prevent proteins from being made. Methylations are chemical additions that are added to some genes that also affect how much protein gets made. Adding methyl groups to a gene can decrease or completely shut down a gene. What this means is that two women can have the same sequence of DNA for a particular gene associated with heart disease; but if one woman has a methylated gene and the other does not, they will have different levels of gene expression and therefore different risk even though they have the same DNA.
Hormone Therapy and CHD
The second study, led by Dr. Rexrode, will use state-of-the-art metabolite profiling techniques to measure metabolites, which are small molecules found within cells and biological systems, with the goal of identifying metabolomics signatures that might predict coronary heart disease.
“Many of the strongest risk factors for cardiovascular disease (CVD) in women, such as obesity, diabetes, and insulin resistance, are associated with altered metabolism, but exactly how these metabolic perturbations directly impact the pathophysiology of CHD is not clear,” Dr. Rexrode explained.
Dr. Rexrode’s team will use extremely sensitive mass spectrometry analysis to analyze a core group of more than 300 compounds – or metabolites – believed to play a role in coronary heart disease development as well as identify new compounds that scientists knew existed but didn’t realize were relevant in this space.
“Up until now, while there has been some research on metabolomics and coronary heart disease, there is very little data that has focused specifically on women and heart disease despite the fact that heart disease is the leading cause of death in women,” Dr. Rexrode said. “There also has been very little research into variations based on race and ethnic groups, and we will be able to examine this in the Women’s Health Initiative.”
In relation to the use of hormones and CHD, Dr. Rexrode’s team plans to look at participants’ metabolic signatures at baseline and compare those with their metabolic signatures a year into hormone therapy treatment to see how those signatures may have changed and to see if particular changes may predict which women end up with bad outcomes. If particular changes can be identified, Dr. Rexrode said that may provide insight into the mechanisms by which postmenopausal hormone therapy increase the risk of heart disease.
“We’ve never been able to explain what mechanisms were causing the increased risk for women assigned to estrogen plus progestin,” she added. “It’s been a medical mystery for a long time.”
The researchers also plan to examine the relationship between the identified metabolomics profiles and previously measured clinical parameters (such as blood pressure, waist circumference, and BMI), biomarkers (including markers of lipoproteins, inflammation, endothelial function, and insulin resistance), and other genomic data.
“Ultimately, if we can identify metabolic signatures that predict CHD, we may be able to find new pathways that we could use to influence treatment and prevention of cardiovascular disease in women,” Dr. Rexrode said.
Dr. Rossouw added that identifying new metabolomics signatures and biomarkers may help doctors identify who is at greater risk for CHD and help doctors provide advice on whether hormone therapy is a suitable treatment option or not for individual patients.
Leukocyte telomere length, Aging and CHD
The third study will focus on a particular biomarker called leukocyte telomere length (LTL) that is important for aging. Telomeres, found at the ends of each chromosome, get small every time a cell divides and shortened telomeres have been linked to age-related diseases such as hypertension, stroke, and diabetes, and to the aging process itself.
“Some evidence has linked LTL to cardiovascular disease, and the Women’s Health Initiative is an opportunity to increase our understanding of the role of telomere length as a biomarker for aging as well as cardiovascular disease,” said principal investigator Dr. Alex Reiner from the Fred Hutchinson Cancer Research Center. “The most recent WHI examination occurred just this past year and because of the length of time since the study’s onset, and the age of the WHI study participants, there is an increased scientific focus on functional aging status and outcomes related to successful aging.
Researchers plan to compare LTL measurements that were taken at two time points 15 years apart and assess the relationship of LTL and, separately, LTL attrition with incident CHD events and mortality.
“We’ll be looking at whether a woman’s telomere length at baseline – when she entered the study – predicts heart disease and mortality in the future and also whether the change in telomere length over time is important as a predictor of aging and heart disease,” Dr. Reiner explained. “That’s one of the more unique aspects of the study and it’s something that hasn’t really been looked at: Does an accelerated rate of telomere shortening predict mortality or heart disease?”
Dr. Reiner also is excited about the multicultural nature of the WHI participants.
“Previous studies on telomere length and risk of heart disease or mortality have largely occurred in Caucasian populations,” he said. “There is very little data on other ethnic groups. Interestingly, there are some studies that suggest that African Americans have longer telomeres than Caucasians. We hope to be able to shed some light on the reasons behind this ethnic difference, whether there are particular genes, for example, or environmental factors or some combination of the two that account for this difference.”
One of the strengths of the three contracts is the integration among them. Each project will utilize a partially overlapping sample set. Because of this, there are a number of potential opportunities for scientific synergy across the individual projects, particularly in the field known as “systems biology.” One example is a discussion underway between Dr. Assimes’ and Dr. Reiner’s teams about a project that would compare data from methylation and telomere length to examine the inter-relationships between methylation status as a “biologic clock” and telomere length, and also compare the ability of these measures of biologic aging to predict health and/or risk.
Dr. Rossouw emphasized that the overlap will allow for a better analysis of the results.
“When you put the results and data from all three together, you’ll get an integrated view, a systems biology view, of what’s going on in the body,” Dr. Rossouw explained. “And if we can get a better understanding of what’s going on, then that ultimately offers the possibility of new treatments or prevention strategies.”