Students Probe Blood Vessels And Heart Cell Signals

Gaurav Gupta, 19, a sophomore from McLean, Va., found that mice deficient in the "anti-aging" gene klotho are a good model for age-related blood vessel stiffening. Chris Gregg, 21, a senior from South Orleans, Mass., discovered a signaling pathway through which cells regulate the heart's ability to contract. These research projects were funded through the university's Provost Undergraduate Research Awards and conducted in the lab of Dan E. Berkowitz, an associate professor of anesthesiology and critical care medicine in Johns Hopkins' School of Medicine with a secondary appointment in biomedical engineering.

Berkowitz praised both Gupta and Gregg for their diligence and drive. "They were the ones always coming to me, asking if I'd seen a recent journal article or suggesting what their next studies should be," he said. "Of all the students I've mentored, the ones that are motivated, resourceful and take the most initiative are the ones that succeed. My role is to focus their energy, like a lens."

Gupta, who has worked in Berkowitz's lab since his freshman year, said he likes its congenial atmosphere. With several undergraduates working there, the students learn from each other, becoming experts in specific laboratory techniques.

Gupta studied the cardiovascular biology of aging. In the aging cardiovascular system, abnormal dilating signals sent by the chemical nitric oxide, as released by the endothelial cells lining blood vessels, contribute to increased risk of heart disease. Among the effects of impaired endothelial function is an increase in vascular stiffness, or arteriosclerosis, a contributor to cardiac-related death.

To study this phenomenon, Gupta compared normal mice to a population of mice bred to be deficient in klotho, a gene with "anti-aging" properties. The latter mice exhibit a syndrome resembling human aging, including vascular stiffness.

Gupta took blood vessels from the mice and sectioned them into rings. He then suspended the rings between two tiny stirrups attached to a machine that measured how tightly the vessels constricted and recorded the information on a computer. Gupta then applied different chemical agents to the rings. He observed a weak response to acetylcholine, a natural chemical responsible for muscle contraction, indicating that the endothelial cells were not working properly, and that the signal pathway for nitric oxide was altered. The appearance of blood vessels in the klotho-deficient mice was consistent with human aging.

The research could help identify therapeutic targets for the treatment of age-related dysfunction of the endothelial cells lining the walls of blood vessels-a major determinant of vascular disease, Gupta said.

"By discovering the pathway interactions in age-related endothelial dysfunction, anti-aging therapies can be developed for the cardiovascular system," Gupta said. "Not only does this research open new doors for drug therapy, but it also will allow for further research into the interaction of cholesterol and vascular stiffness in conditions such as atherosclerosis and coronary artery disease."

Gupta plans to build on this work by evaluating the ability of other medications, such as statins or cholesterol-lowering drugs, to improve vascular stiffening. He is applying for funding from the Howard Hughes Medical Institute.

Gregg, the lab's other undergraduate research award recipient, studied the heart's beta adrenergic receptors. These chemical messengers are critical for intervening in responses to physical activity or stress. The receptors also respond to drugs used to modulate functions such as heart rate. Disregulation of these receptors and related signaling pathways is associated with congestive heart failure.

Initially, Gregg studied the ability of the beta-3 receptor to regulate overstimulation to the heart. He applied to mouse blood vessels an enzyme solution that released individual heart cells that still functioned as if they were part of a whole organ. He then exposed the cells to fluorescent dyes that would reveal under a high-powered microscope the amount of calcium being released during each heart beat-a read-out of the cells' response to stimuli. Next, he applied to the cells agents that either stimulate or inhibit specific proteins to disrupt signal pathways involved in contraction.

Along the way, he found a pathway related to the beta-2 receptor, whereby stimuli regulate the heart's ability to contract. The knowledge could eventually lead to better treatments for heart failure, Berkowitz said.

Gregg presented his research results March 6 at the American College of Cardiology's annual meeting in Orlando, Fla., and is submitting a manuscript of his work to a peer- reviewed heart journal.

"He has really become an integral member of our lab effort and has continued to inspire and train other students," Berkowitz said of Gregg.

"It was a great overall experience," said Gregg. "I think I was really lucky, and I learned a lot."

After graduating in May, Gregg plans to enter a doctoral research program.

Gregg and Gupta also pursued other areas of interest while working in the lab. As a side project, Gregg wrote a software program that integrated his data in a new way to look at the relationship of calcium release and contraction length in heart cells. Ion Optix Corp., manufacturer of one of the instruments used in Gregg's study, is adapting the software for its own use. And Gupta's laboratory techniques to prepare blood vessels and examine their function helped cardiology faculty member Kaiko Irani complete his work characterizing the vascular appearance of a mouse deficient in a protein called redox-factor 1. For his efforts, Gupta was second author on a paper published in Circulation Research last year.

On March 10, Steven Knapp, university provost and senior vice president for academic affairs, hosted the 12th annual Provost's Undergraduate Research Awards ceremony, which honored the 45 winners who conducted their projects in the summer and fall of 2004. Since 1993, about 40 students each year have received PURA grants of up to $3,000 to conduct original research, some results of which have been published in professional journals. The awards, funded through a donation from the Hodson Trust, are an important part of the university's commitment to research opportunities for undergraduates.

The Johns Hopkins University is recognized as the country's first graduate research university, and has been in recent years the leader among the nation's research universities in winning federal research and development grants. The opportunity to be involved in important research is one of the distinguishing characteristics of an undergraduate education at Johns Hopkins.

The Provost's Undergraduate Research Awards program provides one of these research opportunities, open to students in each of the university's four schools with full-time undergraduates: the Krieger School of Arts and Sciences, the Whiting School of Engineering, the Peabody Conservatory and the School of Nursing.