Research matters

News from the Harvard Medical School research community.
Research matters
By Harvard Medical International
Fri 30 May 2008 04:00 AM

News from the Harvard Medical School research community.

Cardiovascular medicine

Simple non-laboratory methods as good as laboratory tests at predicting cardiovascular disease risk

In a new study from Brigham and Women's Hospital (BWH), researchers show that methods using non-laboratory-based risk factors predict cardiovascular events as accurately as more costly laboratory-based tests. These results are published in the March 14, 2008 issue of Lancet.

"Using non-laboratory tests could simplify risk assessment in countries where laboratory testing is inconvenient or unavailable," said Thomas Gaziano, MD of the Division of Cardiovascular Medicine at BWH and lead author of the study.

Using a previous cohort developed in the early 1970s and comprised of 14,407 US participants between the ages of 25 -74, Gaziano and colleagues re-analysed the NHANES I study.

The follow-up study population included 6186 participants who did not report a history of cardiovascular disease (heart attack, heart failure, stroke, angina), or cancer.

The lab-based method, which required blood-tests, looked at age, systolic blood pressure, smoking status, total cholesterol, reported diabetes status, and current treatment for high blood pressure. The non-lab method substituted body-mass index for cholesterol.

Researchers found that in the 6186 people initially not reporting a history of CVD, there were 1529 first-time cardiovascular events and 578 deaths due to cardiovascular disease over a 21 year period.

Both lab and non-lab methods calculated a number called the c-statistic to assess cardiovascular risk prediction, and for both men and women, lab and non-lab methods gave similar c-statistics.

Furthermore, the non-lab method correctly classified patients at the same rate as the lab method across four commonly used levels of risk used in guidelines around the world, suggesting good calibration.

Study authors add that the cost for developing nations to perform cholesterol tests on patients who were at risk for the development of cardiovascular disease could use more than 10 percent of the nation's health care budget, which adds little benefit to non-lab tests.

Non-lab test are effective at collecting the appropriate information to determine risk quickly and in a non-invasive way.

"Approximately 80 percent of cardiovascular deaths occur in developing countries where assessment of patients at high risk is an important strategy for prevention. Since developing countries have limited resources for laboratory testing, cheap, simple and effective, non-lab methods of testing would help immensely," said Gaziano.

Cancer treatment

Regulator of microRNAs is key to cell reprogramming and carcinogenesis: Could provide target for enhancing stem-cell generation or inhibiting cancer

MicroRNAs are a recently discovered class of RNAs that encode no proteins but instead regulate gene activity. Researchers at Children's Hospital Boston and the Harvard Stem Cell Institute have discovered the first protein to block the processing of immature microRNAs in cells to mature forms.

Drugs that target this protein, known as Lin-28, have the potential to influence the creation of stem cells and provide a new approach to treating some cancers, the authors say.

The discovery, reported online February 21 in ScienceExpress, "provides a key insight into two fundamental processes in biology--stem cell generation and carcinogenesis," says Richard Gregory, PhD, the study's senior author.

Lin-28 regulates the let-7 family of microRNAs, known to be key players in cancers of the lung and breast, and was also recently shown to help reprogram skin cells to pluripotent stem cells resembling embryonic stem cells.

Because both cellular reprogramming and carcinogenesis entail cellular de-differentiation (reversion of a mature cell to a less mature state), Lin-28 provides a missing link between stem cell generation and cancer.

"We are actively seeking drugs that mimic or block the effect of Lin-28 on microRNAs, as these drugs will either enhance stem cell generation, or inhibit cancers, respectively," says Gregory.

In 2006, Scott Hammond's group at University of North Carolina, Chapel Hill, discovered that microRNAs were made as immature forms in embryonic cells and cancer cells, but the mechanism for the blocking of their maturation remained elusive.

Srini Viswanathan, a graduate student in the laboratory of George Daley, MD, PhD, at Children's Hospital Boston, was interested in how microRNAs might dictate tissue formation from embryonic stem cells, which show a block in processing of a specific family of microRNAs called let-7.

Viswanathan and Gregory hypothesized that some protein must bind to the immature form of let-7 microRNAs to block their processing, and set out to purify the protein. They identified a number of proteins associated with microRNAs, but only Lin-28 was uniquely expressed in embryonic tissues.

They then demonstrated that Lin-28 was directly responsible for the blockade of let-7 microRNA in embryonic cells: expressing Lin-28 in non-embryonic cells reproduced the block in let-7 processing, and inhibiting Lin-28 expression in embryonic tissues caused upregulation of let-7.

Unbeknownst to the Children's researchers, Lin-28 was being studied by James Thomson's group at the University of Wisconsin as a factor that enhanced reprogramming of skin cells back to an embryonic state.

Thomson's paper (published while the Children's paper was under review) did not provide an explanation for how Lin-28 could contribute to stem cell generation. "Our study helps explain how Lin-28 works--by blocking microRNAs that promote cell differentiation," says Daley. The study also provides a key insight into cancer.

A characteristic of cancer is low expression of microRNAs. In their paper, Viswanathan, Daley and Gregory show that a close relative of Lin-28, Lin-28b, which has been linked to hepatocellular carcinoma, likewise blocks processing of let-7 microRNAs. Loss of the let-7 microRNA is critical for breast and lung cancer.

Therefore, the study suggests that activating Lin-28 may promote cancer formation, whereas blocking it may be protective, the researchers say.

"The discovery of how Lin-28 works was an unexpected ‘Eureka' moment," says Viswanathan. "We are looking forward to finding drugs that antagonize Lin-28, as these might be an important weapon against cancer.

The circulatory system

Another way to grow blood vessels: Scientists find alternate pathway to angiogenesisResearchers at Dana-Farber Cancer Institute have found a previously unknown molecular pathway in mice that spurs the growth of new blood vessels when body parts are jeopardized by poor circulation.

At present, their observation adds to the understanding of blood vessel formation. In the future, though, the researchers suggest it is possible that the pathway could be manipulated as a means of treating heart and blood vessel diseases and cancer. The paper appears in the Feb. 21 issue of the journal Nature.

Bruce Spiegelman, PhD, and his colleagues at Dana-Farber discovered that PGC-1alpha - a key metabolic regulatory molecule - senses a dangerously low level of oxygen and nutrients when circulation is cut off and then triggers the formation of new blood vessels to re-supply the oxygen-starved area - a process known as angiogenesis.

A similar response to hypoxia, or oxygen deprivation, has been observed before.

The response is regulated by a group of proteins known as Hypoxia Inducible Factors (HIF) that detect hypoxia and activate the production of VEGF (vascular endothelial growth factor). VEGF, in turn, stimulates angiogenesis.

The newly discovered pathway provides "an independent way of getting there," says Spiegelman, who is also a professor of cell biology at Harvard Medical School.

Along with lead author Zoltan Arany, MD, PhD, and colleagues, Spiegelman found that HIF was completely left out of the loop when PGC-1alpha accomplished the same feat in single cells and in live mice using a different regulator, known as ERR-alpha (estrogen-related receptor-alpha).

When the scientists knocked out the activity of PGC-1 alpha (which was first identified in the Spiegelman lab) in cells and live mice, the hypoxia-induced response and angiogenesis were sharply decreased.

"We were surprised to find this novel mechanism," comments Spiegelman. "It was apparently there all along," adds Arany. "That means there is now a second pathway that you need to know about if you are trying to activate or inhibit angiogenesis."

Angiogenesis occurs in the normal development of the body, but it's also an on-call service when an injury or an artery blockage - the cause of heart attacks and strokes leaves normal tissues starved for blood.

Generating a new network of small vessels to nourish the area can protect against further injury. Muscle-building exercise also triggers angiogenesis to provide circulation for the enlarging muscle tissue.

On the downside, cancer has evolved the ability to commandeer VEGF and other angiogenic factors to encourage blood vessel growth around tumors that have outgrown their oxygen supplies.

In recent years, companies have developed a number of drugs that manipulate the angiogenic pathway - in both directions. Among them are anti-angiogenic cancer drugs, including thalidomide and Avastin, which are designed to starve tumors by blocking the formation of blood vessels.

Avastin is also used to dampen the abnormal growth of small vessels in the retina that causes macular degeneration in the eye.

Conversely, researchers have tried using VEGF and other compounds to improve the circulation in the legs and feet - and even heart muscle - of patients with poor blood supply.

"We're still far from having good drugs to modulate angiogenesis through the HIF pathway," commented Arany. The discovery of a second, alternate pathway, involving PGC-1 alpha and ERR-alpha, leading to angiogenesis may offer new opportunities for therapy "in any situation where angiogenesis is a factor," he said.

Preventing infection

Study identifies mechanism underlying multidrug resistance in fungi: Finding could improve treatment of dangerous infections in immune-compromised patients

A team of researchers led by Anders Näär, PhD, of the Massachusetts General Hospital (MGH) Cancer Center has identified a mechanism controlling multidrug resistance in fungi.

This discovery could help advance treatments for opportunistic fungal infections that frequently plague individuals with compromised immunity, such as patients receiving chemotherapy, transplant recipients treated with immunosuppressive drugs, and AIDS patients. The findings appear in the April 3 issue of Nature.

Almost 10 percent of bloodstream infections are caused by pathogenic fungi, such as the Candida species; and the mortality of such infections is approaching 40 percent.

Just as many bacterial strains have become resistant to important antibiotics, resistance to common antifungal drugs is an increasing phenomenon in pathogenic fungi.To better understand the molecular pathways controlling multidrug resistance in fungi, the research team first investigated drug resistance in baker's yeast, a common genetic model for observing biological processes.

Using detailed genetic, biochemical, and molecular approaches, the researchers found that yeast induce multidrug resistance via a molecular switch similar to one that removes drugs and other foreign substances from human cells.

When the yeast protein Pdr1p binds to antifungal drugs or other chemicals, it switches on molecular pumps that remove the drugs from the cell. The research team showed that this chemical switch also controls drug resistance in an important human pathogenic fungus, Candida glabrata.

In humans, a protein called PXR is the drug sensor that turns on genes involved in detoxifying and removing drugs from cells.

"This intriguing similarity between the regulatory switches controlling multidrug resistance in fungi and drug detoxification in humans will allow us to take advantage of the extensive knowledge of the human molecular switch and identify new therapies for resistant fungal infections in patients with compromised immunity," says Näär, an assistant professor of Cell Biology at Harvard Medical School (HMS).

The researchers also found exactly how Pdr1p turns on the multidrug resistance program.

After binding to drugs, the Pdr1p protein partners with another key mediator of genetic switches called Gal11p. In-depth molecular and structural studies - in collaboration with the team of co-author Gerhard Wagner, PhD, Elkan Blout Professor of Biological Chemistry and Molecular Pharmacology at HMS - identified the specific area of Gal11p that binds to Pdr1p to induce multidrug resistance.

"This detailed understanding of the interaction between these proteins will allow screening for small-molecule inhibitors of protein binding. Such inhibitors may lead to novel co-therapeutics that will sensitize multidrug-resistant fungal infections to standard antifungal therapy," says Wagner.

To further investigate the relevance of their findings, the researchers used a C. elegans roundworm model system - recently developed by co-author Eleftherios Mylonakis, MD, PhD, of MGH Infectious Disease - to study fungal pathogens.

They found that worms infected with Candida glabrata that lacked either the Pdr1p or Gal11p proteins could be successfully treated with typical antifungal medications, suggesting that targeting the gene switch controlled by those proteins' interaction could restore the effectiveness of standard drugs.

"Fungal infections have a serious impact on immunocompromised patients, and the development of resistance is particularly worrisome, since targets for antifungal drugs are limited," says Mylonakis, an assistant professor of Medicine at HMS.

Given these concerns, having the opportunity to use our model system for the in vivo investigation of this resistance mechanism has been a particularly fulfilling endeavor.

Immunology

Study helps explain how allergic reactions are triggered

In demonstrating that a group of calcium ion channels play a crucial role in triggering inflammatory responses, researchers at Beth Israel Deaconess Medical Center (BIDMC) have not only solved a longstanding molecular mystery regarding the onset of asthma and allergy symptoms, but have also provided a fundamental discovery regarding the functioning of mast cells.

Their findings appear in the January 2008 issue of Nature Immunology.

A group of immune cells found in tissues throughout the body, mast cells were once exclusively known for their role in allergic reactions, according to the study's lead author Monika Vig, PhD, an investigator in the Department of Pathology at BIDMC and Instructor of Medicine at Harvard Medical School.

"Mast cells store inflammatory cytokines and compounds [including histamine and heparin] in sacs called granules," she explains. "When the mast cells encounter an allergen - pollen, for example - they ‘degranuate,' releasing their contents and triggering allergic reactions.

But, she adds, in recent years, scientists have uncovered numerous other roles for mast cells, suggesting they are key to a number of biological processes and are involved in diseases ranging from multiple sclerosis and rheumatoid arthritis to cancer and atherosclerosis.

In order for mast cells to function, they require a biological signal - specifically, calcium. Calcium moves in and out of the cells by way of ion channels known as CRAC (calcium-release-activated calcium) currents. Last year, several research groups, including Vig's, identified CRACM1 as being the exact gene that was encoding for this calcium channel.

"With the identification of this long-elusive gene, we were able to create a knockout mouse that lacked CRACM1, and [as predicted] these animals proved to be resistant to various stimuli that usually cause severe allergic reactions," she explains.

Further experiments demonstrated that mast cells removed from the CRACM1 knockouts were not able to take in calcium, and therefore, were unable to provoke allergic responses when they were exposed to allergens.

"These findings provide the genetic demonstration that CRAC channels are essential in mast-cell activation," notes senior author Jean-Pierre Kinet, MD, BIDMC Professor of Pathology at Harvard Medical School.

This provides the proof of concept that an inhibitor of the CRAC channel should be able to impact mast-cell related diseases, including asthma and allergic diseases.

Adds Vig, "Since mast cells are also known to contribute to the progression of several other debilitating diseases, including multiple sclerosis, rheumatoid arthritis and cancer, an inhibitor of the CRAC channel could, in the future, help in slowing the progression of these diseases as well as alleviate disease symptoms."

Research Matters brings together selected research being conducted at Harvard Medical School's 18 affiliated institutions. For more information, visit the Harvard Medical School website at www.hms.harvard.edu.

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