Research presented at the 2010 ACR Research and Education Foundation (REF) Investigators’ Meeting in June covered subjects on the pathogenesis and treatment of rheumatoid arthritis (RA) as diverse as the expertise of those involved. These presentations included new research on the role of periodontal disease in promoting systemic inflammation, the underlying mechanisms involved in heart dysfunction, and the influence of lipid-metabolizing enzymes in immune regulation to name a few.
Held in Fort Worth, Texas, the meeting allowed physicians and basic scientists to discuss their research programs as well as network and initiate collaborations across disciplines, said E. William St.Clair, MD, president of the REF, which funds the Within Our Reach: Finding a Cure for Rheumatoid Arthritis grants received by all attendees. Dr. St.Clair is professor of medicine and immunology at Duke University Medical Center, Durham, N.C.
By the middle of 2010, $24 million in grant funds will have been awarded, totaling more than 50 grants, Dr. St.Clair said. “We have increased the critical mass of rheumatoid arthritis investigators,” he says, noting that roughly 25% of those funded hadn’t previously conducted rheumatoid arthritis (RA) research. “The overarching goal is to stimulate innovative research.”
Some grant recipients have already made scientific advances that helped them obtain even larger National Institutes of Health grants, Dr. St.Clair said. Since 2007, 65 publications in peer-reviewed journals have stemmed directly from Within Our Reach–funded projects. Here are some key research highlights presented at June’s meeting.
Proteins and Heart Disease Risk
Subramaniam Pennathur, MBBS, a nephrologist and assistant professor in the Department of Internal Medicine at the University of Michigan, Ann Arbor, was already pursuing research into lipoproteins and cardiovascular disease when he became interested in the risk of cardiovascular disease in RA, including underlying mechanisms.
Despite the higher risk of cardiovascular disease in the RA population, prior research has indicated that traditional heart risk factors—as identified by the Framingham Heart Study—aren’t as predictive in RA patients compared with the general population, he said. This discrepancy raises the question of whether other risk factors might play a role in increasing cardiovascular disease and if unique biomarkers could be identified to predict which individuals are more susceptible to this complication.
Although much research to date has focused on the role of low-density lipoproteins (LDL) in promoting cardiovascular disease, Dr. Pennathur has always been intrigued by the role of their high-density counterparts, HDL. Despite their reputation as the so-called “good cholesterol,” high levels of HDL have not prevented heart disease in some individuals, he said. “Maybe the HDL is getting somehow dysfunctional, and it’s not working as well as it should,” said Dr. Pennathur.
Dr. Pennathur, working in collaboration with his colleague at the University of Michigan, Mariana Kaplan, MD, has hypothesized that the inflammatory factors associated with RA might inhibit the cardioprotective benefits typically found with high-density lipoproteins. In his ongoing research, Dr. Pennathur and colleagues are trying to unravel specifically what it is about oxidized, dysfunctional HDL and its related proteins that might contribute to a higher risk of cardiovascular disease.
To date, they have enrolled 70 RA patients, of whom 25 individuals have completed the study, he said. Preliminary results show that the levels of oxidized HDL are “markedly elevated” in people with RA compared with healthy individuals, he said.
Protein composition analyses in a subset of patients revealed roughly three dozen shared proteins in the two groups, as well as several proteins that are distinct to the RA patients studied. These include proteins involved in inflammation, lipid metabolism, oxidation reactions, and immune response. Dr. Pennathur said that the final tally might be higher or lower, based on the confirmatory studies. One possibility that will be studied is whether the dysfunctionality of the HDL is related in some way to those RA-specific proteins, he said.
If researchers could identify a “protein signature” that is associated with a higher risk of developing cardiovascular disease, then RA patients could be screened with a blood test early in their disease development, he said. “I think this could be an early application of this research, to risk-stratify patients,” said Dr. Pennathur in comments following the meeting. In the future, once there is better insight into the HDL biology, targeted treatments could be developed, he said.
Targeting Extracellular Cyclophilins
In her laboratory work at the George Washington University in Washington, D.C., immunologist Stephanie Constant, PhD, has been working to understand better the immune factors that regulate inflammation, with an initial focus on the lung. Over time, she has increasingly delved into the role of cyclophilins, specifically extracellular cyclophilins, investigating their function in RA as well as asthma and lung injury.
Although cyclophilins are typically intracellular, in times of bodily stress, cells will secrete the proteins, allowing them to be detected outside the cell, said Dr. Constant, associate professor in the Department of Microbiology, Immunology, and Tropical Medicine. She noted that a prior study identified high levels of extracellular cyclophilins in the synovial fluid of individuals with RA.
Based on studies showing that extracellular cyclophilins can attract proinflammatory leukocytes from the circulation into tissues, Dr. Constant is investigating whether targeting extracellular cyclophilins will reduce the recruitment of proinflammatory leukocytes into inflamed tissues, she said. To that end, Dr. Constant has been working with two analogs, both derivatives of
cyclosporin A, that have been altered to be non-immunosuppressive. These derivatives are known to inhibit the leukocyte attracting function of cyclophilins. Dr. Constant hopes to learn whether they reduce joint inflammation and to gain insights into the mechanisms involved.
At the June meeting, Dr. Constant presented data comparing changes in different inflammatory markers after treatment with the two analogs, using mouse models of inflammation. One of the analogs penetrates cells and therefore targets both intracellular and extracellular sources of cyclophilins; the second has been modified to work only on extracellular cyclophilins. “So there will be no accumulation of the drug inside the cells,” she said.
In mice with allergic asthma, the analog targeting only extracellular cyclophilins produced a greater reduction in a variety of inflammatory markers, she said. The next step is to test this analog, which Dr. Constant has “high hopes for,” in collagen-induced arthritis in mice. That study will be conducted within the next year, she said.
The parent drug involved, cyclosporin A, has already been well studied in patients, she said. “We believe it will have fewer side effects because it will not have any impact on intracellular cell function, like signaling, and so it will only target cellular events that are regulated outside the cell.”
Inhibiting PAD Activity
Paul R. Thompson, PhD, presented the work that his research team has been pursuing—first at the University of South Carolina in Columbia and more recently at Scripps Florida in Jupiter, Fla.—to decipher the role that protein arginine deiminases (PADs), and specifically PAD4, plays in the development of RA. Increased PAD4 activity has been identified in RA and dysregulated activity of the enzyme family has been associated with a number of other diseases, including cancer, multiple sclerosis, and ulcerative colitis.
Dr. Thompson’s ongoing research, conducted with collabortors at the University of Colorado, has been looking at whether specific compounds can inhibit PAD4 activity as a strategy to treat RA. At the meeting, he presented findings on disease incidence and severity in mouse models following injections with one of two PAD inhibitors, Cl-amidine and F-amidine.
In these experiments, the mice were immunized with type II collagen (CII) in complete Freund’s adjuvant (CFA) and boosted at Day 15 in induce collagen-induced arthritis. Then they were monitored for at least 40 more days. The disease severity was lower in the group that received daily injections of Cl-amidine compared with F-amidine.
In a subsequent study, the mice received the CII/CFA immunization initially, were boosted at 21 days, and then were tracked for at least 14 more. Meanwhile, the effectiveness of Cl-amidine treatment was assessed at various dose levels. In a dose-dependent way, the compound decreased the disease severity by as much as 55% percent, said Dr. Thompson, a biochemist and associate professor in the Department of Chemistry at Scripps Florida.
Subsequent studies demonstrated decreased bone damage and inflammation, as well as decreased PAD activity and citrullination, in the arthritic limbs of the Cl-amidine-treated mice. “The key thing we’ve done is to show that the PADs are a target,” Dr. Thompson said in comments following the meeting. “It’s also possible that the compound could potentially be a therapeutic.”
However, there was no response when Cl-amidine was used in another model of arthritis called antibody-induced arthritis. In some respects, the lack of a response in the effector phase of the disease is worrisome, Dr. Thompson said. It is also unclear how closely this model mimics all aspects of arthritis in humans, he said.
Blocking Sphingosine Kinase 1
Gary Gilkeson, MD, was sitting on a graduate school committee several years ago when he heard a student involved in lipidomics research discuss the role of sphingosine kinase 1 (SphK1) in signaling by tumor necrosis factor a (TNFα). “Because of the critical role of TNF in rheumatoid arthritis, that got me interested in whether sphingosine was playing a role in rheumatoid arthritis and whether blocking it could be a potential therapy,” said Dr. Gilkeson, a rheumatologist and professor of medicine at the Medical University of South Carolina in Charleston.
His research group subsequently began working with transgenic mice in an effort to better understand the inflammatory role of the lipid-metabolizing enzyme. They obtained transgenic mice that expressed a modified copy of the human TNFα gene and thus were vulnerable to limb swelling and deformity. Then they crossed those mice with a second group, which lacked functional copies of the SphK1 gene.
In his presentation, Dr. Gilkeson discussed findings involving the two groups of mice, both of which carried a TNFα gene, but one with the SphK1 gene and the second with it knocked out.
Through scoring of pathology, the researchers determined that periarticular inflammation was reduced at both three months and six months in the mice which lacked the SphK1 gene. Bone erosion, measured at five months, also declined. The mice also demonstrated a reduction in synovial proliferation, but to a less significant degree.
It may be that SphK1 does not work on that synovial proliferation, while it does work on the pathway involved with inflammation and bone erosion, Dr. Gilkeson said in comments following the meeting. “We are trying to figure out where exactly sphingosine kinase is acting and how exactly it’s doing it,” he said.
The mice also showed an increase in the factor SOCS-3 which regulates immune responses. One possible explanation for this finding might relate to the link between blocking SphK1 and a resulting increase in ceramide, which may in turn stimulate SOCS-3 production, Dr. Gilkeson said. “We postulate, but we haven’t proven it, that the higher levels of ceramide that are present in the knockout mice are increasing the SOCS-3,” he said.
At this point, there have been no significant side effects identified in the knockout mice, Dr. Gilkeson told meeting attendees. “There have not been any toxicities—the mice themselves look fine,” he said.
Pharmaceutical companies are already pursuing treatments that can inhibit sphingosine kinase in other diseases, including cancer, he said. “I think we will soon have a plethora of agents that can address this pathway.” Dr. Gilkeson also noted that it remains to be seen whether a drug that blocks a particular enzyme works better or worse than research conducted in mice with the knocked-out gene.
Charlotte Huff is a medical journalist based in Texas.
