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Researchers at the Medical University of South Carolina (MUSC) have used viruses to infect neurons with genes that allow them to switch on brain receptors involved in suppressing addiction relapse. Results of these preclinical studies were published in the September 28th, 2016 issue of the Journal of Neuroscience. The technology, called designer receptors exclusively activated by designer drugs, or DREADDs, is one of the most promising gene therapies for the future treatment of addiction in humans.

 The brains of people who use cocaine become hijacked by drug cues. Powerful memories are formed between these cues–such as the using environment and drug paraphernalia–and the dopamine flood that occurs from using the drug itself. In users trying to quit, these drug cues activate an intense desire to seek cocaine again.

 Resistance to relapse is partly mediated in the ventromedial prefrontal cortex–the brain region slightly above and behind our eyes, where previously learned associations are broken. This region of the brain stores extinction memory, which works to suppress the emotional response to drug cues, according to Jamie Peters, Ph.D., Research Assistant Professor in the MUSC Department of Neuroscience.

 “Extinction doesn’t overwrite the original memory,” explained Peters. “It just helps suppress the pathological component of the response.” 

 Peters and her colleague Peter W. Kalivas, Ph.D., Chair of the MUSC Department of Neuroscience, wanted to know if the response to drug cues associated with the dopamine rush of cocaine could be suppressed when the extinction memory region was activated. To test their hypothesis, they obtained viruses carrying the DREADD gene from Bryan L. Roth, M.D., Ph.D., in the Department of Pharmacology at the University of North Carolina Chapel Hill. The DREADD technology is openly accessible to researchers around the world through the National Institutes of Mental Health Psychoactive Drug Screening Program, where Roth serves as director.

 The viruses work by inserting the DREADD gene directly into the genome of cells, causing them to grow receptors on their surface that are normal except for a slight alteration. These receptors express a protein encoded by the DREADD gene that allows them to be activated by a single drug designed to bind that protein. In this case, the Peters lab infused a virus carrying a DREADD gene designed to change surface receptors on neurons. After the neurons were infected, they would fire in response to administration of the designer drug. Because the body’s other cells had not been infected with the DREADD gene, they would remain unaffected.

 “This new approach for treating drug addiction is exactly what is needed because it is targeted to a specific circuit in the brain regulating addiction,” said Kalivas. “This may allow the circuit to be selectively regulated with minimum side effects on other circuits and brain functions.”

 The researchers allowed rats to self-administer cocaine by pressing one of two levers, one active and one inactive. Once a rat pressed the active lever, cocaine was delivered along with a brief audio tone and a pulse of light that would serve as the drug cues. After a series of daily cocaine exposure sessions, the rats had learned to associate the simple drug cues with cocaine availability. Then they were removed from the drug. Next a surgical technician infused virus carrying the DREADD gene directly into the rats’ ventromedial prefrontal cortices. After two weeks of cocaine abstinence, the rats were placed back in front of the two levers in ten daily sessions, but this time the levers produced neither cues nor cocaine. The next day, rats were subjected to a relapse test where the cues were returned. Before testing, half of the rats were given designer drug and half were not. Next, rats underwent an additional relapse test where they were given a low dose of cocaine to trigger relapse.

 The experiments worked. Rats that were given the designer drug relapsed less in the presence of drug reminder cues. However, when exposed to cocaine again, rats relapsed regardless of whether they were given the designer drug. In other words, Peters’ hypothesis was correct: rats with activated extinction memories weren’t as susceptible to relapse triggered by cocaine-associated cues but were still vulnerable when exposed to cocaine again. This meant that extinction memory retrieval reduced relapse triggered by reminder cues.

 This study shows that it is possible to use this technology to target a small population of cells in the brain that is important for regulating addiction, thereby inhibiting the drive to relapse to addictive drug use. In the future, Peters hopes that safe and effective viruses of this kind can be infused into the brains of human addicts during neurosurgery. A person would simply take a pill to activate the extinction memory region of their brain, helping them to suppress the urge to seek out drug in the face of those reminder cues. Since extinction memory isn’t as powerful as the emotional response to a drug, this strategy could work when paired with effective psychological counseling approaches such as cognitive behavioral therapy.

Clinicians interested in using DREADDs in humans will have to remain patient, however. DREADDs have to be designed to match drugs that suppress only memories of drug cues while leaving other memories unaffected. And the crystal structure of newer human-appropriate designer drugs bound with the special receptors is being actively investigated in order to visualize exactly how they might work some day in patients with cocaine addiction.

 “Certainly within my lifetime I would expect to see these virus-mediated gene therapies start to be used in the brain, in a neurosurgical setting,” said Peters. “You can envision a person ultimately taking a pill to activate this very specific part of his or her brain.”

Image licensed from iStock.com.

Induced Pluripotent Stem Cell

Researchers from the Medical University of South Carolina and elsewhere devise new method to enhance genome-wide association studies for liver disease.

Caption: This image shows induced pluripotent stem cells expressing a characteristic cell surface protein called SSEA4 (green).Image courtesy of Stephen A. Duncan of the Medical University of South Carolina.

A research team including developmental biologist Stephen A. Duncan, D. Phil., SmartStateTM Chair of Regenerative Medicine at the Medical University of South Carolina (MUSC), has found a better way to purify liver cells made from induced pluripotent stem cells (iPSCs). Their efforts, published August 25, 2016 in Stem Cell Reports,1 will aid studies of liver disease for the National Heart, Lung, and Blood Institute (NHLBI)’s $80 million Next Generation Genetic Association Studies (Next Gen) Program. The University of Minnesota (Minneapolis) and the Medical College of Wisconsin (Milwaukee) contributed to the study.

 This new methodology could facilitate progress toward an important clinical goal: the treatment of patients with disease-causing mutations in their livers by transplant of unmutated liver cells derived from their own stem cells. Previous attempts to generate liver-like cells from stem cells have yielded heterogeneous cell populations that bear little resemblance to diseased livers in patients.

NHLBI’s Next Gen was created to bank stem cell lines sourced from patients in genome-wide association studies (GWAS). The goal of the NHLBI Next Gen Lipid Conditions sub-section—a collaborative effort between Duncan and Daniel J. Rader, M.D., and Edward E. Morrisey, Ph.D., both at the University of Pennsylvania—is to help determine the genetic sources of heart, lung, or blood conditions that also encompass the liver. These GWAS studies map the genomes in hundreds of people as a way to look for genetic mutation patterns that differ from the genomes of healthy individuals.

A GWAS study becomes more powerful—more likely to find the correct genetic mutations that cause a disease—as more genomes are mapped. Once a panel of suspected mutations is built, stem cells from these individuals can be “pushed” in culture dishes to differentiate into any of the body’s cells, as for example liver-, heart-, or vascular-like cells. The cells can be screened in high-throughput formats (i.e., cells are expanded and cultured in many dishes) to learn more about the mutations and to test panels of drugs that might ultimately help treat patients harboring a disease.

 The problem arises during the “pushing.” For example, iPSCs stubbornly refuse to mature uniformly into liver-like cells when fed growth factors. Traditionally, antibodies have been used to recognize features of maturity on the surfaces of cells and purify cells that are alike. This approach has been crucial to stem cell research, but available antibodies that recognize mature liver cells are few and tend to recognize many different kinds of cells. The many types of cells in mixed populations have diverse characteristics that can obscure underlying disease-causing genetic variations, which tend to be subtle.

“Without having a pure population of liver cells, it was incredibly difficult to pick up these relatively subtle differences caused by the mutations, but differences that are important in the life of an individual,” said Duncan.

Instead of relying on antibodies, Duncan and his crew embraced a new technology called chemoproteomic cell surface capture (CSC) technology. True to its name, CSC technology allowed the group to map the proteins on the surface of liver cells that were most highly produced during the final stages of differentiation of stem cells into liver cells. The most abundant protein was targeted with an antibody labeled with a fluorescent marker and used to sort the mature liver cells from the rest.

The procedure was highly successful: the team had a population of highly pure, homogeneous, and mature liver-like cells. Labeled cells had far more similar traits of mature hepatocytes than unlabeled cells. Pluripotent stem cells that had not differentiated were excluded from the group of labeled cells.

“That’s important,” said Duncan. “If you’re wanting to transplant cells into somebody that has liver disease, you really don’t want to be transplanting pluripotent cells because pluripotent cells form tumors called teratocarcinomas.”

Duncan cautions that transplantation of iPSC-derived liver cells is not yet ready for translation to the clinic.  But the technology for sorting homogeneous liver cells can be used now to successfully and accurately model and study disease in the cell culture dish.

“We think that by being able to generate pure populations, it will get rid of the variability, and therefore really help us combine with GWAS studies to identify allelic variations that are causative of a disease, at least in the liver,” said Duncan.

Reference

Mallanna SK, et al. Mapping the cell-surface N-glycoproteome of human hepatocytes reveals markers for selecting a homogeneous population of iPSC-derived hepatocytes. Stem Cell Reports. ePub August 25, 2016. doi: http://dx.doi.org/10.1016/j.stemcr.2016.07.016

SUMMARY: A genomics approach at the Medical University of South Carolina (MUSC) has unmasked genetic signatures in breast cancer cells that predict their sensitivity to certain drugs. The findings, published in the May 2, 2016 issue of Oncotarget, provide proof of concept for personalized pharmaceutical therapies that target the genes responsible for driving tumor growth.

Drug treatments for breast cancer patients might soon be designed based on the unique genetic autograph of their tumor.

A genomics approach at the Medical University of South Carolina (MUSC) has unmasked genetic signatures in breast cancer cells that predict their sensitivity to certain drugs. The findings, published in the May 2, 2016 issue of Oncotarget, provide proof of concept for personalized pharmaceutical therapies that target the genes responsible for driving tumor growth.

Dr. Stephen EthierCertain oncogenes drive solid tumor growth in some breast cancer patients but are just passenger genes in others—expressed but not essential for growth. As a result, tumors in different breast cancer patients may respond differently to the same treatment depending on which oncogenes are active and which are just along for the ride. Identifying the panel of active genes in a patient’s tumor—called the functional oncogene signature—could help an oncologist select therapies that target its growth, according to Stephen P. Ethier, Ph.D., Interim Director of the Center for Genomic Medicine at MUSC and senior author on the study.  

“In order to move the field forward, we now need to be able to use oncogene signatures to tailor therapies using combinations of targeted drugs so that multiple driving oncogenes can be targeted at once,” said Ethier.  “Doing this successfully requires the identification of the oncogenes to which the cancer cells are addicted, as this allows the use of targeted drugs at very low doses. Low doses are essential if we are to use combinations of different targeted drugs.”

Ethier’s group identified unique functional oncogene signatures in four different human breast cancer cell types. Using next-generation genome sequencing and genome silencing as each cancer cell type grew and multiplied, they assembled a list of genes for each cell type’s functional oncogene signature—those genes that were copy number amplified or point mutated, and most essential to cancer cell survival. Although thousands of candidate oncogenes were screened during experimentation, only a handful made the list—fewer than 20 for each cell type.

The brevity of each list facilitated selection of the best oncogene for pharmaceutical targeting. Because lower doses of targeted drugs can be highly effective, side effects could be reduced. For example, Ethier’s group found that targeting two or more members of a signature with much lower total drug concentrations in combination still killed cancer cells better than one higher-concentration drug alone.

Remarkably, a BCL2L1-targeted drug  that worked in one cell line also then worked in a fifth breast cancer cell line with a similar oncogene signature containing BCL2L1, an oncogene not normally associated with breast cancer. This work demonstrates that one signature-targeting treatment can be extended to more than one cancer cell type. This means that patients with other types of cancer who have a similar functional oncogene signature might benefit from drugs that target BCL2L1, which are already in development.

Ethier thinks that oncogenes identified in a tumor biopsy might one day soon provide a rational and individualized approach to pharmaceutical treatment with targeted drug combinations. Meanwhile, these findings from his laboratory—showing the importance of considering a patient’s functional oncogene signature before testing a new drug— could provide a rationale for redesigning clinical trials for breast cancer.

Stephen T. Guest, Ph.D., of the MUSC Department of Pathology & Laboratory Medicine, was first author on the study.

Stylized DNA replication fork. Illustration by Madelaine Price Ball

Mechanism of genome replication arrest provides pioneering insight about cell life span and aging.

A research collaboration between the Medical University of South Carolina, the Institute of Human Genetics in France, and Howard Hughes Medical Institute at Rockefeller University has revealed the means by which cells accomplish programmed DNA replication arrest. Their results in the June 13, 2016 issue of the Proceedings of the National Academy of Sciences describe the conditions that require a replication fork to stop, and in doing so explain why terminator sites on DNA don’t always successfully stop a replication fork. It is a matter of different proteins working together to calibrate fork movement.

In a process similar to a rail system in which trains follow a coordinated schedule of stops, cells use programmed fork arrest to halt the replication machinery at predetermined places along the DNA strand called terminator sites. Terminator sites minimize collision between replication machinery and transcription machinery traveling along the same track of DNA by blocking both processes at the halted fork. A collision might otherwise cause the DNA strand to break or become unstable. Programmed fork arrest also prevents replication and transcription machinery from running constantly, which helps conserve the amount of energy a cell needs to function.

These measures control cell life span and preserve genome stability, according to Deepak Bastia, Ph.D., Endowed Chair for Biomedical Research in the MUSC Department of Biochemistry and Molecular Biology and co-senior author of the study.

“Programmed fork arrest interconnects DNA replication with aging, transcription and gene differentiation,” says Bastia. “You have to maintain the genome so that genetic integrity and life span is maintained.”

During DNA replication and transcription, DNA polymerases travel along the double helix. During replication, one enzyme, a helicase, unwinds the double-stranded DNA into two single strands that travel behind it as it moves. DNA polymerases serve as templates on each single strand, allowing synthesis of two double-stranded daughter copies from one parent DNA strand. The junction where double-stranded DNA is separated into two single strands is aptly called the fork.

Only large proteins called histones that bind tightly to DNA are guaranteed to stop a replication fork in its tracks. The replication fork machinery easily sweeps other DNA-bound proteins out of the way. In one sense, this process keeps replication moving smoothly along the DNA strand. But in order to fine-tune their life cycle, cells need a more precise measure to stop replication other than the bulky histones. It turns out that a protein called Fob1 resides at terminator sites on DNA and works intermittently to halt fork progression, much like a gate. Its biochemical signal is phosphorylation.

It is this process that Bastia and his colleagues worked out. DDK, one of the two major cell cycle dependent kinases that sense which phase of life a cell is in, is responsible for assembling a replication fork blockage on terminator sites where Fob1 is bound. During active replication, the replication machinery easily pushes Fob1 off the DNA track and continues past the terminator sites. However, when DDK phosphorylates the helicase that unwinds double-stranded DNA at the head of the fork, it initiates the formation of a protein-based landing pad that connects to the helicase. An enigmatic protein named “Timeless” then docks on the landing pad and restrains other helicases that would normally sweep  off of the DNA terminator sites ahead of the moving fork. The Fob1 gate then stops the replication fork as programmed.

Bastia’s group showed this in yeast by genetically inactivating a component of DDK that is responsible for phosphorylating the helicase. In “chromosome combing” microscopy experiments, where single-stranded and double-stranded DNA were labeled with different colored fluorescent molecules and gently extended on coverslips, inactivated DDK failed to stop the replication fork. When active DDK was blocked from phosphorylating the helicase, Timeless protein could not reach the landing pad and the replication fork proceeded uninhibited. This physiologic program, which is similar across many organisms, is also likely to be conserved in humans, according to Bastia.

Bastia states that this new understanding will inform research on aging. Deciphering the means to prolong programmed fork arrest in healthy cells might eventually extend healthy life span in humans. “Aging is a disease,” he says, “not a natural process.”

Image caption: Stylized DNA replication fork with nucleotides matched, 5'->3' synthesis shown, no enzymes in diagram. Illustration by Madeline Price Ball. Obtained via wikimedia (Creative Commons License).

Reb-1Researchers at the Medical University of South Carolina and elsewhere resolve the first protein structure in a family of proteins called transcription terminators that could provide insight into aging and cancer. The work reveals the protein Reb1 to be a traffic signal for coordinating transcription and gene replication, rather than a passive roadblock as previously thought.

Image Caption: Space-filling model of Reb1 bound to DNA. Reused with permission from PNAS.

 

In a study published on 28 March 2016 in the Proceedings of the National Academy of Sciences, researchers at the Medical University of South Carolina (MUSC) and Virginia Commonwealth University have resolved the first protein structure in a family of proteins called transcription terminators. The crystal structure of the protein, called Reb1, provides insight into aging and cancer, according to Deepak Bastia, Ph.D., Endowed Chair for Biomedical Research in the MUSC Department of Biochemistry and Molecular Biology and co-senior author of the study.

During transcription, large molecular machines read genes by traveling along double-stranded DNA. This machinery simultaneously reads out the gene code in continually lengthening chains of single-stranded RNA. The RNA code is then used to assemble proteins that cells use for growth and division. At certain times during the life of a cell, transcription must be stopped–in order to conserve cellular energy or prevent uncontrolled growth, for example. At other times, cells may be preparing to divide, during which period trouble can arise.

Before a cell can divide, the DNA must be exactly replicated for use in the new cell. During part of this process, two types of machinery are now moving along the DNA strand–transcriptional machinery and replication machinery. In regions where the two machines are moving in opposite directions, collisions can occur and DNA broken, causing mutations. Harmful gene mutations can be passed into the new cell. That’s where Reb1 comes in.

One way to prevent genome instability is to prevent replication from colliding with transcription,” says Bastia. That’s what these terminator proteins do.”

Bastia’s group knew that there are specific sites on the DNA strand called terminator regions to which Reb1 binds itself. Reb1 was thought of as a simple physical barrier that sits on DNA and blocks both the transcriptional and replication machinery from moving further along the DNA strand and colliding with each other. Then Bastia’s group did an experiment to cut the transcription terminator region (tail) off of Reb1. Intriguingly, Reb1 was no longer able to halt the transcription machinery without its tail but was still able to bind to DNA. Therefore, the simple roadblock theory couldn’t be correct.

The insight came when they solved the crystal structure–a laborious process during which Carlos R. Escalante, Ph.D., Bastia’s co-senior author from Virginia Commonwealth University, made monthly drives transporting freshly made crystals from MUSC to the X-ray crystallography facility at Brookhaven National Laboratory in New York. The crystal structure showed that, when bound to DNA, the transcription terminator tail of Reb1 can interact with a specific part of the transcriptional machinery, acting as a tether between the two.

The work illuminates Reb1 as a traffic signal for coordinating transcription and gene replication, rather than as a simple roadblock as previously thought.

Though the tether between Reb1 and the transcriptional machine is clear, the team is still not sure exactly how terminator proteins stop transcription, a question which drives their current work. And the connection between terminator proteins and colorectal cancer has been made, but work in other cancers and in aging has yet to be undertaken.

Still, Bastia suspects that this coordination prevents the type of gene errors that lead to many types of cellular aging and tumor growth, both of which are processes that result from uncontrolled transcription and replication. The group is currently researching another type of terminator protein, work which Bastia hopes will lend further knowledge to the diseases of aging.

 

Photograph of veteran on tranIn an article published in the March 2016 issue of the Journal of Anxiety Disorders, investigators in the Department of Psychiatry and Behavioral Sciences at the Medical University of South Carolina (MUSC) report that veterans who fall just below the threshold for a diagnosis of post-traumatic stress disorder (PTSD) respond to a psychotherapy regimen better than those with full PTSD. The study highlights the need to recognize veterans suffering from an overlooked condition called subclinical PTSD. “The study shows not only that we can treat those experiencing subclinical presentations of PTSD, but also that those with subclinical PTSD may actually respond better to treatment than those with more severe forms of the disease,” says MUSC investigator Kristina Korte, Ph.D., who is the first author on the article. MUSC co-authors include Ron Acierno, Ph.D., Daniel F. Gros, Ph.D., and Nicholas P. Allan, MS.

Just like patients with full PTSD, those with subclinical PTSD have experienced a traumatic event and are regularly re-experiencing it, often in nightmares or flashbacks. Patients with full PTSD also experience hyperarousal (i.e., they are easily startled) and avoid reminders of the event, for example by withdrawing from social interaction or turning to substance abuse. In addition re-experiencing the event, patients with subclinical PTSD may exhibit either hyperarousal or avoidance, but not both.

Psychologists began noticing this pattern more frequently in the nineties in veterans returning from the first Iraq War, and even more frequently in veterans returning from Iraq and Afghanistan in the last decade. As researchers have learned more about these patients over time, varying and sometimes conflicting symptoms have provided an incomplete picture of the disorder and how to treat it. Further confounding the issue is that those with subclinical PTSD are often excluded from clinical trials testing treatments for PTSD—patients with only some symptoms of PTSD commonly aren’t included in the healthy control group or in the group with full PTSD. As a result, there is still no standard psychotherapy for treating subclinical PTSD as there is for full PTSD.

The researchers devised an intuitive approach—Why not treat subclinical PTSD patients with one of the standard evidence-based psychotherapy tools already being used in PTSD patients? They enrolled 200 patients with combat-related PTSD symptoms from the Ralph H. Johnson VA Medical Center located adjacent to MUSC, identifying those with either subclinical or full PTSD. For eight weeks, patients received intensive weekly sessions of behavioral activation and therapeutic exposure therapy, designed to lessen their PTSD symptoms by helping them safely re-experience and resolve elements of the original trauma. Psychologists rated the patients’ PTSD symptoms and had patients rate their own symptoms before, during, and after the eight weeks.

The results were encouraging. Those with subclinical or full PTSD each experienced a real drop in PTSD symptoms after treatment. The striking result was in how much those symptoms dropped: 29% in those with subclinical PTSD as compared to 14% with full PTSD.

It may seem obvious that patients with a less severe form of PTSD would respond better to standard psychotherapy, but the implications for treatment extend beyond that. PTSD symptoms often worsen over time; as they do, treatments become less effective at reducing symptoms. In this context, subclinical PTSD could be seen as “early-stage” PTSD, in that treatment might be more effective when the disorder is caught early.

Gros’ group hopes these early studies can move beyond men in combat to civilians of both sexes.

“It is our hope that providing treatment for subclinical PTSD could have a significant impact on the cost-effectiveness of treating this common disorder,” says Korte. “It could lead to the prevention of more intractable forms of PTSD that can occur when subclinical PTSD goes untreated.”

Image Caption:  Licensed from iStock. Copyright: Mie Ahmt.

fatty liver disease image 3

In results published on October 19, 2015 in the Journal of Lipid Research (http://dx.doi.org/10.1194/jlr.M063511), a team of translational scientists at the Medical University of South Carolina (MUSC) report a new reason why non-alcoholic steatohepatitis (NASH) worsens in people who are obese. The results may help prevent cirrhosis and liver cancer, according to co-senior authors Kenneth D. Chavin, M.D., Ph.D., a transplant surgeon in the MUSC Health Department of Surgery, and Lauren Ashley Cowart, Ph.D., Associate Professor in the Department of Biochemistry and Molecular Biology and Co-Director of the MUSC Center of Biomedical Research Excellence in Lipidomics and Pathobiology.

 NASH (also called non-alcoholic fatty liver disease) has become a major cause of liver disease requiring transplant. “In my 17 years of doing liver transplants, it’s gone from 4% of patients to around 20% of patients who get transplants because of NASH,” says Chavin. “In 10-15 years, because of advances with Hepatitis C, it will probably become the number one reason why patients get transplants.”

When excess dietary fats are consumed over time, fat deposits form in the liver and NASH can develop. Early-stage NASH is typically not associated with any physical symptoms; nearly 30% of people in the U.S. have it. Though obesity is correlated with the development of NASH, the team wanted to know exactly why NASH worsens to a stage requiring transplant in certain obese people. “Obesity doesn’t cause disease in every obese person and we don’t understand why it does in some but not others,” explains Cowart.

The team suspected that inflammation stemming from a lipid molecule called sphingosine-1-phosphate (S1P) might be responsible. They’d previously discovered in other organs that S1P is increased by excess dietary saturated fat.

Chavin took biopsies from human livers during transplant surgery and supplied them to Cowart, who determined the levels of sphingosine kinase 1, the enzyme that makes S1P. They found double the normal amount of sphingosine kinase 1 in livers of obese people with non-alcoholic steatohepatitis.

The team wanted more understanding of why S1P causes inflammation, but NASH has previously been difficult to mimic in the laboratory setting. They developed a new and highly improved preclinical model of NASH, wherein mice were fed on custom-designed diets of either high saturated fat or high unsaturated fat. Curiously, mice on each type of diet became obese, but only mice on the saturated fat diet developed inflammation and NASH-like pathology stemming from S1P. Taking the human and pre-clinical studies together, it’s likely that saturated fat, but not unsaturated fat, raises S1P levels in obese people, and it’s S1P that unleashes the inflammation that characterizes NASH.

Performing lipid studies in the laboratory is not easy—most biochemistry is water-based, and fat and water don’t easily mix. The group relied on the MUSC Sphingolipidomics Core laboratory, one of only a handful of such facilities in the country capable of developing the new methods needed to examine S1P for their study. Without lipidomics, we never would have understood that saturated fats activate this pathway,” says Cowart. The team is working to identify the S1P receptors responsible for inflammation in NASH, with the ultimate goal of designing treatments to prevent the need for a liver transplant in obese patients with NASH.

Does this work support the idea that it’s the type of fat, but not all fat, that leads to health problems? After all, mice fed a high unsaturated fat diet still became obese but were metabolically healthy. “Because the unsaturated fat diet didn’t cause NASH, it may provide a clue as to what actually links obesity to disease,” says Cowart. “Even if it’s difficult to lose weight, dietary modifications might prevent some disease associated with obesity.”

 MUSC researchers Tuoyu Geng, Ph.D., Alton Sutter, M.D.,Ph.D., Arun Palanisamy Ph.D., and Michael D. Harland also contributed to this study.

 This work was supported by a Veterans Affairs Merit Award, National Institutes of Health Grants 1R01HL117233 and 5P30GM103339-03 (L.A.C.), and National Institutes of Health Grant 1R01DK069369 to K.D.C .

External carotidIn an article published ahead of print on November 24, 2015 in the journal Diabetes (available at http://dx.doi.org/10.2337/db15-0930), researchers from the Medical University of South Carolina (MUSC), the American University of Beirut (AUB), and Case Western Reserve University report that a molecule called pre-kallikrein (PK) could be a target for the vascular complications associated with type 1 diabetes. PK has been formerly suggested as a marker for diabetic vascular disease of the kidneys, but the new work supports the idea that increased plasma PK levels are an independent risk factor for whole-body diabetic vascular disease, similar to the risks of high triglycerides or high blood pressure in heart disease. Ayad A. Jaffa, Ph.D., who holds a dual appointment at MUSC and AUB, led the study. Other MUSC investigators included Miran A Jaffa, Ph.D., Deirdre Luttrell, Ph.D., Richard L. Klein, Ph.D., Maria Lopes-Virella, M.D., Ph.D., and Louis M Luttrell, M.D., Ph.D.  

PK is a member of the kallikrein-kinin system, a group of molecules that frequent the walls of blood vessels. In healthy vessels, circulating PK reaches the vessel surface and activates a sequence of molecular signals that travel inward to the inner vessel layers, called the intima-media, causing momentary changes in dilation and tension. The types of blood vessel malfunction seen in patients with diabetes causes the cells of the intima-media to spread to the surface, allowing PK to contact them directly. This contact closes the circuit of an alternative pathway of chronic inflammation. Scientists who study the kallikrein-kinin system suspect that this chronic inflammation is responsible for the blood vessel thickening observed in diabetic kidney disease, retinopathy, and atherosclerosis.

Jaffa’s team wanted to know if these suspicions were relevant to patients. Specifically, are levels of PK in the blood associated with the blood vessel thickening commonly seen in people with type 1 diabetes?

They started by examining patient samples housed at MUSC and collected as part of a multi-center observational study, called the Epidemiology of Diabetes Interventions and Complications, designed to track the complications and progression of vascular disease in hundreds of people with diabetes. They focused on levels of PK in blood samples paired with ultrasounds taken to measure the thickness of the intima-media of their carotid arteries.

Relevance was found: patients with higher levels of PK in their blood do have thicker layers of intima-media in the vasculature of their carotids.

It isn’t clear if high levels of PK cause arteries to thicken or if thicker arteries release more PK. In other words, Jaffa’s group can’t say yet if PK causes vascular disease or not. Their work, though, is an important first step to developing a treatment for the vascular complications that seem unavoidable for patients with type 1 diabetes. Their next steps involve developing drug candidates to target PK in preclinical experiments. “These preclinical studies not only will give us insights into the involvement of plasma PK in vascular disease,” says Jaffa, “but will also contribute to development of novel treatment strategies for diabetic vascular disease.”

Image Caption: Arteries of the neck - right side. The external carotid artery arises from the common carotid artery - labeled Common caroti on the figure. From Gray's Anatomy 1918. Public Domain Available at http://www.bartleby.com/107/Images/large/image520.gif

heart made out of rope to represent fibrotic heartIn patients with heart failure with a preserved ejection fraction (HFpEF), the prescribed treatments for managing comorbid hypertension do not seem to improve mortality as they do in other heart failure patients. Now MUSC researchers want to know why. In patients with HFpEF, who account for about half of all heart failure cases, the ventricles gradually thicken and stiffen, preventing normal relaxation from beat to beat. The underlying myocardial changes responsible for HFpEF development have proven elusive, providing a major challenge for cardiologists who seek to treat HFpEF patients. Using a translational approach, MUSC researchers and their colleagues are the first to address this challenge directly.

 MUSC Health cardiologists Michael R. Zile, M.D., and John S. Ikonomidis, M.D., Ph.D., along with their MUSC colleagues Catalin Baicu, Ph.D. and Amy Bradshaw, Ph.D., suspect that changes in certain fibrous proteins contribute to left ventricle relaxation deficits in HFpEF patients. Emerging data from a study led by Zile and published in the April 7, 2015 issue of Circulation1 examined changes in collagen and titin, two major fibrous proteins that constitute the physical scaffold necessary for normal relaxation in the heart. Using small myocardial muscle strips extracted from the hearts of 70 cardiac bypass surgery patients, Zile’s group discovered that a measure of ventricular muscle tension during relaxation, called passive stiffness, was pathologically increased in those patients with HFpEF. Just as suspected, this increase was dependent on changes in both collagen and titin. Importantly, these changes were only detected in patients with both hypertension and HFpEF. Moreover, biomarkers in patient plasma reflecting changes in collagen correlated with the presence and severity of HFpEF.

This work, undertaken at MUSC in collaboration with other centers, is the first to use tissue from HFpEF patients to pinpoint specific changes in titin and collagen as important underlying drivers of HFpEF development. How can this new information be used to help patients? Zile states that MUSC scientists are already collaborating with major pharmaceutical partners to develop new biomarker tools for HFpEF detection. “Proteins and peptides that indicate changes in collagen in the heart can be easily measured in small amounts of blood,” says Zile. “These biomarkers can be used to help make early diagnosis and predict prognostic outcomes in HFpEF patients. The arrival for these novel applications is just over the horizon.”

 Reference

 1Zile MR, et al. Myocardial stiffness in patients with heart failure and a preserved ejection fraction: contributions of collagen and titin. Circulation. 2015 Apr 7;131(14):1247-59.

 

In patients with HFpEF, thicker and stiffer ventricles impair normal relaxation and filling.

 

Fibrotic heart

The summer of 2015 saw approval of the first new drug for heart failure in almost two decades. The angiotensin receptorneprilysin inhibitor Entresto (formerly LCZ696, Novartis Pharmaceuticals) was approved for heart failure with reduced ejection fraction (HFrEF) on the basis of the results of the PARADIGM-HF trial published in the September 11, 2014 issue of the New England Journal of Medicine.1 MUSC Health cardiologist Michael R. Zile, M.D., who served on the national steering committee of the PARADIGM-HF trial, predicts that the new therapy “will  replace ACEs and ARBs as the cornerstone of standard therapy for patients with HFrEF.”

 However, patients with HFrEF account for only about half of the 3 million heart failure cases in the U.S. each year. No agent has been shown to improve morbidity and mortality in patients with heart failure with preserved ejection fraction (HFpEF), who make up the other half of heart failure cases. In patients with HFrEF, the left ventricle does not fully contract, while in those with HFpEF, the ventricle does not fully relax. The two largest clinical trials to date showed that traditional heart failure therapy with angiotensin receptor blockade (Irbesartan or Candesartan) did not improve heart function in patients with HFpEF. According to Zile, “The biggest unmet need in cardiology today is a treatment for HFpEF.”

That could be changing. Zile is on the national steering committee of the PARAGON-HF trial, which will assess the efficacy of Entresto in patients with HFpEF, and MUSC Health is one of the sites enrolling patients into the trial. Results are expected in 2017. “PARAGON-HF will be largest clinical trial in patients with HFpEF,” says Zile. “We hope that Entresto will form the foundation for novel and effective treatment that reduces symptoms and increases survival in HFpEF.”

For more information about the trials, read "A New Paradigm for the Treatment of Heart Failure."

 Reference

1 McMurray JJ, et al. Angiotensin-neprilysin inhibition versus enalapril in heart failure. N Engl J Med. 2014 Sep 11;371(11):993-1004. doi: 10.1056/NEJMoa1409077. Epub 2014 Aug 30.

Genetic Origin of Mitral Valve ProlapseAs part of a multi-center investigation recently reported in the journals Nature1 and Nature Genetics,2 researchers at the Medical University of South Carolina (MUSC) and Harvard/Massachusetts General Hospital as well as other international institutes have discovered genetic and biological causes for MVP. The investigators identify that MVP can be a result of heritable genetic errors that occur during embryonic cardiac development and progress over the lifespan of affected individuals.

Mitral valve prolapse (MVP) affects 1 in 40 individuals making it one of the most prevalent human diseases. Many individuals with MVP develop potentially life-threatening cardiac arrhythmia and heart failure.

In MVP, one or both flaps of the mitral valve bulge backward into the left atrium causing it to close improperly upon termination of atrial systole. Mitral valve prolapse is often detected as a heart murmur and is usually asymptomatic, but in roughly 10% of cases mitral valve regurgitation intensifies to a clinically severe stage. In severe cases, arrhythmic heartbeats develop, which increases the risk of stroke, heart failure and sudden cardiac death. In fact, the risks are high enough in MVP to make it the leading indication for mitral valve surgery.

In the Nature article,1 investigators used linkage analyses and capture sequencing technology to examine protein-coding genes on chromosome 11 in four members of a large family segregating non-syndromic MVP. They discovered a missense mutation in the DCHS1 gene, which codes for the protein dachsous homolog 1, a member of the calcium-dependent cell-cell adhesion family of cadherins. Another DCHS1 mutation was found in additional families segregating deleterious MVP. Both mutations reduce DCHS1 protein stability in mitral valve interstitial cells (MVICs), a finding corroborated with the discovery of the original mutation in MVICs in a human patient with MVP that underwent mitral valve repair surgery. Dchs1 mutant mice displayed similar pathology, along with scattered migration of MVICs during growth, suggesting that protein stability is essential to maintaining cues for cell polarity during mitral valve development.

In a subsequent manuscript published in Nature Genetics,2 the investigators performed a genome-wide association study (GWAS) to identify genetic variants in a population of  more than 10,000 subjects.  Single nucleotide variants (SNPs) with genome-wide significance were identified in the patient cohorts and genes surrounding these SNPs were functionally evaluated in multiple in vivo models. 

The results from both studies highlight a potential unifying biological cause for MVP in the population.

“We have found a genetic and biological reason for one of the most common diseases affecting the human population," says MUSC researcher Russell A. (Chip) Norris, Ph.D., who was a co-senior author on the studies. "This is a critical initial step as we transform this discovery into new remedial therapies to treat the disease.”  Roger R. Markwald, Ph.D.,  and Andy Wessels, Ph.D. both of the Department of Regenerative Medicine and Cell Biology at MUSC, were also co-authors.

If you are interested in supporting medical research, visit donorscure.org, an MUSC-affiliated 501(c)(3) nonprofit organization that allows you to fund biomedical research projects led by researchers across the United States.

References

1 Durst, et al. Mutations in DCHS1 cause mitral valve prolapse. Nature. 2015 Aug 10 [Epub ahead of print]. Available at http://dx.doi.org/10.1038/nature14670

2 Dina C, et al. Genetic association analyses highlight biological pathways underlying mitral valve prolapse.
Nat Genet. 2015 Aug 24.  [Epub ahead of print] Available at http://dx.doi.org/10.1038/ng.3383.

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