Newly installed equipment in the USF Health Heart Institute Preclinical Imaging Suite.

The USF Health Heart Institute has installed new variable 7T-3T MR/PET and PET-CT integrated imaging systems – establishing the first comprehensive preclinical imaging suite at the USF Health Morsani College of Medicine.

The 520-square-foot suite includes three pieces of leading-edge MR Solutions equipment operated from one console – a magnetic resonance imager (MRI), a computed tomography scanner (CT), and a positron emission tomography (PET) machine.  It also houses an existing state-of-the-art cardiac ultrasound machine used for preclinical studies.

The compact multimodal systems can be used to visualize small animal models (rodents) from the molecular/cellular level to entire organs, including the blood flow of a beating heart. It combines PET’s sensitivity in tracking the metabolism of living cells and uniquely labeled cell types with MRI’s and CT’s strength in high-resolution anatomical imaging of soft tissue (i.e., heart, brain, blood vessels, nerves) and bony structures like the skull and spine. The PET technology can be used independently, or sequentially with either the MRI or the CT machine.

“This new preclinical imaging technology enables us to capture more detailed data critical for cardiovascular studies where we need to see exactly how the heart and vascular system respond to diseases and various targeted therapies,” said USF Health Heart Institute Director Samuel Wickline, MD. “It will enhance our competitiveness for National Institutes of Health grants and can also help advance research on cancer, neurodegenerative diseases and many other conditions.”

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The 7T MR/PET imaging system uses pioneering dry magnet (liquid helium-free) technology, making the equipment much smaller, lighter and less costly than conventional wet magnet systems. The 7T MR scanner is self-shielded to prevent noise interference.

“Quantitative research methods developed for use in the powerful preclinical imaging systems are directly applicable to larger imaging platforms used in clinical medicine”, said Dr. Wickline, a professor of cardiovascular sciences who focuses on building nanoparticles to deliver drugs, genes, or other therapeutic molecules to specific cell types, or targets.

“It’s basically the same physics and engineering on a smaller scale, which transfers nicely to the imaging devices we use for patients.”

The Heart Institute ultimately plans to operate the suite as a core research facility, allowing biomedical researchers across USF as well as collaborators outside the university to access its imaging services for reasonable fees, Dr. Wickline said.

Diabetic mouse heart with 60 frames per cardiac cycle. | Image courtesy of Mariah Daal, MSc-PhD candidate, AMC, Amsterdam, The Netherlands: MRS* DRYMAG 7T

Cardiac muscle fibers (left ventricle, mouse heart) acquired with an MRI technique known as diffusion tensor imaging tractography.  The reconstructed image can  improve understanding of cardiac remodeling due to heart muscle disease. | Courtesy of Pr Gustav Strijkers, AMC, Amsterdam, The Netherlands: MRS* DRYMAG 7T.

The USF Health Heart Institute at the University of South Florida is committed to translating their findings from the laboratory to the clinic to directly benefit patients. Scientists and physicians work together with the common goal of creating new therapies for cardiovascular disease, generating biomedical inventions leading to patents and licenses, and attracting biotech and pharmaceutical companies with their innovations.

The USF Health Heart Institute is housed in the newly constructed USF Health Morsani College of Medicine, which opened earlier this year in the Water Street Tampa wellness district.

-Photos of preclinical imaging systems equipment courtesy of Gilberto Prudencio, MRS Solutions

A USF Health-Johns Hopkins Medicine team shows that nanoparticles targeting the blood-clotting protein reduces necrotizing enterocolitis-like injury in neonatal mice

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TAMPA, Fla. (May 4, 2020) — Necrotizing enterocolitis (NEC), a rare inflammatory bowel disease, primarily affects premature infants and is a leading cause of death in the smallest and sickest of these patients. The exact cause remains unclear, and there is no effective treatment. Since no test can definitively diagnose the devastating condition early, infants with suspected NEC are carefully monitored and administered supportive care, such as IV fluids and nutrition, and antibiotics to fight infection caused by bacteria invading the gut wall. Surgery must be done to excise damaged intestinal tissue if the condition worsens.

A new preclinical study by researchers at the University of South Florida Health (USF Health) Morsani College of Medicine and Johns Hopkins University School of Medicine offers promise of a specific treatment for NEC, one of the most challenging diseases confronting neonatologists and pediatric surgeons.  The team found that inhibiting the inflammatory and blood-clotting molecule thrombin with targeted nanotherapy can protect against NEC-like injury in newborn mice.

Their findings were reported May 4 in the Proceedings of the National Academy of Sciences.

“Our data identified the inflammatory molecule thrombin, which plays a critical role in platelet-activated blood clotting, as a potential new therapeutic target for NEC,” said coauthor Samuel Wickline, MD, professor of cardiovascular sciences at Morsani College of Medicine and director of the USF Health Heart Institute. “We showed that anti-thrombin nanoparticles can find, capture and inactivate all the active thrombin in the gut, thereby preventing or reducing the small blood vessel damage and clotting that accelerates NEC.”

The PNAS paper’s senior author is Akhil Maheshwari, MD, professor of pediatrics and director of neonatology at the Johns Hopkins University School of Medicine. Before joining Johns Hopkins Medicine (Baltimore) in 2018, Dr. Maheshwari’s group at USF Health was the first to demonstrate that platelet activation is an early, critical event in causing NEC, and therapeutic measures to block these platelets might be a new way to prevent or reduce intestinal injury in NEC.

The nanotherapy platform created by Dr. Wickline and USF Health biomedical engineer Hua Pan, PhD, delivers high drug concentrations that specifically inhibit thrombin from forming blood clots on the intestinal blood vessel wall without suppressing the (clotting) activity needed to prevent bleeding elsewhere in the body. This localized treatment is particularly important for premature infants, Dr. Wickline said, because the underdeveloped blood vessels in their brains and other vital organs are still fragile and susceptible to rupture and bleeding.

Samuel Wickline, MD, and Hua Pan, PhD, of the USF Health Heart Institute, created the treatment platform used to deliver anti-thrombin nanoparticles to the site of gut damage.

For this study the researchers used a model they created — infant mice, or pups, induced to develop digestive tract damage resembling human NEC, including the thrombocytopenia commonly experienced by premature infants with NEC. Thrombocytopenia is characterized by low counts of blood cell fragments known as platelets, or thrombocytes, which normally stop bleeding from a cut or wound by clumping together to plug breaks in injured blood vessels.

The molecule thrombin plays a key role in the bowel inflammation driven by overactive platelets. While investigating role of platelet depletion in NEC-related thrombocytopenia, the USF-Johns Hopkins researchers were surprised to find that thrombin mediates platelet-activated blood clotting early in the pathology of NEC-like injury – before bacteria leaks from inside the gut to circulating blood or other organs. This clotting clogs small blood vessels and restricts blood flow to the inflamed bowel. Eventually, the lining of the damaged intestinal wall can begin to die off.

The investigative therapy essentially works “like a thrombin sponge” that is exponentially more potent than current agents used to inhibit clotting, Dr. Wickline explained. “It literally puts trillions of nanoparticles at that damaged (intestinal wall) site to sponge up all the overactive thrombin, which tones down the clotting and inflammation processes promoted by thrombin.”

“We are so excited about finding this new way to attenuate intestinal injury in NEC,” Dr. Maheshwari said.

The same approach has also been shown in preclinical studies to inhibit the growth of atherosclerotic plaques and certain kidney injuries without causing systemic bleeding problems, Dr. Wickline added. “The nanoparticles can be tailored to other inflammatory diseases highly dependent on thrombin for their progression.”

The study authors conclude that their experimental targeted treatment for NEC merits further evaluation in clinical trials. Grants from the National Institutes of Health supported the project.

A leading expert on the role of monocytes and macrophages (types of immune cells) in atherosclerosis and other chronic inflammatory conditions delivered the keynote address Nov. 7 at the USF Health Heart Institute’s 2^nd Annual Scientific Colloquium.

Gwendalyn Randolph, PhD, an immunologist by training, began her career by studying how innate immune cells travel around the body and along the way began discovering connections between cardiovascular disease, lipid metabolism and the gut.

USF Health Heart Institute Director Samuel Wickline, MD, with speakers at the Institute’s 2nd Annual Scientific Symposium. From left: David Lominadze, PhD, USF Health professor of surgery; Gwendalyn Randolph, PhD, Unanue Distinguished Professor of Pathology and Immunology at Washington University School of Medicine in St. Louis; Dr. Wickline; and Travis Jackson, PhD, USF Health associate professor of molecular pharmacology and physiology.

For the Heart Institute talk, Dr. Randolph, the Emil R. Unanue Distinguished Professor of Pathology and Immunology at Washington University School of Medicine in St. Louis, focused on research investigating what drives inflammation in atherosclerosis – the most common cause of heart attacks.  She shared her work on the trafficking of immune cells and the lipoproteins that carry cholesterol through the bloodstream to deposit inside the artery walls.

“Dr. Randolph’s work in the field of atherosclerosis has produced novel and important insights into the critical cell types that are responsible for forming atherosclerotic plaques in patients with heart disease,” said Samuel Wickline, MD, professor of cardiology and director of the USF Health Heart Institute. “She also has elucidated the molecular factors that attract these cells to plaques and cause them to grow and become unstable, which leads the plaques to break down and clot. This process can ultimately result in blockage of vessels that supply blood to the heart and brain, causing heart attacks and strokes.”

Research by Dr. Randolph, this year’s keynote speaker at the colloquium, has yielded new insights into how immune cells drive inflammation contributing to atherosclerotic plaques in heart disease.

Mouse model studies by Dr. Randolph and others have shown white blood cells, known as monocytes, contribute to the initial build-up of atherosclerotic plaques.

The cascade of events leading to atherosclerosis can take decades.  Initial damage to the inner wall (endothelium) of arteries under the influence of high cholesterol levels triggers a molecular signal that attracts monocytes to travel from the bloodstream into developing plaques. These recruited monocytes are converted into macrophages that take up (eat) the cholesterol trapped in blood vessels and eventually die off. But before that happens, they stay busy secreting molecules that drive plaque inflammation  and weaken the vessel wall, leading to plaque rupture, clotting, and coronary artery obstruction.

In terms of developing new therapies to halt or reverse atherosclerosis, Dr. Randolph said, her research suggests that upstream targeting of recruited monocytes — either before or just after these immune cells arrive in plaques — may be more beneficial than targeting the fat-laden macrophages known as foam cells. Experiments with fluorescent tracers indicate that monitoring endothelial cells lining the arterial wall may be a way to track monocyte migration, she added.

Thomas McDonald, MD, a professor in the USF Health Morsani College of Medicine’s Department of  Molecular Pharmacology and Physiology and member of the Heart Institute, listens to Dr. Randolph’s talk.

In addition to Dr. Randolph, two new faculty members recruited this summer to the USF Health Heart Institute – Travis Jackson, PhD, and David Lominadze, PhD — provided overviews of their National Institutes of Health-funded research.

Dr. Jackson, an associate professor in the Department of Molecular Pharmacology and Physiology, discussed his translational work in therapeutic hypothermia — investigating ways to optimize cold-shock proteins and cold-stress hormones to increase the benefits of cerebroprotective cooling for traumatic brain injury. Dr. Lominadze, a professor in the Department of Surgery, presented research looking into the interactions of blood cells and the endothelium, with the aim of better understanding the microcirculatory disorders associated with cardiovascular and cerebrovascular diseases.

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The USF Health Heart Institute is scheduled to move to the new USF Health Morsani College of Medicine building in Water Street Tampa in late February 2020.  Its annual scientific colloquium will be held in the new home next year, and continue to evolve with the growth of the Institute, Dr. Wickline said.

“We will expand the program to cover other topics of interest to the cardiovascular community such as genetic heart diseases, heart failure, peripheral vascular disease, and gene therapy.”

-Photos by Allison Long, USF Health Communications and Marketing

A Washington University-USF Health preclinical study finds that the novel nanoparticle delivers gene-level treatment to suppress KRAS-driven cancer without adverse effects

Despite advances in cancer survival, more than 90 percent of people with pancreatic cancer die within five years. Most patients with pancreatic tumors (and half of those with colorectal cancers) carry a mutation in the KRAS gene, which normally controls cell growth and death.

The KRAS oncogene was discovered more than 35 years ago and is considered one of the most desirable targets in cancer biology — particularly for cancers (like pancreatic) often diagnosed late and in desperate need of improved therapies to prolong survival. Yet KRAS has earned a reputation as being “undruggable” by researchers who continue searching for effective ways to inhibit the mutated form of RAS proteins driving the growth of deadly tumors.

Now a preclinical study led by Washington University School of Medicine in St. Louis, including co-investigators at the University of South Florida Health (USF Health) Morsani College of Medicine, Tampa, Fla., has demonstrated that specially designed peptide-based nanoparticles can suppress pancreatic cancer growth without the toxic side effects and therapeutic resistance seen in drug trials. The findings were published July 30 in Oncotarget.

The nontoxic, peptide-based p5RHH nanoparticles designed by USF Health researchers Samuel Wickline, MD, and Hua Pan, PhD, MBA, deliver an RNA molecule known as small interfering RNA, or siRNA (also known as silencing RNA). The molecules silence the chemical signal (message) telling the KRAS oncogene to make more mutated KRAS proteins that cause pancreatic cells to grow uncontrollably and largely resist existing cancer-killing drugs.

Confocal microscopy image shows fluorescent-tagged nanoparticles (pink) carrying siRNA diffusely taken up by mouse colorectal cancer cells.| Photo courtesy of Washington University School of Medicine in St. Louis

“We’ve developed a nanoparticle system that gets enough of the therapeutic molecule from the bloodstream to the tumor cell without (the molecule) being metabolized or excreted,” said coauthor Dr. Wickline, professor of cardiovascular sciences and director of the USF Health Heart Institute. “The nanoparticle is actively taken up by the targeted tumor cells and then the molecule escapes and does its job to prevent production of mutated KRAS proteins.”

“Approaches that precisely target tumors with various therapies are the future of cancer care. Nanoparticle delivery allows higher concentrations of drugs to reach their target while sparing normal tissues any side effects,” said senior author Ryan Fields, MD, professor of surgery and chief of surgical oncology at Washington University School of Medicine. “In pancreatic cancer, where breaking through the resistant tumor microenvironment is a current unmet need, this approach has the potential to improve therapeutics and patient outcomes.”

Compared to control cells, nanoparticle treatment of both pancreatic and colorectal cells was shown to deliver KRAS-specific siRNA, decrease KRAS RNA expression and lead to increased tumor cell death. Using a genetically engineered mouse model for spontaneously arising pancreatic cancer, the researchers also demonstrated that the intravenously administered nanoparticles could selectively target silencing RNA against the KRAS oncogene to ultimately slow KRAS-driven pancreatic cancer growth.  Their system effectively delivered this nanotherapy even in the “stroma-rich” environment of pancreatic tumors.

3D reconstruction of a single pancreatic cancer cell treated with siRNA nanoparticles. Confocal microscopy depicts accumulation of strong fluorescent silencing RNA signaling (pink) within the cell membrane (cyan) boundaries, but separate from lysosomes (yellow) that can degrade the nano-delivered molecules. | Photo courtesy of Washington University School of Medicine in St. Louis

Pancreatic cancer is so difficult to treat in part because fibrous tissue, or stroma, that surrounds the solid tumor “like the shell of a clam” is much denser than the stroma surrounding other more treatable tumors, Dr. Wickline explained. This protective stromal barrier can complicate nanoparticle-delivered treatment.

“Our p5RHH peptide nanoparticle is relatively small and it can squeeze in and out of tight spaces to get (treatment) safely into tumor-specific cells while staying out of normal tissue,” he said.  “It avoids adverse ‘off-target’ effects.”

Cancer is very efficient at evading treatments that target one, or even a few mutated genes or proteins, Dr. Wickline said. “The advantage of this nanoplatform carrying siRNA is that it’s easy to change out the target you want silenced, or add many more targets for simultaneous treatment in the same tumor cell.”

The nanoparticles formulated by Dr. Wickline and Dr. Pan have also shown promise for siRNA treatment in mouse models of atherosclerosis and arthritis.

The Oncotarget reported study was supported in part by grants from the National Cancer Institute.

Samuel Wickline, MD, and Hua Pan, PhD, researchers at the USF Health Heart Institute, designed the siRNA nanoparticle system tested in the study using tumor cells and a genetically engineered mouse model for pancreatic cancer. | Photo by Eric Younghans

Two USF Health Morsani College of Medicine research-focused institutes rank among the Top 10 Institutes and Centers within Florida’s State University System (SUS).  The ranking was derived from the SUS 2018 survey of its 536 university institutes and centers engaged in scientific research, education, community service and other scholarly activity supported by public and private funds.

The USF Health Informatics Institute (HII) was the No 1 institute with $69.6 million in total expenditures. HII is led by Distinguished University Professor Jeffrey Krischer, PhD, who ranks in the top 1 percent of all National Institutes of Health-funded principal investigators worldwide (Blue Ridge Institute for Medical Research, 2018). He has made USF an international hub for NIH epidemiological research initiatives in both type 1 diabetes and rare diseases.

The USF Health Heart Institute, directed by Samuel Wickline, MD, professor of cardiovascular sciences, attained the No. 7 spot, with $13.4 million in total expenditures. The Heart Institute, created with the support of state and county funding, brings together NIH-funded laboratory researchers and physician-scientists to pioneer new discoveries for heart attacks, stroke and other cardiovascular diseases.

Jeffrey Krischer, PhD, leads the USF Health Informatics Institute

Virtually every major university conducting type 1 diabetes research is linked to Dr. Krischer’s institute at USF Health. The HII team coordinates, analyzes and maintains data from several international NIH-sponsored clinical networks investigating the causes and outcomes of type 1 diabetes, including The Environmental Determinants of Diabetes in the Young (TEDDY), TrialNet, the Rare and Atypical Diabetes Network (RADIANT), and the Trial to Reduce IDDM in the Genetically at Risk (TRIGR). Members of the Institute also have funding from industry, the Patient-Centered Outcomes Research Institute (PCORI) and the NIH for studies in oncology, type 2 diabetes, molecular biology and “big data” (‘omics).

“The Health Informatics Institute has been able to design and implement an infrastructure to support high performance computing and big data and create a platform for scientific advances yet to come,” Dr. Krischer said.

Samuel Wickline, MD, is founding director of the USF Health Heart Institute.

The USF Health Heart Institute will be housed within the new Morsani College of Medicine building now in the final stages of construction in downtown Tampa. By bridging basic science and clinical translational research to create new therapies for heart disease, generating biomedical inventions leading to patents and licenses, and attracting biotech and pharmaceutical companies with its innovative work, the Heart Institute is expected to be a major driver of economic activity in the Tampa Bay region.

“USF has put forward a significant investment to pursue solutions to cardiovascular disease, the number one cause of death and health care expenditures worldwide,” said Dr. Wickline, a pioneer in harnessing nanotechnology to combat all types of inflammatory diseases. “It’s vital to the public good for universities to undertake applied translational research that achieves useful bench-to-bedside successes.”

USF Health Heart Institute scientists developed nanoparticles deployed to suppress inflammation in the bowels of anemic infant mice, preventing transfusion-associated intestinal injury

Babies born prematurely who are severely anemic can develop necrotizing enterocolitis, or NEC, within 48 hours after receiving a red blood cell transfusion.

Physicians have long suspected that red blood cell transfusions given to premature infants with anemia may put them in danger of developing necrotizing enterocolitis, or NEC, a potentially lethal inflammatory disease of the intestines. However, solid evidence for the connection has been difficult to obtain in part because of the lack of a practical animal model able to accurately represent what physically occurs when a baby gets NEC.

Now, a research team led by Johns Hopkins Medicine, including USF Health Morsani College of Medicine collaborators, describes the development of a new model — using infant mice, or pups, that are first made anemic and then given blood transfusions from neonates of a different mouse strain. The method, the researchers say, mimics what happens when blood transfusions are given to human babies from a non-related donor.

“We needed a working live mouse model in order to learn if a blood transfusion alone leads to NEC or does it only happen if the transfusion is given when anemia is present,” says Akhil Maheshwari, MD, professor of pediatrics at the Johns Hopkins University School of Medicine, director of neonatology at Johns Hopkins Children’s Center, and senior author of the research paper.

A description of the mouse model, along with significant findings and potential benefits from its first uses, is published in a new paper in the journal Nature Communications.

Maheshwari, a professor of pediatrics at USF Health before joining Johns Hopkins Medicine (Baltimore) in 2018, conducted much of the research published in the paper while he was at the USF Health and Tampa General Hospital. Nanoparticles designed by USF Health coauthors Samuel Wickline, MD, and Hua Pan, PhD, were employed to test whether immune cells (macrophages) in the intestines of the anemic infant mice must undergo inflammatory activation to cause bowel injury.

More common in severely anemic preemies after transfusion

Seen in approximately 10% to 12% of infants weighing less than 3.5 pounds at birth, NEC is a rapidly progressing gastrointestinal emergency in which bacteria invade the wall of the colon, causing inflammation that can ultimately destroy healthy tissue at the site. If enough cells are necrotized (killed) that a hole results in the intestinal wall, fecal material can enter the bloodstream and cause life-threatening sepsis.

Since 2004, Maheshwari says, research studies have repeatedly shown that babies born prematurely who are severely anemic — those with a proportion of red blood cells to total blood volume between 20% and 24% at birth — can develop NEC within 48 hours after receiving a red blood cell transfusion. By comparison, the American Academy of Pediatrics says that babies delivered at term normally have red blood cell volumes between 42% and 65%, dropping to between 31% and 41% at age 1.

In search of a useful and practical mouse model, Maheshwari and his colleagues had to overcome a size problem. “Newborn mouse pups are about the size of a quarter and weigh less than an ounce, so it’s extremely difficult to remove enough blood from them for laboratories to analyze,” Maheshwari says.

A new working mouse model was used to investigate whether blood transfusions alone leads to NEC, or if the potentially deadly inflammatory disease only occurs only when tranfusions are administered in the presence of anemia.

To get past that obstacle, a private medical diagnostic equipment company donated the use of its advanced blood analysis system that only requires a 5 microliter (5 millionths of a liter) sample instead of the 50 microliters — 60% of a mouse pup’s total blood supply — that most testing labs require.

Next, the researchers designed a procedure to induce severe anemia in the pups by removing about half of their blood volume every other day for 10 days after birth. This dropped their red blood cell counts to levels approximating those in severely anemic newborn babies.

Seven days after birth, the researchers introduced bacteria that had been isolated and cultured from a premature infant with NEC. Finally, red blood cell transfusions were given on the 11th day after birth.

Over the next 48 hours, the researchers looked for development of NEC-like symptoms in their experimental group and three other sets of mouse pups: (1) a control group without any intervention, (2) a group without anemia that received transfusions and (3) a group with anemia but not transfused.

“Only the severely anemic pups who received blood transfusions showed intestinal damage that resembled human NEC with necrosis, inflammation and separation of the tissues supporting the lining of the colon,” Maheshwari says. “The next step was to see if we could find a mechanism for why this occurred.”

A “double whammy” on the intestinal wall

Examining the blood of the pups with NEC-like conditions after they were transfused, the researchers discovered that it contained three components not seen in the blood of the other test mice: (1) a large number of macrophages, the immune cells that engulf and digest cellular debris, bacteria and viruses, (2) freely circulating hemoglobin, the iron-based molecules that normally carry oxygen throughout the body when attached to red blood cells, and (3) elevated levels of inflammation-inducing proteins, indicating that the macrophages had been activated even without a biological threat to the intestine.

The researchers also observed that levels of haptoglobin, a protein that removes free hemoglobin from the blood, were extremely low.

“These findings suggest that anemia reduces the amount of haptoglobin in the neonate, preventing the free hemoglobin that comes in via transfusion from being properly removed as it normally would,” Maheshwari says.

What apparently happens, he says, is that free hemoglobin attaches to a protein receptor on the intestinal wall that is the same site where bacterial poisons bind. As a result, the immune system mistakenly believes the intestine is being attacked and activates the macrophages.

Once those immune cells go to work, Maheshwari explains, they trigger release of the inflammatory proteins seen in the blood of the anemic, transfused mice. “That event starts a double whammy on the intestinal wall,” he says. “First, the macrophage proteins inflame and weaken the tissues, making them vulnerable, and then, bacteria move in and produce endotoxins that kill the individual cells.”

Johns Hopkins Medicine senior author Akil Maheshwari, MD, says he hopes the findings from the current study can be used to develop blood biomarkers that could indicate which human newborns are most at risk of developing NEC.

With evidence for a probable mechanism to explain the connection between anemia and transfusion in the development of NEC, the researchers next sought to confirm it by seeing if they could block two of its stages, and perhaps, advance the search for potential therapies.

“In one trial, we gave haptoglobin to our model anemic mice before transfusing them and blocked macrophage activation, so they did not develop NEC-like symptoms,” Maheshwari says.

Controlling a master regulator of inflammation in NEC

In another test, nanoparticles that Samuel Wickline, MD, and his colleagues at the University of South Florida  Health developed were used to deliver a genetic interruption — an RNA molecule known as a small interfering RNA, or siRNA — that blocks the chemical signal telling macrophages to start producing inflammatory proteins. The nanoparticles were tagged with a fluorescent dye to track their movement and included a non-toxic compound derived from honeybee venom.

Maheshwari says the macrophages in the blood of anemic mouse pups engulfed the nanoparticles and enclosed them within vacuoles. The bee venom derivative, he explains, broke open the vacuoles so that the siRNA could be released inside the macrophages.

The genetic signal blocker worked well, Wickline says, protecting the anemic mouse pups from intestinal inflammation after red blood cell transfusions.

“Because we showed that the inhibitory nanoparticles with siRNAs were able to control a master regulator of inflammation in NEC, perhaps this technology can one day be applied to not only treat or prevent NEC, but other diseases where inflammation plays a key role such as arthritis and atherosclerosis,” he says. “We had previous shown in preclinical studies that while these peptide siRNA nanoparticles can suppress harmful inflammation, they do not affect levels of an inflammatory signaling molecule (NF-kB) important in protecting normal tissues. That sets the stage for clinical testing in the near future.”

Maheshwari says he hopes the new mouse model and the findings from the current study can be used to develop blood biomarkers that could indicate which human newborns are most at risk of developing NEC.

Research paper coauthors Samuel Wickline, MD, and Hua Pan, of the USF Health Morsani College of Medicine, developed the nanoparticles used in the study.

Funding for this investigation came from National Institutes of Health (NIH) awards HL124078 and HL133022, and American Heart Association award 14GRNT20480307, given to Maheshwari; and NIH awards HL073646, DK102691 and AR067491, given to Wickline. Sysmex America donated the use of its automated veterinary hematology analyzer that made possible the mouse model developed in this study.

Collaborating on the study were researchers from the Johns Hopkins University School of Medicine, the Morsani College of Medicine at the University of South Florida, the Yale School of Medicine and the University of Alabama at Birmingham. Along with Maheshwari and Wickline, the team members included lead author Mohan Kumar Krishnan, Kopperuncholan Namachivayam, Tanjing Song, Byeong Jake Cha, Andrea Slate, Jeanne Hendrickson, Hua Pan, Joo-Yeun Oh, Rakesh Patel, Ling He and Benjamin Torres.

-This story is an edited version of a news release by Johns Hopkins Medicine. 

The peptide-siRNA nanoparticle technology applied in the preclinical study was developed by USF Health Heart Institute researchers

Advanced ovarian and uterine cancers are deadly diseases. Ovarian cancers, in particular, present with vague symptoms common to other diseases, and often are not diagnosed until a late stage when cancer has spread throughout the abdomen.  More options are needed to effectively treat these metastasized gynecological cancers and improve patient survival rates.

A preclinical study published recently in Scientific Reports demonstrates that nanoparticle-delivered small interfering RNA (siRNA) targeting production of the protein AXL (AXL siRNA) inhibits metastasis of ovarian and uterine cancer cells.  The study was conducted by researchers at Washington University School of Medicine, St. Louis, Mo., and the USF Health Heart Institute, University of South Florida Morsani College of Medicine, Tampa, Fla.

3D illustration of ovarian cancer

The research team used a new nanoparticle system developed by USF Health co-investigators Samuel Wickline, MD, and Hua Pan, MBA, PhD, to test the experimental nanotherapy in human uterine and ovarian tumor cells and in immunodeficient mice implanted with these cancer cells.

“We’ve figured how to package in a simple peptide all the critical steps needed to efficiently get this particular small interfering RNA (also known as silencing RNA) into tumor cells and then release the siRNA so it can do its job,” said Dr. Wickline, a professor of cardiovascular sciences who direct the USF Health Heart Institute. “The nanoparticle basically hijacks the tumor cells’ biological machinery to get the siRNA where it needs to go – without being destroyed along the way, or creating harmful side effects.”

The nanoparticle combines two components in one delivery package 100 times smaller than a red blood cell: AXL siRNA and the peptide p5RHH.  AXL siRNA is designed to target and silence the expression of AXL, a key molecule that drives uterine and ovarian cancers. The p5RHH nanoparticles are derived from a major substance of bee venom called melittin, detoxified and selectively modified to facilitate timely escape of AXL siRNA from the nanostructure once the silencing RNA is delivered inside the targeted tumor cells.

Among the findings of the Washington University-USF Health study:

• In cell culture, treatment with p5RHH-siAXL nanoparticles decreased the ability of uterine and ovarian cancer cells to migrate and invade neighboring normal tissues.
• Mice with established uterine and ovarian tumors were intravenously and abdominally (intraperitoneally) injected with nanoparticles containing p5RHH and fluorescent control siRNA. The peptide nanoparticles localized to and released their contents into both tumor cell types regardless of the injection route, but fluorescent imaging showed that intraperitoneal administration was more effective than IV administration.
• In the mouse models, p5RHH-siALX treatment significantly reduced metastasis of both uterine and ovarian cancer without toxic effects.

USF Health Heart Institute Director Samuel Wickline, MD, and biomedical engineer Hua Pan, PhD, build nanoparticles to safely and efficiently deliver drugs or other therapeutic agents to specific cell types.

Overall, the study demonstrates this nanoparticle approach shows promise for treating patients with ovarian or uterine cancers, the authors conclude.

Challenges in translating preclinical successes into patient care remain, but Dr. Wickline believes nanoparticle-mediated delivery of siRNA has applications beyond just suppressing one target (AXL) implicated in other cancers in addition to uterine and ovarian.

The power of harnessing tiny nanotechnology for gene therapies lies in its flexibility, he said.

“As we identify new disease-modifying targets, it offers the potential to attack multiple different targets at the same time.  So, one nanoparticle could deliver a whole host of genetic materials – a combination of RNA interference drugs, or other types of synthetic RNA or DNA-based drugs —  to hit any specific cell types where treatment is needed.”

Preclinical cancer study has implications for inhibiting atherosclerotic plaque growth

Uncontrolled angiogenesis (new blood vessel growth) plays a critical role in the growth and spread of cancer. | Image courtesy of The Angiogenesis Foundation

The formation of new blood vessels, known as angiogenesis, is essential for embryonic tissue development, wound healing and regenerative functions.  But when this normal process goes awry, the oxygen and nutrients carried though blood vessels can spur tumor cells to multiply uncontrollably and lead to cancer in distant tissues.

Cancer medications inhibiting excessive growth of new blood vessels frequently target vascular endothelial growth factor (VEGF), the main driver of blood vessel formation in tumors.  While VEGF inhibitors have been a breakthrough in treating many cancers, the medications also cause cardiotoxic side effects, such as hypertension and blood clots, in some patients.

Seeking to improve selective targeting of tumor-associated vessels and the safety profile of anti-angiogenesis therapy, a team of scientists, including USF Health Heart Institute Director Samuel Wickline, MD, investigated the role of the developmentally critical transcription factor Etv2 in tumor angiogenesis. VEGF signaling helps blood vessels survive and maintain stability when disease is not present in addition to regulating tumor angiogenesis. But, Etv2 appears to be expressed only in cancer tissue in adults, making the molecular control mechanism an attractive target, said Dr. Wickline, a professor of cardiovascular sciences and medical engineering at USF Health Morsani College of Medicine.

Findings of the preclinical study, led by Washington University School of Medicine in St. Louis, were reported last year in JCI Insight.

Testing a new target

The researchers found that optimal tumor growth requires reactivation of dormant Etv2, and Etv2-deficient tumor blood vessels resembled normal, stable vessels when mice without tumors were compared to those in which the Etv2 gene was knocked out (disabled). They then injected into tumor-bearing mice nanoparticles carrying small interfering RNA (siRNA) specifically designed to hit and silence Etv2, with the intent of switching off the signaling needed to feed the tumor’s blood supply and fuel VEGF-induced tumor growth.

The treatment worked to block tumor angiogenesis without creating cardiovascular side effects associated with VEGF inhibitors.  More research is needed, but the study authors concluded that combining Etv2 inhibition with chemotherapy or immunotherapy may improve future cancer treatments.

Samuel Wickline, MD, director of the USF Health Heart Institute, and USF Health biomedical engineer Hua Pan, PhD, design nanoparticles to precisely deliver therapeutic molecules to specific cell types associated with inflammatory diseases.

A balance of signals circulating in the microenvironment around each cell tightly regulates angiogenesis.  When no longer needed to establish the cardiovascular system and drive blood supply to the embryo’s developing organs, angiogenesis switches off. In adults, angiogenesis primarily switches on for a limited time to restore blood flow to tissues or organs after injury.  A hallmark of cancer, however, is an angiogenesis switch that never turns off.

“While a tumor (early cancer) is still growing slowly we want to turn off angiogenesis to prevent new blood vessel growth and metastasis,” Dr. Wickline explained. “The idea behind this particular nanotechnology targeting Etv2 is that the treatment selectively inhibits the angiogenesis associated with cancer progression, and not other physiological angiogenesis processes that are needed for repair of tissues anywhere else in the body.”

Potential applications for atherosclerosis

Dr. Wickline’s laboratory built the nanoparticle peptide-siRNA structure capable of precisely delivering a message to silence Etv2 in tumors.  That same nontoxic delivery system can carry other therapeutic molecules to various specific cell types in the body associated with inflammatory diseases, including cardiovascular disorders.

The researchers plan to test whether Etv2 may also be a good nanotherapy target for blocking angiogenesis in atherosclerosis, which leads to fatty plaques building up in the arteries. That’s because abnormal blood vessel growth also provides nutrients to the inflammatory cells inside atherosclerotic plaques and plays a crucial role in plaque rupture that can cause heart attacks and stroke.

“Just like inhibiting angiogenesis starves a tumor of its blood supply,” Dr. Wickline said, “we also want to find ways to starve plaques of the blood supply to keep them from getting bigger and more dangerous.”

March 25, 2019 — USF Health Heart Institute Director Samuel Wickline, MD, a pioneer in nanotechnology targeting heart disease and other disorders, has been inducted into the American Institute for Medical and Biological Engineering (AIMBE) College of Fellows Class of 2019.  Election as an AIMBE Fellow is one of the highest professional distinctions; the College of Fellows is comprised of only the top 2 percent of medical and biological engineers.

Samuel A. Wickline, MD, founding director of the USF Health Heart Institute

Dr. Wickline is also the Tampa General Hospital Endowed Chair in Cardiovascular Medicine, associate dean for cardiovascular research, and a professor of cardiovascular sciences, molecular pharmacology and physiology, and medical engineering at the University of South Florida.  He was among the 156 new Fellows inducted at a ceremony held March 25 during the AIMBE Annual Meeting at the National Academy of Sciences, Washington, DC. The inductees also included Norma Alcantar, PhD, professor of chemical and biomedical engineering at USF.

Dr. Wickline was nominated, reviewed, and elected by peers and members of the College of Fellows for “pioneering advancements in molecular imaging with ultrasound and magnetic resonance imaging/magnetic resonance spectroscopy, and biocompatible nanotechnologies targeting myriad diseases.”

Much of Dr. Wickline’s pioneering research has investigated the molecular basis of disease-causing processes using novel imaging methods to detect the genetic signature of cells and deploying nanoparticles to treat a variety of cardiovascular conditions, including targeting atherosclerotic plaques that cause heart attacks. His academic entrepreneurial work led to the development of advanced cardiac imaging techniques, such as magnetic resonance imaging (MRI) of the heart to assess coronary artery disease.

During his tenure with Washington University in St. Louis, Dr. Wickline led a consortium that works with academic and industry partners to develop broad-based clinical applications for nanotechnology and imaging.  He also worked with corporate partner Philips Medical Systems to establish one of the first clinical and research programs for cardiac MRI and was a founding member of the International Society for Cardiovascular Magnetic Resonance.

In 2016 Dr. Wickline joined USF to be founding director of the USF Health Heart Institute, now under construction as part of the university’s new Morsani College of Medicine in downtown Tampa. He has been instrumental in helping recruit scientists, forge industry partnerships and design new basic research laboratories and clinical research space that will accelerate the Institute’s graduate student training and pursuit of translational medicine and entrepreneurial activities when the facility opens in late 2019.

Dr. Wickline’s current research centers on designing and evaluating new nanotechnology  approaches to understand underlying molecular mechanisms of disease and to deliver more precise, safer treatments for arthritis, cancers, kidney injury and other conditions as well as cardiovascular diseases. Continuously funded by the National Institutes of Health for more than 30 years, he has started four biotechnology companies, holds over 50 issued or filed patents, and has authored more than 315 peer-reviewed research papers.

The AIMBE College of Fellows has inducted Fellows representing 30 countries and employed in academia, industry, clinical practice and government. These Fellows include two Nobel Prize laureates, 17 Fellows who received the Presidential Medal of Science and/or Technology and Innovation, and 158 also inducted to the National Academy of Engineering, 72 inducted to the National Academy of Medicine and 31 inducted to the National Academy of Sciences.

With its two new honorees, USF is now home to a total of 15 AIMBE Fellows, including four from the USF Health Morsani College of Medicine.

The USF Health Heart Institute recently hosted its inaugural scientific conference, marking another milestone in the young Institute’s short history and setting a standard for future collaborative work that seeks to halt cardiovascular disease.

The 1^st Annual Scientific Colloquium was held Sept. 24 on the USF campus and welcomed several dozen faculty researchers from throughout the USF research community.

At the inaugural Heart Institute Scientific Colloquium are, from left, Sam Wickline, Lee Sweeney, Sami Noujaim and Charles Lockwood.

“This is a key moment for the USF Health Heart Institute and we are proud to launch an event that showcases impactful cardiovascular research,” said Samuel Wickline, MD, professor and the Tampa General Hospital Endowed Chair for Cardiovascular Research, interim chair of the USF Health Department of Cardiovascular Sciences, and director of the USF Health Heart Institute.

“This inaugural Heart Institute Scientific Colloquium welcomed scientists from across our field to hear about new approaches to heart research. We also know this event is a great example of our collaborative, multidisciplinary approach to scientific discovery. Real progress in cardiovascular research happens when the scientific community works together.”

The Heart Institute in the USF Health Morsani College of Medicine (MCOM) conducts several collaborative projects with researchers from Duke, Stanford, Albert Einstein College of Medicine (NY), University of Michigan, Washington University in St. Louis, MO, and others, he said.

“This Colloquium offers a glimpse of that, showing how our Heart Institute researchers are working with experts from other institutions.”

The keynote speaker for the Colloquium was Lee Sweeney, PhD, professor of pharmacology and therapeutics and director of the Myology Institute at the University of Florida. His talk, titled “The Dilated Cardiomyopathy Associated with Duchenne Muscular Dystrophy,” addressed ways to improve heart failure in children with Duchenne muscular dystrophy (DMD), the most common fatal genetic condition in children. DMD affects mostly boys and is caused by a genetic mutation that prevents the body from producing dystrophin, a protein essential for strong muscle fibers, including those by the heart.

Dr. Lee Sweeney presents his work on cardiomyopathy associated with Duchenne muscular dystrophy.

“Dr. Sweeney’s work dovetails beautifully with efforts taking place in the Heart Institute,” Dr. Wickline said.

In addition to Dr. Sweeney, two current Heart Institute research scientists offered overviews of their current research: Jerome Breslin, PhD, and Sami Noujaim, PhD.

Dr. Breslin, professor of molecular pharmacology and physiology at MCOM, presented “Targeting S1P Receptors to Reduce Inflammation and Microvascular Permeability.”

“Our work focuses on finding new ways to reduce the negative impact of inflammation after injury or during disease,” Dr. Breslin said. “Specifically, we are identifying the molecular signaling pathways that initiate, sustain, and resolve leakage of plasma proteins from the blood into the surrounding tissues, which is what causes swelling. To date our work suggests a key role for a compound known as sphingosine-1-phosphate, which is normally released by circulating blood cells, as a key contributor to maintain the walls of blood vessels and reduce plasma protein leakage.”

And Dr. Noujaim, associate professor of molecular pharmacology and physiology at MCOM, presented “Antiarrhythmic Block of Potassium Inward Rectifiers in Atrial Fibrillation.”

“Atrial fibrillation (AF) is the most common arrhythmia seen in the clinic and is increasingly recognized as a disease of aging, and as a significant cause of morbidity and mortality,” Dr. Noujaim said. “For instance, it has been found that AF independently increases mortality and that it is associated with dementia, and is a major risk factor for stroke. AF is very challenging to treat with currently used antiarrhythmic drugs. It has been found that the aberrant working of a class of proteins called cardiac potassium channels perpetuates AF. My lecture showed how we used a widely prescribed antimalarial drug to correct the function of these potassium channels, and consequently stop AF. Our studies used mathematical modeling, and sophisticated experiments, from the level of the single molecule, all the way to the patient’s heart to understand how this antimalarial drug could stop AF and restore the heart’s normal rhythm. We hope that such studies will help us to find a novel class of therapies that can potentially be effective in treating AF.”

Dr. Lee Sweeney’s work focuses on cardiomyopathy associated with Duchenne muscular dystrophy.

The Colloquium was also a celebration of what’s to come, Dr. Wickline said.

“We’ve had a busy year so far with progress on our building in downtown Tampa, great success in recruiting more prominent scientists to our Heart Institute roster, and increases in research funding,” Dr. Wickline said. “This momentum, and these close collaborations, indicate that people are perking up their ears about USF Health, the Heart Institute and all of our developments here.

“But we aren’t stopping here,” he added. “This is an exciting time for cardiovascular disease research and, over the next year, we will see more research emphasis on regenerative medicine, heart failure, bioinformatics, and molecular and functional imaging. As our Heart Institute continues to outfit our building and our team, we continually seek foundational research and philanthropic funding and collaborative opportunities.

“All of this will help us attract the best and the brightest and truly impact cardiovascular disease.”

Photos by Torie Doll, USF Health Communications