USF Health researcher Mack Wu, MD, studies what happens when the microvascular endothelial barrier controlling blood-tissue exchange is compromised during ischemia-reperfusion injury, a condition that can lead to irreversible tissue damage. He also investigates the molecular control of gut permeability, also known as “leaky gut,” in tissue injuries caused by trauma and severe burns.

His group’s work has broad implications for a variety of conditions including stroke, heart attack, thrombosis, sepsis, trauma or other inflammatory diseases associated with microvascular injury.

Mack Wu, MD, is a professor of surgery and molecular medicine at USF Health Morsani College of Medicine and a research physiologist at James A. Haley Veterans’ Hospital. On the monitor next to him are images of microvessels in the small intestine injected with fluorescent dye.

The closely connected endothelial cells lining the interior of blood vessel walls play a critical role in limiting the how much fluid, proteins and small molecules cross the wall of the tiny blood vessels, or microvessels. However when this protective endothelial barrier is damaged, excessive amounts of blood fluid, proteins and molecules leak outside the microvessels into nearby body tissue – a process known as microvascular hyperpermeability. If this breech of endothelial barrier is associated with a body-wide inflammatory response, it can trigger a chain of events leading to edema (swelling), shock from severe blood and fluid loss (hypovolemic shock), and ultimately multiple organ failure.

Pinpointing potential solutions for ischemia-reperfusion injury

Previous research by Dr. Wu’s laboratory and other groups discovered that ischemia-reperfusion injury can cause endothelial barrier damage leading to vascular hyperpermeability, or abnormally leaky blood vessels.

Ischemia-reperfusion injury is typically associated with conditions like organ transplantation, stroke, heart attack, or cardiopulmonary bypass where blood supply to a vital organ is temporarily cut off (ischemia), resulting in oxygen deprivation. For instance, a period of ischemia occurs while a donor organ is transported to a recipient in the operating room, or when a clot interrupts blood circulation to the brain. When blood supply is re-established with new blood returned to the previously oxygen-deprived area (reperfusion), tissue injury can worsen because the reperfusion itself causes inflammation and oxidative damage rather than restoring normal function. It its severest form, ischemia-reperfusion injury can result in multiple organ failure, or even death.

“I believe endothelial barrier injury is one of the key elements of ischemia-reperfusion injury, so my group is trying to find out which molecule is ultimately responsible for the endothelial barrier damage,” said Dr. Wu, a professor of surgery and molecular medicine at USF Health Morsani College of Medicine and a research physiologist at James A. Haley Veterans’ Hospital.

Dr. Wu with some members of his laboratory team. From left, Rebecca Eitnier, research assistant; Shimin Zhang, Department of Molecular Medicine graduate student; Ricci Haines, research associate; and Fang Wang, research assistant.

With the support of a $1.49-million, four-year R01 grant from the National Heart, Lung and Blood Institute, Dr. Wu’s team is zeroing in on a molecule known as focal adhesion kinase, or FAK, an enzyme that may play a role in weakening the microvascular endothelial barrier during ischemia-reperfusion injury.   Using cell models and a newly developed mouse model in which the endothelial-specific gene for FAK is knocked out, the USF researchers are testing whether selectively inhibiting FAK activity can rescue the endothelial barrier from such injury.

The work is critical because no FDA-approved treatment exists to prevent tissue damage following reperfusion. Identifying a new mechanism for the injury would provide potential targets for drug development, Dr. Wu said. So for instance, he said, after an initial stroke a new intravenously administered drug selectively targeting endothelial cells in the brain’s microvessels might stop further harmful swelling of the brain caused by stroke.

Defining molecular control of “leaky gut” in severe burn trauma

A second grant from the U.S. Department of Veterans Affairs funds Dr. Wu’s studies to define the underlying molecular mechanisms of leaky guts induced by traumatic injury associated with thermal (fire, scald or chemical) burns.  Massive burn trauma is a significant cause of injury and death in American soldiers. With a $960,000 VA Merit Award, Dr. Wu focuses on how intestinal epithelial barrier damage happens during severe burns, with the aim of developing targeted therapies to prevent posttraumatic complications.  In particular, he is working to determine the pathways by which the protein palmitoylation in gut epithelial cells are stimulated by burn injury.

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Epithelial cells line the interior of the small intestines, and after severe burn injury, this protective epithelial barrier commonly breaks down, causing bacteria and toxins to flow from the intestine into the circulating blood.  The result of this abnormal epithelial permeability, or “leaky gut,” can be deadly if sepsis ensues – a bacterial infection in the bloodstream sets up a body-wide inflammatory response leading to multiple organ failure.

While the role gut barrier failure plays in posttraumatic complications is well recognized, its cellular and molecular mechanisms remain poorly understood.  Currently, pushing IV fluids to help prevent hypovolemic shock and administering antibiotics and anti-inflammatories are the only therapies, mostly supportive, Dr. Wu said.

“More effective early therapeutic interventions to prevent leaky gut and systemic inflammatory response will be key to preventing sepsis,” he added, whether in soldiers with trauma or VA patients with inflammatory bowel diseases.

From industry to academia

Dr. Wu joined USF Health and the Haley VA Hospital in 2011.  He came from Sacramento, Calif, where he was an associate professor of surgery at the University of California at Davis School of Medicine and a research physiologist at Sacramento VA Medical Center.   Previously, Dr. Wu was a faculty member in the Department of Medical Physiology at Texas A&M University Health Science Center. He screened pharmaceutical compounds as a toxicologist in a biotechnology laboratory before joining Texas A&M, moving from industry to academia in 1995.

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Dr. Wu received his MD degree from Second Military Hospital in Shanghai, China, and conducted an internship at Shanghai Second Hospital.

One of his earliest and most highly cited studies, published in the American Journal of Physiology (1996), was first to report nitric oxide’s role in contributing to cardiovascular injury. The study showed an increase in nitric oxide induces vascular endothelial growth factor (VEGF) to promote leakage in tiny coronary veins.

Another more recent study in Shock (2012) provided direct evidence that thermal burn injury causes intestinal barrier disruption and inflammation characterized by intestinal mucosal permeability (leakage) and an infiltration of immune system cells known as neutrophils.

Something you may not know about Dr. Wu:

He loves deep-sea fishing. Dr. Wu has fished for sharks off the Golf coast of Texas, rockfish off the Pacific coast of California, and grouper off the west coast of Florida.

Dr. Wu is a member of the USF Health Heart Institute. His team’s work has broad implications for a variety of conditions including stroke, heart attack, thrombosis, sepsis, trauma or other inflammatory diseases associated with microvascular injury.

Photos by Eric Younghans, USF Health Communications and Marketing

Jerome Breslin studies what happens when the endothelial barrier is breeched by traumatic injury and inflammation

Traumatic injury is the leading cause of death among people ages 1 to 44 in the United States. The body’s inflammatory response accompanying massive injury can severely complicate the resuscitation of trauma victims, worsen clinical outcomes and often lead to multiple organ failure.

In his laboratory at the USF Health Morsani College of Medicine, Jerome Breslin, PhD, and colleagues study microvascular hyperpermeability, that is, the “excessively leaky” small blood vessels that are a hallmark of systemic inflammation.  Their aim is to find new, more effective ways to treat trauma and prevent early death, but their work also has implications for the treatment of lymphedema, wound healing and arteriosclerosis.

Jerome Breslin, PhD, can do live imaging of vascular endothelial cells under a microscope that he helped build.

In particular, Dr. Breslin, an associate professor in the Department of Molecular Pharmacology and Physiology, looks at what happens when the protective barrier of endothelial cells forming an interface between circulating blood and tissues outside the blood vessel network is compromised by traumatic injury and inflammation.

Leaky blood vessels: The soaker hose analogy

“These capillaries are like soaker hoses used to water plants, that leak out fluid carrying proteins and other nutrients in addition to delivering oxygen to surrounding tissue,” Dr. Breslin said. “In patients who have undergone trauma or major surgery, blood pressure drops in part because the wall of the hose becomes too leaky. There is less fluid in the blood vessels and more flowing out into nearby tissues, which can cause damage and impair the function of some organs.”

In addition to investigating ways to prevent excessive blood vessel leakage, Dr. Breslin’s lab focuses on how to return the leaked fluid back into the blood by the lymphatic vessels.  As a result, his team spends a lot of time studying the pumping function of the lymphatic system, which manages fluid levels in the body. Swelling, or edema, occurs when it fails to drain off excess fluid.

Dr. Breslin’s work is currently supported by two National Institutes of Health RO1 grants totaling more than $2 million.

Dr. Breslin with two undergraduate students who conduct research in his laboratory: Andrea Burgess (American Physiological Society IOSP Summer Fellow) and Sara Spampinato, center (NIH Diversity Grant recipient).

 Dr. Breslin comments on his approach to research problems.

The most recent award from the NIH’s National Institute of General Medical Sciences focuses on testing whether a class of drugs that activate the S1P1 receptor may keep blood vessels from leaking too much and stabilize blood pressure following trauma.

In this project, Dr. Breslin will use the first rat model combining alcohol intoxication and hemorrhagic shock to induce excessive leakiness in small blood vessels. He will evaluate whether fluid containing sphingosine-1-phosphate (S1P) reduces the blood vessel permeability, thereby restoring normal blood pressure and fluid balance. If so, Dr. Breslin said, drugs similar to S1P, a bioactive lipid that prevents cell death, may offer a more effective way for paramedics and physicians to resuscitate trauma patients than the standard IV fluid therapy now administered.  That standard fluid resuscitation protocol works particularly poorly in alcohol-intoxicated victims suffering major blood loss, a significant portion of all trauma cases coming through emergency rooms, he said.

With the second award, a competitive renewal from the NIH’s National Heart, Blood and Lung Institute, Dr. Breslin and colleagues are studying the molecular and cellular mechanisms that may regulate and resolve microvascular leakage following inflammation caused by traumatic injury.

Dr. Breslin points to a human heart valve suspended in a test tube solution. His group plans to study the microvessels within heart valves.

Unexpected finding leads to “new way of thinking”

Previous work by his group using live imaging of vascular endothelial cells under a microscope demonstrated that when the edges of these cells make contact with their neighboring cells they appear very active and are constantly remodeling, or changing shape — rapidly opening up holes at cell junctions and then closing back up. This finding, published in the journal PLOS One, countered one of the conventional theories that endothelial cells were more rigid at the junctions where they connect and adopted a contracted state during inflammation.

“It was an unexpected finding that changed our thinking about how these cells behaved,” Dr. Breslin said.

This led the researchers to begin to question the prevailing view about the role actin stress fibers — threadlike structures involved in cell stability, adhesion and movement — play in disrupting the endothelial barrier function.

Further preclinical studies by Dr. Breslin and others over several years showed that in response to an inflammatory agent actin stress fibers cause endothelial cells to spread out, not contract, at the junctions. The USF researchers published evidence in the American Journal of Physiology: Cell Physiology that actin stress fiber formation may be a reaction to, rather than a cause of, reduced integrity of the endothelial barrier that protects against excessive fluid leakage.

Earlier this year, Dr. Breslin was first author on a study appearing in the Journal of the American Heart Association showing that the signaling protein Rnd3 reduced leakage of small blood vessels when delivered a new way in a rat model of hemorrhagic shock. The researchers suggested Rnd3 (or analog drugs) might offer an anti-inflammatory treatment to repair the endothelial barrier compromised by prolonged and uncontrolled inflammation.

  Dr. Breslin talks about his most exciting experiment

//www.youtube.com/watch?v=lm2ag8m1QqQ

Live imaging of endothelial microvascular cells at 600x magnification shows the dynamic movement of the protruding cell edges (local lamellipodia). Videoclip courtesy of Jerome Breslin, PhD. 

Heart Institute, former mentor a draw to USF

Dr. Breslin joined USF in 2012 from Louisiana State University Health Sciences Center in New Orleans, where he was an assistant professor of physiology.  He received his PhD in pharmacology and physiology from Rutgers University – New Jersey Medical School in Newark, NJ.  His postdoctoral training was conducted at both Texas A&M and the School of Medicine at the University of California Davis, where he was mentored by Sarah Yuan, MD, PhD, the chair of Molecular Pharmacology at Physiology at Morsani College of Medicine who is nationally recognized for her translational research on the regulation of microcirculation.

The opportunity to be part of a growing university, join core faculty who will help build a Heart Institute advancing bench-to-bedside cardiovascular research, and work again with Dr. Yuan attracted him to USF Health, Dr. Breslin said.

“Dr. Yuan was a great mentor to me when I was a postdoctoral fellow,” he said. “This has reopened our scientific collaborations and now we’re mentoring a student together.”

Dr. Breslin, center, with some members of his laboratory.

  His advice to emerging scientists

Dr. Breslin is a fellow of the American Physiological Society Cardiovascular Section and a member of The Microcirculatory Society and the American Heart Association.  He is associate editor of the journal Microcirculation and a member of the editorial board of PLOS One.  He has authored or co-authored nearly 40 articles in peer-reviewed journals.

Dr. Breslin serves on two NIH special emphasis panels, one on lymphatics and another for the Intramural Postdoctoral Research Associate Program.  He is also a grant reviewer for the Association of American Medical Colleges (AAMC) Innovations in Research and Research Education Awards.

Something you may not know about Dr. Breslin

To help pay for tuition while earning his master’s degree in biology, Dr. Breslin worked as a park ranger in Somerset County, N.J, for a couple of summers.

No stranger to outdoor activities, including camping, as a teen Dr. Breslin attained the rank of Eagle Scout, the highest achievement in the Boy Scouting program.  His connection with scouting continues today as committee chair for his 13-year-old son’s Boy Scout troop.

Dr. Breslin’s Scouting experiences included learning wilderness survival skills, such as how to build a shelter from scratch in the woods or navigating a group of boys through the wilderness without a map and compass, or a smartphone for that matter. They were instrumental, he said, in helping him develop the resourcefulness and leadership skills he hopes to impart to the emerging scientists he mentors in his laboratory

In case you’re wondering, one of the most challenging of the merit badges he earned as a Boy Scout: bugling.

Photos and audioclips by Sandra C. Roa, USF Health Communications and Marketing