USF Health-UT Southwestern Medical Center preclinical study suggests inhibiting PPP1R3G/PP1γ may protect against tissue damage from heart attacks, other diseases linked to inflammation

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TAMPA, Fla. (Feb. 16, 2022) – Cell death plays an important role in normal human development and health but requires tightly orchestrated balance to avert disease. Too much can trigger a massive inflammatory immune response that damages tissues and organs. Not enough can interfere with the body’s ability to fight infection or lead to cancer.

Zhigao Wang, PhD, associate professor of cardiovascular sciences at the University of South Florida Health (USF Health) Morsani College of Medicine, studies the complex molecular processes underlying necroptosis, which combines characteristics of apoptosis (regulated or programmed cell death) and necrosis (unregulated cell death).

During necroptosis dying cells rupture and release their contents. This sends out alarm signals to the immune system, triggering immune cells to fight infection or limit injury. Excessive necroptosis can be a problem in some diseases like stroke or heart attack, when cells die from inadequate blood supply, or in severe COVID-19, when an extreme response to infection causes organ damage or even death.

A new preclinical study by Dr. Wang and colleagues at the University of Texas Southwestern Medical Center identifies a protein complex critical for regulating apoptosis and necroptosis — known as protein phosphatase 1 regulatory subunit 3G/protein phosphatase 1 gamma (PPP1R3G/PP1γ, or PPP1R3G complex). The researchers’ findings suggest that an inhibitor targeting this protein complex may help reduce or prevent excessive necroptosis.

The study was reported Dec. 3, 2021, in Nature Communications.

Zhigao Wang, PhD, associate professor of cardiovascular sciences, in his laboratory at the USF Health Heart Institute. Images on the monitor depict two types of cell death: apoptosis (left) and necroptosis. — Photo by Allison Long, USF Health Communications

“Cell death is very complicated process, which requires layers upon layers of brakes to prevent too many cells from dying,” said study principal investigator Dr. Wang, a member of the USF Health Heart Institute. “If you want to protect cells from excessive death, then the protein complex we identified in this study is one of many steps you must control.”

Dr. Wang and colleagues conducted experiments using human cells and a mouse model mimicking the cytokine storm seen in some patients with severe COVID-19 infection. They applied CRISPR genome-wide screening to analyze how cell function, in particular cell death, changes when one gene is knocked out (inactivated).

Receptor-interacting protein kinase (RIPK1) plays a critical role in regulating inflammation and cell death. Many sites on this protein are modified when a phosphate is added (a process known as phosphorylation) to suppress RIPK1’s cell death-promoting enzyme activity. How the phosphate is removed from RIPK1 sites (dephosphorylation) to restore cell death is poorly understood. Dr. Wang and colleagues discovered that PPP1R3G recruits phosphatase 1 gamma (PP1γ) to directly remove the inhibitory RIPK1 phosphorylations blocking RIPK1’s enzyme activity and cell death, thereby promoting apoptosis and necroptosis.

Dr. Wang (back) and laboratory associate Ken Chen. — Photo by Allison Long, USF Health Communications

Dr. Wang uses the analogy of a car brake help explain what’s happening with the balance of cell survival and death in this study:  RIPK1 is the engine that drives the cell death machine (the car). Phosphorylation applies the brake (stops the car) to prevent cells from dying. The car (cell death machinery) can only move forward if RIPK1 dephosphorylation is turned on by the PPP1R3G protein complex, which releases the brake.

“In this case, phosphorylation inhibits the cell death function of protein RIPK1, so more cells survive,” he said. “Dephosphorylation takes away the inhibition, allowing RIPK1 to activate its cell death function.”

The researchers showed that a specific protein-protein interaction – that is, PPP1R3G binding to PP1γ — activates RIPK1 and cell death. Furthermore, using a mouse model for “cytokine storm” in humans, they discovered knockout mice deficient in Ppp1r3g were protected against tumor necrosis factor-induced systemic inflammatory response syndrome. These knockout mice had significantly less tissue damage and a much better survival rate than wildtype mice with the same TNF-induced inflammatory syndrome and all their genes intact.

Overall, the study suggests that inhibitors blocking the PPP1R3G/PP1γ pathway can help prevent or reduce deaths and severe damage from inflammation-associated diseases, including heart disease, autoimmune disorders and COVID-19, Dr. Wang said. His laboratory is working with Jianfeng Cai, PhD, a professor in the USF Department of Chemistry, to screen and identify peptide compounds that most efficiently inhibit the PPP1R3G protein complex. They hope to pinpoint promising drug candidates that may stop the massive destruction of cardiac muscle cells caused by heart attacks.

The research was supported by grants from the Welch Foundation and the National Institute of General Medical Sciences, a part of the National Institutes of Health.

Graphic created with Biorender app by Zhigao Wang, USF Health Heart Institute.

Using an innovative cellular model, the USF Health-led study also discovers drug combinations to prevent degeneration of these tiniest blood vessels

Many diseases arise from abnormalities in our capillaries, tiny exquisitely branching blood vessel networks that play a critical role in tissue health. Researchers have learned a lot about the molecular communication underlying capillary formation and growth, but much less is understood about what causes these critical regulators of normal tissue function to collapse and disappear.

“Capillary regression (loss) is an underappreciated, yet profound, feature of many diseases, especially those affecting organs requiring a lot of oxygen to work properly,” said George E. Davis, MD PhD, a professor in the Department of Molecular Pharmacology and Physiology, University of South Florida Health (USF Health) Morsani College of Medicine, Tampa, Fla.

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Videoclip courtesy of E. George Davis, MD, PhD, USF Health Morsani College of Medicine

“If we know how blood vessels are altered or begin to break down we should be able to fix it pharmacologically,” said Dr. Davis, a member of the USF Health Heart Institute.

A team led by Dr. Davis advanced the understanding of how capillaries regress in a study published Dec. 19 in the American Heart Association journal Arteriosclerosis, Thrombosis, and Vascular Biology.  The USF Health researchers worked with the laboratory of Courtney Griffin, PhD, at Oklahoma Medical Research Foundation.

The researchers discovered that three major proinflammatory mediators – interlukin-1 beta (IL-1β), tumor necrosis factor alpha (TNFα) and thrombin – individually and especially when combined, directly drive capillary regression (loss) known to occur in diseases such as hypertension, diabetes, cardiovascular diseases, neurodegenerative diseases and malignant cancer. They also identified combinations of drugs – neutralizing antibodies to specifically block IL-1β and TNFα or combinations of pharmacologic inhibitors – that significantly interfered with capillary regression.

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Videoclip courtesy of George E. Davis, MD, PhD, USF Health Morsani College of Medicine

Capillaries, our body’s tiniest and most abundant blood vessels, connect arteries with veins and exchange oxygen, nutrients and waste between the bloodstream and tissues throughout the body.  The Davis laboratory grows three-dimensional human “blood vessel networks in a dish” under defined, serum-free conditions to delve into the complexity of how capillaries take shape to sustain healthy tissues. Lately, his team has begun applying what they’ve learned using this innovative in vitro model to attack, and possibly protect against, diseases.

For this study the researchers cultured two types of human cells: endothelial cells, which line the inner surface of capillaries, and pericytes, which are recruited to fortify the outer surface of the endothelial-lined tubes. Cross-communication between these cells controls how the blood vessel networks emerge, branch and stabilize. Macrophages, a type of immune cell, were activated in the cell culture media to simulate a tissue-injury environment highly conducive to capillary regression.

Among the key study findings:

– Macrophage-derived molecules IL-1β and TNFα, combined with thrombin, selectively cause endothelial-lined capillary tube networks to regress; however, pericytes continue to proliferate around the degenerating capillaries. Why the pericytes are spared remains an intriguing question to be answered, but Dr. Davis suggests these more resilient cells may be left behind to help repair tissue damaged by inflammation.

– IL-1β and TNFα, combined with thrombin, induce a unique set of molecular signals contributing to the loss of blood vessels. This “capillary regression signaling signature” is opposite of the physiological pathways previously identified by Dr. Davis and others as characterizing capillary formation and growth.

– Certain drug combinations (two were identified by the researchers) can block the capillary loss promoted by IL-1β, TNFα and thrombin.

USF Health cell biologist George Davis, MD, PhD, grows three-dimensional human “blood vessel networks in a dish” under defined, serum-free conditions to study how capillaries take shape | Photo by Allison Long, USF Health Communications and Marketing

The USF Health researchers found several other proinflammatory molecules that promoted capillary loss, but none proved as powerful as IL-1β, TNFα and thrombin, especially when all three were combined.

Antibodies to counteract the effects of IL-1β and TNFα are already used to treat patients with some inflammatory diseases, including atherosclerosis, rheumatoid arthritis, and Crohn’s disease. And physicians prescribe direct thrombin inhibitors for certain patients with atrial fibrillation, deep vein thrombosis and pulmonary embolism.

“These drugs are out there and they work. Our data suggests that, if combined, they may actually prevent vessel breakdown (earlier in the disease process) and improve outcomes,” Dr. Davis said.

The USF Health team plans to investigate how abnormal capillary response may influence the loss of cells and tissues specific to disease states like sepsis, ischemic heart disease and stroke. Their model of 3D blood vessel networks can also be easily used to screen more potential drug candidates, Dr. Davis said. “We’ve identified some promising (existing) drugs to rescue capillary regression — but there may be more therapeutic opportunities.”

Above: Endothelial cell-lined tubes (red) with associated pericytes (green). Both cell types co-assemble to create the capillary networks vital to the health of tissues throughout the body.  Below: Capillary networks following exposure to the three proinflammatory molecules, IL-1 beta, tumor necrosis factor and thrombin, which caused marked loss of the endothelial tubes while sparing pericytes.  Images courtesy of George E. Davis, MD, PhD, USF Health.

The study was supported by grants from the National Institutes of Health, National Institutes of Health, National Heart, Lung, and Blood Institute, and the Oklahoma Center for the Advancement of Science & Technology.