Give credit to your dad’s gene for keeping you safe during those long months in your mother’s womb.

Because without this genetic warrior, you might have succumbed to any number of viral infections that otherwise could be fatal to a fetus. A new paper published this week in the journal Cell Host & Microbe explains the mechanisms behind this anti-viral protection.

Hana Totary-Jain, PhD, associate professor of Molecular Pharmacology and Physiology and Heart Institute at the USF Health Morsani College of Medicine

“What’s unique about this gene is how it produces a form of defense for the baby in the womb,’’ said Hana Totary-Jain, PhD., associate professor of Molecular Pharmacology and Physiology and Heart Institute at the USF Health Morsani College of Medicine and senior author of the paper.

Their research focused on viruses that affect a pregnant mother and consequently her fetus, which are highly vulnerable to infection because their immune systems are immature. Some viruses, including Zika, rubella, and other serious infections, are rarely transmitted from mother to fetus in utero and can cause devastating consequences.

But the biological processes that protect a fetus from most viral infections are less clear. In the new paper, titled “SINE RNA of the imprinted miRNA clusters mediates constitutive type III interferon expression and antiviral protection in hemochorial placentas,’’ Dr. Totary-Jain and her team describe how a certain gene in the placenta is always armed for the battle.

“The placenta, in human and in mouse, is the first organ the fetus develops, and it is constantly exposed to maternal blood. This increases the chances of transmitting viral infections from the mother to the fetus. Therefore, the placenta has evolved robust defense mechanisms to prevent this transmission. We discovered a gene in the placenta that is expressed only from the paternal allele and produces a viral mimicry response. It tricks the placenta into thinking it’s infected and induces a constant state of antiviral defense”, Dr. Totary-Jain explained.

“So when we turned on this gene in other cells, we could protect the cells from several viruses. This is evolution’s way of protecting the baby. Without it, chances are you wouldn’t have made it into childbirth.’’

Ishani Wickramage, a PhD candidate in Dr. Totary-Jain’s laboratory and a lead author of the study added: “This research fills the gap in our knowledge about how many viruses that may infect a pregnant mother, including SARS-CoV-2, only rarely affect the fetus.’’

“Learning more about how the placenta shields the fetus from viruses also has important implications beyond childbirth,” said Dr. Charles Lockwood, MD, MHCM, one of the paper’s authors, who also is dean of the Morsani College of Medicine and executive vice president of USF Health.

“This is a novel placental mechanism that protects the developing fetus from transplacental transmission of most viruses,” Dr. Lockwood said. “This is the kind of knowledge that could lead to the development of new anti-viral medications to fight viruses that can be deadly for fetuses and newborn babies.”

This work was supported by a grant from the National Institutes of Health. Dr. Totary-Jain and a team of researchers at USF spent five years investigating this intriguing phenomenon in collaboration with Dr. Thomas Tuschl’s lab at Rockefeller University, who performed the sRNAseq and bioinformatic analysis, including researcher Klaas Max and Kemel Akat; and Drs. Kimiko Inoue and Atsuo Ogura from RIKEN and University of Tsukuba, Japan, who provided the mouse model that was used to show that the mouse placenta also developed the same mechanism to protect the fetus from viral infections.

Other USF Health members of the research team are: Jeffrey VanWye; John H. Lockhart; Ismet Hortu; Ezinne F. Mong; John Canfield; Hiran M. Lamabadu Warnakulasuriya Patabendige; Ozlem Guzeloglu-Kayisli; and Umit A. Kayisli.

— Story by Kurt Loft for USF Health News

A USF Health preclinical study indicates nanoparticles containing a micro-RNA switch offers promising biotechnology to advance the fight against atherosclerotic cardiovascular disease

Illustration of angioplasty with a stent

Tampa, FL (Feb. 9, 2021) – Percutaneous coronary intervention (PCI), commonly known as angioplasty with a stent, opens clogged arteries and saves lives. Despite its benefit in treating atherosclerosis that causes coronary artery disease, this common minimally invasive procedure still poses severe complications for some patients.

Angioplasty involves inflating a balloon at the tip of a catheter to compress fatty deposits (plaques) against the artery wall, thereby restoring blood flow to the narrowed or blocked vessels. The image-guided procedure is often combined with the placement of either uncoated stents — tiny expandable mesh devices– or stents coated with slowly-released antiproliferative drugs. The drug-eluting stents help avert the growth of scar tissue (smooth muscle cell proliferation) in the artery so that the vessel does not eventually close again, known as restenosis.

However, current antiproliferative drugs indiscriminately inhibit the growth of all nearby cells, including the layer of endothelial cells lining the blood vessels. These endothelial cells prevent blood clots (thrombosis) within the stent and the formation of more plaques (neoatherosclerosis), which can trigger a heart attack or sudden cardiac death.

Focused on tackling this treatment complication, University of South Florida Health (USF Health) Morsani College of Medicine researchers recently developed a next-generation nanotherapy. Their preclinical findings are detailed in a study published Feb. 2 in Molecular Therapy.

Hana Totary-Jain, PhD, USF Health associate professor of molecular pharmacology and physiology, was principal investigator for the nanotherapy study.

The nanotherapy comprised of a nontoxic peptide known as p5RHH and a synthetic messenger RNA (mRNA) that carries the genetic instructions, or code, needed by cells to make proteins. By simply mixing up the p5RHH with the mRNA, they spontaneously self assemble into compacted nanoparticles that specifically target the injured regions of the arteries in mouse models mimicking angioplasty. The nanoparticles contain an microRNA switch added to the mRNA.

“One of the main challenges of cardiovascular disease remains the delivery of targeted therapies specifically to the plaque regions and the cells that form plaques, including the smooth muscle cells and inflammatory cells — without affecting the endothelial cells or the healthy regions,” said the study’s principal investigator Hana Totary-Jain, PhD, an associate professor of molecular pharmacology physiology at USF Health Morsani College of Medicine.

To do this, the researchers used mRNA that encodes for p27 protein, which blocks cell growth, and added to the mRNA an endothelial cell-specific microRNA to generate a microRNA switch. The design of this microRNA switch allowed the researchers to turn on the mRNA in smooth muscle cells to inhibit their growth and the formation of restenosis. It also enabled them to turn off the mRNA in endothelial cells so these cells could grow uninhibited and quickly heal the damaged blood vessel.

John Lockhart, PhD, was the paper’s lead author.

“If we can come up with an antiproliferative therapy that specifically targets the cardiovascular smooth muscles cells and the infiltrating inflammatory cells but spares the endothelial cells – which we’ve done with the design of our microRNA switches – then we should be able to achieve the therapeutic effects of drug-eluting stents without the downside of thrombosis and neoatherosclerosis,” said the paper’s lead author John Lockhart, PhD, who worked on the study as a doctoral student at USF Health Molecular Pharmacology and Physiology. Dr. Lockhart is continuing his postdoctoral training at Moffitt Cancer Center.

The latest study builds upon previous research by Dr. Totary-Jain, indicating that a microRNA-based therapy worked better than drug-eluting stents in a rat model of angioplasty. That work used an adenovirus vector to carry the cell-selective therapy to injured arteries. In this study the viral vector was replaced with a nanoparticle alternative – a change needed to avoid safety concerns and advance the therapy toward use in patients.

The investigational nanoparticles were injected into mice with arteries mimicking post-angioplasty vessel injury every three days for two weeks (5 doses total). Mice treated with the nanoparticles containing the miRNA switch had significantly reduced restenosis and completely restored endothelial cell growth in the injured artery, compared to animals treated with nanoparticles containing mRNA without the miRNA switch, the researchers report.

Above: Injured control artery treated with near infrared florescent protein, depicts restenosis in center. Below: Injured artery treated with the microRNA switch nanotherapy shows open artery (no restenosis) and clear endothelial cell layer marked in green. | Images courtesy of Hana Totary-Jain, USF Health

In addition, the nanoparticles efficiently delivered its mRNA cargo, without degradation, solely to regions of the artery where endothelial cells were damaged. The particles did not toxically accumulate either in the cells of healthy organs (the liver, spleen. lungs or kidneys), or in uninjured arteries adjacent to those requiring treatment. The researchers observed no adverse reactions or outcomes in mice treated with the nanoparticles.

Overall, the findings suggest that the miRNA-switch nanoparticles could be applied clinically to selectively prevent restenosis after PCI by specifically targeting areas of endothelial cell damage to allow quicker cell regrowth and repair of injured arteries.

The USF Health researchers next plan to investigate the potential of the microRNA-switch nanoparticles to directly treat atherosclerotic plaques, thereby eliminating the need for PCI.

“Cardiovascular disease is still the number one cause of death,” said Dr. Totary-Jain, a member of the USF Health Heart Institute. “This research offers promise for the development of novel biomolecular therapies to advance the fight against coronary artery disease and peripheral artery disease,”

One person dies of cardiovascular disease every 36 seconds in the U.S., according to the Centers for Disease Control and Prevention.

The USF Health research was supported by grants from the National Institutes of Health. Samuel Wickline, MD, director of the USF Health Heart Institute, and Hua Pan, PhD, assistant professor at the Heart Institute, collaborated on the study.

USF Health researchers applied CRISPR technology to study the very large human non-protein coding gene expressed only in placenta, stem cells and certain cancers

TAMPA, Fla (March 16, 2020) — The placenta, an organ which attaches to the lining of the uterus during pregnancy, supplies maternal oxygen and nutrients to the growing fetus. Abnormal formation and growth of the placenta is considered an underlying cause of various pregnancy complications such as miscarriages, stillbirth, preeclampsia and fetal growth restriction. Yet, much remains to be learned about molecular mechanisms regulating development of this blood-vessel rich organ so vital to the health of a pregnant woman and her developing fetus.

Hana Totary-Jain, PhD, an associate professor of molecular pharmacology and physiology in the USF Health Morsani College of Medicine, was senior author of the study published in Scientific Reports.

University of South Florida Health (USF Health) Morsani College of Medicine researchers recently discovered how a very large human non-protein coding gene regulates epithelial-to-mesenchymal transition (EMT) – a process that contributes to placental development during early pregnancy, but can also promote cancer progression.

During the first trimester, fetal-derived placental cells known as trophoblasts invade the maternal uterine lining and modify its blood vessels to allow oxygenated blood to flow from the mother to fetus. However, trophoblast invasion requires tight regulation of EMT. If inadequate, trophoblast invasion is too shallow to adequately remodel the maternal blood vessels, and adverse pregnancy outcomes can occur. Conversely, excess EMT can cause exaggerated trophoblast invasion through the uterine wall leading to placenta accreta, a condition that can cause hemorrhage and often requires hysterectomy at delivery.

The USF Health researchers used a powerful genome editing technology called CRISPR (shorthand for “CRISPR-dCas9) to activate all of the chromosome 19 microRNA cluster (known as C19MC), so they could study the gene’s function in early pregnancy. C19MC — one of the largest microRNA gene clusters in the human genome — is normally turned off but becomes expressed only in the placenta, embryonic stem cells and certain cancers.

Dr. Totary-Jain discusses the molecular aspects of placenta development and pregnancy complications with research collaborator Umit Kayisli, PhD, a professor of obstetrics and gynecology at USF Health.

In their cell model study, published Feb. 20 in Scientific Reports, a Nature research journal, the USF Health team showed that robust activation of C19MC inhibited EMT gene expression, which has been shown to reduce trophoblast invasion.

But when trophoblast-like cells were exposed to hypoxia – a lack of oxygen similar to that occurring in early placental development — C19MC expression was significantly reduced, the researchers found. The loss of C19MC function causes differentiation of trophoblasts from stem-like epithelial cells into mesenchymal-like cells that can migrate and invade much like metastatic tumors. This EMT process helps explain trophoblast invasion and early placental formation.

“We were the first to use CRISPR to efficiently activate the entire gene, not just a few regions of this huge gene, in human cell lines,” said the paper’s senior author Hana Totary-Jain, PhD, an associate professor in the Department of Molecular Pharmacology and Physiology, USF Health Morsani College of Medicine. “Our study indicates C19MC plays a key role in regulating many genes important in early implantation and placental development and function. The regulation of these genes is critical for proper fetal growth.”

Above: Chromosome 19 microRNA cluster (stained purple) expressed in first-trimester placenta.  Below: In preparation for pregnancy, fetal trophoblast cells (brown) from which the placenta arises invade maternal decidual cells (pink) in the uterus lining. | Images courtesy of Hana Totary-Jain, originally published in Scientific Reports: doi.org/10.1038/s41598-020-59812-8

“You need EMT, but at some point the process needs to cease to prevent adverse pregnancy outcomes,” Dr. Totary-Jain said. “You really need a balance between not enough invasion and too much invasion, and C19MC is important in maintaining that balance.”

Dr. Totary-Jain and others in her department collaborated with colleagues in the medical college’s Department of Obstetrics and Gynecology on the project.

“The USF Health study offers new insight into how trophoblasts interact with the maternal uterine environment to become more invasive or less invasive in the formation of the placenta,” said coauthor Umit Kayisli, PhD, a USF Health professor of Obstetrics and Gynecology. “More research on microRNA expression and how it inhibits EMT may help us better understand the causes and potential prevention of preeclampsia and fetal growth restriction, which account for 5-to-10 percent of all pregnancy complications as well as spontaneous preterm births.”

Investigating the effects of altered C19MC expression on cell differentiation and trophoblast invasion has implications not only for a better understanding of normal and abnormal placental development, but also for cancer and stem cell research, Dr. Totary-Jain added.

Dr. Totary-Jain and Dr. Kayisli

Photos by Freddie Coleman, USF Health Communications and Marketing

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

Hana Totary-Jain, PhD, an assistant professor of molecular pharmacology and physiology at USF Health Morsani College of Medicine, is among leading scientists from across the country who appear in the Research!America “Reasons for Research” video series to discuss why federal support for medical and health research is crucial to saving lives and reducing health care costs.

The National Institutes of Health-funded research team led by Dr. Totary-Jain — including students at all levels — is part of the USF Health Heart Institute.  They are working on solutions for heart disease and other cardiovascular disorders.  The team’s research focuses on developing new nanotherapies to reverse arterial damage caused by atherosclerosis. These investigational therapies, designed to target diseased cells without harming healthy cells, could be applied to other diseases, including cancer.

“Today we have the tools to treat many diseases, but it takes many years of training and hard work to make the right discoveries that lead to treatment,” Dr. Totary-Jain said.  “That is why it is crucial that the U.S. government continues to invest in biomedical research.”

Hana Totary-Jain, PhD, far right, with her research team

Research!America is the nation’s largest not-for-profit public education and advocacy alliance committed to making research to improve health a higher national priority.

-Video and photo by Sandra C. Roa, University Communications and Marketing

Preclinical study shows microRNA approach inhibited re-narrowing while healing vessels

Tampa, FL (Aug. 18, 2014) — A new therapy developed by researchers at the University of South Florida (USF) Morsani College of Medicine and Columbia University Medical Center (CUMC) may help reduce the life-threatening complications of interventional cardiovascular disease treatment.

The researchers demonstrated in a rat model that the novel molecular therapy could selectively inhibit blood vessel re-narrowing and simultaneously promote vessel healing following a medical procedure using a balloon catheter to open narrowed or blocked arteries.

Their preclinical study was published in Sept. 2, 2014 in the Journal of Clinical Investigation.

Hana Totary-Jain, PhD, assistant professor of molecular pharmacology and physiology at the USF Health Morsani College of Medicine, was principal investigator for the study.

“This innovative microRNA-based strategy can be used to combine anti-proliferative and pro-healing mechanisms for improved repair of coronary arteries,” said the study’s principal investigator Hana Totary-Jain, PhD, assistant professor of molecular pharmacology and physiology at the USF Health Morsani College of Medicine, who came to USF Health from CUMC last year to join the USF Health Heart Institute.

“The most significant finding of our study is that for the first time we were able to achieve in one fell swoop both the inhibition of cells responsible for re-narrowing of the vessel, and preserving the ‘good’ endothelial cells that protect against thrombosis,” said lead author Gaetano Santulli, MD, PhD, a cardiologist working at CUMC’s College of Physicians & Surgeons.

Angioplasty, the world’s most common medical procedure, opens a narrowed or blocked artery by inserting a small balloon into the blood vessel. If the artery is blocked, a tiny wire-mesh tube, known as a stent, is mounted on the end of the balloon to leave in the vessel when the balloon is removed. The stent holds the artery open and maintains blood flow after angioplasty clears the vessel of fatty deposits. Physicians performed 560,500 angioplasties in the United States in 2011, according to a recent report by the Agency for Healthcare Research and Quality,and, Dr. Santulli said, 70 to 90 percent of all angioplasty patients receive one or more stents.

Together, angioplasty and stenting have helped advance the field of interventional cardiology and save lives.

Drug-eluting stents, first approved for use in the United States in 2003, dramatically reduced rates of restenosis compared to earlier bare metal stents. Medications coating these stents thwart the development of scar tissue causing the treated coronary artery to re-narrow, a complication often requiring another procedure.

While the drug-eluting stent overcame the obstacle of restenosis, research eventually showed that the medications released by the device were not specific — meaning they failed to discriminate between destructive and beneficial cells. The drugs blocked proliferation and migration of vascular smooth muscle cells leading to artery re-narrowing, but they also blocked regrowth of endothelial cells indispensable to healing blood vessel walls disrupted by stent implantation.

Formation of blood clots several months or even years after initial implantation remains a severe, though rare, increased risk associated with the lack of endothelium covering the treated vessel. This risk for late stent clotting, or thrombosis, requires patients to stay on prolonged dual antiplatelet therapy to help prevent life-threatening heart attacks — but not without increasing the odds of major bleeding.

Gaetano Santulli, MD, PhD, of Columbia University Medical Center, was lead author.

With this history in mind, researchers at USF and CUMC harnessed the intrinsic power of microRNAs — master regulators of gene expression affecting many biological processes including cell proliferation — to create a more selective therapy.

Their goal was to inhibit blood vessel re-narrowing and, at the same time, allow endothelial cells to regrow and heal the vessel. They tested the experimental therapy in a rat model of balloon angioplasty injury, and discovered it worked.

Among the findings:

–          As soon as two weeks following arterial injury induced by balloon angioplasty, the injured arteries in the rats receiving microRNA-based therapy were 80 percent covered with new endothelium. In the group receiving a molecular therapy that mimicked drug-eluting stents, endothelial cell coverage remained below 30 percent even after one month. “The difference was quite amazing,” Dr. Totary-Jain said.

–          Measures of blood clotting in the microRNA-based therapy group at two weeks post-injury were reduced to the same levels as in the uninjured control animals.

–          In addition to helping protect against thrombosis-associated clotting, the endothelial cells restored in the treated group appeared to work as well in helping dilate blood vessels as endothelial cells in the vessels of the healthy, untreated control group. “From a clinical point of view, reduced thrombosis and functional vascular responses represent the most promising aspects of the whole study,” Dr. Santulli said.

Dr. Totary-Jain works with Jamie Chilton, PhD, one of the study’s co-authors.

More studies are needed, including implanting stents to test the therapy in other models of atherosclerosis and diabetes.

“This is just the first step, but we are working on tailoring the strategy to be more effective,” Dr. Totary-Jain said. “The combination of this selective therapy with a better stent platform and biodegradable polymer has the potential to revolutionize the future of vascular interventional medicine.”

The USF/CUMC study was supported by the American Heart Association and the National Institutes of Health/National Heart, Lung and Blood Institute.

Article citation: Gaetano Santulli, Anetta Wronska, Kunihiro Uryu, Thomas G. Diacovo, Melanie Gao, Steven O. Marx, Jan Kitajewski, Jamie M. Chilton, Kemal Marc Akat, Thomas Tuschl, Andrew R. Marks, Hana Totary-Jain; “A selective microRNA-based strategy inhibits restenosis while preserving endothelial function;”Journal of Clinical Investigation: 2014;124 (9):4102-4114. DOI: 10.1172/JCI76069.

-USF Health-

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