Roughly 8.5 million people in the United States suffer from peripheral artery disease (PAD), a narrowing of the arteries in the legs or arms (frequently due to the buildup of fatty plaque) that can cut off blood flow to the limbs, causing tissue death, gangrene, and even amputation. Strategies to combat PAD by delivering compounds that promote angiogenesis (the growth of new blood vessels) to bypass the blocked arteries have been investigated, but have largely failed to improve outcomes. More recently, there has been increasing interest in using the body's immune system to treat ischemia as some immune cells are known to secrete blood-vessel-promoting compounds. However, getting therapeutic immune cells to concentrate and secrete a sufficient amount of the desired compounds where new vessels are needed remains a challenge.
A new approach from researchers at Harvard's Wyss Institute for Biologically Inspired Engineering and John A. Paulson School of Engineering and Applied Sciences (SEAS) takes advantage of the surprising combination of implantable biomaterial scaffolds and childhood vaccines to solve this problem. In models of mice with hindlimb ischemia (a severe form of PAD), their technique increased the concentration of T cells at the ischemic site and stimulated angiogenesis, blood flow, and muscle fiber regeneration for up to two weeks. The study is reported in Science Advances.
"One of the most exciting aspects of this work is that it provides a new method of enhancing blood vessel formation that does not rely on traditional biologics, such as cells, growth factors, and cytokines, that are typically used to promote vascularization," said first author Brian Kwee, Ph.D., a former graduate student at the Wyss Institute and SEAS who is now a postdoctoral research fellow at the FDA. "Also, it more broadly suggests that advances in bioengineered T-cell therapies, which have traditionally been used to treat cancers, may be utilized to promote wound healing and regeneration."
The biomaterial scaffold Kwee and the rest of the team used has been in development at the Wyss Institute for several years, and has been used to successfully modulate the immune system for a variety of purposes, including treating cancer and trapping T cells that have gone rogue and are attacking the body. For treating ischemia, they focused on a specific type of immune cell called T helper 2 (TH2) cells, which have been found to secrete molecules that promote blood vessel growth in addition to producing cytokines that initiate immune responses.
TH2 cells are also the crucial "memory" element of vaccinations against pathogens, as they recognize the microbe that is introduced by the vaccine and help the body mount an immune response against it in the future. For reasons that are not yet fully understood, delivering a small amount of aluminum in a vaccine greatly enhances TH2 cell formation, and nearly all Americans receive aluminum-containing childhood vaccines that protect them from a variety of diseases. The Wyss team had a hunch that vaccinated people could potentially mount a stronger TH2 cell response if the right triggering antigen was introduced; and, if that antigen was incorporated into a biomaterial scaffold located near a blocked artery, TH2 cells could be recruited to the scaffold and release their angiogenesis-promoting compounds where they are needed to help treat ischemia.
"This method essentially takes advantage of the fact that standard vaccines 'prime' the immune system to recognize specific antigens. By reintroducing an antigen via a scaffold in a very localized place, we're able to attract and retain enough TH2 cells that they can effectively treat ischemic tissue and promote the growth of new blood vessels," said senior author David Mooney, Ph.D., who is a Founding Core Faculty member at the Wyss Institute as well as the Robert P. Pinkas Family Professor of Bioengineering at SEAS.
Mooney's team injected mice with ovalbumin, the primary protein found in egg whites, to create a mild immune reaction, along with aluminum hydroxide to mimic a human childhood immunization vaccine. Two weeks later the mice got a "booster" of the same vaccine, and four weeks later were implanted with an ovalbumin-containing scaffold in their ischemic hindlimbs. These mice displayed higher numbers of ovalbumin-specific TH2 cells and eosinophils (angiogenesis-promoting cells that are activated by TH2 cells) in their ischemic muscles than mice that received the implant without the priming vaccine.
Vaccinated mice also displayed a lower level of tissue death, higher blood vessel density, greater blood perfusion, and more regenerating muscle fibers in their ischemic hindlimbs after two weeks than unvaccinated mice that received the implant. To confirm that the primed TH2 cells were indeed responsible for this improvement, the researchers introduced antibodies against the TH2 cells into vaccinated mice that received the implant, and observed that neutralizing TH2 cells reversed the benefits observed.
Further work will investigate how changes in the timing between vaccination and implantation affect the TH2 cell response, as humans are often vaccinated when they are very young but develop PAD and ischemia later in life.
"We are very excited about this proof-of-concept study because it demonstrates the novel idea that memory T cells can be used to promote the growth of blood vessels, and it supports the premise that biomaterials can manipulate T cells to enhance regeneration of damaged tissues," said Mooney.
"We are at the dawn of a new age of beginning to understand the extent to which the immune system impacts human health and disease. This work from our Immuno-Materials Platform here at the Wyss Institute provides yet another example of how a bioinspired materials approach to reengineer the immune system can potentially lead to disruptive medical breakthroughs," said Wyss Institute Founding Director Donald Ingber, M.D., Ph.D., who is also the Judah Folkman Professor of Vascular Biology at Harvard Medical School and the Vascular Biology Program at Boston Children's Hospital, as well as Professor of Bioengineering at SEAS.
Additional authors of the paper include Bo Ri Seo, Ph.D., Alexander Najibi, Aileen Li, Ph.D., and Tin-Yu Shih from the Wyss Institute and Harvard SEAS; and Des White from the Wyss Institute. This research was supported by the National Institutes of Health, the National Science Foundation, and the Wyss Institute for Biologically Inspired Engineering at Harvard University.