Blood-vessel-on-a-chip provides insight into new anti-inflammatory drug candidate

January 15, 2018, Harvard University
Credit: Wyss Institute at Harvard University

One of the most important and fraught processes in the human body is inflammation. Inflammatory responses to injury or disease are crucial for recruiting the immune system to help the body heal, but inflammation can also cause an increase in the production of thrombin, which can lead to dangerous blood clots and other conditions. Activated protein C (APC) is a naturally occurring anti-coagulant protein with anti-inflammatory and other protective effects that has been used medically to treat severe blood infections and wounds; however, its use is limited because its inhibition of thrombin also impacts the blood's ability to clot, increasing bleeding risk.

Now, a collaborative team of researchers from the Division of Hemostasis and Thrombosis at Beth Israel Deaconess Medical Center (BIDMC) and the Wyss Institute at Harvard University have discovered that synthetic APC-mimicking small molecules called "parmodulins" provide anti-inflammatory and anti-thrombotic protection to endothelial cells on par with APC's without interfering with clotting, making them attractive new drug candidates. This work was enabled by leveraging the Institute's Organ-on-a-Chip technology to model coagulation within a human blood vessel in vitro. The results are reported in this week's issue of Proceedings of the National Academy of Sciences.

"We essentially performed a mini pre-clinical trial of parmodulins' effect on the endothelium, and not only determined the pathway through which parmodulins function, but also demonstrated that they help protect endothelial cells from inflammatory damage," says former Wyss postdoc Abhishek Jain, Ph.D., who is now an Assistant Professor and director of the the Bioinspired Translational Microsystems lab at Texas A&M University.

The target protein on which both APC and parmodulins act is the transmembrane protein protease-activated receptor 1 (PAR1), which is present on both endothelial cells that line blood vessels and platelets that circulate through the blood and promote clotting, making mechanistic analysis difficult. PAR1 was originally identified as a receptor for thrombin, which is a crucial part of the inflammatory process. However, when PAR1 is activated by APC on endothelium, it triggers anti-inflammatory, anti-apoptotic, and barrier-fortifying pathways, all of which help protect cells from the negative effects of inflammation.

In addition to activating PAR1, APC also independently inhibits the generation of thrombin, which is an essential component of healthy blood clotting - but inhibiting thrombin too much leads to uncontrolled bleeding. Knowing that parmodulins bind to PAR1, the team of scientists and clinicians set out to find a way to activate endothelial PAR1 and reduce thrombic responses without thinning the blood, and thus provide a better alternative to APC.

To evaluate the activity of parmodulins on endothelium, Karen De Ceunynck, Ph.D., postdoctoral research fellow at BIDMC and first author of the paper, incubated human endothelial cells with parmodulin 2 in vitro for 4 hours and then exposed them to the thrombin-inducing inflammatory agents lipopolysaccharide (LPS) or tumor necrosis factor-α (TNF-α). In the parmodulin-exposed cells, both agents' ability to generate thrombin was reduced by over 50% compared with non-parmodulin-exposed cells. However, parmodulin 2 did not inhibit the activity of factor V or factor X, proteins that function in blood coagulation. "We were intrigued by the notion that parmodulin 2 inhibited LPS- and TNF-mediated prothrombotic effects on the endothelial surface without impairing blood clotting" says De Ceunynck.

Credit: Wyss Institute at Harvard University

To confirm this theory, the team used a Wyss-developed blood-vessel-on-a-chip consisting of microfluidic channels embedded in a clear polymer chip, coated with collagen, and lined by . Whole blood was perfused through the chip to simulate the flow conditions within human blood vessels, to which were added different pro- and anti-inflammatory compounds to evaluate the response of the endothelium.

When the endothelial cells were exposed to TNF-α before being perfused with whole blood, platelets accumulated on the endothelium in a typical inflammatory response; if the were first exposed to parmodulin 2 and then TNF-α, platelet accumulation was inhibited and the endothelium resumed its normal function. These results indicated that parmodulin exposure blocks the thrombotic response of to inflammatory stimuli without affecting blood coagulation in humans - a significant improvement over APC.

A series of tests in vitro performed by co-first author Christian Peters, Ph.D. at BIDMC, confirmed that parmodulin 2's activation of PAR1 also induces cytoprotective responses in by inhibiting apoptosis (programmed cell death) induced by thrombin, TNF-α, and the apoptotic alkaloid staurosporine through a signaling pathway that begins with parmodulin 2's binding to a specific site on the cytoplasmic side of PAR1. "We observed that the cytoprotective response induced by parmodulin 2 happened very quickly, and confirmed its rapid onset in time course and gene expression assays," says Peters.

Furthermore, in vivo studies in mice showed that parmodulin 2 reduces the binding of to and impairs platelet and fibrin accumulation at injury sites during the inflammatory response, confirming the anti-thrombotic and anti-coagulant activity of parmodulin 2 observed in vitro. Additionally, parmodulins do not interact with many of APC's other binding partners, making it much more targeted to PAR1 and reducing other side effects.

"The discovery of an anti-inflammatory molecule that prevents endothelial thrombosis but also preserves normal is a major step toward an alternative and better approach to treating inflammatory disease," says Rob Flaumenhaft, M.D., Ph.D., Professor of Medicine at Harvard Medical School, Chief of the Division of Hemostasis and Thrombosis at BIDMC, and corresponding author of the paper. "Furthermore, nearly all other pharmaceuticals that target G-protein coupled receptors like PAR1 bind to the exterior of the receptor; parmodulin 2 represents a paradigm shift for compounds targeting GPCRs because it acts on internal domains. We are excited to see if we can advance it to clinical trials."

"This work provides another example of how organ-on-a-chip technology can enable faster and safer development and evaluation of drugs that could help patients around the world," says co-author and Wyss Institute Founding Director Donald Ingber, M.D., Ph.D., who is also the Judah Folkman Professor of Vascular Biology at HMS and the Vascular Biology Program at Boston Children's Hospital, as well as Professor of Bioengineering at Harvard's John A. Paulson School of Engineering and Applied Sciences (SEAS).

Explore further: Pulmonary Thrombosis-on-a-Chip provides new avenue for drug development

More information: Karen De Ceunynck el al., "PAR1 agonists stimulate APC-like endothelial cytoprotection and confer resistance to thromboinflammatory injury," PNAS (2018). www.pnas.org/cgi/doi/10.1073/pnas.1718600115

Related Stories

Pulmonary Thrombosis-on-a-Chip provides new avenue for drug development

May 23, 2017
The average human pair of lungs is permeated by a network of about 164 feet of blood vessels (roughly the width of a football field), including microscopic blood capillaries, which facilitate the diffusion of oxygen into ...

'Fixing' blood vessel cells to diagnose blood clotting disorders

August 9, 2016
When in dysfunction, the vascular endothelium—the tissue that lines the blood vessels throughout our body's entire circulatory system—plays a big role in the development of many human diseases, including diabetes, stroke, ...

Scientists unravel likely causes of blood vessel leakage in severe dengue

November 9, 2017
A protein secreted by cells infected with dengue virus can cause dangerous leakage of fluid from blood vessels, and new research published in PLOS Pathogens supports a primary underlying mechanism: disruption of a molecular ...

Researchers shed light on anti-adhesive molecule in vascular endothelium

February 2, 2016
Researchers from the Harvard Medical School (HMS) Department of Ophthalmology and the Schepens Eye Research Institute of Massachusetts Eye and Ear have gained new insight into how a non-inflammatory state is maintained in ...

Blood vessel 'master gene' discovery could lead to treatments for liver disease

October 16, 2017
Scientists have identified a key gene in blood vessels which could provide a new way to assess and potentially treat liver disease.

Recommended for you

New blood test to detect liver damage in under an hour

May 24, 2018
A quick and robust blood test that can detect liver damage before symptoms appear has been designed and verified using clinical samples by a team from UCL and University of Massachusetts.

Selective neural connections can be reestablished in retina after injury, study finds

May 24, 2018
The brain's ability to form new neural connections, called neuroplasticity, is crucial to recovery from some types of brain injury, but this process is hard to study and remains poorly understood. A new study of neural circuit ...

Space-like gravity weakens biochemical signals in muscle formation

May 23, 2018
Astronauts go through many physiological changes during their time in spaceflight, including lower muscle mass and slower muscle development. Similar symptoms can occur in the muscles of people on Earth's surface, too. In ...

Eating at night, sleeping by day swiftly alters key blood proteins

May 21, 2018
Staying awake all night and sleeping all day for just a few days can disrupt levels and time of day patterns of more than 100 proteins in the blood, including those that influence blood sugar, energy metabolism, and immune ...

Hotter bodies fight infections and tumours better—researchers show how

May 21, 2018
The hotter our body temperature, the more our bodies speed up a key defence system that fights against tumours, wounds or infections, new research by a multidisciplinary team of mathematicians and biologists from the Universities ...

Deep space radiation treatment reboots brain's immune system

May 21, 2018
Planning a trip to Mars? You'll want to remember your anti-radiation pills.

0 comments

Please sign in to add a comment. Registration is free, and takes less than a minute. Read more

Click here to reset your password.
Sign in to get notified via email when new comments are made.