Ripla Arora, PhD - phaware® interview 562

Why the Clues to Pulmonary Hypertension Start Before Birth

When Ripla Arora first studied TBX4 as a young PhD student, no one imagined this gene held secrets to life-threatening lung diseases. Now, years later, her early work is the foundation of a global effort to unlock the fetal origins of pulmonary hypertension.

My name is Ripla Arora and I’m an Associate Professor in the OB-GYN department and the biomedical engineering department at Michigan State University. I also am part of the Institute of Quantitative Health Science and Engineering. I’m a developmental biologist and a geneticist by training. I was born in Africa and I grew up in India learning biochemistry. I wanted to learn genetics and developmental biology, so I moved to the U.S. 20 years ago. 
 
The first thing I worked with is a gene called TBX4 during my PhD. During fetal development, TBX4 is actually really important for endothelial cells, or the cells that line your blood vessels to come together to make a blood vessel. When I joined my PhD lab, we were trying to figure out why these cells won’t come together to make a vessel. We discovered TBX4, that protein, is not there in the cells that need to come together. It’s actually around the surrounding cells, which we call the mesenchymal cells.
 
If you have a defect in the surrounding cells, they can’t talk properly to these cells that line the blood vessels, and that actually results in a phenotype. I also, then, started exploring the role of this protein in lung development. In 2012, I published this paper where I showed that this gene, along with a very closely related protein, TBX5, both of them are absolutely necessary for lung development. If you start removing pieces of these proteins from lung cells, the lungs don’t develop and the fetuses struggle at birth. 
 
This paper came out in 2012, and at that time it was not known that TBX4 causes pulmonary hypertension or affects lung development in human patients. It was only known that it affects skeletal development or the feet. It was related to syndrome called small patella syndrome. So, if you had feet abnormalities, people would predict that you may have a mutation in this gene and then they would sequence your genome, etc. But nobody ever knew that if you had lung disorders that you may also be suffering from a problem with this TBX4 protein. 
 
Then, I graduated, I moved on. I went to UCSF to do my postdoc. My PhD was at Columbia. I moved to do my postdoc at UCSF where I got involved in learning about reproductive organs. I developed an imaging technique to study early pregnancy. I’m very fascinated by fetal development. So, throughout my career, I have jumped around working with different organs but during fetal development. Here I was studying how the embryo connects with the uterus for the first time and generating images for that.
 
Using that, I actually started my faculty position at Michigan State University and studying early pregnancy. Then, I spoke with one of my collaborators on the lung project during my PhD. His name is Ross Metzger, who also works on lung development. He told me that my paper in TBX4 and lung development was very highly cited because now TBX4 is associated with developmental lung disorder or pediatric pulmonary hypertension. He said that if I was interested, there is room for somebody to understand the fetal basis. I already had a lot of experience studying embryos or fetal mice that had problems with these proteins.
 
This is where I started connecting with people. I got in touch with Eric Austin. My collaborator Maya Kumar who’s at Stanford, she told me about Eric Austin. I reached out. Eric connected me to Anton Morkin. They all had read my 2012 paper, because other than that paper, there was no mouse model for this disease. I was now interested in making a mouse model which would actually survive birth. The actual phenotype, when you have a TBX4 mutation, is that sometimes the lungs are so malformed that the babies won’t make it, but some of them will make it and then they will develop pulmonary hypertension. So, that’s when I started connecting. From 2012 to now, we’ve had many more resources. We have had advances in imaging technologies, where you can image the lungs and look at them in three dimensions, which was not there when I was doing my PhD.
 
Where I am right now is we have made a mouse model where we have removed, again, TBX4 and TBX5 proteins. This model, where about 30% of the mice will die at birth, about 30% will make it through the first week and then die. About 30 to 35% will go on to adulthood or young adulthood and then will develop pulmonary hypertension. So, that’s where we are at. Our goal is to determine what is the fetal basis of the disease, what goes wrong in early lung development that then forms the basis of pulmonary hypertension later in life. Or is it that later in life is a separate function for these proteins, which is distinct from what happens in the fetus? So, that’s my connection to pulmonary hypertension and TBX4Life, which is the organization that Eric and Anton are part of.
 
When I finished my PhD, my PhD mentor was retiring. Normally, when you do your graduate work, once you leave, somebody in your PhD lab finishes or takes over and follows through on it. That was the first study on this. Typically, you would have somebody else from the lab who would continue the work and determine the basis of the phenotypes that we saw. But my PhD mentor retired soon after. When I was early in my postdoc, I even considered, an NIH funding mechanism called DP1, (Director’s Pioneer Award), where if you have a good idea, you can apply to be… It’s sort of a bridge to your faculty position and you forego the postdoctoral training. 
 
I went back to my PhD mentor and I asked her and she was like, “Yep, I am retiring. So whatever you need, if you want to continue working on.” I always in my heart was like, I wanted to know more because I did these initial studies and I was very excited. I actually kept following who was citing my work, because usually, if people are citing my work, that means they’re either working, making a new mouse model. I would see all these studies coming out with human patients that would then cite my publication. So, I was always connected to it. I think it was just the right moment to go back to it that I was working on uterine biology. There was actually an NIH funding mechanism. It’s called the Stephen Katz funding mechanism, that says if you want to pivot and develop a new line of investigation within your lab that’s different from your current line of investigation, that they would fund it.
 
Actually, I started applying for that funding and I reached out to Ross and I said, “Will you talk to me about this?” He said, “This is the perfect opportunity and you should do this,” because he knew about the TBX4Life and the field. He said that “a lot of people are working on the adult models of pulmonary hypertension and they’re looking at the blood vessels, but nobody’s actually looking at what happens in the fetal lungs. So, given your previous experience, you might be well positioned to do this.” So, that’s how I acted on it. But I always in my heart wanted to know why we saw the things we saw in those mice that did not have those proteins.
 
I think in 2021, COVID times, I think is when Anton reached out to Eric. Anton is a father of an affected child, and Eric Austin is this pediatric pulmonologist with experience with pulmonary hypertension. They connected to form this organization, TBX4Life. I think a lot of these not-for-profit organizations that are started by families, their goal is to bring scientists and clinicians and geneticists and pathologists all together in the same room. As scientists, we are working with our mice. We are on our own and we’re discussing. We connect with other people, but I think if somebody facilitates it, it makes our lives easier and then we are all in the same room and then it’s more exciting.
 
I think the idea with organizations like TBX4Life is generally that there are basic scientists who are working with cells or animal models. The data that they collect could very well inform preclinical studies such as therapy, drug targets. Also, the basic scientists, the kind of phenomena they see might inform what kind of clinical course the doctor should take depending on what knowledge is gained from the basic models. So, TBX4Life came about, and it was probably initially 25 people. Now we are an organization of about 100 people. There are working groups. There’s a clinical working group, and then there is a biology of TBX4 working group.
 
The clinical working group is, obviously, trying to get what are the TBX4 mutations that are known to show pulmonary hypertension. Sometimes, you get the same mutation, but the parent does not have pulmonary hypertension, but the children have pulmonary hypertension. Or another thing that TBX4 causes is diffused lung developmental disorder, which means that the lungs are not developing properly. Sometimes, you will get pulmonary hypertension, which is associated with an inefficient lung, because the lungs are not well-developed, there’s not enough oxygenation. Then, the heart is working harder, and that’s when you have pulmonary hypertension. So, TBX4 mutations are shown to cause both kinds, ones that are associated with inefficient lungs in terms of growth and oxygenation, but also just a pulmonary hypertension phenotype.
 
I think the clinical working group, which is led by Drs. Matina Prapa and Olivier Danhaive, their goal is to bring together the clinicians and the clinical presentations and the geneticists who have all the information on where those mutations are in the protein. Currently, this is a rare disease, which means if you look at it worldwide, you are going to collect more data versus in individual sites. The biology of the TBX4 working group is asking the question, what does TBX4 as a protein do? What other proteins does it turn on? What proteins it might shut off? Then, actually, very similar to the first project I work on, TBX4 is not expressed in the cells that line the airways in the lung, and it’s not expressed in the cells that line the blood vessels of the lung, but it affects both of their function. So, people want to know, does it affect something in the surrounding cells and then those cells fail to talk to the blood vessels or the airways? Or what is the mechanism of how TBX4 is controlling these different processes?
 
One more thing to know is that the TBX4 protein comes on very early on in lung development. So, as soon as your lungs exist in the fetus, the TBX4 protein is expressed there. It may regulate different kinds of processes during development. So, the biology of the TBX4 working group, which is led by Jeff Whitsett and myself, our goal is we try to bring scientists every month together. The goal is to share unpublished data. A lot of scientists, although are little worried, because it’s unpublished and you’re sharing it with the community, so we try to keep it very honor-based. People share their research. The goal is something that another group may find might help speed up my work and something that I can contribute may help speed up somebody else’s work. The idea is to collaborate rather than compete, and also to see, okay, if someone else is working on one aspect of it, I could work on a different aspect of it, because that means that now we are teamed up not working on the same thing and actually discovering new things that might converge at some point. 
 
That is one of the achievements, to get all these people in a room. It’s very easy to buy into the vision because it’s for the children. I think it gives everybody a purpose. But at the same time, I think that since I’ve been part of this for about two and a half years, it makes it more exciting. Because now you don’t have to just do it all by yourself, you have people you can brainstorm with and then pull things together, and then ideas grow faster. Hopefully, the end goal is what we learn from the biology of TBX4 can be given to people who are maybe trying to develop new drugs or therapeutics or targets, and that targeting can eventually go to the clinic. That’s the long-term goal.
 
I think we are at the stage where the basic scientists are coming together and figuring out where are we, what do we need. The clinician scientists are doing the same thing and the geneticists are doing the same thing. I think information has started to flow across. We are a very open group and we invite people from all over. Something that we worked on, which we are very proud of is the roadmap, your goal setting for the short-term and the long-term. That’s what we were able to achieve. We got our group of people in the same room. Then, we had some mini-groups. We decided on priorities. Now, we’re going to collate everybody’s ideas to try to get it into a roadmap that will be shared with the whole community so that we can all get on the same page and contribute to it in whatever our capabilities are. 
 
Thanks for listening. My name is Ripla Arora, and I’m aware that I’m rare.

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