Dr. Anna McGuire, Dr. Brianna Slatnick, and Dr. Jenny Yu - Research Fellowship in Review

It's nice to see everybody with their white coats nearby and their ties on no scrubs for our department photo today. So, get through to me the red call by delaying us for the rain date because we've got a beautiful day. All right. All right. Just a reminder, no scrubs. Go ahead. Okay, perfect. All right. So good morning everyone and welcome to Grand Rounds. It's my pleasure to introduce today's speakers in the first of two weeks of our research fellows who are graduating and finishing their time with us. So, we have three speakers today and they'll each speak for about 15 minutes and then we'll have time for a question or two and some comments from their mentors. We will stay on time as our department pictures are to follow at 8 a.m. So, first step, it's my pleasure to introduce Dr. Anna McGuire who graduated cum laude with a BA in anthropology and sociology and BS in neuroscience from Lafayette College followed by medical school at University of Rochester where she stayed on for two years of surgical residency before joining us for her research time. She has spent her time here over the last two years primarily working with Dr. Fishman and Dickie in the Vascular Anomaly Center on a variety of clinical research projects and we'll be sharing some of our work here with us today. Thank you. Good morning. I will share my screen. Thank you. Everyone see that okay? Good morning. Thank you, Hester, for that introduction and thank you to everyone for being here this morning to give me the chance to present my work. This Hester said I'm the Vascular Anomaly's research fellow. I work with Dr. Fishman and Dr. Dickie. I've also had the unique privilege of being the first fellow to be based in Dr. Daniel Cohane's lab. The work I'm presenting this morning is a director's result of this new inter-part mental collaboration. Dr. Cohane's lab focuses on biomaterials and drug delivery with a focus on novel local anesthetics and drug delivery systems. There are multiple types of local anesthetics. There's your pro-kine, tetra-kine and cocaine as well as amides which include betybocaine and lydocaine. These bind to the intercellular portion of the sodium channel blocking sodium influx into the cell and preventing action potential propagation. They exhibit both systemic and local toxicity. Troxins can also be used as local anesthetics. These are site one sodium channel blockers that block sodium influx from the extra cellular portion of the channel. They exhibit less local toxicity but exhibit systemic respiratory depression at higher doses. This audience is intimately aware of the benefits of local anesthetics. They provide local pain control while minimizing systemic effects. At the height of the opioid epidemic, they have been an integral part of multimodal pain control to minimize narcotic use. However, they aren't perfect. They exhibit both local and systemic toxicity. They can damage nerve and muscle at the injection site as well as exhibit cardiac respiratory depression and seizures at higher doses. They also never seem to last as long as their patients would like. That's why we're always looking for new ways to improve local anesthesia. This led us to look to benzana-tate or test-lon pearls as they're more commonly known clinically. This is a drug that was FDA approved in 1958 as a non-narcotic cough medicine. It's an ester type local anesthetics and more tetra-kine, cocaine and cocaine and structure. It was initially thought to act selectively on the vagus nerve. Until 2016, paper sought to determine if benzana-tate was selective for the voltage-gated sodium channel 1.7. This is the primary sodium channel expressed on the vagus nerve. That paper drew several important conclusions that helped to guide our work. The first was that it is not in fact selective for NAV 1.7. They found that it also binds to NAV 1.3. Importantly, again, as it's an orally administered medication, it was not the metabolites that were blocking the sodium channels. This led to our hypothesis that benzana-tate will exhibit local anesthetic properties that is decreased sensory and motor function by blocking sodium channels on peripheral nerve-swelling local injection. To test this hypothesis, we used a sciatic nerve block model. This model has been validated and used in Dr. Kohani's lab for many years. We perform a percutaneous nerve block at the left sciatic nerve with the right side serving as a control. The effectiveness and duration of the block is then assessed. We use a hot plate to assess for thermal latency and motor function is assessed by the amount of weight the animal can bear on its hind paws. We monitor for signs of toxicity by testing for a control lateral nerve block on the right side, as well as monitoring clinically for systemic symptoms. We also send samples for histological assessment. For all experiments, I will discuss this morning. We used male rats between 300 and 400 grams. This weight was chosen to allow us to better assess for systemic toxicity. Four to eight animals were used per group. Based on power analysis, it was indicated that a sample size of four rats would provide 80% power to detect a 50% difference between groups. Eight rats were used when a smaller difference was expected. Prior to injection, we ensure we were not altering the structure of benzana tape by dissolving it in solution. NMR and LCMS were used to verify this. There is also what you're shown here. Rats were injected with increasing doses of benzana tape as shown here. A block rate of 18 minutes to 3.5 hours for sensory and 39 minutes to 5 hours for motor was obtained with increasing doses from 3.25 mx per mil all the way up to 120 mx per mil. There is an incomplete block rate seen at lower doses, with 30 mx per mil being required to obtain a 100% block rate. There were no control letter, block seen, or any systemic side effects seen at any dose. This was our dose response curve. We then sought to further explore the properties of benzana tape and potentially extend our duration of block by using chemical permeation enhancers or CPEs. CPEs are compounds that improve the drug's ability to permeate the nerve as well as prolonged duration of block. We looked specifically at fupivocane and terrodotoxin or TTX. These were chosen due to their different sites of action on the sodium channel with fupivocane blocking intercellularly while TTX acts on the extracellular portion of the sodium channel. We also chose to look at Tween80 or Polysorbate 80. This is a surfactant that aids in my cell formation. Looking specifically at benzana tape, co-injected with fupivocane, we chose a dose of 7.5 mx per mil of benzana tape. We chose this dose piece that did not have a 100% block rate and would allow us to more easily see an enhanced effect. As you can see here, the duration of block was enhanced by the addition of fupivocane with the sensory block more than doubling and we now achieved a 100% block rate for both sensory and motor. Looking now at TTX, we again chose 7.5 and 3 micrograms of TTX was used. This and the, we use, sorry, I forgot to mention, we use point 4 mix per mil of Bpivocane, both of these independently do not block sensory or motor function. So looking again, we see enhancement this time with a threefold increase in our block time. So we did not able, we were not able to achieve 100% block, it was increased from 50 to 87 and 75 to 87. We then chose to look at co-injection with TTX, again 7.5 was used along with 10 microleaders of TTX which does not independently block. Again, we saw improvement in our duration of block with our time yearly doubling and we reached 100% block rate. We then chose to look at cytotoxicity in vitro. This is our cytotoxicity at 24 hours and it expresses the expected decrease in percent viability and proportion to concentration. This was also seen at 48 hours. We sent samples for histological assessment at four days we saw inflammation with some superficial myotoxicity. At 30 days this was resolved, the muscle is healthy appearing again on the side and at 60 days the samples were back to baseline. So in conclusion, benzonitate exhibits a dose dependent motor and sensory nerve block. It's enhanced by the addition of bupivocane TTX and TN80. It does exhibit dose dependent myotoxicity at four days. This was resolved at 30 days. In future steps, we can look at mapping the specific site of binding. We co-injected with compounds with known binding sites but we don't know where on the channel specifically benzonitate acts whether it's on the intercellular or extracellular portion. We could not see any systemic toxicity even at our highest dose of 120 mix per meal. We could continue to increase dosing until systemic side effects were seen to know what those doses were. So this slide is grown over my two years with all the amazing people who have helped and taught me with this project and many others over time and it still does not encompass everyone. Thank you very much. Thank you very much. I'm very grateful to Dr. Fishman and Dr. Dickie for giving me this opportunity and for being such amazing mentors over the past two years. To Dr. Kohane, who welcomed me into his lab and has treated me as one of his own post-docs. To Dr. Collian, who never wavered in her, belief that I could do chemistry and worked hard to ensure that my projects were successful. I'm also a member of the VAC faculty and the amazing MPs who have helped me so much with clinical projects as well as learning about the care of these complex patients. And then importantly in this column, the amazing postdocs and research assistants who trained me on the model you saw me present on today, as well as answered all of my questions and helps me troubleshoot problems. I've been doing this for many of these past two years, a lot of fun. So thank you to everyone for coming this morning and for all the help and that you've taught me over time and on call. I'm eternally grateful. I welcome any questions or comments? Thank you, Anna. I believe a couple of minutes for questions if anybody has any and then Dr. Sishman or Dickey want to make any comments. Well, I'd like to comment at some questions that obviously what Anna just presented is something that Dr. Dickey and I have no expertise in learning and watching and sharing on the side. As Anna said, she is the first to participate in a usual partnership. I have a long ago given up and the fact that I could be a scientific mentor, but I believe that it's a wonderful opportunity to partner with real scientists. Anna has become a clinical expert in the Ashkanami's, the status for certain and we're going to miss her tremendously in the Ashkanami Center. We're looking forward to all the clinical projects that she didn't have time to present today. But she's a spectacular physician. I think from all this she's a spectacular scientist, but I'm not one so I don't know. I want to really thank Dan Kahani for making this opportunity of this kind of partnership and also to Kate, a colleague who's really been an unbelievable mentor hands on with Anna and Kate was kind enough to join us this morning. Kate, I'd like to make a comment. I would love to. I speak for Dan and myself when I say that Anna was exceptional in the lab as a growing scientist. I feel comfortable to make that comment. So it's hard to really put into words all the effort that it takes to get a project like this to to grow. You know, Anna doesn't put this piece into the presentation, but we started with, you know, not knowing anything about this compound. We have to figure out even how to dissolve it, which took weeks, believe it or not, just because of the viscosity of the compound. And in doing that, she learned how to do really sophisticated chemistry to properly identify what this structure really is. And of course, we knew that she was going to be exceptional in the, you know, the sciatic nerve block technique, but then when she became a master at understanding sodium channels and how they work to be able to pick different chemical permeation enhancers, that was really next level. This project will be ultimately, it's a completely novel, local anesthetic. There's talks about pattening this agent and certainly pubs to come. So we're really proud of Anna an excellent job and a very short amount of time in the science world. Kudos for a surgeon to take on that kind of science and honestly, I want to thank Kate and Dan so much for their willingness to partnership with our department with a surgeon and to demonstrate the positive power of interdisciplinary collaboration. And Anna, you deserve all the credit for diving right in and taking up things that the doctor to get I couldn't, couldn't begin to to understand. Thank you, everyone. All right, so we'll move on to our next speaker. It's my pleasure to introduce Dr. Brie Slatnik. She graduated Madna Cum Laude with a BS in microbiology from UC Davis, followed by medical school at University of Toledo. She then completed two years of her surgical residency at Rutgers, Robert Wood Johnson, before joining us for her research time. She spent her time here over the last two years, primarily working with doctors Kim and Demeri in the surgical innovation fellowship and was selected for the chairman's fellowship by our department this year. She'll be sharing some of her exciting innovation work today. Thanks, Esther. Good morning, everyone. I'm looking forward to sharing a small portion of my work here with you today. I don't have any commercial affiliations, but I will be discussing some off label use of hospital supplies. And I like to think of our fellowship generally in terms of pillars. And when I started here, our training primarily focused on these three pillars, which are devices, procedures and digital health care platforms. And one pillar that we've added since my time here has been an innovation research pillar and work that I've done under this are some of the projects that I'm most proud of during my time is I'll talk a little bit about them today. Additionally, my time here was particularly impacted by the pandemic. And our lab was fortunately able to pivot last spring to direct our energy to work on a few devices during the surge capacity. And they learned a lot of lessons from this. So today I'll touch on a couple of the projects from the innovation research pillar that I was able to see from start to finish for finalizing the manuscripts on them now. And then I'll discuss how the COVID pandemic in particular has acted as an accelerant to novel innovation over the past year. And then I'll share some of our early work developing reusable PPE from readily available hospital supplies and a couple other COVID specific projects that we've been working on and in some lessons that I've learned from them. So starting with the first objective, as I mentioned the past couple years, we've been laying the groundwork for some initial studies in research and surgical innovation. And this project started when we got access to the US patent database. And at first we just looked simply at the number of surgical patents that were filed per year over the last 25 years. We're able to make this graph, which you can see has exponential growth after 2008. And as a surgical innovation group working to patent some of our own devices, this beg the question to us that as these patent numbers are rising has this increase in patent filings been driven by an increase in certain inventors. So to answer this, we did a query of the publicly available patent database from the years, 1993 to 2018. And the patent database is complicated, but to explain it to quickly it's organized by something called the CPC code, which is the cooperative patent classification code. And this is a classifying system such that when a patent is filed, it's placed into one of these separate categories. And broadly the categories go from A to H like it's shown here and include things like transportation, engineering and chemistry. And for medical patents, these fall under the human necessity category. So each category is broken down further into subgroups and we define the surgically related patents as those that fell under these four CPC codes, which are in the medical branch of the health category and these were surgery implantables, dressings and introducers. And we then pull the list of all US patents with these codes on them for the past 25 years. And from this we identified a group of about 275,000 surgical patents. We then separately took the complete list of almost 57,000 active 2019 fellows of the American College of Surgery. And we matched the names of the surgeons to the listed inventors of each one of these surgical patents to find out which surgeons had been named as an inventor during this period. So as an overall cohort from 1993 to 2018, we found that there was this overall increase in the number of surgical patents filed per year with this more dramatic increase in the last 10 years. And among all surgical patents, 15,690 or about 5.7% of them had a surgeon as a co-inventor. The percentage of patents per year that had a surgeon as a co-inventor actually decreased. So it went from 2.2% in 1993 to 1.7% showing that this increase in patents has occurred without a significant increase in surgeon contribution. And those 15,000 patents were held by only 3877 surgeons of that almost 57,000 surgeon cohort who are fellows of the American College. We also found that only 2% of patents were held by women. And when we looked at the likelihood of holding a patent by gender using logistic regression and adjusting for specialty, we found that men were 3.5 times more likely to hold a patent than women. So to follow up this initial study, we looked more closely at an outlier group of surgeon patent holders, specifically those who are in the top 1%. And in this study, we aimed to investigate characteristics of these highly successful surgeon innovators to evaluate any association between the number of patents that are held publications and any revenue received from medical device companies. And our methods from this project were the same where we cross index the patent database over that same 25 year period to identify surgeon inventors and we selected for the same surgical patent CPC codes. So we identified the top 1% of patent holders and this was 25 surgeons who held 25 or more patents and these surgeons hold 16% of all the surgeon held patents. We then collected information about their specialty gender academic rank, executive titles, number of publications, total NIH funding and total payments. And by payments, I'm referring to general payments. These are from drug and medical device companies and they're defined by the Centers for Medicare and Medicaid Services through the open payments program and we took fees from 2013 to 2019. So among this top 1% of patent holders, we found that 96% were subspecialists, 96% were male, 40% of them had no academic affiliation, 60% of them were publicly listed as either a founder, a CEO or a CMO of a medical device company. There was a median of 49 publications, 28% of them had NIH funding in this range from 22,000 up to 8.1 million. And the mean total payments received was 54,000 in this range up to 106 million. And when you look closely at the breakdown of the payments, the columns on this graph, each represent a single surgeon to orient you and each a surgeon patent holder. And the colors represent the percentage breakdown of the type of payments that they received and 23 of those 25 surgeons received some form of payment and overwhelmingly the majority of the payments 96% of them were from royalties. We also found a moderate positive correlation between the number of patents held and the number of payments received, but we did not find any correlation between the number of patents and the number of publications. So these studies were just a first step in a new field of research and the patent database has not been widely published on, especially not in surgery. And so we're just laying the groundwork here for much more work that can be done with this data and in this space. The third project I want to quickly touch on under this same innovation research pillar is a survey that we distributed to the apps membership where we were evaluating opinions of typical innovation fellowship outputs among candidates who are applying for pediatric surgery fellowship. And we asked surveys to report how valuable a list of typical innovation achievements were in comparison to more traditional research outputs. And we found that although innovation experiences generally viewed positively for fellowship selection committee members, the most valuable achievements are more traditional academic outputs such as publishing as a first author and presenting at a national meeting. And this study in particular has put things into perspective for me that it's important for our training to strike a very fine balance between pursuing projects and the business and the industry world, while also still finding ways to generate typical academic outputs concurrently so that the trainees can be competitive for fellowship selection. So moving on through my objectives now to numbers two and three and specifically focusing on COVID. I want to talk about how the pandemic actually provided an ideal scenario for innovation and how we were able to harness this. So this last year really drove home this quote for me that where there is necessity innovation is born. And when you think about it, COVID provided really the ultimate combination of necessity and in environment that was very resource rich in terms of time and funding and some decreased regulatory barriers and this allowed innovation to take off at a hyper speed. And this very accelerated environment ended up teaching me some of the most important lessons that I learned during my time here. This brings me to one of the best examples of brainstorming and teamwork that I've personally ever been a part of and this was a hackathon that we held last year for our reusable respirator project, which I know many of you have are familiar with. And for this project, we identified a problem, which was the PPE shortages that we experienced last March. And from that, we identified a need to create reusable PPE, usually using readily available hospital equipment that would be comfortable to wear. And the device that we ultimately proposed was developed through a collaborative effort. It was a multi departmental hackathon. And it included members from our innovation fellowship as well as representatives and leadership from the Department of Surgery from respiratory care and from environmental health and safety. And the video that we published ended up calling viral, which allowed us to connect with people all over the country and the world everywhere from large universities like our neighbors at BID and Cedar Sinai and University of Chicago to several universities abroad and Turkey and the UK. And in Brazil, and we're also able to connect with several small community hospitals and private practices across the country. And this project was a tremendous multi departmental effort and it really highlights the benefit and importance of building a strong multi faceted team because it adds incredible value to your prototyping into your project implementation. And I am extremely grateful to have been a part of this team. So after the respirator project, our team moved on to tackling other COVID problems, including working on an ergonomic and collapsible intubation shield. And we developed a pruning device and a back to school screener with the digital health accelerator program that was published online through the hospital website. And it's interesting because each of these projects requires you to learn a new skill set and to take on new responsibilities and really each taught me how to pick up on the needs of different stakeholders and how to adapt in different scenarios. Another lesson that I've learned is the importance of knowing when to move on from a project when it's no longer benefiting you. So for example, last summer, we had an idea to pair an in a nausea screener with our back to school screener. And for this, we plan to make scratch and sniff cards coupled with the survey to assess more objectively whether somebody would be experiencing was experiencing some early signs of COVID. But we ultimately realized after a pretty significant amount of research and submitting an invention disclosure form that we were not really in a position to be experts in the field of an nausea. And so we weren't really well equipped to take on that project. And I bring up this point because I learned that it's just as important to identify when a project is not moving along the way that you expected to and to have the courage to pivot it or to move on to something else that may be better suited for you. So with that, I want to give my sincere gratitude to my lab and especially to my mentor, a sector Kim and Dr. D'Amerri who have taken me under their wing and helped us pivot our project. And mentored me over the past couple years and I also want to thank the entire department for being so dedicated to educating the research residents because it really makes a difference for us to have the extra protected time with the with the faculty and I really do appreciate it. And I want to thank my previous co-fellows Robin Kyle and my junior fellow John and our phenomenal medical student Alex Yang and Nicole when he was our research assistant she joined us here and she's been very helpful getting proud of me. And I'm happy to take any questions. Thank you, Bri. We have time for a question or two if anybody has one or Dr. Kim or D'Amerri want to make any comments. I think I can start things off. I want to thank Bri for a great two years. As you can see, she really has a really innovative spirit. She really brought that to our team. The COVID and them really allowed her to shine actually and she is the one that really taught us together for this hackathon. Pretty much within a couple of days of us shutting down. That hackathon really led to all of the projects that resulted in the pandemic. It was a great experience for me. I think for our whole team, the teamwork during that time was incredible to watch actually. And Jenny, thank you to the Perfee and Perfee and part of that. I'm enjoying our team for a very time. It was really fun to watch. Again, thank you for your work. That's the luck going forward in your career, hopefully in plastic surgery or to see you in the future. Thanks Dr. Kim. And Bri is a pro. I also echo what HB said. I think the opportunity to work with you has been a real pleasure. And honestly, to get a chance to work with residents like you is, you know, if you're so excited to work here because it's been truly inspiring. My first day after fellowship pretty much when I was still on a month off, I remember you sending a message out saying, hey, if I'm this patent database, no one seems to be like looking into this. This looks interesting. What can we do with this? And I just, you know, recovered from fellowship and this is really cool. And this was those were idea. I think it was one everyone know that this was something that Bri just said, let's look into this. And we just watch her run with it and really has opened up sort of a new field of inquiry, which really has not been explored and it is going to be a lot of cool things that come out of that. And as HB said, the COVID project some were really inspiring. And importantly, you know, the milestones that you've had, you know, starting a visual art career and then soon getting married. So I think it's been really fun watching your life blossom during this time in the report to see you in the next chapter. So I want to thank you for teaching me about Twitter to. Thank you watching the video viral. Thank you. Thank you. She also taught us how many WhatsApp messages could be sent in a single hour during during the hackathon. I had the pleasure of three in the morning, four in the morning, seeing the continuous innovation going on amongst this group. Bri became a social media phenomenon when her video went viral and represented us well and really did provide a solution to people in need around the world. And you will never know how many people were impacted by your starrgum. So thank you for your contributions. Bring that to us. I'm teaching a few of us old dogs on pay attention to Twitter and WhatsApp and all the rest. Thank you. All right, then. So we'll move on to our last speaker for the for the morning. So it's my pleasure to introduce Dr. Jenny you should graduate at summa cum laude with a BA in cell biology and neuroscience and minor in psychology at Rutgers university followed by medical school at Rutgers rubber with Johnson. She then completed two years of surgical residency at Beth Israel Deakinis Medical Center before joining us for her research time. She has spent her time over the last three years primarily working with Dr. Puder in our department and the vascular biology program. She received the Sandra and Richard Cummings research fellowship from the B.I. as well as the Joshua Ryan Rappaport fellowship from our department. She'll be sharing some of our work today, which is when several awards including the Excellence in Research Award from the American College of Surgeons. Thanks, Esther. You just share my screen. And thank you for the opportunity this morning to speak to you about some of our work on the role of anti-clagulation and the management of pulmonary hypoplasia. I have no disclosures, but a patent has been filed by Boston Children's Hospital on behalf of Dr. Cudra and numbers of our lab for the use of the Jeff in pulmonary hypoplasia. In general, diaphragmatic fremia or CDH occurs with an incidence of one in 2500 newborns per year is characterized by a whole diaphram and the protrusion of abdominal content such as the stomach and intestines into the thoracic cavity. This equality of CDH include pulmonary hypoplasia pulmonary hypertension and cardiac dysfunction. On a histological level, pulmonary hypoplasia is characterized by reduction in lung thrincoma and alveolarization as seen in the slide on the right of lung from a patient with pulmonary hypoplasia compared to lung from an aged matched control on the last. Pulmonary hypoplasia is a key contributor to the morbidity and mortality of the CD's process and the small underdeveloped lungs are associated with concurrently underdeveloped fast lager, which could also be a key factor for the patient. The patient can contribute to pulmonary hypertension as such therapeutic studies in our lab typically aim to accelerate pulmonary growth and concomitant vascular growth. In development, airway and vascular branching are intimately associated likely as a setup for the formation of the alveolar tathleria units where gas exchange occurs. Vascular endothelial growth factor or VHS is a major regulator of angiogenesis and as lung development is driven strongly by endothelial epithelial interaction is also an essential player in this process. VHS is accreted by lung masoncumol and alveolar epithelial cells and drives endothelial cell proliferation with subsequent autocrine and periprin signaling for coordinated bronchovascular growth. In CDH, decreased expression of VHS in the lung rankama is observed. Here, the mRNA expression level of VHS in lung tissue from CDH patients versus H-match controls is demonstrated with substantially lower levels of VHS mRNA observed in CDH patients in the alveolar stage lung development during which there's the most substantial expansion of gas exchange area. To study CDH and pulmonary hypoplasia and vivo, we use a mirroring model of left humanectomy induced compensatory lung growth. While in this model, there's no diaphragmatic defect and no true pulmonary hypoplasia removal of one lung induces a regenerative process that includes re-albularization and expansion of the functional lung volume available for gas exchange, which is a process that's characterized by the same molecular patterns as developmental albularization. This allows for the study of modulation of growth in pulmonary hypoplastic diseases. Our group and others have previously demonstrated that compensatory lung growth after a new manectomy stimulates the remaining right lung to approach the original volume of both lungs in just eight days. Knowing that VHS is decreased in lungs of CDH patients, the initial hypothesis was that enhancing VHS may rescue coordinated bronchovascular development. In a compensatory lung growth model, our group showed that systemic vegeotherapy accelerated compensatory lung growth with the remaining right lung reaching growth completion by post-operative day four after left new neck to me. Further studies demonstrated increased perencomal volume, alveolar volume, septal surface area, just the functional area available for gas exchange, as well as total alveolar count. Together, indicating that increased functional lung was existing on post-up day four with vege treatment compared to controlled treated mice. By post-operative day 10, most of these effects even out between control and vege retreated mice, indicating that vege does not necessarily cause additional growth or rather accelerates the process. There's an additional subset of patients with CDH who fail conservative medical management and may require ECMO to sustain adequate profusion of oxygenate blood. The first-genetic-based pulmonary growth wall on ECMO's support of care is imperative for improved morbidity mortality, which is as high as 50% in the patient population is not changed much in the past several decades despite significant advances in the surgical and intensive care abuse infants. With ECMO canulation comes the need for systemic anti-cogulation to prevent clotting in the circuits. Because unfractionated heparin, which is the most commonly used anti-cogulate in ECMO circuits, is well known to interact with many major angelogenic growth factors, including vege F, which could see the effect of the infection. The stimulates the lung growth that is essential in CDH are grouped and investigated the impact of systemic cavernization on pulmonary growth. In this model, after left-newmentectomy, heparin impaired compensatory lung growth at post-operative day 4, the halfway point to lung growth completion, and this is demonstrated by lung volume measured by the water displacement method. And on more phonometric analysis, this correlated with significant decreases in perincomal volume, alveolar volume, and septal surface area, the functional area available for gas exchange. In these representative histological images, decreased alveolarization and septation can be appreciated in the sample from the heparin treated lung tissue seen on the right compared to that from saline treated lung tissue on the left. In addition to critically ill CDH patients requiring ECMO and therapeutic anticoagulation, a much larger population of patients receives subtherapeutic anticoagulation to maintain the agency of venous catheters and prevent venous thrombosis. They may concomitantly have pulmonary pathology. And so in reconsidering anticoagulation as a factor in the management of pulmonary hypoclasia, we aim to determine the effects of different doses of heparin, and the commonly used low molecular weight heparin lowvenox, lung endophilial cells in vitro, and uncompensatory lung growth in vivo. Further, we determine the molecular, histological, and functional effects of bivalrydine, a direct thrombin inhibitor, to assess its value as a potential alternative interquigalant to be used in patients with pulmonary hypoplasia. First, in vitro, human microvascular lung endophilial cells were treated with increasing doses of unfrashinated heparin or lowvenox and allowed to grow for 72 hours. Cell proliferation in apoptosis were assessed using chlorometric acids. After a threshold of 0.5 units per amel, unfrashinated heparin inhibited human microvascular lung endophilial cell proliferation in a dose-dependent manner. Similarly, lowvenox also inhibited lung endophilial cell proliferation, and although this might be an overly simplistic way of interpreting this data, note that the therapeutic anticoidulation level in serum of these drugs are as indicated by the arrows here, suggesting that clinically relevant doses of both types of heparins may have some intrinsic inhibitory effect on lung endophilial cell proliferation. However, heparin and lowvenox treatment have minimal effects on apoptosis or programmed cell death, indicating that these interquigalants may slow the growth of lung endophilial cells and thus affect downstream processes, but does not necessarily halt the process altogether. Next, the effects of heparin and lowvenox on structural and functional outcomes were assessed in the Meary and Compensatory Lung Growth Model. Eight-week-old male C57-Black-6 mice that did not undergo surgery were treated with three doses of low-dose unfrashinated heparin, high-dose unfrashinated heparin, or lowvenox, while you matched the handling was used as a control. Plasma, PT and antifactor 10A levels were measured after the last dose to determine the degree of anticoagulation achieved. A second cohort of mice underwent left-newmenectomy then received treatment with anticoagulation or volume-edge handling for eight days. On post-opera update 8, they underwent pulmonary function testing, lung volume measurement, and more phonometric analysis. High-dose unfrashinated heparin significantly elevated PTT over control while low-dose heparin did not. Similarly, high-dose unfrashinated heparin and lowvenox significantly increased antifactor 10A levels over control while low-dose unfrashinated heparin did not. On post-opera update 8, both low and high-dose unfrashinated heparin, as well as lowvenox, impaired compensatory lung growth as measured by significantly decreased lung volume by water displacement compared to their respective controls. On pulmonary function testing, both unfrashinated heparin and lowvenox displayed a trend toward decreased total lung capacity. While only low-dose unfrashinated heparin significantly decreased inspiratory capacity, high-dose unfrashinated heparin and lowvenox again trended towards a decrease in inspiratory capacity. On post-operative date 8, more phonometric analysis, unfrashinated heparin and lowvenox trended toward decreasing peranglimal volume in the remaining right lung compared to controls. However, both anti-quite lengths significantly decreased alveolar volume and sepulsurface area, the functional areas available for gas exchange. Unfrashinated heparin affected alveolar volume and sepulsurface area in a dose-dependent manner. Low-dose unfrashinated heparin and lowvenox also significantly increased mean-sepulfitness, which may impair effective gas exchange. Representative H&E's stain lung sections from mice treated with control, low-dose unfrashinated heparin and high-dose unfrashinated heparin demonstrate impaired alveolarization with heparin treatment, which appears dose-dependent. Similarly, representative H&E stain lung sections from mice treated with control and low-dose unfrashinated heparin again demonstrate impaired alveolarization and seputation with low-dose unfrashinated heparin treatment. To determine the functional effects of anti-plagulation in compensatory lung growth, a third cohort of mice underwent treadmill exercise tolerance testing at baseline two days prior to left-numinectomy. They were then subject to systemic antifagulation either with low or high-dose unfrashinated heparin or low-venox, and isovolumetric saline was used as a control. Fouring eight days post-operatively mice again underwent compulsory exercise on a treadmill to determine if antifagulation influenced exercise tolerance. Time spent running and distance run were compared in each mouse pre and post-intervention. During exercise tolerance testing, mice are first acclimated to a treadmill apparatus prior to compulsory running until exhaustion, which is defined as sitting on the shock grid at the end of the treadmill for greater than five seconds despite receiving multiple low-voltage shocks. When exercise distance and time were adjusted according to each mouse's pre-operative baseline exercise tolerance, both low-dose and high-dose unfrashinated heparin significantly reduced exercise tolerance on post-up days four and eight. Low-venox did not differ from control on post-operative day four, but significantly impaired exercise distance by post-operative day eight. Together, these data indicate that therapeutic and sub-theraputic unfrashinated heparin as well as low-venox, plus structural and functional deficits in mirroring compensatory lung growth, possibly by inhibition of pulmonary endophilial self-puliferation. Finally, both the in vitro and in vivo experiments were performed with bivalryrudent, a direct thrombin inhibitor, to determine if the different mechanism of action of this anticoagulant may render it more appropriate for use in patients with pulmonary hypoplasia for dependent on lung growth for survival. While both unfrashinated heparin and low-venox inhibited pulmonary endophilial self-puliferation in vitro, bivalryrudent particularly at a dose equivalent to therapeutic levels in serum, did not impair long endophilial self-puliferation. However, bivalryrudent did inhibit pulmonary endophilial self-apoptosis or program cell death, while heparin and low-venox did not have this effect. These results suggest that bivalryrudent may preserve the rate of compensatory lung growth in vivo and may promote some yet unidentified survival signaling. In vivo, bivalryrudent dosing achieved therapeutic interquitulation as measured by plasma PTT. At this dose, bivalryrudent preserved lung volume measured by water displacement on post-operative date 8 after left-name andectomy. On pulmonary function testing, bivalryrudent also preserved both total lung capacity and inspiratory capacity compared to ceiling control on post-operative date 8. On morphometric analysis, bivalryrudent preserved perencomal volume and alveolar volume compared to controls. There was also a small increase in septal surface area, the functional area available for gas exchange, and a decrease in mean septal thickness, which could potentially improve the gas exchange in bivalryrudent treated subjects. Represented H&E stained lung sections for mice treated with control and bivalryrudent demonstrate preserved alveolarization and septation with treatment. Finally, as a readout of functional outcomes, bivalryrudent preserved exercised tolerance as measured by percent change in exercise distance and time from baseline on post-operative date 8 after left-name andectomy, or recall that both hebron and lovenox impaired exercised tolerance at this time point. To summarize, in contrast to hebron and lovenox, bivalryrudent preserved pulmonary alveolarization and functional outcomes in a muring compensatory lung growth model. Now, it's important to consider the clinical implications of the state. Neonates with CDH require therapies to accelerate lung growth to overcome their pulmonary hypoplasia and wean from ECMO expeditiously, particularly as longer ECMO runs are associated with worse outcomes. A result indicate that unfractionated hebron, the most commonly used anticoagulant in ECMO circuits, inhibits lung endophilial self-puliferation and impairs post-numinectomy lung growth, alveolarization, and exercise tolerance. At therapeutic and sub-theraputic doses, and that lovenox also has similar effects. In these data suggests that hebrons as a class of anticoagulants mean worse in outcomes for muonates with CDH and other pulmonary hypoplastic diseases through inhibiting pulmonary endophilial proliferation and impairing pulmonary function. Meanwhile, bivalryrudent has been demonstrated to preserve lung endophilial self-puliferation in vitro and preserve pulmonary structural and functional outcomes in vivo in a compensatory lung growth model. Accordingly, the use of bivalryrudent and direct-thrombin inhibitors as a class of anticoagulants deserves further attention and the choice of drug, dose, and duration of therapy for muonates with hypoplastic lung disease requiring anticoagulation should be reconsidered. This was but a snapshot of the full body of work that I've been a part of in the Peter Lab in the past three years, and I've been fortunate to have had the mentorship and support of many in the vascular biology program and in the Department of Surgery. I'm also grateful to have had the support from the Sandra and Richard Cummings Research Fellowship of the Department of Surgery at BI and the RAP Report Fellowship from the department here. I also owe a special thank you to Dr. Alex Coenka, who is my fellow when I wrote to you through as an intern. So not quite sure what you saw on me, but I'm here today because you told me I could and encouraged me to take a shot and for that, I'll always be grateful. The members of the Peter Lab, past and present, have contributed a significant amount to the work presented today both in conceptualization and actualization. My fellows, my co-fellows, Tori Co and Jordan Seacore, my senior fellows, Dewey and Lorenzo, and my now junior fellows, Savas Tichis and Scott Fligger, who be carrying on some of the ongoing projects in the lab that I have started. Dr. Peter has of course been an unparalleled mentor in many regards, but as I look forward to pursuing translational research in my future career, you've taught me independence and how to scientifically identify and approach the important and important. And clinically relevant questions from the bench side. So thank you for a busy three years, and I'm sure this is not the last you'll hear from me. Finally, it's been a great privilege to have met so many incredible people here at Boston Children's Hospital, both in the clinical and research settings to whom surgery, science, and caring for children are passions and not just jobs. I'm grateful to all of you, but especially to those who have become more like family than friends. So thank you, and I'm happy to take questions at this time. Thanks, Jenny. All right. We can open up to questions or Dr. Peter wants to make any comments. Well, this, Jenny, you did just an amazing job over the last three years. You know, this is just a small part of what you did and each one of these things you did, the normal amount of work just to get controls, get the methods worked out. I mean, you did basic mechanisms, a lot of that, which you didn't show. He translated it to the animals and also did clinical research also. In the field of angiogenesis, you are now an expert and you've certainly surpassed me. You worked so hard, a very meticulous worker. You've also been a great teacher to other fellows throughout the vast of biology and surgical world, and we appreciate what you've done. And I really, really, and I don't usually say this hate to see you go. Thank you. Well, I would second that. We're not going far and we have enjoyed having you here for such an extended period of time. And I think I didn't know the part about Alex, but I think we should thank Alex and I'm sure you will fulfill both your dreams as well as the expectations that you have. That Dr. Scooter and Quenka have set up for you the world of shores. Any comments or questions? I'll just briefly highlight something that should be obvious, but it doesn't hurt to point out that the diverse of projects that he's fell of tackle was on display here and was just a part of it as it was said. And we'll see a little more in a couple of weeks. And this makes for a very vibrant research environment, which I'm very happy to be proud of and to be a part of. And but that also means that they have to tackle very different challenges and these kids and I'm old enough to call you kids have tackled them with great boys and it's a lot of fun to see you grow. I am very proud of you, along with your mentors and look forward to following your career. Thank you very much for everything you've done here. I would second that it's really important for us and the faculty to see growth and development of the trainees both clinically, academically and scientifically and the three of you have really provided a brilliant demonstration of what can be accomplished and we will continue to support you and your successors. And I want to thank Tom, Jacksick and Dariel, for helping lead the scientific effort and for all the mentors more to come next week. Thanks to all of you. If there's no more questions, I want to remind everybody that our department photo will be immediately following this session over at the medical school on the steps and street clothes and white coats, no scrubs. Craig, I mentioned about the rest of the day. You bet Steve and once again, congratulations for it is fun to see what you guys have done. Yes, this afternoon we'll have M&M is at noon and then that will be followed at 130 by the Dr. Wiles going to educate us further about additional tumors. We've got a professor rounds at 230 and actually journal club is at 330 days. So lots of activities for the afternoon. We look forward to seeing you then. See you down the street. You You You You