Uh, good morning everybody. We'd like to welcome all of you to, uh, the M. Judah Folkman, uh, uh, Research Day. This, uh, uh, was named in his honor, um, after he, uh, passed away. Uh, it's been 10 years now that his name has been affiliated with the, um, uh, this research forum for the day. It actually started 19 years ago, um. When we got the idea of having the Boston Children's Hospital Community come together for research to find out what's being done in your own department and in other departments around the hospital, and this has grown over the past 20 years. This year we had 121 abstracts. We had so many abstracts we had to divide up the 6 judges into looking at um. Uh, parts of all the abstracts to get these nominees for a presentation this morning at 120 and at 3:30. So, uh, first I'd like to thank Louanne Posey for all her administrative efforts to make this possible. I'd like to thank my co-host Jordan Kriedberg and all the judges who did a tremendous amount of work. Um, so we'll start off with the first presentation by David Blitzner from cardiac surgery, delayed mitochondrial transplantation by intracorporeal injection for myocardial salvage after ischemia reperfusion injury. OK. Great. I. Thank you very much. It's a privilege to be presenting today when we honor Doctor Faulkman. So mitochondria is present in almost every cell in the body. The heart is an obligate aerobic organ and has abundant mitochondria, and the heart is dependent on these to provide all of the energy for contraction and homeostasis. To support this function, mitochondria utilize 75% of the coronary oxygen delivered at rest. This makes the heart and the mitochondria extremely sensitive to loss of blood flow and oxygen delivery. Previously, we and others have shown that loss of oxygen delivery results in severe damage to the mitochondria and ultimately in cell death and decreased cardiac function. To treat this mitochondrial dysfunction, we have pioneered a protocol in which the mitochondria damaged by ischemia and reperfusion are replaced by viable mitochondria obtained from a non-ischemic area in the patient's own body. This process is termed autologous mitochondrial transplantation. In brief, two small pieces of tissue are obtained from a non-ischemic skeletal muscle such as the pectoralis major or rectus abdominus using a 6 biopsy punch. In that top picture, you can see that the two small pieces can fit on the face of a dime. These pieces are homogenized, subjected to digestion for 10 minutes, and then filtered through a series of filters. The mitochondria can then be collected by centrifugation. And they can be delivered to the heart either by direct injection or by vascular infusion. The advantages of this methodology are this isolation process is rapid, taking less than 30 minutes as compared to previous methods requiring 90 to 120 minutes. Through this process, we can isolate mitochondria that are intact and viable. The mitochondria are isolated from the same patient and therefore, there is no immune response. And finally, the process can be performed in the same operation and so importantly, it falls under the FDA's same procedure exemption. Previously, we have shown that the efficacy of mitochondrial transplantation when used directly after the ischemic event. In these studies, mitochondria were injected either at the end of the ischemic period or just at the start of reperfusion. To extend these investigations, we now examine the use of delayed mitochondrial transplantation. In our studies, we've used the clinically relevant swine model with an N equals 10 in each group. Animals were sedated and anesthetized. A median tsunami was performed, and the left anterior descending artery, just distal to the second diagonal branch was snared to allow for temporary occlusion. The snare was tightened and the heart was made ischemic for a period of 30 minutes. After these 30 minutes were over, the snare was released and all hearts were reperfused for 2 hours. Following this 1st 2 hours of reperfusion, the hearts received either vehicle alone or vehicle containing mitochondria. The concentration of mitochondria used in these studies was determined from preliminary experiments indicating that 1 x 109 mitochondria provided the greatest efficacy. All hearts were then re-perfused for an additional 2 hours. At the end of this 2-hour period, the hearts were harvested for histologic and biochemical analysis. Global and regional function were measured throughout the course of the study. In this particular study, the mitochondria were delivered by vascular infusion. The right carotid artery was accessed, an angiography catheter was advanced to the left coronary ostium, and the mitochondria were thus delivered to the left ventricle. To demonstrate specificity and localization of intravascular mitochondrial injection, we have used PET CT imaging. In this slide, you see mitochondria labeled with 18F rhotamine which appear in white. The PEET CT on the far left shows the whole pig with these mitochondria in the left ventricle. It also shows the syringe and catheter which were used to deliver the mitochondria, which were also radioactive and remained attached to the animal during the imaging. Importantly, we do not see spread of mitochondria to other end organs. In this study, we use sixfold the mitochondrial dose we normally use simply to demonstrate the specificity of uptake with injection. Coronal and transverse imaging show diffuse distribution in the left ventricle. And on the far right, we see that once delivered, the mitochondria are rapidly taken up by the myocardial cells. Our results show that ischemia and reperfusion significantly compromise cardiac function. In this graph, you see N-diastolic pressure, a measure of compliance in the heart. Increased end-diastolic pressure is associated with decreased compliance and therefore decreased function. Our results here show that ischemia and reperfusion significantly increased end-diastolic pressure in both subgroups prior to injection. There was no significant difference between the groups at the end of these first two hours of reperfusion. Following 2 hours of reperfusion. The vehicle only group received an injection of the vehicle, and our results show here that the vehicle had no effect on end diastolic pressure. However, an injection of mitochondria plus vehicle significantly decreases end-diastolic pressure, enhancing compliance of the heart. These effects were evident 5 to 10 minutes following the injection. Similarly, with left ventricular ejection fraction, a measure of cardiac function, it was decreased in both groups following ischemia and the initial reperfusion period. Once again, the injection of vehicle only had no effect on left ventricular ejection fraction. However, injection of mitochondria significantly enhanced left ventricular ejection fraction. Also, similarly, when looking at left ventricular fractional shortening, a measure of function similar to ejection fraction, we see significantly decrease following ischemia and at the end of the 1st 2 hours of reperfusion. Once again, there's no significant difference between either group at the end of these 1st 2 hours. Vehicle-only injection had no effect on left ventricular fractional shortening. And our results show that the injection of mitochondria significantly increased left ventricular fractional shortening. To specifically show the effect within the area at risk, that is the ischemic area of the heart, we have used systolic shortening, which is a more sensitive measurement of regional myocardial function. Following ischemia and reperfusion, myocardial systolic shortening was significantly decreased in both groups. There was no significant difference observed between the groups. Injection of vehicle only failed to enhance systolic shortening. However, the injection of mitochondria significantly enhanced systolic shortening within the area at risk or the area made ischemic. This effect occurred rapidly and was evident in 5 to 10 minutes following the injection and persisted for the remaining 2 hours of reperfusion. To further evaluate the effects of delayed mitochondrial transplantation, we measure the area at risk and the infarct size within that area to allow for estimation of cell viability. No difference between groups was found in the area at risk, indicate indicating that the degree of injury was the same for both groups. Here we actually see that the risk, area at risk was slightly larger in the mitochondrial group, though not significantly so. Infarct size within the area at risk, as determined by TTC staining, was 38% in the vehicle group compared to 7% in the mitochondrial group. These results show that in addition to enhanced functional recovery, there is enhanced cellular viability when with delayed mitochondrial transplantation. In conclusion, our results show that delayed mitochondrial transplantation enhances cardiac function and cell viability. The mechanisms allowing for the efficacy of mitochondrial transplantation have been partially elucidated. However, we're currently continuing with studies on changes affecting the necrosome based on the above data. Our data recapitulates the experience from clinical application to humans performed here at Boston Children's Hospital. We believe this study would also be particularly relevant to patients presenting in delayed fashion and undergoing percutaneous core intervention for STEMMI. And also that mitochondrial transplantation could apply to other organs following ischemia reperfusion injury, such as the lungs, kidneys, and liver. I'd like to thank all of the people that made this study possible, specifically Doctors Delnido and Doctor McCulley for their support, and I'd also like to thank philanthropy that has allowed us to do this work. Thank you very much and I look forward to any questions. Great. That was wonderful. Are there any questions, uh? Without the Blitzer. David, great, great way to start. I was wondering when you mentioned the efficacy of protection of the mitochondria. If you look at echo. And contractility and second shortening how much advocacy how much anticipate. Does that depends on actually where you target the injection, the mitochondria. It's regional, uh, systemic, localized. How much improvement do you anticipate? Um, so we saw in general that, um, Within 1 hour of injection, we were seeing a difference between the two groups and then by 2 hours of reperfusion after the injection, we were back to baseline, uh, for the most part. And um in terms of your question, we've only done injection either by direct injection into the myocardium itself or by vascular infusion where we're directly targeting the left coronary osteum and that left ventricle, which is the regional area we're, in which we're creating ischemia. Um, as we saw in the imaging, the mitochondria are rapidly taken up and just to the end organs. So, um, in terms of your question about, uh, sort of global distribution or, uh, global delivery, I think that, uh, might not be as advantageous. Have you tried any experiments where you let the animals? Um, we, uh, have done that in, not in swine models, which is what we use here, but we've done that in, uh, smaller models such as rabbits, uh, where we've survived them up to 28 days and we've seen, um, A, the, the function remains uh improved over that course and also we've seen that the mitochondria delivered are also still intact and viable uh after that time course. What does the mitochondria get inside. Uh, we know it's by a process of actin-dependent endocytosis. Uh, they've, uh, Dr. Cowan's lab in conjunction with Dr. McCauley's have done this study, uh, using blockers. Uh, only cytocholain D, which is a blocker of actin-dependent endocytosis, was able to stop the, um, entry of mitochondria into the cells. Um, otherwise, it's an incredibly rapid process with upwards of 70% of the mitochondria delivered, entering the cell within half an hour. So, um, it's limiting in terms of the ability to study it in that way. I, I had one question. Uh, I may have missed it, but could you have maintained a, uh, lengthened the the time of, uh, ischemic time to an hour, an hour and a half? Is that, would that make a difference? Uh, we use a stunning model of, uh, Um, myocardial injury, uh, just to make sure that there would be some ability for liability to recover. Um, based on what we've seen here, we're, and kind of what I mentioned during our studies of the mechanism, we're, uh, hypothesizing that mitochondria might affect, uh, protein expression within the necrozome which would lead down that cell death pathway. So it's something we could consider, but, uh, we kind of use the model that we've used in the past. Thank you. Thank you very much. It was great. The next presentation is by Da Dao, paradoxical method to enhance compensatory lung growth utilizing the VEGF inhibitor from vascular biology. Let's see, I'm getting better at this. Yeah, OK. Great. OK. Perfect. Good morning and thank you for the opportunity to present our work. This is a disclosure Vascular, um, uh, vascular endothelial growth factor of VESF is a master regulator of angiogenesis. It interacts with two tyrosine kinase receptors. VSF receptor 2 is a key receptor for angiogenic signaling. The other receptor, VESF receptor 1 or FLIT1, interestingly, can exist in a soluble form where it can bind and sequester VESF, thus inhibiting angiogenesis. VESF is a critical factor in lung growth and development. This regulation of the VESFF pathway is a prominent feature in many ne neonatal lung diseases. One of them is congenital diphhegmatic hernia or CDH, a lethal disease for a newborn characterized by herniation of abdominal organs into thoracic cavity and pulmonary hyperplasia. Even today, it still carries a 30 to 40% mortality and significant pulmonary morbidity. The other one is bronchopulmonary dysplasia or BPD, a chronic lung disease of prematurity that affects more than 10,000 neonates in the US every year. In a previous study from our group, we also showed the exogenous administration of VESF enhanced compensatory lung growth after left pneumonectomy, as evidenced by an increase in total alveolar count and septal surface area on morphometric analysis. So, if VESF is such a critical factor in lung growth and development, we hypothesize that inhibiting VESFF in the form of FLID would impair lung growth by inhibiting angiogenesis. So again, to prove that theory, we use a model of compensatory lung growth after left pneumonectomy. For this study, we use a version of FLIT that has been conjugated with the FC portion of human IgG-1, a strategy to both prolong its half-life and to facilitate its detection in the bloodstream. So using an analyzer that can detect human IgG-1 FC, we determined the half-life of this molecule in mice to be 10 hours. The stability of FLITFC therefore allowed us to administer this medication to the mice on a daily basis. So, We started with a dose response study to determine the effect of FLIT on lung growth. Mice will undergo left pneumonectomy, followed by the administration of FLT ranging from 0 to 180 mcg per kilogram. They were then euthanized and post-op they fall for lung volume measurement. At a low dose, 5 mcg per kilogram, FLIT did what we expected it to do, inhibiting lung growth. To our surprise, however, at a medium dose of 20 mcg per kilogram, there was actually an increase in lung volume. This paradoxical improvement in lung growth caught our attention, and we decided to further investigate this phenomenon. So we repeated the experiment. Mice would again undergo left pneumonectomy followed by daily administration of either saline or FLIT at 20 mcg per kilogram. They were euthanized and post-operate 4 or 10 for lung volume measurement, pulmonary function tests, immunohistochemistry, quantitative PCR, and protein expression studies. After 3 repetitions, we in fact confirmed that FLIT at 20 mcg per kilogram increased lung volume on post-op day 4. On post-op day 10, when compensatory lung growth had finished in mice, this difference expectedly disappeared. To corroborate lung volume data, we also use uh a a system called Flexivent to measure pulmonary mechanical properties of these mice and post-op late fall, and we saw that FLIT treatment, in fact, also increased total lung capacity and pulmonary compliance after left pneumonectomy. But perhaps the most surprising result of all was a paradoxical increase in endogenous levels of HF and its receptors in the lung, suggesting that FLIT actually increased angiogenesis. So to confirm that, we use an angiogenesis antibody array, which demonstrated that FLEIT treatment up-regulated a host of other angiogenic and mitogenic factors. Among those are important factors that stimulate epithelial cell growth and differentiation, including epidermal growth factor EGF and heparin-binding EGF-like growth factor or HBEGF. As a result, FLIT treatment also increased the production of surfactant protein B and C, important markers of lung growth and maturation. So to revisit our initial hypothesis, FLIT at the right dose can actually enhance compensatory lung growth and stimulate angiogenesis. Of course, the question at this point is why? What is the mechanism And to answer that question, we asked another one. What is the single most important factor that controls angiogenesis, that can drive the expression of HF? Hypoxia, hypoxia-induced factor or HIV. Now, HIF is a family of transcription factors that include HIP1 alpha and HIF2 alpha. When dimerized with HIP1 beta, they activate a series of cellular responses aimed at restoring homeostasis in the face of low oxygen. Among these are glucose metabolism, erythropoiesis, cellular migration, in addition to angiogenesis. So using immunohistochemistry, we confirmed that FIIT treatment. In fact, increase the expression of PIF2 alpha, the major isoform of PIP in the lung. Furthermore, using different markers to distinguish different cell populations in the lung, we saw that HIF2 alpha co-localized with surfactant protein C, a marker for alveolar epithelial cell type 2. In another word, we identify alveolar epithelial cell type 2 as a site of production of HIP2 alpha in the lung after left pneumonectomy. But to take that one step further, we also use quantitative PCR to probe for downstream factors under the control PIV, and we saw the FLITT treatment, in fact, increase the expression of angiogenic factors including VAHF and PDGF alpha as expected, but also glucose metabolic enzymes, including pyroba hydrogenase kinase and hexokkinase, chemokine receptor CXCL4, and regulatory proteins of HIF including von Hippel Lindau and polyhydroxylase, proteins that participate in a breakdown of HIF. So to recap, our theory is that FLIT by sequestering VESFF removes a negative feedback brake on HIP2 alpha, thus activating the hypoxic responses. This process most likely originates from alveolar epithelial subtype 2 in the lung. But to be absolutely convinced that it is HIP2 alpha that mediates the effect of FLIT on lung growth, we next aim to inhibit it. And we hypothesize that inhibiting HIP2 alpha would abolish the effect of FLIT on lung growth. So for this purpose, we use PT23A5, a small molecule inhibitor of HIP2 alpha that can be given via oral gastric gavvage. PT23A5 specifically prevents a dimerization between HIP2 alpha and HIP1 beta. So, again, mice will undergo left pneumonectomy followed by randomization into one of the four experimental groups. The V group or the control group received vehicle gavage. The V+ F group received vehicle gavage and FLIT at 20 mcg per kilogram. The I group received the inhibitor PT 2385 via oral gastric gavage, and the IF group received the gavage, uh, the inhibitor and FLIT at 20 mcg per kilogram. They were then euthanize and post-op the fall for lung volume measurement, immunohistochemistry, and physical activity measurement. First, on lung volume measurement, compared to the vehicle control group, the addition of FLIT again, did what we have seen all along, increasing lung growth. In the presence of the HIF2 alpha inhibitor, FLIT had no effect on lung growth. The lung volume of the I and I+F groups were the same as the vehicle control group. The trend on lung volume measurement was also mirrored by the trend on immunohistochemistry. On these micrographs, lung endothelial cells were stained green, and KI-67 cells and microproliferation was then red, so cells that double stain and appear yellow are proliferating endothelial cells. As clearly shown by these pictures, compared to the vehicle control group, the addition of FLIT significantly increased endothelial proliferation. Additional evidence that FLIT actually increased angiogenesis in the lung. In the presence of the HIF2 alpha inhibitor, this effect was neutralized. In addition, we also measure physical activity of these mice in post-op day 4. Mice would be placed in an open field and their movements were quantified for 6 minutes using infrared tracking system. Physical activity was used as a surrogate for pulmonary function in this case. Again, compared to the vehicle control group, the addition of Fleet. Decreased rest time, increased walking distance, and increased movement counts. So in the absence of the HIP2 alpha inhibitor, FLIT, improved physical activity in mice after left pneumonectomy. When the when the inhibitor was introduced, all of these differences disappeared. So FLIT had no effect on physical activity in the presence of the HF2 alpha inhibitor. So in conclusion, FLI at 20 mcg per kilogram paradoxically enhanced compensatory lung growth as evidenced by increased lung volume, pulmonary angiogenesis, and surfactant production. These effects are most likely mediated by an upregulation and hypoxia-induced factor HIP2 alpha. FLIT therefore could be a promising therapy to induce endogenous production of VF and increase lung growth for the treatment of neonatal lung diseases such as CDH and BPD. And finally, I just want to say that this study would not have been possible if it wasn't for the Department of Surgery and the Vascular Biology Program, a legacy of Doctor Judah Folkman. So it truly is standing on the shoulders of giants that allowed us to pursue our research. With that, I thank you for your attention and I'm happy to take any question. OK, questions? Were you able to show decreased levels of circulating VEGF after the uh the treatment? So that would have to be done in the measure in the, in the plasma, and um that's actually something that we still need to confirm. Yeah, so that is actually a very good point. Other questions? In this model, we created a sudden uh Injury low anybody uh so far uh back to that. That's aggressive. I know OK. Right, so that's actually a great question. So the, the primary goal of all of these therapies are to increase alveolar regeneration or alveolar growth in these lungs. The problem with CDH, as you know, is that, um, the lung is hypoplastic, meaning the branching of the lung also doesn't develop normally. The problem is that the, all of the branching happens in around before 20 weeks of gestational age. So there's nothing we can do really, because the disease is not diagnosed until around 2 weeks. Um, so there's nothing we can do about a branching. So really, our goal is just to create more alveoli for these, uh, for these babies. And studies in mice, transcriptomic, uh, data in mice have shown that the compensatory lung growth process, even though it is distinct from neonatal lung growth, it does actually recapitulate the alveolar phase of lung growth. So if we can show alveolar growth in this model, our hope, of course, it's a leap, but our hope is that it will also increase alveolar regeneration and growth in neonates. Any other questions? Do great Thank you. I was kind of curious about the sound. It seems kind of paradoxical as well when you induce therapeutically. That's a great little problem. Uh, either in the presence or absence of hypoxia, what do you typically see as a gray HD. Sorry, the what, what about sorry the yeah. So in a, uh, different study when we administer VCF to the mice, uh, in this model, we actually saw that it also induced expression of HBHF. So actually, we, and then we did a series of individual, uh, studies and we actually show that What happens is VESF first works on endothelial cells, activates endothelial cells, and endothelial cells then secrete HBEGF among other factors, but we think that HBHGF is, um, one of the main factors, and that HBEGF then go and work on epithelial cells. So you can imagine lung. is a bag of endothelial and epithelial cells. You can't just have endothelial proliferation. You have to have concurrent proliferation of both. So when endothelial cells proliferate, they sort of signal and they secrete HBGF and tell epithelial cells, Hey, come with me. So, they both proliferate. So we think the HBEGF sort of serve as a link between angiogenic signaling. And, um, epithelial cell proliferation. So in this model, we again saw that EGF and HBHGF also increased with FLIT. So we think that FLEIT increased angiogenesis, increase endogenous production of VESF, and that VESF then activates endothelial cells which then secrete HBEGF and EGF2, so that epithelial cell can proliferate with them. So that's a link that we sort of proposed. When you reduce the lung growth. Yes, yes, correct. OK, thank you very much. Thank you. OK. I want to, uh, remind everyone that there will be additional presentations at medical grand rounds at 120 here and at, uh, from 3:30 to 4:30, we will have 4 more presentations. And then I hope that as many of you as possible will be able to join us, uh, after that, uh, starting at 4:30 for a poster session at the Galleria where We will have uh uh well over about 130 posters on display as well as lots of great refreshments. Our third speaker is Christian Schwartz from sports medicine, who will be speaking on the comparison of running gait mechanics and female runners with sacroiliac joint pain versus healthy female runners. Hi, everyone. Um, thank you so much for the opportunity to be here today and speak with you about my research. Um, as I mentioned, uh, my project, uh, was focused on comparing, um, running gait mechanics in, um, female runners with sacroiliac joint pain, um, compared to healthy controls. Uh, my interest and experience in this area, uh, does expand beyond the clinical setting, um, into the community. I'm a co-founder of Forest Hills Runners, which is a large community running group in the area. Um, I run for the Boston Athletic Association, um, and I coach for, um, Girls on the Run. Unfortunately, none of these come with a big paycheck, so I have no financial disclosures. Um, so the background and relevance of this issue stems from the, um, the enormous surge in running's popularity in recent years, largely fueled by increased participation by women. Um, 17 million people every year participate in running events, um, and in recent years, um, females have started to um Um, become more, uh, uh, involved in running and have outnumbered their, their male counterparts at the starting lines. Um, some will recall back in 1967, um, Katherine Switzer, um, being, uh, uh, muscled off the, uh, Boston Marathon race route, um, and then in comparison to 50 years later, um, she was able to finish the Boston Marathon, um, in a field of, um, thousands and thousands of women around her. So, um, uh, despite the numerous running, um, uh, benefits on health, there are, um, certain drawbacks in terms of, um, risks from musculoskeletal injuries. Um, Between 19 and 79% of runners report MSK injuries, um, and between 25% and 35% of those involve the lower back, the hips, or the pelvis. Which brings us to uh SI joint pain. It's one of the top 20 most reported um running injuries and it's thought to be underdiagnosed due to the clinical complexity um in diagnosis, um, and it has a female predominance with up to 77% of cases of SI joint pain being reported by women. So just to review a little bit regarding the SI joint, so it's thought to have the formation of a keystone and an arch. It is the base of the spine and it's a key player in force transmission and the dissemination of forces from the lower extremities to the trunk and vice versa. Um, there's two, portions of the joint. The ventral portion is a synovial joint. Um, it's covered in a thin fibrous capsule, um, and is, uh, uh, has articular cartilage, whereas the dorsal portion of the joint does not have a capsule, um, and it's fortified by these very robust, um, interosseous and dorsal SI ligaments. Um, it does, uh, it does show evidence of multiplanar movement, um, in terms of rotation, translation, mutation, and counter mutation. Um, and it's found to be innervated, um, from the L5 to S3 nerve roots, and, um, there's been evidence of small, no susceptive fibers, um, C fibers and A delta fibers, um, present in the cartilage of the joint, um, giving some potential for, um, generation of pain from the joint. So, uh, pathology in the joint, um, there's, um, numerous potential contributors to SI joint pain, um, with, um, the possibility for repetitive torsional forces or sheer forces stressing the joint, um, joint dysfunction in terms of hypomobility or hypermobility, um, repetitive minor trauma, abnormal mechanics, um, extraneous compression on the joint, ligamentous strain, um, uh, all resulting in potential synovial inflammation or bony arthritis. So diagnostic criteria is thought to be threefold. One, pain, um, present in the region of the SI joint with the caveat that, um, there can be some referred pain outside the joint. Um, number 2, so positive provocative physical exam tests, and number 3, full, um, pain relief after diagnostic injection with, um, an anesthetic. So, um, female SI joint. So, the, the, um, female SI joint is actually more susceptible to mechanical stresses and pain degeneration in comparison to, um, the male SI joint. Um, first of all, it has a smaller surface area. Um, it has, um, a more horizontal orientation of the sacrum. Um, the joint contour itself and the articular surface texture of the joint is different in comparison to males. It has, um, less ridges and less grooves in it, um, causing it to have a lower coefficient of friction, um, resulting in, uh, increased susceptibility to shearing forces. Um, it has lower ligamentous tensile strength, um, in part, uh, secondary to hormonal influences on joint mobility, um, which are manifested during puberty and during pregnancy. Um, uh, one key player being the hormone relaxin, um, which softens, um, the ligaments of the pelvis, um, during pregnancy and, um, during, um, ovulation in non-pregnant females. So those are some key anatomic and physiologic um pieces of why females are more at risk for SI joint pain. Um, but what's been less studied is the role of mechanics in the generation of SI joint pain. So just to review our schematic of the running gait cycle, um, it can be broadly divided into a stance phase followed by a double float into a swing phase with the double float component where both feet are off the ground, being the key differentiator between running and walking. Um, and a key area of focus, um, has been the point of initial contact where the, um, foot first hits the ground, um, when it's, when the individual is coming into stance phase. So, um, so, initial contact, um, or the point of foot strike has been looked at in terms of its, um, the, uh, variable strike patterns and the magnitude and the loading rate of ground reaction force that is generated as a result of different foot strike patterns. Um, there's, um, broadly three different groups of foot strike patterns being 4-foot strike, mid-foot strike, and rear foot strike, um, with, um, uh, proven differences in the generation of ground reaction force. Not only in terms of magnitude of ground reaction force being higher in individuals that strike first with their rear foot, um, but also a steeper, um, and faster impact peak of, um, force generation at the point of initial contract. It's been found to be clinically relevant in that um higher ground reaction forces, um, and loading rates have um had a clinical have been clinically correlated with um lower limb stress fractures, plantar fasciitis, medial tibial stress syndrome, and patella femoral pain. So, we sought to look at um whether um differences in running game mechanics um in healthy female runners um are evident in comparison to those with sacroiliac joint pain. So, for this, we performed a retrospective case control, um, analysis of running gait videos. We recruited subjects from our, um, sports medicine clinic. Um, we have a special clinic for injured runners and, um, also at the McKay Center for Injury Prevention. Um, runners who had completed video gate analysis and carried ICD 10 codes of, um, SI joint pain were identified for retrospective chart review. Um, with ultimately case criteria, um, being, um, patient reported pain in the SII region plus greater or equal to two positive provocative physical exam tests and or a positive response to diagnostic injection, which are performed under our ultrasound in our clinic. So we had a case group of 19 individuals, um, and we used a control group of age, height, mass, and BMI match controls of healthy female runners with an N of 63. So, for these individuals, we looked at their running gait mechanics at that point of initial contact, um, in, um, measured in um stance phase inside view of runners that were on a treadmill. So we looked at a variety of different angles. We looked at their foot strike angle, their ankle angle, um, and a degree of overstride or how far the foot um comes anteriorly um in advance of the knee while moving through stride, and then also the knee angle. Um, and we looked at hip angle, um, or also known as pelvic tilt and trunk posture angle as they were moving through the gait cycle. For statistical analysis, we use chi square, um, for categorical variables we used, um, Mann Whitney U test for continuous variables. We, um, calculated, calculated Cohen's D to look at effect size. We use a cutoff of um P of less than 0.05 for statistical significance, all using SPSS IBM software. So the results, so there were not any statistically significant differences um in those with SI pain birth controls in terms of their age, their height, their weight, um, or their BMI. We did find that there was a significant difference in casing controls in terms of two key variables, their foot strike angles and their ankle angles. So runners with SI joint pain were um likely to have higher foot strike angles and um lower ankle angles. So yeah, they're just meaning that they had a higher degree of dorsi flexion at the point of initial contact in comparison to um the healthy controls. Um, and CNC did indicate that this was a strong effect size, um, on these two variables. In terms of categorization of foot strike types at initial contact, in terms of rear foot, mid-foot, or 4 ft strike, um, there was not any statistical statistically significant difference. Um, we think that the lack of statistically significant difference in the actual foot strike pattern between rear foot, mid-foot, and fore-foot strike is likely just, um, due to our study sample. Um, we had a very low number of individuals who actually um struck with their mid-foot or fore-foot. Um, 80% of the general population is rear-foot strikers, so that's not surprising. Um, but was, what was of interest was that, um, uh, even for all of those who were rear-foot strikers, um, the greater degree of dorsiflexion when they hit the ground, um, did predispose them to SI joint pain. Um, this is consistent with past studies that have looked at the kinematics of, um, gait and, uh, particularly the degree of dorsiflexion at, um, initial contact, which does show that, um, those that are more dorsiflex show an increased, uh, rate of loading, um, both in barefoot and shod runners. Um, and the thought is that this increased loading rate, um, causes a maladaptive, um, forced transmission up the kinetic chain, um, which can cause SI joint pain. Um, not only that, but, um, the thought is also that, um, so those with, um, higher ankle angles at initial contact, um, uh, they actually have an altered way of, um, attenuating the rate of loading. Um, individuals that have a more plantar flex foot when landing, um, they have greater soft tissue involvement and better compliance in, in the foot and that the mid-foot arch, um, is a bigger player in their landing, um, in comparison to the more bony rear foot. Um, in contrast to a more rigid dorsiflex landing where there's less force distribution at the foot and ankle and thus higher impact forces are directed through the lower extremities. Um. Um, the thought is that, um, in the high, the more highly dorsiflexed, um, ankle at initial contact, um, there's more um translational kinetic energy which is converted just to the impact force, which ultimately upped the chain given the SI joint's key role in force transmission, um, stresses the SI joint. Um, Lieberman et al. did, um, note, um, in terms of kinetics at the, uh, point of initial contact for angle dorsiflexion, um, versus plantar flexion. The more plantar flexed, um, ankle has a more translational kinetic energy that's converted into rotational kinetic energy, um, and the ground reaction force actually serves to torque the foot around the ankle, um, thus reducing the magnitude of impact transient forces. Limitations of our study was that um our video gate analysis was um two-dimensional rather than three-dimensional, um, a significant limitation in terms of, um, thinking about the SI joint in particular, given the complex bony anatomy of the pelvis. Um, also, the translation of mechanics from the treadmill to the ground, um, in, in terms of running mechanics does remain unclear. Um, and of course, the retrospective study design as opposed to a prospective, um, study sample. So, in conclusion, um, so to our knowledge, this is the first study which has actually identified running gait mechanics that are associated with SI pain. Um, female runners with SI, uh, joint pain, uh, did demonstrate greater ankle dorsiflexion at the point of initial contact compared to healthy controls. Um, and these findings are consistent with prior studies that have observed that a more dorsiflex, uh, foot at initial contact. Um, it creates a higher loading rate, um, and a higher magnitude of ground reaction force on impact, um, which is a likely pathway for injury. Um, so, gait mechanics, um, may have implications on the magnitude and the uh distribution of ground reaction force of the kinetic chain. Um, altogether, um, the, these findings do suggest that there is a role for, um, gait retraining and changes in gait mechanics and injury prevention, um, as a way to, um, prevent the development of this common, um, low back, common cause of low back pain. And this is Des winning the marathon this year. And thank you very much to um my co-authors on this project, Dai Sukamoto, um Doctor Demcourt, um, and thank you very much to uh my many mentors through the Department of Sports Medicine. Questions. Yeah, that's that's really like um. Um, so what are the potential interventions? I mean, are, are the health votes doing something, uh, to maintain their Um, and our protection, uh, in a way that the brothers. Other sort of uh yoga or some other kind of. That's a great question. Um, so, Um, there's a lot that can be done in terms of, um, looking at gait retraining, um, through, for example, at the McKay Center for Injury Prevention, um, and other, um, other centers. Um, individuals are able to have this type of video analysis done, um, and go through, um, step by step analytically and looking at their mechanics, um, and get that type of biofeedback in terms of, um, what What factors are in play, which could be predisposing them to injury. Um, and then individuals uh sort of trained, um, a running coach or an athletic trainer might be able to coach these individuals in terms of certain um strength or flexibility, um, exercises to focus on. To correct some uh maladaptive biomechanics, um, and, um, repeated sessions with repeated um videos and biofeedback um can help them to incorporate um changes in, um, key gate, um, key, key parts of their gait, um, that could potentially correct it over time. Mm. Doctor Kasser, yeah, how much do the running up or down hills change those ground reaction forces, and how much can you offer with uh shoe wear? That is a great question. Um, so, as far as alteration with, with footwear, um, there, the research that I've, um, looked at, um, has actually looked at runners in shoes and runners, um, that are barefoot. Um, and if anything, it's been found that, um, runners that are actually wearing shoes, um, demonstrate Um, even higher ground reaction forces, they're more likely to be more dorsiflex when they're coming into landing, um, versus the, um, an individual who's running barefoot, um, the, um, individual has a way of actually self-correcting and repositioning their foot, um, on, on, on almost like automatically, um, uh, and coming into landing so that they are landing in a more plantar flex position. Um, I think that, um, all things being equal, um, adding some adding some cushion, um, if you come, or you're coming into the same, um, uh, landing, um, angle, I think that adding a little bit of cushion would reduce the ground reaction force that is transmitted through the body, but habitually, um, when coming through the gate cycle, the, um, runner actually, um, retrains themselves almost automatically to be more. Plantar flex when they're coming in on to landing, which is part of the, um, sort of part of the minimalist um shoe movement, um, and some of these things that we hear about, um, in popular media, um, in terms of barefoot running and, and minimalist shoes, um, because the, um, runner actually, um, automatically would, will reposition their foot to a more plantar flex position. Um, as far as changes in, um, the like degree of slope, I actually don't know, um, the response to that. Um. I think that's a good question, and I don't think there's a lot of research out there. Unfortunately, a lot of this research has been done on, um, treadmills that are, um, in a flat position, um, just speaking further to the degree of limitation that we have in terms of the actual practical implications of this research, um, in the everyday setting. OK Thank you Thank you much. OK. Our final speaker this morning is uh Yanfei Wang uh from anesthesiology, and her talk is intravenous treatment of choroidal neovascularization by phototargeted nanoparticles. OK, good morning everybody. Um, so I want to first thank the organizers of this meeting for this invitation and thank you all for being here. Um, I'm excited to share a new drug delivery system that we developed to treat neurovascular retina diseases, and this work is done in collaboration with Doctor Chen's lab in the ophthalmology department. So in the United States, the leading causes of vision loss in the three age groups, children, adults, and the elderly are retinopathy of prematurity, diabetic retinopathy, and vascular or wet AMD respectively. These diseases of different ideology are all characterized by pathogenic neurovascularization, which causes retinal function and, and structure loss and leading to irreversible vision loss. So currently the standard therapies for neurovascular eye diseases are ablation therapy or repeated intravitro injections of anti-vegic drugs. These treatments are invasive and have serious side effects. For example, the standard treatments for ROP are laser or cryotherapy, which destroy the peripheral vision of the patient to save the central vision, and so far there's no drug therapy approved for ROP. And the standard therapy for diabetic retinopathy or wet AMD is repeated intravitro injections, which is unpleasant for the patients and their risks of inflammation, infection, or retina detachment. So it would be highly beneficial to have less invasive means of administration of the drug, for example, by intravenous injection. However, drug delivery to the back of the eye, which is a small area through systemic administration is challenging because compared to local injection, systemic administration often results in a lower proportion of therapeutics deposited in the back of the eye. Which necessitates a substantially higher injection dose to maintain therapeutic levels in the diseased area. However, increasing the injection dose would lead to systemic side effects and toxicity. So the goal of our research is to overcome this challenge of drug delivery by systemic route, which would allow treatment of new vascular retinal diseases by the intra intravenous route. And our strategy is to target intravenously administered nanoparticles as the drug delivery system to the diseased area by light or phototargeting. We developed a system whereby inactivated nanoparticles are injected intravenously and they're only converted to a tissue targeting state upon irradiation in the eye. Our strategy would allow targeted accumulation of drug-containing nanoparticles in the diseased area uh locally in the eye while minimizing drug deposition at off-target sites. So central to our strategy is the design of nanoparticles which is formed from ophilic polymers. Specifically, this flock of polymer consisting of hydrophobic PLA and hydrophilic pack is modified with a targetingli and CPP or cell penetrating peptide, which is a short peptide that facilitates the uptake of its cargo. But, however, the targeting ability of the CPP is initially inactivated by photocaging group DACM. And when it's mixed with the baseline polymer, which is unmodified, uh, they will self-assemble into this hydrophobic core hydrophilic shell nanostructure. And our hypothesis is the hydrophobicity afforded by the Kadian group DEACM would place CPP in the core of the nanoparticles, thereby concealing it from the surface in the beginning. Upon radiation, The DACM or the kin group would be removed by light triggered photo cleavage or bond cleavage, making this polymer chain hydrophilic again, and it will extend to the surface of nanoparticles. So this way CPP is exposed and the nanoparticles can readily bind to the nearby cells. And this is how we built the building block of the nanoparticles. We first attached the caging group to the self-penetrating pipeide by a nucleophilic reaction and then connected this caged CPP to the dye-block of polymer by the melamide biochemistry. And this nanoparticles is formed from self-assembly of polymers have a narrow size distribution and the average diameter of 19 nanometers as shown here by dynamic light scattering and TM image. So, uh, having formed these nanoparticles, we want to test whether they would function as per our design and we confirmed that with flow cytometry, which measures the cells associated fluorescence. And in this study, we fluorescent labeled our nanoparticles by including 10% of EMF conjugated polymer in our formulation. We observed that the cells that are incubated with active CPP nanoparticles. Have tenfold higher fluorescence than the cells are incubated with peptide-free nanoparticles which demonstrated the ability of CPP to target cells, to target nanoparticles to the cells. The cage nanoparticles have minimal cell binding. And irradiation increased the cell uptake level to uh comparable to the active CPP group. So this study confirmed that our caging strategy is able to prevent CPP mediated nanoparticle cell interaction and irradiation is able to reverse that effect. So having confirmed that our photoactivation works in cell culture, we then sought to investigate whether it worked in vivo. And for that study, we used a laser-induced mouse model of choroidal neurovascularization or CMV, which is a disease model for the disease, uh, wet AMD. So in this model, CMV is formed following a laser photocoagulation-induced rupture of group membrane, and we use fluorescent angiography to monitor the development of CMV. One week after the photocoagulation, mice were injected intravenously with fluorescent labeled groups uh in the same four groups as in the previous slide. And 24 hours after the nanoparticle injection, we euthanize the mice and flat mounted the coroids to evaluate the accumulation of nanoparticles in the lesions. We observed that the nanoparticles that are irradiated have a higher fluorescence in the lesions than the other groups, confirming that our photo-targeting strategy can enhance nanoparticle accumulation in the CMV lesions. And then in order to demonstrate that the therapeutic efficacy of our strategy, we included doxorubicin, our nanoparticles as a model drug because it has been shown to inhibit CMV when injected intraocularly. So doxorubicin was effectively encapsulated in the nanoparticles, and the individual release dynamics shows that 90% of doxorubicin was released in the 1st 48 hours. Therefore, we injected. 3 times the nanoparticles to constitute a week of treatment. And after the week of treatment, we evaluated the size of CMV lesions by fluorescent imaging of the flat-mounted choroids, uh, which are stained with isolectin. And here is what we saw. So the group of mice treated with drug containing nanoparticles that are phototargeted had 48 46% reduction in the lesion size compared with the group of mice treated with PBS and the free drug group or the drug continuum group without the radiation had almost half of the inhibitory effect. Moreover, our formulation didn't really cause any tissue toxicity and the irradiation parameters we use didn't cause damage to the mouse eyes. In conclusion, we've developed an intravenously injected phototargeting nanoparticles um as a treatment regimen and demonstrated its efficacy and safety in the standard mouse CMV model. Um, there are a lot of further developments that can be made to improve the therapeutic efficacy of our nanoparticticles, for example, by changing the characteristics of the nanoparticle itself or changing the drugs or, or the irradiation parameters. We also think that this strategy could allow intravenous treatment of other retinal diseases. And with that, I want to thank my collaborator, Zoey and Doctor Cheng in the ophthalmology department and everybody in the Kohani Lab, especially the people who contributed to this work that are listed here. We got a lot of help from some core facilities and of course the funding agencies. And thank you for your attention. Uh, I'm happy to take questions. OK, questions for this stuff. Marsha, that's very nice. Thank you. Yes, so we did see, let me go, I have a slide for that. So we did see accumulation of these nanoparticles in the major organs. However, what we did is uh we calculated the ratio of the nanoparticles in the lesion versus um their distribution in the, in the organs. So the top figure is the absolute accumulation of nanoparticles in all organs and the bottom, the bottom graph shows the ratio. And you can see that with the um last group, the, the yellow bar. It is our phototargeting no party group. It has a higher ratio in the eye versus the organs, so that means our phototargeting can enhance at least the ratio of drug. Any other questions? Yes. Put the self at night. What is this? Yeah, so, um, the, the major part is, uh, the TAT 4748257. Um, I added a few sequences in the beginning, uh, the CGGF, um, the phyanala is so that we can track the nanoparticle with NMR because it has a phenel group and the CGG is so that it can have a spacer between the polymer and the CTP. Yeah, which other um ways. uh. shrimp bread like raw beef. Other, another allergies. it's biopsy. Yeah, so with our current, with our current design, the group we used is sensitive to 400 nanometer light. Um, but we can also use two photons to trigger it, which would require a high intensity laser, uh, 800 nanometer laser. We did test its stability in other conditions. So for example, we let it sit on the benchtop for a while and it did, it didn't hydrolyze for a week. So it is stable and it's triggered only by a specific wavelength of light. OK. Thank you very much. OK, thank you all, and I again, I want to remind everyone we will have a poster session and reception at 4:30 in the galleria, and I hope many of you will attend. Oh. Something's happening. I. That. you I. You so No. I do not. OK. OK. She I. Yeah.
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