D. Banerjee1, N. Sodha1, A. Ehsan1, J. Feng1, F. Sellke1 1Brown University School Of Medicine, Department Of Surgery, Providence, RI, USA
Introduction: Cardiac thromboxane A2 induces vasoconstriction of coronary microvascular resistance arterioles and has been implicated in no-reflow phenomenon and hemodynamic deterioration following coronary revascularization. Aging independently alters microvascular reactivity and has been associated with worse postoperative outcomes, but the precise underlying mechanisms are unknown and human studies are lacking.
Methods: Human atrial tissue was examined from patients before and after cardioplegic arrest and cardiopulmonary bypass (CP/CPB). Patients were divided into younger (age <65) and older (age ≥65) cohorts (N = 42). Ex vivo response of coronary microvessels (<200μm in diameter) dissected from myocardial samples to U46619, a stable thromboxane A2 analogue, was assessed using video microscopy. Molecular signaling was assessed using RNA deep-sequencing and immunoblotting.
Results: Viable coronary microvessels isolated from older patients exhibited increased contraction to U46619 when compared to responses in younger counterparts pre-CP/CPB (p = 0.038). This difference did not persist post-CP/CPB (p = 0.5). RNA deep-sequencing of myocardial samples showed significant differences in gene expression of enzymes involved in thromboxane signaling upstream of the thromboxane A2 receptor. Older patients had decreased expression of gene PLA2G2A that encodes phospholipase A2 (p = 0.02) and relatively increased expression of gene PLCG1 that encodes phospholipase C gamma 1 subunit (p = 0.08) pre-CP/CPB. Post-CP/CPB, expression of gene PTGS1 was decreased (p = 0.02), while changes in gene expression of PTGS2 (p = 0.1) and PLCG1 (p = 0.1) approached significance. Older patients had decreased myocardial expression of thromboxane A2 receptor pre-CP/CPB (p = 0.04), while there was a trend toward decreased receptor expression post-CP/CPB (p = 0.1). There were no differences in total myocardial levels of downstream signaling molecules involved in vasoconstriction pathways between the cohorts either pre- or post-CP/CPB, including protein kinase C-α (PKC-α), phosphorylated PKC-α (p-PKC-α), p-PKC:PKC ratio, phospholipase C β3 (PLC β3), phosphorylated PLC β3 (p-PLC β3), p-PLC:PLC ratio, Erk1/2, phosphorylated Erk1/2 (p-Erk1/2), and p-Erk/Erk ratio.
Conclusion: Older patients (≥65) have pronounced coronary microvascular contractile response to U46619, with compensatory decrease in myocardial thromboxane A2 receptor at baseline. Altered gene expression related to thromboxane-specific vasoconstrictive signaling may partially explain this observed phenotype. Older patients undergoing CP/CPB are predisposed to myocardial malperfusion resulting from increased thromboxane-induced vasospasm.
J. Day1, A. Espinoza Orias3, M. Supanich2, M. Kocak2, K. Stenson4, V. Stubbs4, M. Akyuz2, S. Wrenn5 1Rush University Medical Center, Chicago, IL, USA 2Rush University Medical Center, Radiology And Nuclear Medicine, Chicago, IL, USA 3Rush University Medical Center, Orthopedic Surgery And Radiology, Chicago, IL, USA 4Rush University Medical Center, Otolaryngology And Head And Neck Surgery, Chicago, IL, USA 5Rush University Medical Center, Endocrine Surgery, Chicago, IL, USA
Introduction: Ultrasound (US) of the neck is the standard of care for patients with goiter (thyroid enlargement) to measure gland dimensions, determine disease severity and develop a treatment plan. US limitations include operator variability, spatial heterogeneity and irregularity, transducer size being smaller than the goiter/nodule, and substernal projection. These can impair goiter visualization and subsequent volume calculation. The purpose of this proof-of-concept study is to investigate the efficacy of creating 3D-printed thyroids based on computed tomography (CT) imaging as a means of accurate thyroid volume calculation and visualization.
Methods: 10 retrospective cases were used in this study: 5 goiter cases and 5 control non-pathological thyroid cases. CT neck imaging with reconstructed slice thickness of 1mm or thinner was performed from June-July 2024 and 9 of the 10 patients received intravenous contrast. For each case, a neuroradiologist used CT neck imaging to calculate thyroid volume utilizing the volumetric ellipsoid method (length x width x height x a correction factor of 0.524 for each lobe). The resulting DICOM slices were segmented with commercially available software (Mimics 26.0), yielding virtual 3D thyroid models and their corresponding calculated volumes. Finally, volumes of printed models were measured using the Archimedes method (water displacement).
Results: Paired t-tests demonstrated there was not a significant difference between radiologist-predicted and Mimics-predicted goiter volumes, nor between radiologist-predicted and Mimics-predicted control volumes (significance defined as p<0.05). The same tests showed there was not a significant difference between Mimics-predicted goiter volumes and 3D goiter volumes measured by the Archimedes method, nor between Mimics-predicted control thyroid volumes and 3D control thyroid volumes measured by the Archimedes method (significance defined as p<0.05). The mean print time was 6.17 hours per model (6.55 for goiters, 5.8 for normal thyroids). The mean cost of material used was $5.95 per model ($9.44 for goiters, $2.47 for normal thyroids).
Conclusion: While this pilot study cannot demonstrate technique superiority, the results suggest 3D-printed thyroids provide a similarly accurate estimate of volume compared with traditional radiology estimates. The study may be underpowered to detect volume differences between techniques and additional investigation is required. Future directions for this research group include a prospective, randomized trial that compares 3D printed volume to both US measures and those of excised thyroid tissue. We plan to qualitatively explore additional benefits of 3D printing such as intervention planning and patient education.
M. Bhatia3, A.G. Pachimatla1, K. Ratnakaram4, S. Kalvapudi2, S. Patnaik1, B. Fitzgerald1, S. Yendamuri1 1Roswell Park Comprehensive Cancer Center, Department Of Thoracic Surgery, Buffalo, NEW YORK, USA 2University of Tennessee, Graduate School Of Medicine, Knoxville, TENNESSEE, USA 3Purdue University, Department Of Biochemistry, West Lafayette, IN, USA 4University Of Florida, Department Of Biology, Gainesville, FL, USA
Introduction:
Glucagon-like peptide-1 receptor agonists (GLP1ra) are oral hypoglycemic drugs used for type 2 diabetes and obesity treatment. Emerging evidence suggests that GLP1ra may exert anticancer effects that are mediated not only by their role in weight loss and glucose tolerance, but also by systemic metabolic and inflammatory properties that induce anti-proliferative and pro-apoptotic effects. In this study, we explored the impact of GLP1ra on lung cancer by analyzing preclinical and clinical outcome data. Our findings aim to elucidate GLP1ra as a therapeutic strategy in lung cancer treatment.
Methods:
In vitro, we evaluated the proliferation of Lewis Lung Carcinoma (LLC) cells treated with liraglutide and semaglutide at concentrations of 0, 10, 30, and 90 nM. In vivo, 150,000 LLC cells were injected subcutaneously into 24 young male mice to induce tumors. The treatment group received daily intraperitoneal injections of 0.2 µg/g liraglutide, while the control group received saline.
We also performed a retrospective cohort analysis of 1185 overweight patients (BMI >25) with early-stage non-small cell lung cancer treated with resection from 2015 to 2024. Using the Cox proportional hazards model, we compared outcomes between patients who received GLP1ra and those who did not.
Results:
The cell proliferation assay showed a significant increase in the viability of LLC cells treated with multiple liraglutide concentrations (day 3 mean absorption values: 0.31, 0.56, 0.58, 0.63 at concentrations of 0, 10, 30, and 90nM respectively, p < 0.001), while semaglutide did not show this trend. In mice, liraglutide treatment significantly decreased body weight, with treated mice losing 1g (paired t-test, p = 0.006) versus a 0.8g gain in control mice (p = 0.001) over 15 days. There was no significant effect on tumor growth (tumor weight: 0.86g vs 0.84g, p = 0.9). In the clinical cohort (n = 1185), patients taking GLP1ra (n = 97) had significantly higher body mass indexes (BMI) than those who did not (n = 1106) – mean BMI: 34.7 ± 6 vs. 30.8 ± 4.9, p = 0.01. A Cox proportional hazards model using age, sex, race, smoking status, stage, and GLP1ra use as covariates showed a trend of improved progression-free survival (HR = 0.47; 95% CI 0.21–1.07, p = 0.07) in patients using GLP1ra. These patients also showed a significant weight loss of 5kg (paired t-test, p < 0.001).
Conclusion:
Although GLP1ra significantly impact cancer cell growth and hold promise as a therapeutic agent, its underlying mechanisms remain inconclusive. The absence of a suppressive effect in the preclinical studies suggests that alterations in the tumor immune microenvironment or obesity-related inflammatory pathways may mediate the effects of GLP1ra.
L.M. Bode1, C. Tragesser1, J. Duess1, K. Tsuboi1, H. Moore1, H. Jang1, D. Scheese1, J. Young1, M. Sampah1, S. William-McLeod1, T. Prindle1, W. Fulton1, S. Wang1, M. Wang1, C.P. Sodhi1, D.J. Hackam1 1Johns Hopkins University School Of Medicine, Pediatric Surgery, Baltimore, MD, USA
Introduction:
Toll-like receptor 4 (TLR4) plays a critical role in the pathogenesis of necrotizing enterocolitis (NEC), a severe gastrointestinal disease of premature infants. As part of the innate immune system, TLR4 detects microbial components like lipopolysaccharide (LPS) from Gram-negative bacteria. Activated TLR4 leads to mucosal disruption and impaired intestinal perfusion resulting in the intestinal ischemia and necrosis seen in NEC. Ischemia contributes to mitochondrial dysfunction upon which mitochondria can release damage-associated molecular patterns (DAMPs) and excessive production of reactive oxygen species. DAMPs, sharing structural similarities with LPS, activate TLR4 and amplify the inflammatory response vice versa. We hypothesized that a depletion of mitochondrial DNA would affect TLR4 signaling. We conducted an in vitro model of lipopolysaccharide (LPS)-induced injury in rat intestinal epithelial cells (IEC6) to examine a potential complementary enhancement.
Methods:
Rat intestinal epithelial cells (IEC-6) were cultured under physiologic conditions i.e. 37 °C and 5% CO2 v/v in complete media containing 1% L-glutamine, 1% penicillin-streptomycin and 10% fetal bovine serum. Enterocytes were pre-treated for 2-6 weeks with ethidium bromide 100nM to deplete mitochondria. Sufficient mitochondrial depletion was proven both quantitatively by decreased mRNA levels of cytochrome B, ATP synthase and mitochondrial transcription factor A and qualitatively by ATP assay. To examine the sensitivity of TLR4, experimental wells were exposed to the bacterial ligand LPS (50 ug/mL) for 6 hours in healthy and mitochondrial depleted conditions. To measure gene expression changes, cells underwent total RNA isolation followed by cDNA synthesis and qPCR. For immunohistochemical analyses, cells were washed in cold phosphate-buffered saline and fixed in 4% PFA prior to subsequent antibody staining.
Results:
Mitochondria were most significantly diminished with a treatment concentration of 100nM ethidium bromide for at least 2 weeks (mtcytB 6772.6 vs. 466.1; p=0.0001). LPS treatment resulted in significantly increased pro-inflammatory (IL-1, TNF, iNOS) gene expression and cytokine release (IL-6) close to a level seen upon LPS stimulation. Strikingly, mitochondrial depletion doubled this effect.
Conclusion:
The interaction between mitochondrial DAMPs and TLR4 can create a feedback loop, where inflammation leads to further mitochondrial damage, resulting in increased DAMP release and severe inflammation.Therapeutic approaches such as inhibitors of TLR4 or mitochondrial enforcing treatments to protect mitochondrial function might help mitigate the severity of NEC.
S.S. Dhara1, M.A. Varsanik1, K. Cao1, J.A. Pugar1, L. Pocivavsek1 1University of Chicago, Department Of Surgery, Chicago, ILLINOIS, USA
Introduction: Endovascular aortic repair, in the form of EVAR/TEVAR, has become increasingly common. Mechanistically, EVAR/TEVAR depressurizes the aneurysm sac by blood flow exclusion via apposition between a stent and segment of non-diseased aorta, creating the seal zone. The seal zone is conceptualized as two cylindrical bodies (the aorta and the stent) interacting through adhesive forces. In reality, the stent never perfectly opposes the inner aortic wall. Thus, there exists a potential space within the seal zone for fluid, causing endoleak. We created a model system with adequate imaging resolution in order to capture this space and performed computational fluid dynamic (CFD) analysis to understand the mechanics of endoleak, a major failure mode for EVAR/TEVAR.
Methods: A model seal zone was constructed by placing a small stent (30 mm x 10 mm) within an 8 mm quartz tube (Figure 1A), filled with fluid, and imaged with microCT (~20 μ m resolution). Our model incorporates a 25% oversizing of the stent. Images were reconstructed and segmented to highlight the quartz tube, metal stent, and stent fabric (Figure 1B). Geometry was imported into XFLOW, a CFD solver, to run simulations. Initial data were generated using constant velocity (inlet, 0.6 m/s) and pressure (outlet, 13333 Pa) boundary conditions while blood was simulated as a Newtonian fluid.
Results: The endoleak potential space can be clearly seen in light green caused by infolding of the oversized fabric (Figure 1B), demonstrating the imperfect apposition inherent to any seal zone. The velocity profiles in Figure 1C show that the bulk lumen flow is consistent with the inlet boundary condition but complex flow patterns exist in the endoleak region. An added consequence of the non-uniform geometry experienced by fluid in this space is development of high regions of static pressure specific to the endoleak (Figure 1D).
Conclusion: Our model seal zone CFD simulation demonstrates a varied endoleak velocity distribution and regions of elevated pressure isolated to the endoleak. We hope our work can advance understanding regarding the relationship between stent oversizing and positioning on endoleak formation. Future directions include varying the degree of oversizing to examine how this clinically tunable parameter impacts endoleak mechanics.
A.A. San Agustin1, K.S. Fischer2, B.D. Lyttle3, A. Apte1, E.H. Bolouvi1, A.S. Hauger1, S. Skopp1, J.B. Canchis Angulo1, K. Chen2, G.C. Gurtner2, K.W. Liechty1,4 1University of Arizona Health Sciences Center, Banner Children’s at Diamond Children’s Medical Center, Laboratory For Fetal And Regenerative Biology, Department Of Surgery, Tucson, AZ, USA 2University of Arizona Health Sciences Center, Department Of Surgery, Tucson, AZ, USA 3Laboratory for Fetal and Regenerative Biology, University Of Colorado School Of Medicine And Children’s Hospital Colorado, Aurora, CO, USA 4Ceria Therapeutics, Inc., Aurora, CO, USA
Introduction: The wound healing impairment in diabetic patients is multifactorial, with chronic inflammation and an oxidant/antioxidant imbalance leading to increased oxidative stress as central features. We developed a novel therapeutic strategy targeting both the dysregulated inflammatory response and elevated reactive oxygen species in diabetic wounds. We used cerium nanoparticles (CNP) for their antioxidant properties, conjugating with a miR-146a mimetic (CNP-miR146a) to target dysregulated inflammatory signaling. However, the exact mechanisms by which oxidative stress and inflammatory signaling affect wound healing at the cellular level remain unclear. We hypothesize that CNP-miR146a improves healing in diabetic wounds by modulating the various cell types within the wound microenvironment.
Methods: Single-cell RNA sequencing was employed to interrogate gene expression across more than 5,000 individual genes. The study included three murine treatment groups: non-diabetic wounds (D3 -DB, D7 -DB), untreated diabetic wounds (D3 +DB -T, D7 +DB -T), and diabetic wounds treated with CNP-miR146a (D3 +DB +T, D7 +DB +T). Wounds were collected on days 3 and 7 post-wounding. Cell-specific markers were utilized to quantify the distribution of various cell populations in each group.
Results: On day 3, non-diabetic wounds exhibit a dominant presence of pro-inflammatory macrophages and neutrophils (Pro I Mps 60%; Pro I Neu 38%), and minimal populations of monocyte, dendritic cells (DCs), and other macrophage types (<1%). In contrast, untreated diabetic wounds show a reduction in pro-inflammatory macrophages (Pro I Mps 26%) and an increase in pro-inflammatory neutrophils (Pro I Neu 72%). CNP-miR146a treatment led to an increased presence of pro-inflammatory neutrophils (Pro I Neu 80%) and a decrease in pro-inflammatory macrophages (Pro I Mps 19%). By day 7, non-diabetic wounds show a high population of pro-regenerative macrophages (44%) and monocytes (40%). Untreated diabetic wounds exhibited upregulated in DCs (14%) and pro-inflammatory neutrophils (9%), along with pro-regenerative macrophages (23%), neuronal signaling cells (28%), and lower expression of monocytes compared to non-diabetic wounds (26%). CNP-miR146a-treated diabetic wounds maintain elevated levels of pro-regenerative macrophages (42%) similar to non-diabetic wounds, while monocyte distribution was similar to that observed in untreated diabetic wounds (28%).
Conclusion: Overall, our data highlights the adverse impact of diabetes on wound healing and suggests that targeting inflammation and oxidative stress with CNP-miR146a treatment can modulate and restore immune cell populations to enhance wound healing.
T. Jain1, S. Iyer1, P. Sahay1, V. Dudeja1 1University Of Alabama at Birmingham, General Surgery, Birmingham, Alabama, USA
Introduction: Chronic pancreatitis (CP) is a fibro-inflammatory disease of the pancreas which leads to significant morbidity in the form of chronic pain and endocrine and exocrine deficiency. The underlying pathophysiology of CP is an area of active investigation. The Hedgehog (Hh) pathway is a developmental signaling pathway that has been implicated in several fibrotic diseases. Here, we evaluate the effects of targeting Hh signaling in CP.
Methods: CP was induced in C57BL/6J male mice by intraperitoneal (IP) injections of L-Arginine (2 doses 1hr apart weekly for 4 weeks). After induction of CP, daily IP injections of Vismodegib, an FDA approved inhibitor of Smoothened, were used for inhibiting Hh pathway. DMSO was used as vehicle control. Some mice were sacrificed after conclusion of L-arginine injections (pre-treatment group) while the rest were sacrificed after 4 weeks of Vismodegib or Vehicle control treatment. Pancreata were examined by IHC, flow cytometry and qPCR. To determine early changes induced by Hh signaling inhibition, CP was induced as described above, and mice were sacrificed after 3 days of Vismodegib treatment. RNA sequencing was conducted on Day 3 pancreas to evaluate transcriptional changes induced by Hh signaling inhibition.
Results: IHC and qPCR confirmed upregulation of Hh signaling with L-arginine induced CP in the pre-treatment group. Flow cytometry revealed increased M2 macrophage (CD45+F4/80+CD206+) infiltration while PCR demonstrated increased expression of fibrotic markers including Col1A2 and TGFβ as compared to uninjured pancreas. After 4 weeks of treatment, Vismodegib group demonstrated significant decrease in M2 macrophage infiltration on flow cytometry and fibrotic marker expression on qPCR, when compared to vehicle control. Histological analysis revealed improved pancreatic atrophy upon Vismodegib treatment. Early sacrifice study (Day 3) revealed no differences in pancreatic atrophy or macrophage infiltration, however, global transcriptional changes were seen on RNA sequencing analysis with downregulation of ER stress pathway genes as well as metabolic reprogramming with decreased adipogenesis and oxidative phosphorylation related gene expression.
Conclusion: Hedgehog pathway is activated in CP and its inhibition can abrogate fibroinflammatory transformation of the pancreas with improvement in pancreatic atrophy. Transcriptional changes in metabolic signaling and ER stress pathways precede morphological changes upon Hh inhibition. Further studies are required to elucidate the mechanism underlying improvement in CP upon Hh inhibition. Hh inhibition can emerge as a novel therapeutic strategy against CP.
C.J. McCauley1,2, H. McDowell2,3, E. Rowell1, M. Laronda1,2,3,4 1Ann and Robert H. Lurie Children’s Hospital of Chicago, Division Of Pediatric Surgery, Chicago, IL, USA 2Ann and Robert H. Lurie Children’s Hospital of Chicago, Stanley Manne Children’s Research Institute, Chicago, IL, USA 3Northwestern University Feinberg School of Medicine, Department Of Pediatrics, Chicago, IL, USA 4Northwestern University Feinberg School of Medicine, Department Of Obstetrics And Gynecology, Chicago, IL, USA
Introduction:
Patients diagnosed with childhood cancer often undergo chemotherapeutic regimens that result in premature ovarian insufficiency (POI) and infertility. The only fertility preservation option for prepubertal female patients is ovarian tissue cryopreservation (OTC). OTC requires unilateral oophorectomy, specialized tissue processing, and cryopreservation for long-term storage. Ovarian tissue transplantation has been successful in restoring fertility in adults but has limitations in terms of eligible patient populations and duration of hormone restoration and fertility function. An alternative approach utilizing a bioengineered matrix that regulates follicle activation could provide a safe, long-term solution for fertility and hormone restoration. Interstitial cells and the matrisome (extracellular matrix proteins) are essential for ovarian hormone production, immune response, and vascularization in vivo; however, how this microenvironment influences the production of good quality eggs and which components are required within a transplantable system are unknown.
Methods:
Ovaries from reproductive-aged cows were utilized as a mono-ovulatory model of the human ovary. Ovaries were sliced in the sagittal and axial planes, decellularized, and proteins were isolated for analysis. Bottom-up shotgun proteomics was performed and Jess Western and immunohistochemistry were used to validate the presence and relative abundance of key proteins. Heat maps with a spatial resolution of 0.5 mm2 were created for matrisome proteins using overlapping data from sagittal and axial planes.
Results:
Proteomic analysis identified 1471 unique proteins, of which 218 (14.8%) represented matrisome proteins. ECM proteins from all six matrisome divisions were identified. 39 (17.9%) matrisome proteins were significantly differentially expressed by depth in the sagittal plane and 35 (16.1%) were differentially expressed in the axial plane. In the sagittal plane, stromal-expressed matrisome proteins including FRAS1, LAMB3, LAMC3, and SFRP4 were cortex-predominant and CST3, MGP, SERPINF2 were medulla-predominant. Key matrisome proteins including VTN, COL1, and FN1 were identified equally across compartments. Validation using Jess Western was performed for VTN, EMILIN1, COL4, and ZP3 and confirmed compartmental differences seen in the proteomic dataset. Matrisome protein heat maps were created to spatially map the ovary.
Conclusion:
We map the matrisome of ¼ of the bovine ovary to a spatial resolution of 0.5 mm2 and found 54 of 218 identified matrisome proteins to be differentially expressed by depth within the ovary. This novel information can be utilized to inform the inclusion of matrisome proteins within a bioengineered matrix to better recapitulate the ovarian microenvironment. Further, this work will inform ongoing research on the incorporation of stromal cells into a bioprosthetic and transplantable scaffold to improve follicle survival and transplant efficacy.
A.E. Kennedy1, Y. Golivi1, R. Guenter1, J.B. Rose1 1University Of Alabama at Birmingham, Department Of Surgery, Birmingham, Alabama, USA
Introduction:
Pancreatic neuroendocrine tumors (PNETs) are rare cancers originating from neuroendocrine cells in the pancreas. Patients with advanced disease present a complex medical challenge due to their poor prognosis and limited treatment options. Notch signaling is a local cell-cell communication mechanism where transmembrane Notch receptors interact with neighboring cells through ligands binding, resulting in a cascade that regulates various developmental processes and cell fate decisions. Dysregulated Notch1 signaling is implicated in PNETs, affecting neuroendocrine tumor biology and potentially interacting with HIF pathways in disease progression. We hypothesized Notch1 in PNET cells provides a survival advantage in hypoxia.
Methods:
This study utilized BON cells, a commonly used cell line for PNET research. To examine the role of Notch1, the Notch1 gene was deleted in BON cells used CRISPR/Cas9 to generate a BON-Notch1-knockout (N1-KO) cell line. Wildtype (WT) and N1-KO cells were plated in duplicate at a density of 1.5*106 cells per 10cm² plate before being exposed to normoxic and hypoxic conditions (3% oxygen) for 48 hours. At the study endpoint, apoptosis and cell viability were analyzed by flow cytometry using 7-AAD and Annexin V staining. In parallel with this experiment, four 96 well plates with 8,000 cells per well and 8 replicates for each cell type were placed under normoxic and hypoxic conditions (3% oxygen). Two plates were incubated for 48 hours, and the other two for 72 hours, after which an MTT assay was performed to measure cell viability.
Results:
The MTT assay confirmed a reduction in viability in N1-KO cells compared to WT cells. WT cells showed greater survival in hypoxic conditions (48hrs: 37.8% viability, 72hrs: 39.44% viability) compared to N1-KO (48hrs: 30.95%, 72hrs: 21.52%) cells under the same conditions (48 Hours: p<0.001; 72 Hours: P<0.001). This was confirmed through flow cytometry, which showed a trend of reduced cell numbers following hypoxic treatment (plated at 1.5 × 106 cells, with an average loss of 1.2 × 106 cells in hypoxia). Flow cytometry results indicated a greater decrease in viability under hypoxia in N1KO cells compared to BON cells.
Conclusion:
Our data supports the hypothesis that Notch1 increases the ability of PNET cells to survive hypoxic conditions. This may lead to increased malignant potential of Notch1 positive NETs as they increase in size. Furthermore, it may lead to increased resistance to therapy. The mechanism of this hypoxia resistance is currently under investigation. Better understanding tumor cell adaptation to hypoxia and targeting processes of adaptation could lead to new therapeutic strategies.
H. Nepomuceno1, A. Thomas1, P. Emengo1, R. Gonzalez2, G.L. Fell1, F.S. Jazi1, T. Rafeeqi1, J.C. Dunn1 1Stanford University, Department Of Surgery, Palo Alto, CA, USA 2Stanford University, Department Of Pediatrics, Palo Alto, CA, USA
Introduction: Mechanical lengthening has been demonstrated as a method to induce enterogenesis in porcine intestine. Although mechano-transductive cell signaling pathways underlying mechanical enterogenesis have been partially described, paracrine and endocrine mechanisms of signaling have not been previously explored. We hypothesized that biomechanical pathways activated during mechanical enterogenesis may lead to increased concentrations of circulating intestinal growth factor glucagon-like peptide 2.
Methods: Juvenile mini-Yucatan pigs underwent mechanical lengthening via spring insertion into the intestinal lumen. Serum samples were obtained pre- and post-mechanical lengthening and analyzed by enzyme-linked immunosorbent assay for GLP-2 levels. Data were evaluated for change in concentration based on differences in the location of mechanical lengthening, i.e. jejunal or ileal. Statistical analysis was performed with t-test; p<0.05 was considered significant.
Results: Serum samples from 38 mini-Yucatan pigs pre- and post-mechanical lengthening were analyzed for GLP-2 levels. 10 pigs had springs implanted in the ileum and 28 pigs had springs implanted in the jejunum. GLP-2 serum levels increased significantly following ileal lengthening (ileum p=0.043, jejunum p=0.37, all pigs p=0.082, Figure 1). Ileal lengthening led to a greater increase in serum GLP-2 levels than jejunal lengthening (p<0.01).
Conclusion: Glucagon-like peptide 2 is a hormone with trophic actions in the gastrointestinal tract, stimulating crypt cell proliferation and enhancement of nutrient absorption. GLP-2 is secreted by enteroendocrine L cells of the intestinal epithelium, with the highest abundance of L cells in the terminal ileum and colon. The observation of increased serum GLP-2 levels in ileal lengthening versus jejunal lengthening supports an endocrine-mediated effect of mechanical enterogenesis and suggests there may be systemic effects of lengthening that depend on the location of the lengthened intestinal segment.
H. Shao1, F.A. Voza1, Y. Li1, L. Osafo1, O.C. Velazquez1, Z. Liu1 1University Of Miami, Vascular SurgerySurgery/School Of Medicine, Miami, FL, USA
Introduction: Fibroblasts play a pivotal role in wound healing. The Notch1 pathway is a key molecular determinant that controls the plasticity and function of fibroblasts in modulating diabetic wound healing. We had established a mouse model with gain-of-function (GOF) of Notch1 signaling specifically in fibroblasts on diabetic background. Using this animal model, we studied how increased Notch1 pathway activity in skin fibroblasts affects the diabetic wound healing in vivo and in vitro.
Methods: Re-epithelialization of diabetic skin wounds in mice is calculated based on immunostaining of skin wound tissues harvest at post operation day 8 (n=6 wounds/group). Immunostaining of activated Notch1 and downstream target Hey1 was performed to determine Notch1 pathway activity in skin fibroblasts. Immunostaining of myofibroblast marker α-SMA, proliferation marker Ki67 and keratinocyte marker were performed on wound tissues to observe the interaction of fibroblasts and keratinocytes. To study how fibroblasts with Notch1GOF regulate keratinocytes, we isolated mouse skin fibroblasts and conducted fibroblast-keratinocyte co-culture experiments and 3D skin reconstruct model in vitro.
Results: Re-epithelialization of diabetic skin wounds in Notch1GOF mice (B6-db+/+; RosaN1IC+/+;Col1a2-CreER+/- with Tamoxifen treatment) is delayed by 67% (n=6 wounds/group, Figure 1) compared to the control (B6-db+/+/ RosaN1IC+/+/Col1a2-CreER+/- without Tamoxifen treatment). In addition, trans-differentiation of fibroblasts into myofibroblasts and proliferation of keratinocytes are suppressed in skin of Notch1GOF mice. Co-culture of fibroblasts and keratinocytes experiments, and 3D skin reconstruct model show that fibroblasts isolated from Notch1GOF mice skin inhibit keratinocyte proliferation and formation of epithelium in vitro.
Conclusion: Notch1 pathway activation in skin fibroblasts of mice skin delays diabetic wound healing by inhibiting keratinocyte re-epithelialization. Our data unveils intracellular Notch1 signaling in skin fibroblasts as potential target for therapeutic intervention in diabetic wound healing.
N. Dobrilovic1 1Northshore University Health System, Surgery, Evanston, IL, USA
Introduction:
Proof-of-concept of a novel ‘two-ring’ mitral (tricuspid) annular sizing device and technique was introduced at AATS Mitral Conclave 2023. This technique has the potential to dramatically improve ring selection during valve repair procedures. Figures 1a/1b provide a visual reference to orient the reader with the general concept. This technology has recently been approved for first-in-human trial at our institution. In preparation for in-human use, the current study aims to validate the manufacturing precision of each of 9 custom sizers designed to correspond specifically to Physio 2 (Edwards Lifesciences, Irvine, CA) annuloplasty rings.
Methods:
For each available Physio 2 ring (sizes #24-40), caliper (Mitutoyo 505-732, Kanagawa, Japan) measurements were obtained to evaluate correspondence between implantable commercial ring and our manufactured sizer. A total of 1,080 measurements was conducted to a level of 1/100th of a mm. Antero-posterior (AP) and width (horizontal) dimensions were measured, comparing each ring sizer with its corresponding Physio 2 ring across all sizes. This was performed 30 times for each unique measurement to validate design accuracy and ability of each sizer to perform (precisely control placement of sutures in the desired position). Bland-Altman analysis with plots was performed to evaluate correspondence of paired sets of measurements. Mean difference with a 95% confidence interval (CI), as well as 95% limits of agreement, are reported.
Results:
Mean difference [95% CI] between sizer and ring measurements indicated that measurements differed by negligible amounts across all 9 pairs. All 9 sets of statistical analyses yielded similar outcomes. Given limited abstract space, a sample, complete statistical analysis is provided for a single (#36) sizer (Figure 1c/1d) where mean differences were 20/100ths mm in the AP dimension (-0.20 [-0.37, -0.04]) and 13/100ths in the width dimension (-0.13 [-0.29, 0.02]). Limits of agreement were from -1.08 to 0.68 for AP and -0.95 to 0.69 for width. Differences do not show heteroscedasticity.
Conclusion:
Novel two-ring sizers designed for use with Physio 2 annuloplasty rings performed as expected. Validation measurements were extremely close and do not suggest any pattern of error. It is anticipated that this novel sizing technique will provide surgeons with precise, intraoperative anatomic (ink test coaptation length) and physiologic ring sizing information as we now enter human trials.
P. Emengo1, C. Chen1, H. Nepomuceno1, C. Celebrezze1, J.C. Dunn1 1Stanford University, Pediatric Surgery Department, Palo Alto, CA, USA
Introduction:
There are multiple types of esophageal atresia, but those classified as “long gap” have two segments of esophagus that are positioned too far from each other for 1-step surgical repair. Previous literature has shown recreation of long gap esophageal atresia in animal models to be challenging. Our goal is to recreate the model in rats to test esophageal atresia treatment strategies.
Methods:
We employed two esophageal atresia models. The first involved transection of the esophagus and anastomosis of the proximal portion of the esophagus to the stomach, thereby creating a blind loop of the distal esophagus to enable future intervention. Our second method involved transection of the esophagus and the creation of two blind ends (Figure 1).
Results:
Although upfront more technically challenging, an esophagogastric anastomosis restored esophageal continuity without the need for sustained researcher enteral feeding. Ligation of the esophagus with a gastrostomy feeding tube insertion allowed for the formation of two atretic segments of esophagus. However, the required monitored daily feedings for several hours was more labor intensive than the former procedure. Both models were able to reach post operative day 6.
Conclusion:
We developed two new rat models for the study of esophageal atresia. While both strategies have varying pros and cons, both enable intra-abdominal access to the esophagus on a more reproducible scale than previously described in the literature. This creates the potential to trial future interventions to treat esophageal atresia.
G.H. Gershner1,2, K.B. Snyder1,2, C.M. Dalton1,2, H.C. Grubbs1,2, D.N. Reuter3, J.F. Papin4, S. Gurung5, K.R. Jonscher6, D.A. Myers5, C.J. Hunter1,2 1University Of Oklahoma College Of Medicine, Pediatric Surgery, Oklahoma City, OK, USA 2University Of Oklahoma College Of Medicine, Surgery, Oklahoma City, OK, USA 3University Of Oklahoma College Of Medicine, Comparative Medicine, Oklahoma City, OK, USA 4University Of Oklahoma College Of Medicine, Pathology, Oklahoma City, OK, USA 5University Of Oklahoma College Of Medicine, Obstetrics And Gynecology, Oklahoma City, OK, USA 6University Of Oklahoma College Of Medicine, Biochemistry And Physiology, Oklahoma City, OK, USA
Introduction: Obesity and consumption of a “Western” diet (WD) have been linked to pathologies including cancers and metabolic diseases. This is partly due to a chronic inflammatory response associated with excess adiposity and weight gain, insulin spikes, and excess cholesterol exposure. The effects of this hyperinflammatory state are currently understudied in fetuses. Using a novel nonhuman primate model of maternal obesity, we hypothesize that WD exposure in baboon dams will lead to a hyperinflammatory state, resulting in decreased intestinal barrier function in fetuses.
Methods: Intestinal tissue was harvested from twenty Papio anubis baboon fetuses. RNA was extracted from the tissue and qPCR used to measure relative gene expression. Enteroids were also generated, plated onto a TransWellTM plate and Transepithelial electrical resistance (TEER) measurements were taken. The maximum TEER measured in each well represented maximal barrier function. TEER measurements and gene expression were compared between maternal control diet (CD) and WD groups.
Results: We found elevations in mRNA expression levels of IL-1β, IL-8, IL-10, IFNγ, TNFα, and TLR4. While there was a positive trend, most were not significantly elevated (IL-1β p=0.4496, IL-8 p=0.4290, IL-10 p=0.0878, TNFα p=0.4201, TLR4 p=0.1449). IL-6 and IFNγ were significantly elevated (p<0.0001 and p=0.0021respectively). There was a significant reduction in the maximum TEER achieved in the WD (353.45 ± 122.93) compared to the CD (575.1 ± 108.19; p=0.0117).
Conclusion: Exposure to maternal WD resulted in significantly increased expression of IL-6 and IFNγ, as well as other markers of intestinal inflammation. The inflammatory state was associated with a statistically significant decrease in WD barrier function when compared to CD counterparts. The combination of a hyperinflammatory state and decreased barrier function may increase neonatal risk for conditions like necrotizing enterocolitis.
C.S. Justo-Jaume1, C.I. Moneme1, O.S. Jung1, M.F. Sobstel1, P. Obidike1, Y. Zhang1, L. Cheng1 1University Of Virginia, Surgery, Charlottesville, VA, USA
Introduction: The Piezo family of mechanosensitive ion channels has recently been discovered to play an important role in the function of several visceral organs, including the gastrointestinal (GI) system. We have previously found that Piezo1 activation/inhibition affects the growth of enteric neurons and that Piezo1 expression in the GI tract is diminished in Hirschsprung disease. However, the role of Piezo1 in GI motility has not yet been established. We hypothesize that Piezo1 loss-of-function in intestinal smooth muscle will lead to GI dysmotility.
Methods: An inducible smooth muscle-specific Piezo1 knockout mouse (Piezo1 fl/fl MYH11 CreERT) was generated and Cre+/Cre- adult mice were treated with tamoxifen to induce the conditional deletion of Piezo1 in smooth muscle cells. To confirm the absence of Piezo1 expression in the intestinal smooth muscle in Cre+ mice, the small intestine and colon were isolated, cryosectioned, and immunohistochemistry was performed for Piezo1 and alpha-smooth muscle actin. Total GI transit time, number of pellets per hour, stool weight, stool water content, and colon length were then compared between Cre+ and Cre- littermates (n=3 each group).
Results: There was no significant difference in GI motility between smooth-muscle specific Piezo1 knockout mice and Cre- controls as measured by several functional assays including total GI transit time (4.5 ± 1.3 vs. 4.5 ± 2.2 hours), stool weight (25.4 ± 2.3 vs. 36.8 ± 14.1 grams/pellet), stool water weight (29.4 ± 9.0% vs. 47.1 ± 29.1%), pellets per hour (1.7 ± 1.2 vs. 1.3 ± 1.2), and colon length (8.0 ± 1.0 vs. 7.2 ± 1.0 cm) respectively.
Conclusion: This is the first study to examine the role of Piezo1 in intestinal smooth muscle. No difference in GI motility was seen in our small sample of smooth-muscle-specific Piezo1 knockout mice. Further study is needed to understand the relationship between Piezo1 and GI motility.
D. Jaishankar1, M. Taylor1, Y. Lee1, K. Madavaraju1, A.P. Strouse1, D.B. Srivastava1, C. Atkinson1, S.N. Nadig1 1Northwestern University, Surgery, Chicago, ILLINOIS, USA
Introduction: The phases of heart transplantation (HTx), from procurement to preservation and implantation into recipients, inevitably result in ischemia-reperfusion injury (IRI). The first target of IRI is the endothelial cell (EC) barrier lining the blood vessels of the donor allograft. The EC barrier is damaged, allowing extravasation of immune cells into the allograft, triggering early alloimmune injury and exacerbating IRI. Therefore, protecting the EC barrier can mitigate IRI and acute alloimmune rejection. Gap junctions are membrane proteins that facilitate cell-to-cell communication. In the heart, connexin 43 (Cx43) is the predominant gap junction protein. The role of Cx43 in cardiac donor allografts during IRI in HTx is poorly understood.
Methods: We hypothesize that EC Cx43 gap junctions are reduced during IRI disrupting the EC barrier and exacerbating IRI-associated sequelae. We further hypothesize that preventing the reduction of EC Cx43 gap junction in donor allografts confers allograft protection after implantation. Firstly, to determine the protein expression of Cx43 gap junctions and evaluate the EC barrier permeability, murine microvascular cardiac EC (MCEC) were subjected to an in vitro model of IRI wherein cells undergo cold storage (CS) in a hypoxic chamber in University of Wisconsin (UW) organ preservation solution at 4oC followed by reperfusion in warm culture medium at 37oC (CS-WR). MCEC under normothermic (NT) conditions (at 37oC) served as a control. Secondly, to improve Cx43 gap junction expression and EC barrier in MCEC, we used the therapeutic peptide alpha-connexin carboxyl-terminus 1 (aCT1), which improves the stability and enhances Cx43 gap junction expression. Lastly, to demonstrate translational relevance in improving the EC barrier and mitigating IRI, donor cardiac allografts were preconditioned with aCT1 during CS and implanted into recipients using an in vivo murine heterotopic HTx model.
Results: We observed that MCEC subjected to the CS condition had significantly reduced protein expression of Cx43 at the gap junction compared to the NT condition, and the expression continued to remain reduced during CS-WR. We also observed significantly increased barrier permeability during CS-WR compared to NT, suggesting a compromised EC barrier. Since Cx43 expression was reduced during CS, we observed significantly enhanced expression of Cx43 gap junctions and improved MCEC health and barrier when aCT1 therapy was administered during CS. Similar results were recapitulated in vivo, where we observed an improved EC barrier, reduced EC death, and mitigated inflammation in donor hearts preconditioned with aCT1 during CS before HTx.
Conclusion: Collectively, our results demonstrate that Cx43 gap junction expression is reduced in EC of donor cardiac allografts, and preconditioning donor cardiac allografts with aCT1 therapy to bolster Cx43 gap junction expression can mitigate IRI after implantation and improve allograft outcomes.
J.T. Davies1, R. Cheddadi1, V. Yeramilli1, C.A. Martin1 1Washington University, Department Of Surgery, Division Of Pediatric Surgery, St. Louis, MO, USA
Introduction:
Maternal stress during pregnancy has been associated with several neonatal outcomes including those that impact intestinal development in the newborn—especially in premature and very low birth weight infants. Elucidating the effects of parental stress and downstream signaling on gut maturation is a critical area of research with significant implications in disease prevention and treatment, namely with respect to necrotizing enterocolitis (NEC) a devastating intestinal disease with very high morbidity and mortality with very limited treatment options. The nematode Caenorhabditis Elegans (C. elegans) is a well-established model for studying stress signaling pathways. In this study, we investigated the impact of heat stress on intestinal integrity and permeability in C. elegans.
Methods:
Eggs from adult wild-type (N2) C. elegans worms were isolated and allowed to hatch into L1 larvae. L1 larvae were then transferred to NGM plates seeded with E. coli OP50 and incubated for 24 hours at an optimal growth temperature of 20°C to reach the L2 larvae stage. Now synchronized worms were then separated randomly into 2 treatment groups (28°C and 30°C) and 1 control group (20°C) with each group containing 2 plates each. Each group was then incubated for 48 hours at their respective temperatures. The worms from each plate were then washed and transferred to and incubated on fresh NGM plates seeded with a mixture of fluorescein isothiocyanate (FITC)-dextran (an intestinal permeability marker) and E. coli OP50 for 18 hours. Worms were washed from their plates, ensuring only intracorporal FITC remain, and single worms were transferred and fixated. The intestinal permeability was then examined using fluorescence high-throughput image analysis.
Results:
The mean fluorescence intensities of worms from the control (20°C) and 28°C-treatment groups were 59.281 ± 10.010 and 88.638 ± 14.678, respectively; there was a significant difference in fluorescence intensities (p < 0.05) between these two groups. The 30°C-treatment group was unable to be analyzed due to the inadequate number and size of worms.
Conclusion:
This study demonstrates the effects of heat stress on intestinal permeability in a wild-type C. elegans model. As evidenced by a significant increased fluorescence intensity in the 28°C-treatment group as compared to control, our results suggest that on some level, exposure to heat stress during development may compromise gut integrity and permeability. Future studies will look at this key finding across generations and will seek to understand the mechanism driving this finding.
E.R. Becker1, G.C. Wetmore1, A.D. Price1, R.R. Schuster1, J.R. Merola2, R.C. Quillin1, M.D. Goodman1 1University Of Cincinnati, Department Of Surgery, Cincinnati, OH, USA 2Cincinnati Children’s Hospital Medical Center, Department Of Surgery, Cincinnati, OH, USA
Introduction: Inflammation causing endothelial injury and coagulopathy has been described in various disease populations, but is not well understood within the field of solid organ transplantation. This study aimed to evaluate circulating biomarkers of endothelial injury acutely following renal transplantation.
Methods: Serum samples were collected from 10/2023 to 04/2024 from 20 renal transplant patients preoperatively, immediately postoperatively, and at 24 and 72 hours postoperatively. Serum was analyzed for 12 markers of endotheliopathy including angiopoietin 1, E-selectin, P-selectin, syndecan-1, thrombomodulin, and vascular endothelial growth factors. A retrospective chart review was completed to collect demographics, etiology of end stage renal disease (ESRD), perioperative creatinine and urine output.
Results: Patients were 50% male (n=10), with a median age of 50.5 years [40, 57.75], and 70% White (n=14). The most common cause of ESRD was diabetes and/or hypertension (n=12, 60%) followed by IgA nephropathy (n=3, 15%). 45% of patients were on peritoneal dialysis (n=9), 40% were on hemodialysis (n=8), and 15% were preemptive transplants (n=3), with a median of 2 years [0.625, 4] on dialysis. 80% underwent deceased donor (n=16) and 20% underwent living donor kidney transplant (n=4). Postoperatively, creatinine decreased (7.7mg/dL preoperatively to 4.6mg/dL 72 hr postoperatively, p=0.02) and urine output increased (0.45mL/kg/hr preoperatively to 1.56mL/kg/hr 72 hr postoperatively, p<0.001). 20% of transplants were complicated by delayed graft function (n=4) without an effect on biomarkers. As for markers of endotheliopathy, P-selectin decreased from preoperative (497,984±485,425 pg/mL) to 24 hr (345,870±240,763 pg/mL, p=0.003) and 72 hr (266,036±227,255 pg/mL, p=0.006) postoperative levels. MMP-1 decreased from preoperative to 24 hr postoperative levels (201±151 vs. 135±92 pg/mL, p=0.03). VEGF-A decreased between all four time points consecutively (5,472±6,795, 3,966±5,527, 3,245±5,003, and 2,841±4,952 pg/mL, p<0.001). VEGF-R2 decreased between preoperative and all post operative levels (14,512±5,386, 13,099±5,107, 11,686±4,527, and 11,936±3,606 pg/mL, p<0.006). Finally, VLA-4 demonstrated a stepwise decrease over the four timepoints (470±283, 381±164, 380±242, 291±153 pg/mL, p<0.04) (Figure).
Conclusion: Serum biomarkers of endothelial injury, including adhesion proteins and mediators of angiogenesis, decrease acutely following renal transplantation. The etiology of these beneficial changes, whether by organ implantation alone or by associated immunosuppression, merit further investigation.
D. Kulkarni1, P. Massie1, B. Coffman3, C. Pace1, N. Kanagy2, R. Clark1,2 1University Of New Mexico HSC, Surgery, Albuquerque, NM, USA 2University Of New Mexico HSC, Cell Biology And Physiology, Albuquerque, NM, USA 3Vetern’s Affairs Hospital, Pathology, Albuquerque, NM, USA
Introduction:
Peripheral arterial disease (PAD) affects millions of Americans and is responsible for significant detriments to mobility, with major limb loss possible at advanced stages. Ischemic myopathy associated with PAD is a recently recognized effect of chronic tissue malperfusion. Advanced age, characterized by defects in the sirtuin (SIRT1) histone deacetylase pathway, has been linked to impaired angiogenesis after ischemic insult. Hydrogen sulfide (H2S) has also recently been described as a key mediator of ischemic tissue recovery and neovascularization. Here we describe the development of reproducible ischemic myopathy in young mice as a comparison for mechanistic studies of tissue recovery in advanced age.
Methods:
Hindlimb ischemia was created in young (9-12 week) mice (C57Bl/6, n=9) under anesthesia. A 1 cm groin incision was created to ligate the femoral/external iliac artery. Animals were serially followed over a 21-day period by laser speckle contrast imaging (LSCI) and planimetric photography. After humane sacrifice, ischemic and contralateral non-ischemic gastrocnemius muscles were collected for histologic and molecular analysis. CD31 immunohistochemistry was performed to evaluate angiogenesis. RT-PCR investigated changes in H2S synthetic enzyme expression as well as age-related markers.
Results:
Mean pre-operative limb perfusion was 86.1 perfusion units (PU). After arterial ligation, flux was reduced to mean 40.1 PU, a 46% decrease from pre-operative values and mean 35% contralateral control limb perfusion. During the post-operative period, reperfusion was observed to increase predictably by post-operative day 21, achieving mean 91% baseline compared to the contralateral control. Histologic studies of myofiber diameter show a mean of 11.2 µm for the ischemic hindlimb and a mean diameter of 19.7µm for control muscle (P<0.01). CD31 immunohistochemistry reveals evidence of neovascularization
Conclusion:
Hindlimb ischemia secondary to arterial occlusion in young mice leads to reproducible perfusion deficits with subsequent tissue recovery. Histological changes consistent with ischemic myopathy and impairment in myofiber diameter mirrors the degree of ischemic injury. These findings have provided a comparison group whereby we can evaluate the effects of arterial occlusion on aging mice as well as the role of H2S and its interplay with SIRT1.
T.Y. Patel1, A. Melukote1, M. Samples2,3, R. Kayed2,3, M. Montalbano2,3 1University Of Texas Medical Branch, John Sealy School Of Medicine, Galveston, TX, USA 2University Of Texas Medical Branch, Mitchell Center For Neurodegenerative Diseases, Galveston, TX, USA 3University Of Texas Medical Branch, Department Of Neurology, Neuroscience, And Cell Biology, Galveston, TX, USA
Introduction:
Alzheimer’s Disease (AD) is a neurodegenerative disorder hallmarked by the pathological accumulation of tau protein aggregates, which are implicated in cognitive decline and neuronal death. Emerging evidence underscores a critical interplay between tau pathology and histone modifications, particularly H3K4me3 (tri-methylation at lysine 4 of histone H3) and H3K9me3 (tri-methylation at lysine 9 of histone H3), which are integral to chromatin remodeling and gene expression regulation. The aberrant regulation of these histone marks is believed to contribute to the epigenetic landscape of AD, potentially driving the progression of neurodegeneration.
Methods:
Primary cortical neurons (PCNs) isolated from wild-type murine models were exposed to recombinant Tau oligomers (rTauO) at a concentration of 0.5 µM for 24 hours. Post-treatment, neurons were fixed in paraformaldehyde and subjected to immunofluorescence staining for the detection of H3K4me3 and H3K9me3. Confocal microscopy was employed to capture high-resolution images, which were subsequently analyzed using ImageJ and GraphPad software to quantify changes in histone modification levels and to assess chromatin structural alterations.
Results:
rTauO treatment induced a significant reduction in H3K4me3 levels, as evidenced by a pronounced decrease in mean fluorescence intensity, indicating a potential downregulation of gene transcription associated with neuronal function. Conversely, a significant increase in H3K9me3 levels was observed, characterized by an elevated mean integrated density, suggesting enhanced heterochromatin formation and transcriptional repression. Notably, three-dimensional analysis revealed that H3K9me3-enriched regions occupied more than 50% of the nucleolar volume in rTauO-treated neurons, further highlighting the extensive chromatin remodeling induced by tau pathology.
Conclusion:
This study provides compelling evidence that tau pathology in Alzheimer’s Disease is closely associated with epigenetic modifications, specifically the depletion of H3K4me3 and the accumulation of H3K9me3. These alterations likely contribute to the neurodegenerative process by facilitating the repression of neuronal genes and promoting heterochromatin formation, thereby exacerbating the disease’s pathological features. Elucidating the role of histone modifications in AD pathogenesis offers promising insights for the development of targeted epigenetic therapies aimed at mitigating tau-induced neurotoxicity and disease progression.
W. Colley1, W. Chen1, Y. Golivi1, R. Guenter1, J. Rose1 1University Of Alabama at Birmingham, Department Of Surgery, Division Of Surgical Oncology, Birmingham, Alabama, USA
Introduction:
Pancreatic neuroendocrine tumors (pNETs) are malignancies arising from pancreatic islets. Surgery can be curative for localized pNETs, but patients with advanced disease have limited treatment options. Notch signaling is a biologic pathway currently investigated as a therapeutic target in cancer. Notch has 4 transmembrane receptors (Notch1-4) that participate in cell-cell communication and regulate growth. The gamma-secretase complex activates Notch via receptor cleavage to form the intracellular domain, which affects transcription of downstream targets such as Hes1. Gamma-secretase inhibitors (GSIs) (e.g. nirogacestat and crenigacestat) can block Notch signaling. To test the impact of GSIs in pNETs, we used in vitro 3D spheroid models instead of 2D models due to increased cell-cell contact. We hypothesized that pNET spheroids treated with GSIs will have reduced Notch signaling and lower proliferation rates.
Methods:
3D spheroids were cultured to study the effects of Notch on pNET cell proliferation and viability. Two pNET cell lines (BON, QGP) were seeded at 1,000 cells per well on agarose-coated plates to form spheroids. Proliferation was monitored over time using pixel quantification of spheroid size. Spheroids exposed to GSIs or control began treatment on day 6. Spheroids were treated with 2 doses (20μM or 40μM) of nirogacestat, crenigacestat, or DMSO controls. After 10 days, viability was measured by CellTiter-Glo 3D ATP-luminescence assays. Protein lysates were analyzed via western blot to validate expression of Notch and Hes1.
Results:
Pixel quantification showed BON and QGP spheroids without drug treatment increased in size over the 10-day period. BON and QGP spheroids treated with the highest dose of nirogacestat (40μM) showed the most significant decrease in viability versus DMSO and the lower dose (20μM). A decrease in spheroid size in cells treated with nirogacestat was observed in BON spheroids. However, BON spheroids treated with crenigacestat showed an increase in proliferation and viability at both doses. This was not observed in QGP spheroids. Western blot analysis did not show increased Notch1 or Notch3 expression in 3D compared to 2D. QGP 3D samples treated with nirogacestat did show a reduction in Hes1 compared to DMSO and crenigacestat samples.
Conclusions:
The reduction of pNET spheroid viability by nirogacestat was consistent in both cell lines, but reduction of size was cell line dependent. Crenigacestat, which selectively inhibits Notch1 in cholangiocarcinoma, unexpectedly increased viability and growth of BON spheroids. Overall, GSIs are influencing cell growth and viability, but the role of Notch signaling in this process will need to be elucidated through further experimentation.
J.A. McKean1, G. Tushoski1, G. Thompson1, K. Herremans1, I. Nassour1, S. Han1, S. Hughes1 1University Of Florida, Department Of Surgery, Gainesville, FL, USA
Introduction: Postoperative pancreatic fistula is more prevalent after distal pancreatectomy than pancreaticoduodenectomy (PD), yet postoperative hemorrhage from vascular pseudoaneurysm is virtually exclusive to PD. We hypothesize that enterokinase, produced by the reconstructive intestinal limb, drives trypsin activation necessary for pseudoaneurysm formation, and determining the distance to minimal enterokinase production in the jejunum could mitigate this devastating complication.
Methods: Fresh PD samples were procured in the operating room from multiple locations of the small intestine: proximal duodenum, ligament of Treitz (LOT), ligament of Treitz +5cm (LOT+5cm), ligament of Treitz +10cm (LOT +10cm), ligament of Treitz +15cm (LOT +15cm) and ligament of Treitz +20cm (LOT+20cm). Separately, terminal ileum was collected as a control. Enterokinase mRNA and protein abundance at these anatomic points was quantified using qRT-PCR and immunohistochemistry. Comparisons were performed using Wilcoxon Rank Sum Test.
Results: By qRT-PCR, we observed a significant difference in the expression of enterokinase mRNA when comparing the LOT+20cm to the proximal duodenum, and LOT (p=0.035, p=0.008 respectively). The terminal ileum had significant differences in enterokinase mRNA expression between all locations. No significant differences were observed between LOT+10cm and LOT+20cm (p=0.309). However, we also observed a marked difference in the abundance of enterokinase immunostaining at LOT+15cm when compared to the proximal duodenum, LOT, and LOT+5cm with staining intensity decreasing distally.
Conclusion: There is differential expression of enterokinase mRNA in the duodenum compared to the proximal jejunum with heterogeneity between patients and discordance with mRNA quantities with decreasing protein moving distal to the LOT. Our data would suggest resection of at least 20 cm of jejunum distal to the LOT to ensure minimal enterokinase activity at the pancreatojejunostomy.
A. Sidabraite1, U. Ahmed2, S.K. Jones2, A.K. Gulla3 1Vilnius University, Faculty Of Medicine, Vilnius, VILNIUS, Lithuania 2Vilnius University, VU LSC-EMBL Partnership For Genome Editing Technologies, Life Sciences Center, Vilnius, VILNIUS, Lithuania 3Vilnius University, Faculty Of Medicine, Institute Of Clinical Medicine, Vilnius, VILNIUS, Lithuania
Introduction: Cholangiocarcinoma (CCA) includes heterogeneous tumours with complex diagnoses, management, and poor prognoses. Diagnosing distal CCA (dCCA) is particularly challenging in clinical cases where it presents a diagnostic dilemma due to its clinical and imaging similarities to pancreatic ductal adenocarcinoma (PDAC) and benign diseases. Current diagnostic methods are inadequate, necessitating the development of more sensitive, specific, and non-invasive biomarker detection techniques. MicroRNAs show promise as biomarkers, and innovative approaches like CRISPR/Cas9-mediated detection may enhance diagnostic capabilities.
Methods: We propose using Rolling Circle Amplification (RCA)-assisted CRISPR/Cas9 cleavage (RACE) to detect miRNA-16 and miRNA-877 panel in exosomes derived from plasma samples. Exosomes will be isolated from patients with CCA, PDAC, benign diseases, and healthy controls. The miRNAs will be analyzed using RACE and results will be compared to those obtained via reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The RACE method involves two key steps: replication, where a long double-stranded DNA (dsDNA) assembly is generated through the interaction between the padlock probe with the PAM domain and the target miRNA; and detection, where the sgRNA/Cas9 complex cleaves the dsDNA, leading to a measurable fluorescence signal.
Results: We anticipate that RACE will exhibit superior sensitivity and specificity compared to RT-qPCR, or at least show high consistency, while offering advantages in processing time and cost-effectiveness.
Conclusion: RACE technology has the potential to significantly improve miRNA detection, offering clinicians a robust tool for differentiating dCCA from other diseases with similar clinical and radiological presentations. This method could reduce the reliance on invasive biopsies, ultimately enhancing patient outcomes through earlier and more accurate diagnosis.
K. Nakatsutsumi1, N. Kemmerer2, K. Pool1, D. Agarwal2, S. Sayeed2, D. Park1, K. Wahlin2, B. Eliceiri1, T.W. Costantini1 1University Of California – San Diego, Trauma, Surgical Critical Care, Burns, And Acute Care Surgery/Surgery, San Diego, CA, USA 2University Of California – San Diego, San Diego, CA, USA
Introduction: Acute lung injury (ALI) remains a leading cause of morbidity among patients who survive the initial insult after severe trauma. Clarifying the molecular mechanism of the development of ALI after severe trauma is crucial to identify potential therapeutic targets. We hypothesized that trauma activates cellular proliferation genes in alveolar macrophages, which is associated with the development of ALI.
Methods: A polytrauma model was conducted in C57BL/6J mice by causing unilateral lung contusion using a cortical impactor followed by laparotomy with liver crush injury. ALI was characterized by measuring histology, lung permeability, and wet/dry analysis (n=3/group). Bronchoalveolar lavage fluid (BAL) was harvested 24 hours post-injury for single-cell RNA sequencing (scRNAseq) using matched numbers of viable cells from mice subjected to polytrauma (n=2) compared with sham controls (n=2).
Results: Polytrauma increased ALI compared to sham with evidence of histologic lung injury, increased lung vascular permeability and lung edema. scRNAseq of cells harvested from BAL identified a population of cells consisting primarily of macrophages based on the expression of macrophage markers CD68 and LYZ2 and the macrophage scavenger receptor, MARCO. Polytrauma increased a specific cluster of cells expressing these macrophage markers (see arrow in Figure). These clusters demonstrated increased expression of several genes related to the activation of cell cycle like cyclin-dependent kinase (CDK1), cyclin A2 (CCNA2), cell cycle associated protein (Cdca3), and the mitosis spindle regulating Kinesin-like proteins genes (KIF23 and KIFC1). Polytrauma was also associated with increased macrophage expression of the apoptosis inhibitor survivin (Birc5). Together, these increases in macrophage cell cycle support a model in which polytrauma induces a macrophage proliferation response.
Conclusion: Polytrauma promotes macrophage proliferation in the alveolar space by increasing the expression of genes that regulate the cell cycle and prevent apoptosis. Targeted therapies to control lung macrophage proliferation may be a strategy to prevent trauma-induced ALI.
D. Petrov1, C. Lin2, M. Mazer2,3, M. Moorman5, K.E. Remy2,6, J.T. Ross2,5 1Northeast Ohio Medical University, School Of Medicine, Rootstown, OH, USA 2Case Western Reserve University School Of Medicine, The Blood, Heart, Lung, And Immunology Research Center, Cleveland, OH, USA 3University Hospital Cleveland, Division Of Pediatric Critical Care Medicine, Cleveland, OH, USA 4University of California Davis, Department Of Surgery, Sacramento, CA, USA 5University Hospitals Cleveland, Department Of Surgery, Cleveland, OH, USA 6University Hospitals Cleveland, Division Of Pulmonary Critical Care Medicine, Cleveland, OH, USA
Introduction: Cell-free hemoglobin (CFH) and free heme mediate endotheliopathy and organ injury in sepsis, but their role in trauma is unclear. Traumatic injury produces early endogenous hemolysis, which temporarily overwhelms scavenging mechanisms. Elevated CFH and heme, detectable at ED arrival after trauma, are associated with longer hospital stays, septic complications and AKI. We hypothesize that CFH and heme mediate septic complications in trauma through a pro-inflammatory effect on circulating leukocytes.
Methods: Venous blood was drawn from six healthy volunteers. Fresh whole blood was incubated at 37°C with phosphate-buffered saline (PBS, negative control), lipopolysaccharide (LPS 100 ng/ml, positive control), 40 μM cell-free hemoglobin (CFH), 20 μM heme, or a combination of CFH + LPS or Heme + LPS. TNFα production was quantified by enzyme-linked immunosorbent spot (ELIspot) assay after 4- and 18-hour incubations. ELIspot allows visualization of TNFα production from individual cells, which each generate distinct spots. Exposures were conducted in triplicate and results are expressed as mean spots per 1,000 cells. This study was approved by our institutional IRB.
Results: Six healthy volunteers were enrolled (median age 28, 83% male; to represent a trauma cohort). We found that both 4 and 18 hour exposures to CFH stimulated a greater than 5-fold increase in TNFα release compared to PBS alone (p = 0.002), Figure 1. Notably, CFH exposure produced a nearly 2-fold greater increase in TNFα production compared to LPS (p=0.002), but the combination of CFH and LPS did not significantly alter TNFα production compared to CFH alone. In contrast, Heme exposure did not stimulate significant TNFα release alone and the combination of Heme and LPS did not significantly alter TNFα production compared to LPS alone.
Conclusions: Our study demonstrates that clinically relevant concentrations of cell-free hemoglobin stimulate a robust pro-inflammatory response in whole blood. While ex-vivo stimulation of whole blood ignores the important role of the vascular endothelium, our findings suggest that circulating leukocytes play a role in mediating the inflammatory response to hemolysis. This is an intriguing avenue for research, as multiple existing therapies block components of the hemolysis pathway. We believe that it is possible to leverage those treatments to moderate the burden of acute and chronic inflammation resulting from traumatic injury and associated intravascular hemolysis.
