Myocardial infarction, stroke, and pulmonary embolism are all deadly conditions associated with excessive thrombus formation. Standard treatment for these conditions involves systemic delivery of thrombolytic agents to break up clots and restore blood flow; however, this treatment can impact the hemostatic balance in other parts of the vasculature, which can lead to excessive bleeding. To avoid this potential danger, targeted thrombolytic treatments that can successfully target thrombi and release an effective therapeutic load are necessary. Because activated platelets and fibrin make up a large proportion of clots, these two components provide ample opportunities for targeting. This review will highlight potential thrombus targeting mechanisms as well as recent advances in thrombolytic therapies which utilize blood cells and clotting proteins to effectively target and lyse clots.
OBJECTIVES/GOALS: An age-dependent restitution defect in our neonatal pig intestinal ischemia model is rescued by unknown factors within homogenized mucosa of weaned pigs. A postnatally maturing network of enteric glia regulates the epithelial barrier, so we aim to show rescue is due to replacement of glial factors. METHODS/STUDY POPULATION: Jejunal tissues from suckling or weaned pigs were assessed by RNAseq and processed for immunofluorescent histology and 3-D volume imaging. Jejunal ischemia was surgically induced in weaned pigs and injured mucosa was recovered ex vivo with or without the glial inhibitor fluoroacetate (FA) while monitoring transepithelial electrical resistance (TER). RESULTS/ANTICIPATED RESULTS: Ingenuity Pathways Analysis of RNAseq data revealed significant suppression of numerous pathways critical for epithelial wound healing in suckling pigs (Z-score <−2 for of nine key pathways). Volume imaging studies confirmed lower density (P≤0.05) and complexity of the subepithelial glial network in suckling pigs. Treatment with FA inhibited TER recovery (P<0.0001) and restitution (P<0.05) in weaned pigs, mimicking the suckling pig phenotype and supporting glia as an important regulator of restitution in our model. DISCUSSION/SIGNIFICANCE OF IMPACT: These findings provide important evidence that a developing glial network may be critical to the postnatal development of intestinal barrier repair mechanisms. Ongoing work will explore glial-epithelial interactions in vitro to further define postnatal development of barrier repair.
In our pig intestinal ischemia model, a neonatal defect in epithelial barrier restitution in the jejunum can be rescued by ischemic mucosal homogenate from weaned pigs. This is associated with an immature enteric glial cell (EGC) network, a known driver of restitution that matures postnatally partly in response to microbial colonization, which can be modulated with dietary prebiotic fiber. Therefore, we hypothesized that dietary oligosaccharide supplementation accelerates postnatal microbial colonization and EGC network maturation in the jejunum and colon, thus enhancing restitution after ischemic injury. After 24‐hours of colostrum, piglets were fed control or oligosaccharide‐supplemented formula (control‐fed or prebiotic‐fed) for 21 days and fecal swabs were sequenced for 16S rDNA. Intestinal samples were collected for western blot, imaging, and EGC culture. Surgically ischemia‐injured jejunal and colonic mucosal samples from select day 14 pigs were recovered ex vivo while monitoring epithelial barrier function by transepithelial electrical resistance (TEER). Migration abilities, calcium responses to ATP, paracrine effects on IPEC‐J2 cell restitution, and protein secretome were assessed in jejunal and colonic EGC cultures. Colonic microbial taxa changed in a time‐ and diet‐dependent manner with the prebiotic‐fed taxa clustering by day 7 and becoming progressively more tightly clustered over time (P<.050). TEER and histology of uninjured jejunum and colon were unaffected by diet. Following ischemia, low initial TEER recovered to control levels in the control‐fed colon but not prebiotic‐fed colon (diet and injury interaction, P=.038), while diet had no effect on jejunal TEER recovery. Prebiotic‐fed colon had lower levels of the EGC markers glial fibrillary acidic protein (GFAP) and S‐100B at day 21 (P<.050), and subjectively reduced density of GFAP+ and S‐100B+ EGC were noted in preliminary volume imaging of prebiotic‐fed jejunal submucosa at days 14 and 21. EGC from prebiotic‐fed colonic submucosa showed decreased chemotactic motility toward sterile‐filtered colonic contents (P=.010), decreased intracellular calcium response to ATP (P=.0075), and their co‐culture with IPEC‐J2 enhanced epithelial restitution versus monoculture (P=.032). Oppositely, EGC from prebiotic‐fed jejunal submucosa showed increased intracellular calcium response (P=.050) and their co‐culture with IPEC‐J2 did not enhance restitution as efficiently (P=.33) as those from control‐fed jejunal submucosa (P=.019). For future study, prebiotic‐fed colonic submucosal EGC differentially secreted 13 proteins of interest versus control‐fed. Preliminary results indicate dietary oligosaccharides in neonates exert different effects in the jejunum versus the colon on EGC network development and phenotype, and on epithelial restitution in vivo and in vitro. Ongoing work to understand microbiome‐ EGC‐epithelial interactions during postnatal development may lead to novel preventative and clinical practices to improve intestinal health in vulnerable neonates.
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