Kathryn L. Fowler

Vascular Endothelial Growth Factor (VEGF) Bioavailability Regulates Angiogenesis and Intestinal Stem and Progenitor Cell Proliferation during Postnatal Small Intestinal Development.

Background Vascular endothelial growth factor (VEGF) is a highly conserved, master regulatory molecule required for endothelial cell proliferation, organization, migration and branching morphogenesis. Podocoryne carnea and drosophila, which lack endothelial cells and a vascular system, express VEGF homologs, indicating potential roles beyond angiogenesis and vasculogenesis. The role of VEGF in the development and homeostasis of the postnatal small intestine is unknown. We hypothesized regulating VEGF bioavailability in the postnatal small intestine would exhibit effects beyond the vasculature and influence epithelial cell stem/progenitor populations. Methods VEGF mutant mice were created that overexpressed VEGF in the brush border of epithelium via the villin promotor following doxycycline treatment. To decrease VEGF bioavailability, sFlt-1 mutant mice were generated that overexpressed the soluble VEGF receptor sFlt-1 upon doxycycline administration in the intestinal epithelium. Mice were analyzed after 21 days of doxycycline administration. Results Increased VEGF expression was confirmed by RT-qPCR and ELISA in the intestine of the VEGF mutants compared to littermates. The VEGF mutant duodenum demonstrated increased angiogenesis and vascular leak as compared to littermate controls. The VEGF mutant duodenum revealed taller villi and increased Ki-67-positive cells in the transit-amplifying zone with reduced Lgr5 expression. The duodenum of sFlt-1 mutants revealed shorter villi and longer crypts with reduced proliferation in the transit-amplifying zone, reduced expression of Dll1, Bmp4 and VE-cadherin, and increased expression of Sox9 and EphB2. Conclusions Manipulating VEGF bioavailability leads to profound effects on not only the intestinal vasculature, but epithelial stem and progenitor cells in the intestinal crypt. Elucidation of the crosstalk between VEGF signaling in the vasculature, mesenchyme and epithelial stem/progenitor cell populations may direct future cell therapies for intestinal dysfunction or disease.

Neural Crest Cell Implantation Restores Enteric Nervous System Function and Alters the Gastrointestinal Transcriptome in Human Tissue-Engineered Small Intestine

Summary Acquired or congenital disruption in enteric nervous system (ENS) development or function can lead to significant mechanical dysmotility. ENS restoration through cellular transplantation may provide a cure for enteric neuropathies. We have previously generated human pluripotent stem cell (hPSC)-derived tissue-engineered small intestine (TESI) from human intestinal organoids (HIOs). However, HIO-TESI fails to develop an ENS. The purpose of our study is to restore ENS components derived exclusively from hPSCs in HIO-TESI. hPSC-derived enteric neural crest cell (ENCC) supplementation of HIO-TESI establishes submucosal and myenteric ganglia, repopulates various subclasses of neurons, and restores neuroepithelial connections and neuron-dependent contractility and relaxation in ENCC-HIO-TESI. RNA sequencing identified differentially expressed genes involved in neurogenesis, gliogenesis, gastrointestinal tract development, and differentiated epithelial cell types when ENS elements are restored during in vivo development of HIO-TESI. Our findings validate an effective approach to restoring hPSC-derived ENS components in HIO-TESI and may implicate their potential for the treatment of enteric neuropathies.

Prolonged Absence of Mechanoluminal Stimulation in Human Intestine Alters the Transcriptome and Intestinal Stem Cell Niche

Background & Aims For patients with short-bowel syndrome, intestinal adaptation is required to achieve enteral independence. Although adaptation has been studied extensively in animal models, little is known about this process in human intestine. We hypothesized that analysis of matched specimens with and without luminal flow could identify new potential therapeutic pathways. Methods Fifteen paired human ileum samples were collected from children aged 2–20 months during ileostomy-reversal surgery after short-segment intestinal resection and diversion. The segment exposed to enteral feeding was denoted as fed, and the diverted segment was labeled as unfed. Morphometrics and cell differentiation were compared histologically. RNA Sequencing and Gene Ontology Enrichment Analysis identified over-represented and under-represented pathways. Immunofluorescence staining and Western blot evaluated proteins of interest. Paired data were compared with 1-tailed Wilcoxon rank-sum tests with a P value less than .05 considered significant. Results Unfed ileum contained shorter villi, shallower crypts, and fewer Paneth cells. Genes up-regulated by the absence of mechanoluminal stimulation were involved in digestion, metabolism, and transport. Messenger RNA expression of LGR5 was significantly higher in unfed intestine, accompanied by increased levels of phosphorylated signal transducer and activator of transcription 3 protein, and CCND1 and C-MYC messenger RNA. However, decreased proliferation and fewer LGR5+, OLFM4+, and SOX9+ intestinal stem cells (ISCs) were observed in unfed ileum. Conclusions Even with sufficient systemic caloric intake, human ileum responds to the chronic absence of mechanoluminal stimulation by up-regulating brush-border enzymes, transporters, structural genes, and ISC genes LGR5 and ASCL2. These data suggest that unfed intestine is primed to replenish the ISC population upon re-introduction of enteral feeding. Therefore, the elucidation of pathways involved in these processes may provide therapeutic targets for patients with intestinal failure. RNA sequencing data are available at Gene Expression Omnibus series GSE82147.

Intestinal adaptation in proximal and distal segments: Two epithelial responses diverge after intestinal separation

Background. In short bowel syndrome, luminal factors influence adaptation in which the truncated intestine increases villus lengths and crypt depths to increase nutrient absorption. No study has evaluated the effect of adaptation within the distal intestine after intestinal separation. We evaluated multiple conditions, including Igf1r inhibition, in proximal and distal segments after intestinal resection to evaluate the epithelial effects of the absence of mechanoluminal stimulation. Methods. Short bowel syndrome was created in adult male zebrafish by performing a proximal stoma with ligation of the distal intestine. These zebrafish with short bowel syndrome were compared to sham‐operated zebrafish. Groups were treated with the Igf1r inhibitor NVP‐AEW541, DMSO, a vehicle control, or water for 2 weeks. Proximal and distal intestine were analyzed by hematoxylin and eosin for villus epithelial circumference, inner epithelial perimeter, and circumference. We evaluated BrdU+ cells, including costaining for &bgr;‐catenin, and the microbiome was evaluated for changes. Reverse transcription quantitative polymerase chain reaction was performed for &bgr;‐catenin, CyclinD1, Sox9a, Sox9b, and c‐Myc. Results. Proximal intestine demonstrated significantly increased adaptation compared to sham‐operated proximal intestine, whereas the distal intestine showed no adaptation in the absence of luminal flow. Addition of the Igf1r inhibitor resulted in decreased adaption in the distal intestine but an increase in distal proliferative cells and proximal &bgr;‐catenin expression. While some proximal proliferative cells in short bowel syndrome colocalized &bgr;‐catenin and BrdU, the distal proliferative cells did not co‐stain for &bgr;‐catenin. Sox9a increased in the distal limb after division but not after inhibition with the Igf1r inhibitor. There was no difference in alpha diversity or species richness of the microbiome between all groups. Conclusion. Luminal flow in conjunction with short bowel syndrome significantly increases intestinal adaption within the proximal intestine in which proliferative cells contain &bgr;‐catenin. Addition of an Igf1r inhibitor decreases adaptation in both proximal and distal limbs while increasing distal proliferative cells that do not colocalize &bgr;‐catenin. Igf1r inhibition abrogates the increase in distal Sox9a expression that otherwise occurs in short bowel syndrome. Mechanoluminal flow is an important stimulus for intestinal adaptation.