Small Bowel Resection Increases Paracellular Gut Barrier Permeability via Alterations of Tight Junction Complexes Mediated by Intestinal TLR4.

BACKGROUND: Short bowel syndrome resulting from small bowel resection (SBR) is associated with significant morbidity and mortality. Many adverse sequelae including steatohepatitis and bacterial overgrowth are thought to be related to increased bacterial translocation, suggesting alterations in gut permeability. We hypothesized that after intestinal resection, the intestinal barrier is altered via toll-like receptor 4 (TLR4) signaling at the intestinal level. METHODS: B6 and intestinal-specific TLR4 knockout (iTLR4 KO) mice underwent 50% SBR or sham operation. Transcellular permeability was evaluated by measuring goblet cell associated antigen passages via two-photon microscopy. Fluorimetry and electron microscopy evaluation of tight junctions (TJ) were used to assess paracellular permeability. In parallel experiments, single-cell RNA sequencing measured expression of intestinal integral TJ proteins. Western blot and immunohistochemistry confirmed the results of the single-cell RNA sequencing. RESULTS: There were similar number of goblet cell associated antigen passages after both SBR and sham operation (4.5 versus 5.0, P > 0.05). Fluorescein isothiocyanate-dextran uptake into the serum after massive SBR was significantly increased compared with sham mice (2.13 ± 0.39 ng/µL versus 1.62 ± 0.23 ng/µL, P < 0.001). SBR mice demonstrated obscured TJ complexes on electron microscopy. Single-cell RNA sequencing revealed a decrease in TJ protein occludin (21%) after SBR (P < 0.05), confirmed with immunostaining and western blot analysis. The KO of iTLR4 mitigated the alterations in permeability after SBR. CONCLUSIONS: Permeability after SBR is increased via changes at the paracellular level. However, these alterations were prevented in iTLR4 mice. These findings suggest potential protein targets for restoring the intestinal barrier and obviating the adverse sequelae of short bowel syndrome.

EGFR in enterocytes & endothelium and HIF1α in enterocytes are dispensable for massive small bowel resection induced angiogenesis.

BACKGROUND: Short bowel syndrome (SBS) results from significant loss of small intestinal length. In response to this loss, adaptation occurs, with Epidermal Growth Factor Receptor (EGFR) being a key driver. Besides enhanced enterocyte proliferation, we have revealed that adaptation is associated with angiogenesis. Further, we have found that small bowel resection (SBR) is associated with diminished oxygen delivery and elevated levels of hypoxia-inducible factor 1-alpha (HIF1α). METHODS: We ablated EGFR in the epithelium and endothelium as well as HIF1α in the epithelium, ostensibly the most hypoxic element. Using these mice, we determined the effects of these genetic manipulations on intestinal blood flow after SBR using photoacoustic microscopy (PAM), intestinal adaptation and angiogenic responses. Then, given that endothelial cells require a stromal support cell for efficient vascularization, we ablated EGFR expression in intestinal subepithelial myofibroblasts (ISEMFs) to determine its effects on angiogenesis in a microfluidic model of human small intestine. RESULTS: Despite immediate increased demand in oxygen extraction fraction measured by PAM in all mouse lines, were no differences in enterocyte and endothelial cell EGFR knockouts or enterocyte HIF1α knockouts by POD3. Submucosal capillary density was also unchanged by POD7 in all mouse lines. Additionally, EGFR silencing in ISEMFs did not impact vascular network development in a microfluidic device of human small intestine. CONCLUSIONS: Overall, despite the importance of EGFR in facilitating intestinal adaptation after SBR, it had no impact on angiogenesis in three cell types-enterocytes, endothelial cells, and ISEMFs. Epithelial ablation of HIF1α also had no impact on angiogenesis in the setting of SBS.

Patient-derived small intestinal myofibroblasts direct perfused, physiologically responsive capillary development in a microfluidic Gut-on-a-Chip Model.

The development and physiologic role of small intestine (SI) vasculature is poorly studied. This is partly due to a lack of targetable, organ-specific markers for in vivo studies of two critical tissue components: endothelium and stroma. This challenge is exacerbated by limitations of traditional cell culture techniques, which fail to recapitulate mechanobiologic stimuli known to affect vessel development. Here, we construct and characterize a 3D in vitro microfluidic model that supports the growth of patient-derived intestinal subepithelial myofibroblasts (ISEMFs) and endothelial cells (ECs) into perfused capillary networks. We report how ISEMF and EC-derived vasculature responds to physiologic parameters such as oxygen tension, cell density, growth factors, and pharmacotherapy with an antineoplastic agent (Erlotinib). Finally, we demonstrate effects of ISEMF and EC co-culture on patient-derived human intestinal epithelial cells (HIECs), and incorporate perfused vasculature into a gut-on-a-chip (GOC) model that includes HIECs. Overall, we demonstrate that ISEMFs possess angiogenic properties as evidenced by their ability to reliably, reproducibly, and quantifiably facilitate development of perfused vasculature in a microfluidic system. We furthermore demonstrate the feasibility of including perfused vasculature, including ISEMFs, as critical components of a novel, patient-derived, GOC system with translational relevance as a platform for precision and personalized medicine research.

Adaptation of extracellular matrix to massive small bowel resection in mice.

BACKGROUND: Extracellular matrix (ECM) affects cell behavior, and vice versa. How ECM changes after small bowel resection (SBR) to support adaptive cellular processes has not been described. Here we characterize changes in ECM following SBR and integrate this with concomitant transcriptional perturbations. METHODS: A 50% proximal SBR or sham surgery was performed on mice. On postoperative day 7, ileal tissue was sequentially depleted of protein components to generate an ECM-enriched fraction. ECM was analyzed for protein composition using mass spectrometry with subsequent Ingenuity Pathway Analysis (IPA) to identify predicted pathways and upstream regulators. qPCR and RNA-sequencing (RNA-Seq) were performed to corroborate these predicted pathways. RESULTS: 3034 proteins were differentially regulated between sham and SBR, of which 95 were significant (P < 0.05). IPA analysis predicted PPARα agonism to be an upstream regulator of the observed proteomic changes (P < 0.001). qPCR and RNA-Seq with KEGG analysis confirmed significant engagement of the PPAR pathway (P < 0.05). CONCLUSION: Transcriptional signatures of adapting bowel predict subsequent ECM changes after SBR. How ECM communicates with surrounding cells to drive adaptation and vice versa merits further investigation. Our findings thus far suggest ECM supports tissue hyperplasia and altered metabolic demand following SBR.

Are surgeons behind the scientific eight ball: Delayed acquisition of the NIH K08 mentored career development award.

BACKGROUND: Surgery residents complete their research training early in residency. Non-surgical trainees typically have research incorporated toward the last two years of their fellowship, conferring an advantage to apply for grants with recent research experience and preliminary data. METHODS: The NIH RePORTER database was queried for K08 awardees trained in medicine, pediatrics, and surgery from 2013 to 2017. 406 K08 recipients were identified and time from completion of clinical training to achieving a K08 award was measured. Data were compared using ANOVA and expressed as mean. P < 0.05 was considered significant. RESULTS: Surgeons took longer to obtain a K08 than those trained in internal medicine (surgery = 3.7 years, internal medicine = 2.58 years p < 0.0001)). All K08 recipients without a PhD took longer to obtain a K08 than recipients with a PhD (MD = 3.50 years and MD/PhD = 2.42 years (p=<0.0001). CONCLUSIONS: Surgeons take longer to achieve a K08 award than clinicians trained in internal medicine, possibly due to an inherent disadvantage in training structure.

Single-Cell Analysis Reveals Regional Reprogramming During Adaptation to Massive Small Bowel Resection in Mice.

BACKGROUND & AIMS: The small intestine (SI) displays regionality in nutrient and immunological function. Following SI tissue loss (as occurs in short gut syndrome, or SGS), remaining SI must compensate, or "adapt"; the capacity of SI epithelium to reprogram its regional identity has not been described. Here, we apply single-cell resolution analyses to characterize molecular changes underpinning adaptation to SGS. METHODS: Single-cell RNA sequencing was performed on epithelial cells isolated from distal SI of mice following 50% proximal small bowel resection (SBR) vs sham surgery. Single-cell profiles were clustered based on transcriptional similarity, reconstructing differentiation events from intestinal stem cells (ISCs) through to mature enterocytes. An unsupervised computational approach to score cell identity was used to quantify changes in regional (proximal vs distal) SI identity, validated using immunofluorescence, immunohistochemistry, qPCR, western blotting, and RNA-FISH. RESULTS: Uniform Manifold Approximation and Projection-based clustering and visualization revealed differentiation trajectories from ISCs to mature enterocytes in sham and SBR. Cell identity scoring demonstrated segregation of enterocytes by regional SI identity: SBR enterocytes assumed more mature proximal identities. This was associated with significant upregulation of lipid metabolism and oxidative stress gene expression, which was validated via orthogonal analyses. Observed upstream transcriptional changes suggest retinoid metabolism and proximal transcription factor Creb3l3 drive proximalization of cell identity in response to SBR. CONCLUSIONS: Adaptation to proximal SBR involves regional reprogramming of ileal enterocytes toward a proximal identity. Interventions bolstering the endogenous reprogramming capacity of SI enterocytes-conceivably by engaging the retinoid metabolism pathway-merit further investigation, as they may increase enteral feeding tolerance, and obviate intestinal failure, in SGS.

Lymphatic network remodeling after small bowel resection.

BACKGROUND: Short gut syndrome (SGS) following massive small bowel resection (SBR) is a major cause of pediatric mortality and morbidity secondary to nutritional deficiencies and the sequelae of chronic total parenteral nutrition use, including liver steatosis. Despite the importance of lymphatic vasculature in fat absorption, lymphatic response after SBR has not been studied. We hypothesize that lymphatic vessel integrity is compromised in SGS, potentially contributing to the development of impaired lipid transport leading to liver steatosis and metabolic disease. METHODS: Mice underwent 50% proximal SBR or sham operations. Imaging of lymphatic vasculature in the lamina propria and mesentery was compared between sham and SBR Prox1 ERCre-Rosa26LSLTdTomato mice. mRNA expression levels of lymphangiogenic markers were performed in C57BL/6J mice. RESULTS: Lymphatic vasculature was significantly altered after SBR. Mesenteric lymphatic collecting vessels developed new branching structures and lacked normal valves at branch points, while total mucosal lymphatic capillary area in the distal ileum decreased compared to both sham and intraoperative controls. Intestinal Vegfr3 expression also increased significantly in resected mice. CONCLUSIONS: Intestinal lymphatics, in both the lamina propria and mesentery, dramatically remodel following SBR. This remodeling may affect lymphatic flow and function, potentially contributing to morbidities and nutritional deficiencies associated with SGS.

Assessing gender bias in qualitative evaluations of surgical residents.

BACKGROUND: There are notable disparities in the training, recruitment, promotion, and evaluation of men and women in surgery. The qualitative assessment of surgical residents may be implicitly gender biased. METHODS: We used inductive analysis to identify themes in written evaluations of residents. We also performed a content analysis of words fitting previously defined communal, grindstone, ability, and standout categories. RESULTS: Differences in themes that emerged from evaluations of male and female residents were notable regarding overall performance, references to the future, professional competency, job domains, disposition and humanism, and overall tone of evaluations. Comments about men were more positive than those about women, and evaluations of men included more standout words. CONCLUSIONS: The more positive evaluations of men may handicap women if they are seen as less likely to perform well based on these evaluations. These differences suggest that implicit bias may play a role in the qualitative evaluation of surgical residents.

Tissue underlying the intestinal epithelium elicits proliferation of intestinal stem cells following cytotoxic damage.

The goals of this study were to document the proliferative response of intestinal stem cells (ISCs) during regeneration after damage from doxorubicin (DXR), and to characterize the signals responsible for ISC activation. To this end, jejuni from DXR-treated mice were harvested for histology, assessment of ISC numbers and proliferation by flow cytometry, crypt culture, and RNA analyses. Histology showed that crypt depth and width were increased 4 days after DXR. At this time point, flow cytometry on tissue collected 1 h after EdU administration revealed increased numbers of CD24(lo)UEA(-) ISCs and increased percentage of ISCs cycling. In culture, crypts harvested from DXR-treated mice were equally proliferative as those of control mice. Addition of subepithelial intestinal tissue (SET) collected 4 days after DXR elicited increased budding (1.4 ± 0.3 vs. 5.1 ± 1.0 buds per enteroid). Microarray analysis of SET collected 4 days after DXR revealed 1030 differentially expressed transcripts. Cross-comparison of Gene Ontology terms considered relevant to ISC activation pointed to 10 candidate genes. Of these, the epidermal growth factor (EGF) family member amphiregulin and the BMP antagonist chordin-like 2 were chosen for further study. In crypt culture, amphiregulin alone did not elicit significant budding, but amphiregulin in combination with BMP antagonism showed marked synergism (yielding 6.3 ± 0.5 buds per enteroid). These data suggest a critical role for underlying tissue in regulating ISC behavior after damage, and point to synergism between amphiregulin and chordin-like 2 as factors which may account for activation of ISCs in the regenerative phase.

Side population sorting separates subfractions of cycling and non-cycling intestinal stem cells.

We report here that side population (SP) sorting allows for the simultaneous isolation of two intestinal stem cell (ISC) subsets from wild-type (WT) mice which are phenotypically different and represent cycling and non-cycling pools of cells. Following 5-ethynyl-2'-deoxyuridine (EdU) injection, in the upper side population (USP) the percentage of EdU+ was 36% showing this fraction to be highly proliferative. In the lower side population (LSP), only 0.4% of cells were EdU+, indicating this fraction to be predominantly non-cycling. Using Lgr5-EGFP mice, we show that Lgr5-EGFP(hi) cells, representing actively cycling ISCs, are essentially exclusive to the USP. In contrast, using histone 2B-YFP mice, SP analysis revealed YFP label retaining cells (LRCs) in both the USP and the LSP. Correspondingly, evaluation of the SP fractions for mRNA markers by qRT-PCR showed that the USP was enriched in transcripts associated with both quiescent and active ISCs. In contrast, the LSP expressed mRNA markers of quiescent ISCs while being de-enriched for those of the active ISC. Both the USP and LSP are capable of generating enteroids in culture which include the four intestinal lineages. We conclude that sorting of USP and LSP fractions represents a novel isolation of cycling and non-cycling ISCs from WT mice.