Grading TESI: Crypt and villus formation in tissue-engineered small intestine alters with stem/progenitor cell source.

The small intestine has a remarkable ability to enhance its absorptive and digestive surface area through the formation of villi, a process known as villification. We sought to learn whether developing mouse and human tissue-engineered small intestine (TESI) followed known developmental biology routes to villification, such as Sonic hedgehog (SHH)/Indian hedgehog (IHH) and bone morphogenetic protein 4 (BMP4)/forkhead box F1 (FOXF1) signaling to identify targets to enhance the development of TESI. After generating TESI from prenatal and postnatal stem cell sources, we evaluated the effect of cell source derivation on villification with a grading scheme to approximate developmental stage. χ2 analysis compared the prevalence of TESI grade from each stem cell source. RNAscope probes detected genes known to direct villification and the development of the crypt-villus axis in mouse and human development. These were compared in TESI derived from various pluripotent and progenitor cell donor cell types as well as native human fetal and postnatal tissues. Prenatal and pluripotent cell sources form mature villus and crypt-like structures more frequently than postnatal donor sources, and there are alternate routes to villus formation. Human TESI recapitulates epithelial to mesenchymal crosstalk of several genes identified in development, with fetal and pluripotent donor-derived TESI arriving at villus formation following described developmental patterns. However, postnatal TESI is much less likely to form complete villus-crypt patterns and demonstrates alternate SHH/IHH and BMP4/FOXF1 signaling patterns. Grading TESI and other cellular constructs may assist discoveries to support future human therapies.NEW & NOTEWORTHY The small intestine can enhance its absorptive and digestive surface area through a process known as villification. Tissue-engineered small intestine achieves mature villification at varying levels of success between differing sources. We have developed a consistent grading schema of morphology and characterized it across multiple developmental pathways, allowing objective comparison between differing constructs and sources.

Wnt signaling inhibition by monensin results in a period of Hippo pathway activation during intestinal adaptation in zebrafish.

Intestinal adaptation (IA) is a critical response to increase epithelial surface area after intestinal loss. Short bowel syndrome (SBS) may follow massive intestinal resection in human patients, particularly without adequate IA. We previously validated a model in zebrafish (ZF) that recapitulates key SBS pathophysiological features. Previous RNA sequencing in this model identified upregulation of genes in the Wnt and Hippo pathways. We therefore sought to identify the timeline of increasing cell proliferation and considered the signaling that might underpin the epithelial remodeling of IA in SBS. SBS was created in a ZF model as previously reported and compared with sham fish with and without exposure to monensin, an ionophore known to inhibit canonical Wnt signaling. Rescue of the monensin effects was attempted with a glycogen synthase kinase 3 inhibitor that activates wnt signaling, CHIR-99021. A timeline was constructed to identify peak cellular proliferation, and the Wnt and Hippo pathways were evaluated. Peak stem cell proliferation and morphological changes of adaptation were identified at 7 days. Wnt inhibition diminished IA at 2 wk and resulted in activation of genes of the Wnt/ß-catenin and Yes-associated protein (YAP)/Hippo pathway. Increased cytoplasmic YAP was observed in monensin-treated SBS fish. Genes of the WASP-interacting protein (WIP) pathway were elevated during Wnt blockade. In conclusion, cellular proliferation and morphological changes accompany SBS even in attempted Wnt blockade. Wnt/ß-catenin, YAP/Hippo pathway, and WIP pathway genes increase during early Wnt blockade. Further understanding of the effects of Wnt and YAP pathway signaling in proliferating stem cells might enrich our knowledge of targets to assist IA. NEW & NOTEWORTHY Intestinal adaptation is a critical response to increase epithelial surface area after large intestinal losses. Inhibition of Wnt/ß-catenin signaling impairs intestinal adaptation in a zebrafish model of short bowel syndrome. There is a subsequent upregulation in genes of the Yes-associated protein/Hippo and WIP pathway. These may be targets for future human therapies, as patients are salvaged by the compensation of increased intestinal epithelial surface area through successful intestinal adaptation.

BMP-SMAD-ID promotes reprogramming to pluripotency by inhibiting p16/INK4A-dependent senescence.

Fibrodysplasia ossificans progressiva (FOP) patients carry a missense mutation in ACVR1 [617G > A (R206H)] that leads to hyperactivation of BMP-SMAD signaling. Contrary to a previous study, here we show that FOP fibroblasts showed an increased efficiency of induced pluripotent stem cell (iPSC) generation. This positive effect was attenuated by inhibitors of BMP-SMAD signaling (Dorsomorphin or LDN1931890) or transducing inhibitory SMADs (SMAD6 or SMAD7). In normal fibroblasts, the efficiency of iPSC generation was enhanced by transducing mutant ACVR1 (617G > A) or SMAD1 or adding BMP4 protein at early times during the reprogramming. In contrast, adding BMP4 at later times decreased iPSC generation. ID genes, transcriptional targets of BMP-SMAD signaling, were critical for iPSC generation. The BMP-SMAD-ID signaling axis suppressed p16/INK4A-mediated cell senescence, a major barrier to reprogramming. These results using patient cells carrying the ACVR1 R206H mutation reveal how cellular signaling and gene expression change during the reprogramming processes.

Short-term and long-term human or mouse organoid units generate tissue-engineered small intestine without added signalling molecules.

NEW FINDINGS: What is the central question of this study? Tissue-engineered small intestine was previously generated in vivo by immediate implantation of organoid units derived from both mouse and human donor intestine. Although immediate transplantation of organoid units into patients shows promise as a potential future therapy, some critically ill patients might require delayed transplantation. What is the main finding and its importance? Unlike enteroids, which consist of isolated intestinal crypts, short- and long-term cultured organoid units are composed of epithelial and mesenchymal cells derived from mouse or human intestine. Organoid units do not require added signalling molecules and can generate tissue-engineered intestine in vivo. ABSTRACT: Mouse and human postnatal and fetal organoid units (OUs) maintained in either short-term culture (2 weeks) or long-term culture (from 4 weeks up to 3 months) without adding exogenous growth factors were implanted in immunocompromised mice to form tissue-engineered small intestine (TESI) in vivo. Intestinal epithelial stem and neuronal progenitor cells were maintained in long-term OU cultures from both humans and mice without exogenous growth factors, and these cultures were successfully used to form TESI. This was enhanced with OUs derived from human fetal tissues. Organoid unit culture is different from enteroid culture, which is limited to epithelial cell growth and requires supplementation with R-Spondin, noggin and epidermal growth factor. Organoid units contain multiple cell types, including epithelial, mesenchymal and enteric nervous system cells. Short- and long-term cultured OUs derived from mouse and human intestine develop into TESI in vivo, which contains key components of the small intestine similar to native intestine.

Is less more? Laparoscopic versus open Ladd's procedure in children with malrotation.

BACKGROUND: With the advent of minimally invasive techniques, laparoscopic Ladd's procedure is increasingly used to treat children with malrotation, yet evidence regarding its safety and efficacy is lacking. We hypothesize that operative and postoperative outcomes with the open technique are superior to the laparoscopic Ladd's procedure. METHODS: We conducted a 5-y retrospective chart review of all patients who underwent Ladd's procedure at our institution from 2010-2015. Exclusion of patients included those with concomitant conditions, such as poor gut perfusion, significant reflux, tracheoesophageal fistula, failure to thrive requiring concomitant gastrostomy, and biliary atresia. Kruskal-Wallis and Mann-Whitney tests were used where appropriate. RESULTS: Between 2010 and 2015, of 130 patients who underwent Ladd's procedure, 77 met inclusion criteria. Sixty-two patients underwent initial open surgery, 15 patients underwent laparoscopy, seven of which were converted to open. Patients undergoing open surgery were younger compared to the laparoscopic groups. Thirty-three of the 77 malrotation patients (43%) presented with volvulus, 27 underwent open surgery, four had laparoscopic converted to open procedures, and two patients underwent laparoscopic Ladd's without incident. Laparoscopy resulted in increased operative time and clinic visits. Patients undergoing laparoscopic to open surgery had longer operative times, time to resume diet, and length of hospital stay. No difference was noted in complications among the groups. CONCLUSIONS: Although minimally invasive approaches are becoming increasingly used, no evidence supports laparoscopic superiority over open Ladd's procedure. We found that open surgery was associated with shorter operating times and fewer clinic visits. Furthermore, laparotomy remains the favored procedure for patients presenting with volvulus.

Fibroblast Growth Factors in the Gastrointestinal Tract: Twists and Turns.

Fibroblast growth factors (FGFs) are a family of conserved peptides that play an important role in the development, homeostasis, and repair processes of many organ systems, including the gastrointestinal tract. All four FGF receptors and several FGF ligands are present in the intestine. They play important roles in controlling cell proliferation, differentiation, epithelial cell restitution, and stem cell maintenance. Several FGFs have also been proven to be protective against gastrointestinal diseases such as inflammatory bowel diseases or to aid in regeneration after intestinal loss associated with short bowel syndrome. Herein, we review the multifaceted actions of canonical FGFs in intestinal development, homeostasis, and repair in rodents and humans. Developmental Dynamics 246:344-352, 2017. © 2016 Wiley Periodicals, Inc.

Marked stem/progenitor cell expansion occurs early after murine ileostomy: a new model.

BACKGROUND: Improving treatment for short bowel syndrome requires a better understanding of how intestinal adaptation is affected by factors like mechanoluminal stimulation. We hypothesized that in mice, luminal diversion via an ileostomy would drive adaptive changes similar to those seen in human intestine after diversion while offering the opportunity to study the immediate events after resection that precede intestinal adaptation. MATERIALS AND METHODS: With Institutional Animal Care and Use Committee approval, a distal ileostomy with a long distal Hartman's was created in 9- to 14-week-old C57/B6 mice (n = 8). Control mice only had a midline laparotomy without stoma formation (n = 5). A rim of tissue from the proximal stoma was resected as a historical control for the proximal segment. Postoperatively, mice received a high-protein liquid diet and water ad libitum. On day 3, tissue from both the proximal and distal limbs were collected for histologic and RNA analysis. Morphometric measures, immunofluorescent antigen detection, and RNA expression were compared with Student paired t-tests with a P value < 0.05 considered significant. RESULTS: At 3 d, survival for mice with an ileostomy was 87% and average weight loss was 12.5% of initial weight compared to 6.05% for control mice. Compared to the distal limb, the proximal limb in mice with an ileostomy demonstrated significantly taller villi with deeper and wider crypts. The proximal limb also had decreased expression of intestinal stem cell markers lgr5, bmi1, sox9, and ascl2. Fewer goblet and enteroendocrine cells per hemivillus were also noted in the proximal limb. In control mice, none of these measures were significant between proximal and distal ileum except for villus height. CONCLUSIONS: This new murine ileostomy model allows study of intestinal adaptation without intestinal anastomosis, which can be technically challenging and morbid.

Alternative splicing regulates mouse embryonic stem cell pluripotency and differentiation.

Two major goals of regenerative medicine are to reproducibly transform adult somatic cells into a pluripotent state and to control their differentiation into specific cell fates. Progress toward these goals would be greatly helped by obtaining a complete picture of the RNA isoforms produced by these cells due to alternative splicing (AS) and alternative promoter selection (APS). To investigate the roles of AS and APS, reciprocal exon-exon junctions were interrogated on a genome-wide scale in differentiating mouse embryonic stem (ES) cells with a prototype Affymetrix microarray. Using a recently released open-source software package named AltAnalyze, we identified 144 genes for 170 putative isoform variants, the majority (67%) of which were predicted to alter protein sequence and domain composition. Verified alternative exons were largely associated with pathways of Wnt signaling and cell-cycle control, and most were conserved between mouse and human. To examine the functional impact of AS, we characterized isoforms for two genes. As predicted by AltAnalyze, we found that alternative isoforms of the gene Serca2 were targeted by distinct microRNAs (miRNA-200b, miRNA-214), suggesting a critical role for AS in cardiac development. Analysis of the Wnt transcription factor Tcf3, using selective knockdown of an ES cell-enriched and characterized isoform, revealed several distinct targets for transcriptional repression (Stmn2, Ccnd2, Atf3, Klf4, Nodal, and Jun) as well as distinct differentiation outcomes in ES cells. The findings herein illustrate a critical role for AS in the specification of ES cells with differentiation, and highlight the utility of global functional analyses of AS.

A Purpose in Liquidity: Perfusing 3D Open Scaffolds Improves "Mini-gut" Morphogenesis and Longevity.

Channeling morphogenic signaling gradients intrinsic to intestinal epithelial stem cells, Nikolaev et al. (2020) optimized a three-dimensional microchip perfusion system that augments growth, maturation, and longevity of tubular intestinal enteroids. Reported in Nature, this system may ultimately pave the way to study human intestinal development and pathophysiology, perhaps for therapeutic discovery.

Role of Mucosal Protrusion Angle in Discriminating between True and False Masses of the Small Bowel on Video Capsule Endoscopy.

The diagnosis of small-bowel tumors is challenging due to their low incidence, nonspecific presentation, and limitations of traditional endoscopic techniques. In our study, we examined the utility of the mucosal protrusion angle in differentiating between true submucosal masses and bulges of the small bowel on video capsule endoscopy. We retrospectively reviewed video capsule endoscopies of 34 patients who had suspected small-bowel lesions between 2002 and 2017. Mucosal protrusion angles were defined as the angle between the small-bowel protruding lesion and surrounding mucosa and were measured using a protractor placed on a computer screen. We found that 25 patients were found to have true submucosal masses based on pathology and 9 patients had innocent bulges due to extrinsic compression. True submucosal masses had an average measured protrusion angle of 45.7 degrees ± 20.8 whereas innocent bulges had an average protrusion angle of 108.6 degrees ± 16.3 (p < 0.0001; unpaired t-test). Acute angle of protrusion accurately discriminated between true submucosal masses and extrinsic compression bulges on Fisher's exact test (p = 0.0001). Our findings suggest that mucosal protrusion angle is a simple and useful tool for differentiating between true masses and innocent bulges of the small bowel.

Approach to the Diagnostic Workup and Management of Small Bowel Lesions at a Tertiary Care Center.

BACKGROUND: Small bowel lesions (SBL) are rare, representing diagnostic and management challenges. The purpose of this cross-sectional study was to evaluate diagnostic modalities used and management practices of patients with SBL at an advanced endoscopic referral center. METHODS: We analyzed patients undergoing surgical management for SBL from 2005 to 2015 at a single tertiary care center. Patients were stratified into gastrointestinal bleed/anemia (GIBA) or obstruction/pain (OP). RESULTS: One hundred and twelve patients underwent surgery after presenting with either GIBA (n = 67) or OP (n = 45). The mean age of our study population was 61.8 years and 45% were women. Patients with GIBA were more likely to have chronic or acute-on-chronic symptoms (100% vs 67%) and more often referred from outside hospitals (82 vs. 44%) (p < 0.01). The most common preoperative imaging modalities were video capsule endoscopy (VCE) (96%) for GIBA and computer tomography CT (78%) for OP. Findings on VCE and CT were most frequently concordant with operative findings in GIBA (67%) and OP (54%) patients, respectively. Intraoperatively, visual inspection or palpation of the bowel successfully identified lesions in 71% of patients. When performed in GIBA (n = 26), intraoperative enteroscopy (IE) confirmed or identified lesions in 69% of patients. Almost all (90%) GIBA patients underwent small bowel resections; most were laparoscopic-assisted (93%). Among patients with OP, 58% had a small bowel resection and the majority (81%) were laparoscopic-assisted. Surgical exploration failed to identify lesions in 10% of GIBA patients and 24% of OP patients. Among patients who underwent resections, 20% of GIBA patients had recurrent symptoms compared with 13% of OP patients. CONCLUSION: Management and identification of SBL is governed by presenting symptomatology. Optimal management includes VCE and IE for GIBA and CT scans for OP patients. Comprehensive evaluation may require referral to specialized centers.

Created of Warm Blood and Nerves: Restoring an Enteric Nervous System in Organoids.

The enteric nervous system (ENS) regulates numerous gastrointestinal functions, including epithelial barrier permeability and motility. In a recent Nature Medicine study, Workman et al. (2016) propose a method for introducing human pluripotent stem cell-derived enteric neural crest cells into developing human intestinal organoids, thereby restoring ENS cell types and contractile function.

Carcinoids and Capsules: A Case Series Highlighting the Utility of Capsule Endoscopy in Patients With Small Bowel Carcinoids.

BACKGROUND: Neuroendocine tumors (NETs) or carcinoids arise at many different sites of the gastrointestinal tract. The small intestine is the most common site for NETs. Diagnosing small bowel carcinoids remains challenging given their non-specific presentations and the overall low incidence of small bowel tumors. Video capsule endoscopy (VCE) has significanly improved our ability to detect small bowel malignancies. We explore the value of VCE in the initial workup and management of a series of small bowel carcinoid patients. METHODS: We retrospectively analyzed adult patients undergoing surgical management for small bowel lesions from July 2005 to September 2015 at a tertiary care center. Patient characteristics, presenting symptomatology, diagnostic workup and surgical management were analyzed among patients with histologically confirmed small bowel carcinoid tumors. RESULTS: Our study identified 16 patients treated surgically for small bowel carcinoids. The majority of patients (87.5%) presented with either occult gastrointestinal bleeding or anemia. Most patients (87.5%) were initially evaluated with various endoscopic and imaging modalities before all ultimately undergoing surgery. Seventy-five percent of patients had a VCE, with 83.3% (10/12) having positive findings that correlated with intraoperative findings compared to 62.5% (5/8) with computed tomography scan, 21.4% (3/14) with colonoscopy, 44% (4/9) with deep enteroscopy, and 0% (0/9) with esophagogastroduodenoscopy (EGD). CONCLUSIONS: In the absence of any contraindications, VCE is an effective endoscopic modality in the diagnostic workup of small bowel NETs. Furthermore, positive VCE findings appear to highly correlate with surgical findings, thus suggesting a valuable role for VCE in the initial surgical assessment of patients with small bowel NETs.

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

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 ß-catenin, and the microbiome was evaluated for changes. Reverse transcription quantitative polymerase chain reaction was performed for ß-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 ß-catenin expression. While some proximal proliferative cells in short bowel syndrome colocalized ß-catenin and BrdU, the distal proliferative cells did not co-stain for ß-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 ß-catenin. Addition of an Igf1r inhibitor decreases adaptation in both proximal and distal limbs while increasing distal proliferative cells that do not colocalize ß-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.

Biochemical activity of reactive oxygen species scavengers do not predict retinal ganglion cell survival.

PURPOSE: Retinal ganglion cells (RGCs) die as a result of axonal injury in a variety of optic neuropathies, including glaucoma. Reactive oxygen species (ROS) act as intracellular signaling molecules and initiate apoptosis in nerve growth factor-deprived sympathetic neurons and axotomized RGCs. Determination of the role of specific ROS relies on the use of small molecule or protein scavengers with various degrees of specificity. The pro- or anti-cell-death effect of several ROS generating and scavenging systems in cultured RGCs was correlated with their activity in cell-free assays. METHODS: Neonatal rat retinas were dissociated and incubated with ROS-generating systems for hydroxyl radical, superoxide anion (O2-), and H2O2. Scavengers tested were catalase, polyethylene glycol-superoxide dismutase (PEG-SOD), manganese (III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), deferoxamine, and U-74389G. Viability of retrogradely labeled RGCs was determined with calcein-AM 24 hours after plating. O2- and H2O2 scavenging in cell-free assays was measured with dihydroethidium and Amplex Red (Invitrogen, Carlsbad, CA), respectively. RESULTS: Systematic differences were found between ROS scavenging in cell-free assays and the ability of scavengers to protect RGCs in cell culture. Furthermore, many ROS scavengers lost specificity and protected against various ROS, whereas others failed to protect against their unique ROS target. These activities stray from commonly recognized specificities of individual ROS scavengers or generating systems and are important in understanding ROS biology. In addition, antioxidant defense mechanisms used by RGCs and other retinal cells interfere with responses expected from ROS scavengers in well-defined systems. Last, H2O2 induced intramitochondrial O2-, whereas paraquat produced O2- outside of the mitochondria, and these areas of generation can mislead interpretations of ROS scavenger activity and effectiveness. CONCLUSIONS: There is discordance between ROS effects in cultured RGCs and cell-free assays, with several mechanisms accounting for this divergence. To identify the roles of ROS signaling in cell death accurately, several approaches should be used. These include using a panel of ROS scavengers and generators, testing the panel in primary neuronal cultures, and quantifying ROS with cell-free assays.

Retinal ganglion cell axotomy induces an increase in intracellular superoxide anion.

PURPOSE: Retinal ganglion cells (RGCs) undergo apoptosis after axonal injury. The time course of cell death is variable and depends in part on the degree of injury sustained. Decreasing reactive oxygen species (ROS) levels or shifting the redox state to reduction promotes the survival of RGCs in tissue culture after axotomy. It was hypothesized that a specific ROS, superoxide anion, acts as an intracellular signaling molecule for RGC death after axotomy. METHODS: Intracellular superoxide levels were measured after dissociation in retrograde-labeled rat RGCs with use of the superoxide-sensitive fluorophores hydroethidium and MitoSOX Red. Having found a significant increase, the effect of axotomy was determined on superoxide levels independent of dissociation with an optic nerve crush model. RESULTS: Optic nerve crush caused RGCs to undergo a superoxide burst. The burst was asynchronous and was manifested in only a fraction of cells at any given time. Neurotrophin deprivation was not responsible for the superoxide burst because it was not prevented by incubation with the neurotrophic factors brain-derived neurotrophic factor, ciliary neurotrophic factor, forskolin, or insulin. Several inhibitors of intracellular superoxide generation were studied, but only antimycin A, which inhibits complex III of the mitochondrial electron transport chain, blocked the increase in superoxide. CONCLUSIONS: These findings suggest that superoxide generated in the mitochondrial electron transport chain could be a parallel system to neurotrophic deprivation for signaling cell death after axonal injury.

Kinase-dependent differentiation of a retinal ganglion cell precursor.

PURPOSE: Cell lines are frequently used to elucidate mechanisms of disease pathophysiology. Yet extrapolation of results with cell lines to neurodegenerative disorders is difficult because they are mitotic and usually have other non-neuronal properties. The RGC-5 cell line has many features of retinal ganglion cells (RGCs). Despite its expression of Thy-1 and NMDA receptors, as found in primary RGCs, this line's ability to proliferate and non-neuronal appearance differentiate it from other central neurons, complicating its use for the study of neuronal survival, electrophysiology, or neurite extension. METHODS: A method was identified for differentiating RGC-5 cells using the nonspecific protein kinase inhibitor staurosporine. Cultures were treated with 100 nM to 3.16 muM staurosporine and assessed for a variety of differentiation markers. RESULTS: Differentiated RGC-5 cells expressed numerous neuronal properties, including arrest of proliferation without inducing apoptosis, induction of a neuronal morphology, upregulation of neuronal markers, and establishment of outward rectifying channels. Differentiation was not dependent on a single kinase-dependent pathway, based on profiling multiple kinase phosphorylation targets and attempts to replicate differentiation with multiple specific kinase inhibitors. CONCLUSIONS: This method for producing an RGC-like cell from a proliferating cell line facilitates the following previously impractical techniques: high-throughput screening for agents that are neuroprotective or affect ionic channels; straightforward transduction of gene expression in central neurons by nonviral transfection techniques, including production of stable transfectants; biochemical and other assays of pure RGC-like cells without purification on the basis of cell-surface antigens or anatomic location.

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.