ANGPTL2 expression in the intestinal stem cell niche controls epithelial regeneration and homeostasis

Abstract The intestinal epithelium continually self‐renews and can rapidly regenerate after damage. Dysregulation of intestinal epithelial homeostasis leads to severe inflammatory bowel disease. Additionally, aberrant signaling by the secreted protein angiopoietin‐like protein 2 (ANGPTL2) causes chronic inflammation in a variety of diseases. However, little is known about the physiologic role of ANGPTL2 in normal tissue homeostasis and during wound repair following injury. Here, we assessed ANGPTL2 function in intestinal physiology and disease in vivo. Although intestinal development proceeded normally in Angptl2‐deficient mice, expression levels of the intestinal stem cell (ISC) marker gene Lgr5 decreased, which was associated with decreased transcriptional activity of β‐catenin in Angptl2‐deficient mice. Epithelial regeneration after injury was significantly impaired in Angptl2‐deficient relative to wild‐type mice. ANGPTL2 was expressed and functioned within the mesenchymal compartment cells known as intestinal subepithelial myofibroblasts (ISEMFs). ANGPTL2 derived from ISEMFs maintained the intestinal stem cell niche by modulating levels of competing signaling between bone morphogenetic protein (BMP) and β‐catenin. These results support the importance of ANGPTL2 in the stem cell niche in regulating stemness and epithelial wound healing in the intestine.


REFEREE REPORTS
Referee #1: Within the manuscript 'ANGPTL2 expression in intestinal stem cell niche controls epithelial regeneration and homeostasis' the authors aim to study the role of ANGPTL2 in response to intestinal damage. To study this response, they have implemented two different well-established in vivo damage models; DSS-induced damage and radiation damage. They compare the regeneration response in wildtype versus ANGPTL2-/-mice. Both models show that regeneration is impaired in the ANGPTL2 null mice, whereas in the absence of damage no significant differences could be observed apart from reduced expression of ISC markers/Wnt target genes, indicating a function for ANGPTL2 during regeneration rather than in normal tissue homeostasis. Subsequently they localized the expression of ANGPTL2 to the intestinal mesenchyme, especially to the intestinal subepithelial myofibroblasts (ISEMFs). The authors uncovered the pathways that are activated within ISEMFs in the presence and absence of ANGPTL2, however unfortunately not the direct relation between ISEMFs and the depletion of ISCs within the niche. The manuscript is clearly written and the data well presented. The experiments appear to be well conducted. Several aspects that might improve the manuscript: • Is ANGPTL2 upregulated in response to damage (DSS and radiation) in WT mice.?
• The organoid cultures they used in figure 7 do not look very viable. Do these represent a regenerative system or homeostasis? This needs to be discussed. • In addition to the above, it would be relevant to see how these organoids would behave in response to e.g. irradiation. Furthermore, the authors should attempt to modificatie the media composition and coculture organoids of various genotype with fibroblasts to investigate if the in vivo findings can be recapitulated in vitro to allow for future complete disentanglement of the signals involved. • The discussion on the role of BMP signaling needs to be expanded and the most recent literature incorporated.
Referee #2: Horiguchi and colleagues study the function of angiopoietin-like protein 2 in the intestine of mice. The authors show that loss of ANGPTLII results in no significant phenotypes under homeostatic conditions, but that there is a decline in stem cell function and proliferation when ANGPTLII mutants are exposed to DSS or gamma irradiation. The authors show that ANGPTLII is expressed in intestinal subepithelial myofibroblasts and that these cells, and not the hematopoietic system, contribute to the niche of intestinal stem cells. These cells over-express BMP2 and BMP7 in ANGPTLII loss of function conditions and the authors propose that this induction of BMPs and the activation of BMP signaling in the epithelium limits ISC activity during regeneration. The authors further show that inhibition of integrin signaling can induce BMP2 and BMP7 in ISEMFs. Since previous data suggest that ANGPTLII binds to integrins, and since a direct effect on Wnt signaling is ruled out here, the authors propose a model in which Integrin/NFkB signaling in ISMEFs regulates BMP expression to influence the ISC niche and promote ISC proliferation in intestinal cypts.
These findings are of broad interest as they describe a new signaling interaction by which the intestinal stem cell niche and ISC quiescence are regulated through an interaction between ISMEFs and the epithelium under stress conditions.
The data presented are of high quality, the experiments are well-designed, and the conclusions are appropriate. Publication can be recommended as is. We thank you for your comments, which were very helpful. We have extensively revised the manuscript and addressed your comments. In the revision, we added data related to co-culture of organoids with ISEMFs of respective genotypes and show a direct relationship between ISEMFs and organoid formation in vitro. We feel that these results strengthen our conclusions.

1-Is ANGPTL2 upregulated in response to damage (DSS and radiation) in WT mice.?;
Yes. We found that ANGPTL2 expression in WT mice was upregulated in response to DSS-or irradiation-induced damage (Fig EV2A and EV3A). We report these findings on page 10, lines 2-4 and page 11, lines 8-10 of the revised manuscript. figure 7 do not look very viable. Do these represent a regenerative system or homeostasis? This needs to be discussed.;

2-The organoid cultures they used in
As you note, organoids shown in Figure 7A (right panel) and Figure 7C in the original paper appear damaged. We conclude that damage is attributable to either Wnt deficiency or in some cases due to passage through a syringe. By contrast, a representative normal organoids shown in Figure 7A  . Thus we feel that the organoid culture used in this paper was a valid system in which to test our hypotheses. Thank you for these comments. Based on your suggestion, we have added data testing organoid response to irradiation (Fig EV6E and EV6F). Although the number of viable organoids decreased after irradiation, we observed no difference in crypt formation capacity with or without rANGPTL2 treatment (Figs EV6C-6F) effects similar to those seen after mechanical damage of organoids caused by passage through a syringe. These observations suggest that ANGPTL2 derived from ISEMFs does not directly alter the IEC regenerative response. These findings are reported on page 17, lines 6-12 of the revised manuscript.
To examine ISEMF function in organoid culture, we co-cultured wild-type or Angptl2 -/mouse crypts with ISEMFs derived from mice of either genotype using two paradigms: either direct contact (Fig 7C) or separated by a transwell membrane (Fig EV7A). We observed no difference in either growth or size of organoids in the presence of wild-type or Angptl2 -/-ISEMFs in either paradigm under optimized culture conditions, which included exogenous Noggin (Nog (+)) (Figs 7D, 7E, EV7B and EV7C). However, we hypothesized that ISEMF-derived BMP might be antagonized by exogenous and ISEMF-derived Noggin. Thus, we conducted the same experiments without exogenous Noggin. We found that the size of organoids of either genotype co-cultured with Angptl2 -/-ISEMFs decreased compared with wild-type ISEMFs in either paradigm under culture conditions lacking exogenous Noggin (Nog (-)) (Figs 7D, 7E Figs EV7B and EV7C). These findings suggest that abundant BMP secreted from Angptl2 -/-ISEMFs might inhibit organoid growth. These findings are reported on page 18, line 3-page 19, line 3 of the revised manuscript.
These results suggest that impaired regeneration seen in Angptl2 -/mouse crypts is likely due to upregulated expression of ISEMF-derived BMP (Fig 7F). These findings are reported on page 19, lines 3-7 of the revised manuscript. Referee #2:

These findings are of broad interest as they describe a new signaling interaction by which the intestinal stem cell niche and ISC quiescence are regulated through an interaction between ISMEFs and the epithelium under stress conditions.
The data presented are of high quality, the experiments are well-designed, and the conclusions are appropriate. Publication can be recommended as is.
Thank you very much for your evaluation of our manuscript. Thank you for submitting a revised version of your manuscript. The manuscript has now been seen by Referee #1, who finds that all his/her concerns and recommendations have been addressed. Therefore I am happy to accept the manuscript in principle. However, before I can officially send you the acceptance letter, there are a few editorial issues concerning the text and figures that I need you to address in a final revision.
Thank you again for giving us the chance to consider your manuscript for The EMBO Journal. I look forward to your final revision. Do the data meet the assumptions of the tests (e.g., normal distribution)? Describe any methods used to assess it.
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