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

Figure 1. The Angptl2 −/− mouse colon shows decreased β‐catenin signaling A, BRepresentative images of H&E (A)‐ and PAS (B)‐stained colon sections from wild‐type and Angptl2 −/− mice. Scale bar = 100 μm.C, DRepresentative images of colon crypts as assessed by Ki67 (C) and pH3 (D) staining. Scale bar = 100 μm.EmRNA levels of indicated proliferation and ISC markers in isolated crypts from wild‐type (n = 4) and Angptl2 −/− (n = 4) mice based on qRT–PCR analysis. Data are represented as means ± SEM. *P < 0.05, unpaired Student's t‐test.FWestern blotting analysis of isolated colon crypts from wild‐type and Angptl2 −/− mice. HSC70 served as an internal control. Numbers below panels represent normalized expression of proteins.GIHC for expression of active β‐catenin in colon or wild‐type and Angptl2 −/− mice. Scale bar = 50 μm. Source data are available online for this figure.

Figure EV1. ANGPTL2 is not required for normal intestinal development and does not appreciably regulate baseline epithelial cell turnover ALength of crypts in wild‐type (n = 12) and Angptl2 −/− (n = 14) mice. Average per mouse is shown as respective dots. ns, difference not statistically significant; unpaired Student's t‐test.B, CRepresentative images of BrdU IHC of colon (B) and small intestinal (C) epithelial cells at 2, 24, and 120 h after BrdU injection. Scale bar = 100 μm.

Figure EV2. ANGPTL2 functions in intestinal regeneration A Angptl2 mRNA levels in wild‐type mouse colon were determined following 6 days of DSS treatment (DSS) and then 6 more days with normal water. Controls were treated with normal water for all 12 days (Untreated). n = 4.BWeight loss in wild‐type (n = 10) and Angptl2 −/− (n = 9) mice after initiation of 2.5% DSS treatment over the 12‐day experiment.COverall disease activity of wild‐type (n = 10) and Angptl2 −/− (n = 9) mice administered 2.5% DSS in drinking water for 6 days and then untreated water for 6 more days.D–FRepresentative images of colon crypts assessed by H&E (D), Ki67 (E), and pH3 (F) staining in wild‐type and Angptl2 −/− mice following DSS treatment (assessed at day 6). Scale bar = 100 μm.G Angptl2 mRNA levels in wild‐type mice colon 5 days after 12‐Gy irradiation (n = 5) compared to untreated controls (n = 4).H, IRepresentative images of crypts of small intestine assessed by H&E (H) and Ki67 (I) staining in wild‐type and Angptl2 −/− mice 3 days after 12‐Gy irradiation. Scale bar = 100 μm.J, KRepresentative images of crypts of colon assessed by H&E (J) and Ki67 (K) staining in wild‐type and Angptl2 −/− mice 3 days after 12‐Gy…

Figure 2. ANGPTL2 functions in intestinal regeneration following DSS treatment ASurvival rates of wild‐type (n = 33) and Angptl2 −/− (n = 33) mice supplied with drinking water containing 2.5% DSS for 6 days and then untreated water for 6 more days, from two different experiments. ***P < 0.001, by log‐rank test.BRepresentative images of colons from wild‐type and Angptl2 −/− mice following DSS treatment for 6 days and then untreated water for 6 more days (at day 12).CColon length of wild‐type (n = 7) and Angptl2 −/− (n = 6) mice following DSS treatment for 6 days and then untreated water for 6 more days (at day 12).D–FRepresentative images of colon crypts as assessed by H&E (D), Ki67 (E), and pH3 (F) staining in wild‐type and Angptl2 −/− mice following DSS treatment for 6 days and then untreated water for 6 more days (at day 12). Scale bar = 100 μm.G, HThe number of regenerating crypts as quantified using Ki67 (G) and pH3 (H) staining. Each data point represents the number of Ki67‐positive viable crypts or the number of pH3‐positive cells in a single field of view. Multiple areas in at least six mice per genotype were quantified. Numbers below panels represent the average.IIHC for expression…

Figure 3. ANGPTL2 functions in intestinal regeneration after irradiation AWeight loss of wild‐type (n = 4) and Angptl2 −/− (n = 5) mice in days following 12‐Gy irradiation.BSmall intestine and colon length from wild‐type (n = 4) and Angptl2 −/− (n = 5) mice 5 days after 12‐Gy irradiation.C, DRepresentative images of crypts of small intestine as assessed by H&E (C) and Ki67 (D) staining in wild‐type and Angptl2 −/− mice 5 days after 12‐Gy irradiation. Scale bar = 100 μm.EThe number of regenerating crypts in small intestine as quantified by Ki67 (D) staining at days 3 and 5. Each data point represents the number of Ki67‐positive viable crypts in a single field of view. Multiple areas in at least three mice per genotype were quantified. Numbers below panels represent average.F, GRepresentative images of colon crypts as assessed by H&E (F) or Ki67 (G) staining in wild‐type and Angptl2 −/− mice 5 days after 12‐Gy irradiation. Scale bar = 100 μm.HThe number of regenerating colon crypts as quantified by using Ki67 (G) staining at days 3 and 5. Each data point represents the number of Ki67‐positive viable crypts in a single field of view. Multiple areas in at least three mice per genotype were…

Figure 4. ANGPTL2 localizes to the intestinal mesenchyme Angptl2 mRNA levels in small intestine (proximal, mid, and distal), cecum, and colon (proximal and distal) of wild‐type mice based on qRT–PCR analysis. n = 5.ANGPTL2 IHC in colon from wild‐type and Angptl2 −/− mice. The Angptl2 −/− colon image serves as a negative control. Scale bar = 50 μm.mRNA levels of indicated genes in wild‐type (n = 4) mice based on qRT–PCR analysis.Representative IF images of distal colon from wild‐type mice as assessed with anti‐ANGPTL2 (red) and anti‐α‐SMA (green). Nuclei are counterstained with DAPI (blue). Scale bar = 100 μm.Data information: Data are represented as mean ± SEM.

Figure EV3. ANGPTL2 localizes to the intestinal mesenchyme IHC for ANGPTL2 in three samples of normal human colonic mucosa obtained from colectomy samples from subjects (P1, P2, and P3) with colorectal cancer. Scale bar = 100 μm.Representative IF images of distal colon tissues from wild‐type mice analyzed with anti‐ANGPTL2 (red) and anti‐E‐CADHERIN (green) or CD31 (green). Nuclei are counterstained with DAPI (blue). Scale bar = 100 μm.Representative IF images of distal colon tissue from wild‐type mice following DSS treatment (assessed at day 6) evaluated with anti‐ANGPTL2 (red) and anti‐CD68, Ly6G, or CD45 (green). Nuclei are counterstained with DAPI (blue). Scale bar = 100 μm.Schematic showing relationships between donors and recipients in the BMT experiment. Mice were reconstituted with donor bone marrow for 21 days, and then, bowel inflammation was induced by 2.5% DSS in drinking water for 6 days followed by restoration of normal drinking water for 6 days thereafter.Survival rates of WT BM >> WT (n = 6), −/− BM >> WT (n = 9), −/− BM >> −/− (n = 6) and WT BM >> −/− (n = 7). P‐values were calculated by log‐rank test.

Figure 5. Bmp2 and Bmp7 transcripts are upregulated in Angptl2 −/− ISEMFs A–CmRNA levels of indicated genes in ISEMFs from wild‐type (n = 4) and Angptl2 −/− (n = 3) mice based on qRT–PCR analysis.DRepresentative staining for P‐Smad1/5 in crypts from wild‐type and Angptl2 −/− mice following DSS treatment for 6 days and then untreated water for 6 more days (DSS) or untreated water for 12 days (Untreated). Dashed lines indicate half of the crypt, and arrowheads show P‐Smad1/5‐positive cells in lower halves of crypts. Scale bar = 50 μm.EThe number of P‐Smad1/5‐positive cells in the crypt base (n = 60) from wild‐type and Angptl2 −/− mice following DSS treatment for 6 days and then untreated water for 6 more days (DSS) or untreated water for 12 days (Untreated). Multiple areas in three mice per genotype were quantified.FWestern blotting analysis of isolated colon crypts from wild‐type and Angptl2 −/− mice. HSC70 serves as an internal control. Numbers below panels represent normalized protein expression.Data information: Data are represented as mean ± SEM. *P < 0.05; ***P < 0.001. (A‐C) Unpaired Student's t‐test, (E) unpaired Welch's t‐test.Source data are available online for this figure.

Figure 6. Integrin α5β1/NF‐κB signaling downregulates Bmp expression AmRNA levels of indicated genes in colon and cultured ISEMFs from wild‐type mice (n = 4) based on qRT–PCR analysis. Intα5, integrin α5; Intβ1, integrin β1.BRepresentative IHC of integrin α5β1 in colon from wild‐type mouse. Scale bar = 50 μm.C, DmRNA levels of Bmp2 and Bmp7 in ISEMFs from wild‐type mice 24 h after treatment with integrin α5β1 antibody or various signaling pathway inhibitors. n = 3. U0126, ERK inhibitor; LY294002, PI3K inhibitor; BAY11‐7085, NF‐κB inhibitor.Data information: Data are represented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. (C) Unpaired Student's t‐test, (D) unpaired Student's t‐test or Welch's t‐test.

Figure 7. Co‐culture with Angptl2 −/− ISEMFs decreases organoid formation in vitro Colon organoid cultures treated with vehicle or rANGPTL2 protein. Scale bar = 200 μm.Organoid growth efficiency and size following treatment with vehicle or rANGPTL2. rA2, recombinant ANGPTL2. n = 4. Data from vehicle/Wnt (+) were set at 1.Schematic illustration of co‐culture of colon organoids with ISEMFs in direct contact.Colon organoid cultures in the presence of ISEMFs with or without Noggin (Nog). Scale bar = 50 μm.Organoid growth efficiency and size in the presence of ISEMFs with or without Noggin (Nog). n = 4. Data from wild‐type organoids/wild‐type ISEMFs/Nog (+) were set at 1.Model of ANGPTL2 activity in the intestinal stem cell niche. ISEMF‐derived ANGPTL2 inhibits BMP signaling in an autocrine manner through the integrin α5β1/NF‐κB pathway to maintain ISC stemness by regulating β‐catenin and Smad 1/5 signaling.Data information: Data are represented as mean ± SEM. *P < 0.05; ns, not statistically significant. (B) Unpaired Student's t‐test, (E) unpaired Student's t‐test.

Figure EV4. ANGPTL2 does not directly activate β‐catenin signaling in epithelial cells in vitro A, BExperimental protocol of organoid regeneration assay.CDissociated organoid cultures treated with vehicle or rANGPTL2. Scale bar = 200 μm.DOrganoid growth efficiency and size after passage treated with vehicle or rANGPTL2 (n = 4). Data from vehicle were set at 1.EOrganoid cultures following 1‐Gy irradiation and treated with vehicle or rANGPTL2. Scale bar = 200 μm.FOrganoid growth efficiency and size after 1‐Gy irradiation and treated with vehicle or rANGPTL2 (n = 12). Data from vehicle were set at 1.GmRNA levels of indicated transcripts in colon and epithelium from wild‐type mice (n = 4) based on qRT–PCR analysis. Intα5, integrin α5; Intβ1, integrin β1.HIntegrin expression in HEK293, Caco‐2, and SW480 cells. Representative profiles obtained by FACS analysis using indicated anti‐integrin antibodies (black line traces) or control IgG (filled gray traces).IWestern blotting analysis of HEK293, Caco‐2, and SW480 cells treated with vehicle or rANGPTL2. rA2, recombinant ANGPTL2. HSC70 serves as an internal control.Data information: Data are represented as means ± SEM. ns, not statistically…

Figure EV5. Organoid formation decreases following co‐culture with Angptl2 −/− ISEMFs or treatment with Angptl2 −/− ISEMF CM Schematic illustration of co‐culture of colon organoids with ISEMFs separated by a Transwell membrane.Colon organoid cultures in the presence of ISEMFs with or without exogenous Noggin (Nog). Scale bar = 50 μm.Organoid growth efficiency and size in the presence of ISEMFs with or without exogenous Noggin (Nog). n = 4. Data from wild‐type organoids/wild‐type ISEMFs/Nog (+) were set at 1.Schematic illustration of colon organoid cultures in the presence of ISEMF CM.Organoid cultures in the presence of ISEMF and treated with or without Noggin (Nog). Scale bar = 50 μm.Growth efficiency and size of organoids treated with ISEMF CM, with or without Noggin (Nog). n = 4. Data from wild‐type organoids/wild‐type ISEMFs CM/Nog (+) were set at 1.Data information: Data are represented as mean ± SEM. *P < 0.05; ns, not statistically significant. (C) Unpaired Student's t‐test or Welch's t‐test, (F) unpaired Student's t‐test.