Table 1.
Genotyping Primer Sequences and Annealing Temperatures Employed.
Table 2.
Immunofluorescence Antibodies Applied for Staining.
Table 3.
Primers and Hydrolysis Probes Used for RT-qPCR.
Fig 1.
VEGF and sFlt-1 mutant phenotypes. VEGF mutant overexpression of VEGF by RT-PCR and ELISA. sFlt-1 mutant expression of sFlt-1 by RT-PCR.
(A) VEGF littermate mice revealed a normal, pink to yellow phenotype of the intestine. (B) VEGF mutants demonstrated a deeper red color of the intestine. (C) VEGF mutant mice revealed a 7.36 ± 1.49 SEM-fold increase of transgenic VEGF transcript compared to littermates. (D) VEGF mutant mice duodenum displayed a 13.92 ± 2.60 SEM-fold increase of VEGF protein compared to the littermates; N = 3 mice per group; *p<0.05; Error bars = SEM. (E) s-Flt-1 littermate mice with a normal intestine. (F) sFlt-1 mutants demonstrated paler coloration of the intestine. (G) sFlt-1 mutant mice duodenum demonstrated a significant decrease in full length Flt-1 and sFlt-1 transcript at 21 days compared to littermates, suggesting a negative feedback response. N = 3 mice per group; *p<0.001; Error bars = SEM. (H) sFlt-1 mutant small intestine enteroid cultures demonstrated increased expression of full length Flt-1 and sFlt transcript after 3 days of doxycycline administration. N = 25 OU per well; 6 wells; *p = 0.019; Error bars = SEM.
Fig 2.
VEGF mutant duodenum displayed longer villi compared to littermates.
(A) Cross-section of littermate duodenum. (B) Cross-section of VEGF mutant duodenum. Scale bars = 200 μm. (C) VEGF mutant mice had significantly taller villus height compared to littermates. (D) There was no significant difference in crypt depth between VEGF mutants and littermates. (E) There was no significant difference in the number of crypts per measured length between VEGF mutants and littermates. (F) There was no significant difference in duodenal circumference between VEGF mutants and littermates. N = 6 mice per group. *p<0.05; Error bars = SEM.
Fig 3.
sFlt-1 mutant duodenum displayed shorter villi, increased crypt depth and fewer crypts per length measured.
(A) Cross-section of littermate duodenum. (B) Cross-section of sFlt-1 mutant duodenum; Scale bars = 100 μm (C) sFlt-1 mutant mice had shorter villi compared to littermates (*p<0.0001). (D) sFlt-1 mutant mice had deeper crypts compared to littermates (*p<0.0001). (E) sFlt-1 mice had fewer crypts per measured length (*p = 0.03); N = 6 mice per group; Error bars = SEM.
Fig 4.
VEGF mutant villi demonstrated increased angiogenesis and vascular permeability.
(A) Cross-section of littermate duodenum. (B) Cross-section of VEGF mutant duodenum; Scale bars = 100 μm (C) Single RBC counts per individual villus were increased in VEGF mutant mice; N = 3 mice per group; 20–35 villi per mouse were evaluated; *p<0.001; Error bars = SEM. (D) RBCs cluster counts (>3 RBCs in close contact) per individual villus were increased in VEGF mutant mice; N = 3 mice per group; 20–35 villi per mouse were evaluated; *p<0.001; Error bars = SEM. (E) Three-dimensional cross-section of littermate duodenum. (F) Three-dimensional cross section of VEGF mutant duodenum. Nuclei labeled with DAPI (Cyan) and vasculature labeled with FITC-dextran (Yellow); Scale bars = 100 μm. (G) VEGF mutant vasculature displays increased volume compared to littermates; N = 4 mice per group; *p<0.001; Error bars = SEM.
Fig 5.
VEGF mutants demonstrated increased VE-cadherin expression compared to littermates, while sFlt-1 mutants demonstrated decreased expression.
(A) VEGF mutants demonstrated a 16.5-fold increase in VE-cadherin expression compared to littermates (*p = 0.04). (B) sFlt-1 mutants had a 22.4-fold reduction in VE-cadherin expression compared to littermates (*p = 0.01); N = 3 mice per group; Error bars = SEM.
Fig 6.
VEGF augmentation increased epithelial cell proliferation in the transit-amplifying zone whereas reduced VEGF bioavailabity restricted proliferation to the crypt bases.
(A) Littermate crypts had typical proliferation in the crypt and transit-amplifying zone of the duodenum. (B) VEGF mutant crypts displayed increased proliferation at higher positions in the transit-amplifying zone compared to littermates. First panel shows nuclei with DAPI staining (Blue), second reveals Ki-67-positive cells (Green) and third panel is the composite (MERGE); N = 6 mice per group; Scale bars = 20 μm (C) VEGF mutants had more Ki-67-positive cells at positions 16 through 25 than the littermates. (D) Littermate crypts had typical proliferation in the crypt and transit-amplifying zone. (E) sFlt-1 mutant crypts displayed decreased proliferation in the transit-amplifying zone compared to littermates. First panel shows DAPI staining (Blue), second reveals Ki-67-positive cells (Green) and third is the composite (MERGE); N = 6 mice per group; Scale bars = 10 μm (F) sFlt-1 mutants had fewer Ki-67-positive cells at positions 10 through 25 than littermates. N = 6 mice per group; *p<0.05; Error bars = SEM.
Fig 7.
sFlt-1 mutant mice demonstrated increased lysozyme-positive cells per hemicrypt.
(A) Immunofluorescence demonstrating lysozyme (Red) staining in littermates. (B) Lysozyme staining in sFlt-1 mutants showing increased staining in the duodenal crypts; Scale bars = 75μm. (C) The average percentage of lysozyme-positive cells per hemicrypt was significantly increased in sFlt-1 mutants (*p = 0.005). (D) No significant difference in the average percentage of lysozyme-positive cells per hemicrypt was appreciated in VEGF mutants; N = 4 mice per group; Error bars = SEM.
Fig 8.
VEGF and sFlt-1 mutants exhibit changes in stem cell marker gene expression by RT-qPCR.
(A) VEGF mutants demonstrated 0.55-fold reduction in Lgr5 expression compared to littermates (*p = 0.04). There were no other significant differences in stem cell marker expression in the VEGF mutants compared to littermates; N = 6 mice per group; Error bars = SEM. (B) Bmi1 expression decreased 0.42 fold (*p = 0.03) and Wdr43 expression decreased 0.60 fold (*p = 0.004) in sFlt-1 mutants compared to littermates. sFlt-1 mutants demonstrated a 2.47-fold increase in expression of EphB2 (*p = 0.02) and a 1.50-fold increase in Sox9 expression (*p = 0.004); N = 6 mice per group; Error bars = STDEV.
Fig 9.
VEGF overexpression in OU culture increased OU size and altered stem/progenitor cell gene expression.
(A) The diameter of VEGF mutant OU were measured every other day during a 10-day culture. The diameter of all OU increased over time; however, VEGF mutant OU treated with doxycycline were larger on day 5 compared to controls (*p = 0.04). N = 25 OU per well, 6 wells; Error bars = SEM. (B) VEGF mutant OU exposed to doxycycline demonstrated significant increase in serum VEGF levels over 5 days in culture (*p<0.05). N = 3; Error bars = SEM. (C) Significant increase in Bmi1 and Atoh1 expression and decrease in EphB2 expression was observed in doxycycline-treated VEGF OU compared to controls at 5 days (*p<0.05). N = 3; Error bars = STDEV.