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Class 3 semaphorins negatively regulate dermal lymphatic network formation.

Uchida Y, James JM, Suto F, Mukouyama YS - Biol Open (2015)

Bottom Line: In contrast, Sema3f;Sema3g double mutants display increased lymphatic branching, while Nrp2 mutants exhibit reduced lymphatic branching.Our results provide the first evidence that SEMA3F and SEMA3G function as a negative regulator for dermal lymphangiogenesis in vivo.The reciprocal phenotype in lymphatic branching between Sema3f;Sema3g double mutants and Nrp2 mutants suggest a complex NRP2 function that regulates LEC behavior both positively and negatively, through a binding with VEGFC or SEMA3s.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Stem Cell and Neuro-Vascular Biology, Genetics and Developmental Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Building 10/6C103, 10 Center Drive, Bethesda, MD 20892, USA.

No MeSH data available.


Related in: MedlinePlus

SEMA3F and SEMA3G induce LEC contraction and inhibit LEC sprouting in culture. (A-C) Cell contraction assay for human LECs with 1 nM of AP control protein (A), AP-SEMA3F(R580A/R582A) (B) or AP-SEMA3G (C). Cell shape is visualized by Phalloidin-AF568 staining and nuclei are stained by the nuclear marker TO-PRO-3. (D) Quantification of total cell area. N=231 cells, AP control; N=278, AP-SEMA3F; N=260, AP-SEMA3G; two independent experiments. (E-G) Sprouting assay for human LECs in response to VEGFC with 0.5 nM AP (E), VEGFC with 1 nM AP-SEMA3F (R580A/R582A) (F), or VEGFC with 0.5 nM AP-SEMA3G (G). Arrowheads indicate each sprout. Cell sprouts are visualized by Phalloidin-AF568 staining and nuclei are stained by the nuclear marker TO-PRO-3. (H) Quantification of total number of LEC sprouts per one aggregate. N=8 from two independent experiments, bars represent mean±s.d. (I-K) Proliferation assay for human LECs in response to VEGFC with 0.5 nM AP protein (I), VEGFC with 0.5 nM AP-SEMA3F(R580A/R582A) (J), or VEGFC with 0.5 nM AP-SEMA3G (K). The cells were stained with the LEC marker PROX1 (red), proliferation marker Ki67 (green) and TO-PRO-3 (blue). Arrowheads indicate PROX1 and Ki67 double positive cells. (L) Quantification of Ki67+/PROX1+ LECs. N=43 images, AP control; N=34, AP-SEMA3F; N=39, AP-SEMA3G; from 2-3 independent experiments were analyzed. Bars represent mean±s.e.m. *P<0.01, one-way ANOVA with Tukey-HSD multiple comparison test. Scale bars: 100 µm.
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BIO012302F1: SEMA3F and SEMA3G induce LEC contraction and inhibit LEC sprouting in culture. (A-C) Cell contraction assay for human LECs with 1 nM of AP control protein (A), AP-SEMA3F(R580A/R582A) (B) or AP-SEMA3G (C). Cell shape is visualized by Phalloidin-AF568 staining and nuclei are stained by the nuclear marker TO-PRO-3. (D) Quantification of total cell area. N=231 cells, AP control; N=278, AP-SEMA3F; N=260, AP-SEMA3G; two independent experiments. (E-G) Sprouting assay for human LECs in response to VEGFC with 0.5 nM AP (E), VEGFC with 1 nM AP-SEMA3F (R580A/R582A) (F), or VEGFC with 0.5 nM AP-SEMA3G (G). Arrowheads indicate each sprout. Cell sprouts are visualized by Phalloidin-AF568 staining and nuclei are stained by the nuclear marker TO-PRO-3. (H) Quantification of total number of LEC sprouts per one aggregate. N=8 from two independent experiments, bars represent mean±s.d. (I-K) Proliferation assay for human LECs in response to VEGFC with 0.5 nM AP protein (I), VEGFC with 0.5 nM AP-SEMA3F(R580A/R582A) (J), or VEGFC with 0.5 nM AP-SEMA3G (K). The cells were stained with the LEC marker PROX1 (red), proliferation marker Ki67 (green) and TO-PRO-3 (blue). Arrowheads indicate PROX1 and Ki67 double positive cells. (L) Quantification of Ki67+/PROX1+ LECs. N=43 images, AP control; N=34, AP-SEMA3F; N=39, AP-SEMA3G; from 2-3 independent experiments were analyzed. Bars represent mean±s.e.m. *P<0.01, one-way ANOVA with Tukey-HSD multiple comparison test. Scale bars: 100 µm.

Mentions: To examine how lymphatic endothelial cells (LECs) respond to SEMA3F and SEMA3G in culture, dermal-derived human lymphatic microvascular ECs (HMVEC-dLy-Neo; human LECs) were treated with AP-SEMA3F and AP-SEMA3G. Both SEMA3F and SEMA3G dramatically induced cell contraction (Fig. 1A-D). We next examined whether SEMA3F and SEMA3G influence VEGFC-mediated LEC behavior in vitro. VEGFC stimulates sprouting and proliferation of human LECs in a dose-dependent manner (data not shown). We found that both SEMA3F and SEMA3G inhibit VEGFC-mediated human LEC sprouting in 3D collagen gel (Fig. 1E-H). We also found that both SEMA3F and SEMA3G inhibit VEGFC-induced human LEC proliferation (Fig. 1I-L). These results demonstrate that SEMA3F and SEMA3G inhibit VEGFC-mediated human LEC sprouting and proliferation.Fig. 1.


Class 3 semaphorins negatively regulate dermal lymphatic network formation.

Uchida Y, James JM, Suto F, Mukouyama YS - Biol Open (2015)

SEMA3F and SEMA3G induce LEC contraction and inhibit LEC sprouting in culture. (A-C) Cell contraction assay for human LECs with 1 nM of AP control protein (A), AP-SEMA3F(R580A/R582A) (B) or AP-SEMA3G (C). Cell shape is visualized by Phalloidin-AF568 staining and nuclei are stained by the nuclear marker TO-PRO-3. (D) Quantification of total cell area. N=231 cells, AP control; N=278, AP-SEMA3F; N=260, AP-SEMA3G; two independent experiments. (E-G) Sprouting assay for human LECs in response to VEGFC with 0.5 nM AP (E), VEGFC with 1 nM AP-SEMA3F (R580A/R582A) (F), or VEGFC with 0.5 nM AP-SEMA3G (G). Arrowheads indicate each sprout. Cell sprouts are visualized by Phalloidin-AF568 staining and nuclei are stained by the nuclear marker TO-PRO-3. (H) Quantification of total number of LEC sprouts per one aggregate. N=8 from two independent experiments, bars represent mean±s.d. (I-K) Proliferation assay for human LECs in response to VEGFC with 0.5 nM AP protein (I), VEGFC with 0.5 nM AP-SEMA3F(R580A/R582A) (J), or VEGFC with 0.5 nM AP-SEMA3G (K). The cells were stained with the LEC marker PROX1 (red), proliferation marker Ki67 (green) and TO-PRO-3 (blue). Arrowheads indicate PROX1 and Ki67 double positive cells. (L) Quantification of Ki67+/PROX1+ LECs. N=43 images, AP control; N=34, AP-SEMA3F; N=39, AP-SEMA3G; from 2-3 independent experiments were analyzed. Bars represent mean±s.e.m. *P<0.01, one-way ANOVA with Tukey-HSD multiple comparison test. Scale bars: 100 µm.
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BIO012302F1: SEMA3F and SEMA3G induce LEC contraction and inhibit LEC sprouting in culture. (A-C) Cell contraction assay for human LECs with 1 nM of AP control protein (A), AP-SEMA3F(R580A/R582A) (B) or AP-SEMA3G (C). Cell shape is visualized by Phalloidin-AF568 staining and nuclei are stained by the nuclear marker TO-PRO-3. (D) Quantification of total cell area. N=231 cells, AP control; N=278, AP-SEMA3F; N=260, AP-SEMA3G; two independent experiments. (E-G) Sprouting assay for human LECs in response to VEGFC with 0.5 nM AP (E), VEGFC with 1 nM AP-SEMA3F (R580A/R582A) (F), or VEGFC with 0.5 nM AP-SEMA3G (G). Arrowheads indicate each sprout. Cell sprouts are visualized by Phalloidin-AF568 staining and nuclei are stained by the nuclear marker TO-PRO-3. (H) Quantification of total number of LEC sprouts per one aggregate. N=8 from two independent experiments, bars represent mean±s.d. (I-K) Proliferation assay for human LECs in response to VEGFC with 0.5 nM AP protein (I), VEGFC with 0.5 nM AP-SEMA3F(R580A/R582A) (J), or VEGFC with 0.5 nM AP-SEMA3G (K). The cells were stained with the LEC marker PROX1 (red), proliferation marker Ki67 (green) and TO-PRO-3 (blue). Arrowheads indicate PROX1 and Ki67 double positive cells. (L) Quantification of Ki67+/PROX1+ LECs. N=43 images, AP control; N=34, AP-SEMA3F; N=39, AP-SEMA3G; from 2-3 independent experiments were analyzed. Bars represent mean±s.e.m. *P<0.01, one-way ANOVA with Tukey-HSD multiple comparison test. Scale bars: 100 µm.
Mentions: To examine how lymphatic endothelial cells (LECs) respond to SEMA3F and SEMA3G in culture, dermal-derived human lymphatic microvascular ECs (HMVEC-dLy-Neo; human LECs) were treated with AP-SEMA3F and AP-SEMA3G. Both SEMA3F and SEMA3G dramatically induced cell contraction (Fig. 1A-D). We next examined whether SEMA3F and SEMA3G influence VEGFC-mediated LEC behavior in vitro. VEGFC stimulates sprouting and proliferation of human LECs in a dose-dependent manner (data not shown). We found that both SEMA3F and SEMA3G inhibit VEGFC-mediated human LEC sprouting in 3D collagen gel (Fig. 1E-H). We also found that both SEMA3F and SEMA3G inhibit VEGFC-induced human LEC proliferation (Fig. 1I-L). These results demonstrate that SEMA3F and SEMA3G inhibit VEGFC-mediated human LEC sprouting and proliferation.Fig. 1.

Bottom Line: In contrast, Sema3f;Sema3g double mutants display increased lymphatic branching, while Nrp2 mutants exhibit reduced lymphatic branching.Our results provide the first evidence that SEMA3F and SEMA3G function as a negative regulator for dermal lymphangiogenesis in vivo.The reciprocal phenotype in lymphatic branching between Sema3f;Sema3g double mutants and Nrp2 mutants suggest a complex NRP2 function that regulates LEC behavior both positively and negatively, through a binding with VEGFC or SEMA3s.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Stem Cell and Neuro-Vascular Biology, Genetics and Developmental Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Building 10/6C103, 10 Center Drive, Bethesda, MD 20892, USA.

No MeSH data available.


Related in: MedlinePlus