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. 2019 Jul 8;11(7):956. doi: 10.3390/cancers11070956

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© 2019 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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TRPA1 promotes in vitro tubulogenesis and in vivo angiogenesis. (a) left panels: TRPA1-overexpressing HMEC display increased in vitro tubulogenesis in Matrigel compared to control cells; right panels: TRPA1-downregulating hTERT PTEC display decreased in vitro tubulogenesis in Matrigel/collagenI substrate compared to control cells; bar graphs show the quantification of number of master segment and the number of isolated segments of control HMECs and TRPA1-overexpressing HMECs. Three independent experiments were performed 24 h after overexpression or after II oligofection pulse. (b) Endogenous TRPA1 is diffusely expressed in postnatal mouse retina but clearly co-localizes with CD31 staining, indicating that TRPA1 is found in ECs. (c, upper panel): analysis of CD31 levels by qPCR in a fraction of CD31-enriched cell population isolated from the mouse retina (CD31+) compared to the depleted cell population (CD31−); (c, lower panel): analysis of TRPA1 mRNA expression by qPCR in a fraction of CD3-enriched cell population isolated from the mouse retina (CD31+) compared to the depleted cell population (CD31−). Values are expressed relative to actin expression. Statistical significance *: p-value < 0.05, **: p-value < 0.005 and ***: p-value < 0.0005 vs. control. (d) Subretinal injection of AITC or HC030031 did not alter the overall development of the vascular plexus 3 days after treatment, but the front of growing vasculature became uneven after injection of the TRPA1 agonist AITC in the subretinal space. (e, upper panel) This was illustrated by a significant increase of 26.8% in the length of sprouts (white bars): quantification of tip cell length during mouse retinal angiogenesis shows that compared to that in controls, tip cell length increased significantly 3 days post-intravitreous injection of 200 µM AITC. (e, lower panel): Treatment with TRPA1 inhibitor (200 µM HC030031) induced a decrease in tip cell length. Cell lengths were either classified into three groups according to ranges (<70 µm, 70–150 µm, >150 µm) or considered altogether (total). Bars represent the mean ± SEM of each length range or total grouping. (f, upper panel): Percentage of tip cells with each length range shows that treatment with AITC induces a decrease in the number of short tip cells (<70 µm) and an increase in the number of medium and long tip cells (>70 µm). (f, lower panel): Inhibition of TRPA1 with HC030031 induces the opposite effect, increasing the number of short tip cells and decreasing the number of medium and long tip cells.

TRPA1 promotes in vitro tubulogenesis and in vivo angiogenesis. (a) left panels: TRPA1-overexpressing HMEC display increased in vitro tubulogenesis in Matrigel compared to control cells; right panels: TRPA1-downregulating hTERT PTEC display decreased in vitro tubulogenesis in Matrigel/collagenI substrate compared to control cells; bar graphs show the quantification of number of master segment and the number of isolated segments of control HMECs and TRPA1-overexpressing HMECs. Three independent experiments were performed 24 h after overexpression or after II oligofection pulse. (b) Endogenous TRPA1 is diffusely expressed in postnatal mouse retina but clearly co-localizes with CD31 staining, indicating that TRPA1 is found in ECs. (c, upper panel): analysis of CD31 levels by qPCR in a fraction of CD31-enriched cell population isolated from the mouse retina (CD31+) compared to the depleted cell population (CD31−); (c, lower panel): analysis of TRPA1 mRNA expression by qPCR in a fraction of CD3-enriched cell population isolated from the mouse retina (CD31+) compared to the depleted cell population (CD31−). Values are expressed relative to actin expression. Statistical significance *: p-value < 0.05, **: p-value < 0.005 and ***: p-value < 0.0005 vs. control. (d) Subretinal injection of AITC or HC030031 did not alter the overall development of the vascular plexus 3 days after treatment, but the front of growing vasculature became uneven after injection of the TRPA1 agonist AITC in the subretinal space. (e, upper panel) This was illustrated by a significant increase of 26.8% in the length of sprouts (white bars): quantification of tip cell length during mouse retinal angiogenesis shows that compared to that in controls, tip cell length increased significantly 3 days post-intravitreous injection of 200 µM AITC. (e, lower panel): Treatment with TRPA1 inhibitor (200 µM HC030031) induced a decrease in tip cell length. Cell lengths were either classified into three groups according to ranges (<70 µm, 70–150 µm, >150 µm) or considered altogether (total). Bars represent the mean ± SEM of each length range or total grouping. (f, upper panel): Percentage of tip cells with each length range shows that treatment with AITC induces a decrease in the number of short tip cells (<70 µm) and an increase in the number of medium and long tip cells (>70 µm). (f, lower panel): Inhibition of TRPA1 with HC030031 induces the opposite effect, increasing the number of short tip cells and decreasing the number of medium and long tip cells.