Human chorionic gonadotropin controls luteal vascular permeability via vascular endothelial growth factor by down-regulation of a cascade of adhesion.

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Human chorionic gonadotropin controls luteal vascular permeability via vascular endothelial growth factor by down-regulation of a cascade of adhesion proteins Daniel Herr, M.D., Hamish M. Fraser, Ph.D., Regina Konrad, B.S., Iris Holzheu, B.S., Rolf Kreienberg, Ph.D., Christine Wulff, Ph.D. Fertility and Sterility Volume 99, Issue 6, Pages 1749-1758.e6 (May 2013) DOI: 10.1016/j.fertnstert.2013.01.120 Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Immunohistochemical detection of VE-cadherin, nectin2, claudin 5, and CD 31 in the human corpus luteum during the mid-luteal phase. (A) Dual staining of CD 31 (red fluorescence) with VE-cadherin, nectin 2, and claudin 5 (green fluorescence) revealed localization to the vessels. (B) Dual staining of VE-cadherin/claudin 5 proving colocalization of the two proteins showing a predominant occurrence of claudin 5. Fertility and Sterility 2013 99, 1749-1758.e6DOI: (10.1016/j.fertnstert.2013.01.120) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 2 (A) Representative Western blot of VE-cadherin, nectin 2, GAPDH, and claudin 5 in HUVEC in coculture with granulosa cells after 48 hours: [1] control; [2] Flt1-Fc; [3] coculture without any treatment; [4] coculture with hCG-treated granulosa cells; [5] coculture with hCG and Flt1-Fc (n = 3; P<.05). (B) Densitometric quantification of VE-cadherin protein production (corresponding to panel A) in HUVEC in coculture with granulosa cells after 48 hours, revealing a significant down-regulation after treatment with hCG (panel 4; n = 3; P<.05). (C) Densitometric quantification of nectin 2 protein production (corresponding to panel A) in HUVEC in coculture with granulosa cells after 48 hours, revealing a significant down-regulation after treatment with hCG (panel 4; n = 3; P<.05). (D) Densitometric quantification of claudin 5 protein production (corresponding to panel A) in HUVEC in coculture with granulosa cells after 48 hours, revealing a significant down-regulation after treatment with hCG (panel 4; n = 3; P<.05). ∗Significant results. Fertility and Sterility 2013 99, 1749-1758.e6DOI: (10.1016/j.fertnstert.2013.01.120) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Relative permeability of HUVEC in coculture with granulosa cells after 72 hours: [1] control; [2] Flt1-Fc; [3] coculture without any treatment; [4] coculture with hCG-treated granulosa cells; [5] coculture with hCG and Flt1-Fc (n = 2; *P<.05). Fertility and Sterility 2013 99, 1749-1758.e6DOI: (10.1016/j.fertnstert.2013.01.120) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Relative permeability of HUVEC after knockdown of VE-cadherin (A), nectin 2 (B), and claudin 5 (C) for 72 hours. The white bar represents the permeability of the control, and the black bar of the cells after knockdown of the protein of interest (n = 9; *P<.05). Fertility and Sterility 2013 99, 1749-1758.e6DOI: (10.1016/j.fertnstert.2013.01.120) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Schematic picture of the coculture system containing LGC and HUVEC. Fertility and Sterility 2013 99, 1749-1758.e6DOI: (10.1016/j.fertnstert.2013.01.120) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 2 Schematic picture of the permeability assay used for measurement of endothelial permeability. Fertility and Sterility 2013 99, 1749-1758.e6DOI: (10.1016/j.fertnstert.2013.01.120) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 3 Western blot of VE-cadherin, nectin 2, GAPDH, and claudin 5 in human granulosa cells and in HUVEC showing protein production of VE-cadherin and claudin 5 exclusively in HUVEC but not in granulosa cells (n = 2 different samples). Fertility and Sterility 2013 99, 1749-1758.e6DOI: (10.1016/j.fertnstert.2013.01.120) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 4 Knockdown of VE-cadherin in HUVEC. (A) Representative Western blot of VE-cadherin and GAPDH in HUVEC after knockdown of VE-cadherin (C = control; KD = knockdown). (B) Real-time reverse transcription–polymerase chain reaction (RT-PCR) quantification (1/delta ct) of VE-cadherin gene expression after knockdown of VE-cadherin (control = white; knockdown = black). Expression is normalized to 18S RNA as the internal standard in HUVEC cells (n = 3; *P<.05). (C) Densitometric quantification (OD) of VE-cadherin in HUVEC after knockdown of VE-cadherin. The knockdown is significant (n = 4; *P<.05). (D) Representative Western blot of nectin 2 and GAPDH in HUVEC after knockdown of VE-cadherin (C = control; KD = knockdown). (E) Real-time RT-PCR quantification (1/delta ct) of nectin 2 gene expression after knockdown of VE-cadherin (control = white; knockdown = black) shows no changes. Expression is normalized to 18S RNA as the internal standard in HUVEC cells (n = 3; *P>.05). (F) Densitometric quantification (OD) of nectin 2 in HUVEC after knockdown of VE-cadherin. The observed down-regulation is significant (n = 4; *P<.05). (G) Representative Western blot of claudin 5 and GAPDH in HUVEC after knockdown of VE-cadherin (C = control; KD = knockdown). (H) Real-time RT-PCR quantification (1/delta ct) of claudin 5 gene expression after knockdown of VE-cadherin (control = white; knockdown = black). Expression is normalized to 18S RNA as the internal standard in HUVEC cells. The observed down-regulation is significant (n = 3; *P<.05). (I) Densitometric quantification optical density of claudin 5 in HUVEC after knockdown of VE-cadherin. The observed down-regulation is significant (n = 4; *P<.05). Fertility and Sterility 2013 99, 1749-1758.e6DOI: (10.1016/j.fertnstert.2013.01.120) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 5 Nectin 2 after knockdown of nectin 2 in HUVEC. (A) Representative Western blot of nectin 2 and GAPDH in HUVEC after knockdown of nectin 2. (B) Real-time RT-PCR quantification (1/delta ct) of nectin 2 gene expression after knockdown of nectin 2 shows a significant knockdown of nectin 2 (control = white; knockdown = black). Expression is normalized to 18S RNA as the internal standard in HUVEC cells (n = 3; *P<.05). (C) Densitometric quantification (OD) of nectin 2 in HUVEC after knockdown of nectin 2 shows a significant knockdown of nectin 2 (n = 4; *P<.05). (D) Representative Western blot of VE-cadherin and GAPDH in HUVEC after knockdown of nectin 2 (C = control; KD = knockdown). (E) Real-time RT-PCR quantification (1/delta ct) of VE-cadherin gene expression after knockdown of nectin 2 does not show any significant changes (control = white; knockdown = black). Expression is normalized to 18S RNA as the internal standard in HUVEC cells (n = 3; P>.05). (F) Densitometric quantification (OD) of VE-cadherin in HUVEC after knockdown of nectin 2. The observed differences are not significant (n = 4; P>.05). (G) Representative Western blot of claudin 5 and GAPDH in HUVEC after knockdown of nectin 2. (H) Real-time RT-PCR quantification (1/delta ct) of claudin 5 gene expression after knockdown of nectin 2 does not show any significant changes (control = white; knockdown = black). Expression is normalized to 18S RNA as the internal standard in HUVEC cells (n = 3; P>.05). (I) Densitometric quantification (OD) of claudin 5 in HUVEC after knockdown of nectin 2. The observed differences are not significant (n = 4; P>.05). Fertility and Sterility 2013 99, 1749-1758.e6DOI: (10.1016/j.fertnstert.2013.01.120) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 6 Knockdown of claudin 5 in HUVEC. (A) Representative Western blot of claudin 5 and GAPDH in HUVEC after knockdown of claudin 5. (B) Real-time RT-PCR quantification (1/delta ct) of claudin 5 gene expression after knockdown of claudin 5 shows a significant knockdown of claudin 5 (control = white; knockdown = black). Expression is normalized to 18S RNA as the internal standard in HUVEC cells (n = 3; *P<.05). (C) Densitometric quantification (OD) of claudin 5 in HUVEC after knockdown of claudin 5 shows a significant knockdown of claudin 5 (n = 4; *P<.05). (D) Representative Western blot of nectin 2 and GAPDH in HUVEC after knockdown of claudin 5 (C = control; KD = knockdown). (E) Real-time RT-PCR quantification (1/delta ct) of nectin 2 gene expression after knockdown of claudin 5 (control = white; knockdown = black) does not show any changes. Expression is normalized to 18S RNA as the internal standard in HUVEC cells (n = 3; *P>.05). (F) Densitometric quantification (OD) of nectin 2 in HUVEC after knockdown of claudin 5. The observed down-regulation is significant (n = 4; *P<.05). (G) Representative Western blot of VE-cadherin and GAPDH in HUVEC after knockdown of claudin 5 (C = control; KD = knockdown). (H) Real-time RT-PCR quantification (1/delta ct) of VE-cadherin gene expression after knockdown of claudin 5 (control = white; knockdown = black). Expression is normalized to 18S RNA as the internal standard in HUVEC cells. The observed down-regulation is significant (n = 3; *P<.05). (I) Densitometric quantification (OD) of VE-cadherin protein production in HUVEC after knockdown of claudin 5. The observed down-regulation is significant (n = 4; *P<.05). Fertility and Sterility 2013 99, 1749-1758.e6DOI: (10.1016/j.fertnstert.2013.01.120) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions