Journal: The Journal of Physiology

Article Title: Adenosine receptors regulate gap junction coupling of the human cerebral microvascular endothelial cells hCMEC/D3 by Ca 2+ influx through cyclic nucleotide‐gated channels

doi: 10.1113/JP273150

Figure Lengend Snippet: A, the increase in the dye diffusion distance induced by 2‐PAA (20 μm, 1 h) was not blocked by the protein kinase A inhibitors Rp‐cAMPS (Rp, 200 μm) and KT5720 (KT, 1 μm). Additionally, the activator of the exchange protein directly activated by cAMP, 8‐pCPT‐O‐Me‐cAMP (100 μm) did not affect the dye diffusion distance. B, the cyclic nucleotide‐gated (CNG) channel inhibitor l‐cis‐diltiazem (L‐cis‐dil., 100 μm) could prevent the increase in the dye diffusion distance induced by 2‐PAA (20 μm, 1 h) relative to the vehicle control (cont., 0.3% ethanol) in scrape loading/dye transfer assays. C, RT‐PCR showed that the CNG channel subunits A1, A2 and B1 were expressed in hCMEC/D3 cells. D, furthermore, the cAMP‐sensitive subunit CNGA2 was confirmed to be expressed at protein level with β‐tubulin (β‐Tb) serving as loading control. E, in whole‐cell patch‐clamp experiments, 2‐PAA (20 μm) increased the current measured in hCMEC/D3 cells. This increased current was completely abolished by simultaneous application of the CNG channel blocker l‐cis‐diltiazem (L‐cis‐dil., 100 μm) with 2‐PAA. All results were analysed using Student's t test. *Significant differences to the vehicle control: * P < 0.05, ** P < 0.01, *** P < 0.001; #significant differences to 2‐PAA: # P < 0.05.

Article Snippet: Anti‐β‐tubulin antibody for the loading control (Sigma‐Aldrich, T4026) was diluted 1:7500, anti‐CNGA2 antibody (Alomone Labs, Jerusalem, Israel, APC‐045) was diluted 1:750 and anti‐Cx37 antibody (Abcam, ab58918) was diluted 1:700 in TBS‐T and applied to the membranes at 4 °C overnight.

Techniques: Diffusion-based Assay, Reverse Transcription Polymerase Chain Reaction, Patch Clamp

Journal: The Journal of Physiology

Article Title: Adenosine receptors regulate gap junction coupling of the human cerebral microvascular endothelial cells hCMEC/D3 by Ca 2+ influx through cyclic nucleotide‐gated channels

doi: 10.1113/JP273150

Figure Lengend Snippet: List of all primer pairs used for gene expression analyses and quantitative real time PCR

Article Snippet: Anti‐β‐tubulin antibody for the loading control (Sigma‐Aldrich, T4026) was diluted 1:7500, anti‐CNGA2 antibody (Alomone Labs, Jerusalem, Israel, APC‐045) was diluted 1:750 and anti‐Cx37 antibody (Abcam, ab58918) was diluted 1:700 in TBS‐T and applied to the membranes at 4 °C overnight.

Techniques: Expressing, Sequencing, Amplification

Journal: Developmental biology

Article Title: Combining Foxc2 and Connexin37 deletions in mice leads to severe defects in lymphatic vascular growth and remodeling

doi: 10.1016/j.ydbio.2015.06.004

Figure Lengend Snippet: Mesenteric lymphatic endothelial cells (LECs) of Cx37 −/− mice are more mitotically active than WT mice. Foxc2 (A) and phospho-Histone H3 (pHH3, B) immunolabeling of WT mesenteric lymphatic vessels. (C) Colocalization of Foxc2 (green) and pHH3 (magenta) signal highlight LECs that are in M-phase of the cell cycle. (D) Segmentation of mitotic LECs (Foxc2+/pHH3+ cells) in (C). Foxc2 (E) and pHH3 (F) immunolabeling of Cx37 −/− mesenteric lymphatic vessels. (G) A greater number of mesenteric LECs show colocalization of Foxc2 (green) and pHH3 (magenta) signal in Cx37 −/− mice. (H) Segmentation of mitotic LECs (Foxc2+/pHH3+ cells) in (G). (I) LEC mitotic index measured as the number of Foxc2+pHH3+ cells divided by total number of Foxc2+ cells; there is a significant increase in the percentage of mitotic mesenteric LECs in Cx37 −/− mice compared to WT. Note that there is non-specific labeling of the cytoplasm of macrophages and mast cells with the pHH3 antibody, seen as the saturated signal from cells in white (B,F) and magenta (C,G). Scale bars: (A–C, E–G) 100 µm. Values are presented as means, with error bars indicating standard error of the mean. Asterisks, p < 0.05.

Article Snippet: Antibodies Primary antibodies used for immunostaining were as follows: rabbit antibodies to Cx37 ( Simon et al., 2006 ), Cx43 (C6219, Sigma), Prox1 (11–002, AngioBio; ab11941, Abcam), pHH3 (06–570, Millipore); rat antibodies to CD31 (550274, BD Biosciences); goat antibodies to Foxc2 (ab5060, Abcam), Vefgr3 (AF743, R&D Systems).

Techniques: Immunolabeling, Labeling

Journal: Developmental biology

Article Title: Combining Foxc2 and Connexin37 deletions in mice leads to severe defects in lymphatic vascular growth and remodeling

doi: 10.1016/j.ydbio.2015.06.004

Figure Lengend Snippet: Craniofacial morphometrics of E18.5 embryos. (A–D) Superior view of the head of littermates with the following genotypes: WT (A), Foxc2 +/− (B), Foxc2 +/−Cx37 +/− (C), and Foxc2 +/−Cx37 −/− (D). (E) Schematic diagram of the embryonic head indicating where snout length (green), snout width (blue), and interpupillary distance (magenta) were measured. Bregma and lambda (λ) are shown on the schematic at the anterior and posterior fontanelles, respectively. Double-headed arrow denotes R (rostral) and C (caudal) directions. (F) Numeric measures of snout length (green points), snout width (blue points), and interpupillary distance (magenta points) normalized to λ –bregma distance are graphed for WT, Cx37 +/−, Foxc2 +/−, Foxc2 +/−Cx37 +/−, and Foxc2 +/−Cx37 −/− mice. The length of the snouts of Foxc2 +/−Cx37 −/−mice were significantly shorter than WT, Cx37 +/−, and Foxc2 +/−Cx37 +/− mice. The width of the snouts of Foxc2 +/−Cx37 +/− mice were significantly greater than WT and Cx37 +/− mice. No statistically significant differences were found for interpupillary distance among genotypes analyzed. Scale bar: (A–D) 2 mm. Values are presented as means, with error bars indicating standard error of the mean. Asterisks, p < 0.05.

Article Snippet: Antibodies Primary antibodies used for immunostaining were as follows: rabbit antibodies to Cx37 ( Simon et al., 2006 ), Cx43 (C6219, Sigma), Prox1 (11–002, AngioBio; ab11941, Abcam), pHH3 (06–570, Millipore); rat antibodies to CD31 (550274, BD Biosciences); goat antibodies to Foxc2 (ab5060, Abcam), Vefgr3 (AF743, R&D Systems).

Techniques:

Journal: Developmental biology

Article Title: Combining Foxc2 and Connexin37 deletions in mice leads to severe defects in lymphatic vascular growth and remodeling

doi: 10.1016/j.ydbio.2015.06.004

Figure Lengend Snippet: Characterization of intestinal lymphatics in WT, Foxc2 +/−, Cx37 −/−, and Foxc2 +/−Cx37 −/− mice at E18.5. Confocal immunomicrographs of the small intestine and colon from E18.5 WT (A,B), Foxc2 +/− (C,D), Cx37 −/− (E,F), and Foxc2 +/−Cx37 −/− mice are shown; samples were probed for Vegfr3. The lymphatic vessels were segmented based on Vegfr3 signal and skeletonized. (I–L) Quantitation of the submucosal lymphatic network of the proximal small intestine. Submucosal lymph vessel diameter (I) and submucosal lymph vessel area density (I) of Foxc2 +/−Cx37 −/− mice were significantly greater than WT, Foxc2 +/−, and Cx37 −/−mice. Submucosal lymph vessel length density (K) and submucosal lymphatic branch point density (L) of Foxc2 +/−Cx37 −/− mice were significantly lower than WT, Foxc2 +/−, and Cx37 −/−mice. Additionally, submucosal lymph vessel area density of Cx37 −/− mice was significantly greater than WT and Foxc2 +/− mice. Scale bar: (A–H) 200 µm. Values are presented as means, with error bars indicating standard error of the mean. Asterisks, p < 0.05.

Article Snippet: Antibodies Primary antibodies used for immunostaining were as follows: rabbit antibodies to Cx37 ( Simon et al., 2006 ), Cx43 (C6219, Sigma), Prox1 (11–002, AngioBio; ab11941, Abcam), pHH3 (06–570, Millipore); rat antibodies to CD31 (550274, BD Biosciences); goat antibodies to Foxc2 (ab5060, Abcam), Vefgr3 (AF743, R&D Systems).

Techniques: Quantitation Assay

Journal: Developmental biology

Article Title: Combining Foxc2 and Connexin37 deletions in mice leads to severe defects in lymphatic vascular growth and remodeling

doi: 10.1016/j.ydbio.2015.06.004

Figure Lengend Snippet: Lacteal development is disrupted in Foxc2 +/−Cx37 −/− mice at E18.5. Vegfr3 whole-mount immunostaining (imaged via confocal microscopy) highlights the submucosal lymphatics and lacteals of the proximal small intestine in WT (A–C), Foxc2 +/− (D–F), Cx37 −/− (G–I) and Foxc2 +/−Cx37 −/− (J–L) mice. Single longitudinal sections (optical) from WT (A), Foxc2 +/− (D), Cx37 −/− (G), and Foxc2 +/−Cx37 −/− (J) mice. Maximum intensity projections of confocal z-stacks in the X-Y and X-Z planes for WT (B,C), Foxc2 +/− (E,F), Cx37 −/− (H,I), and Foxc2 +/−Cx37 −/−(K,L) mice. In X-Y projections (B,E,H,K), lacteals are seen as intense regions of Vegfr3 signal. In X-Z projections (C,F,I,L), individual lacteals resemble ropes hanging from the top of each image. Intestinal lumen is directed towards the bottom of the image in (A,C,D,F,G,I,J,L). Measurements of lacteal density (M) and lacteal length (N) for WT (black bars), Foxc2 +/− (dark grey bars), Cx37 −/− (light grey bars), and Foxc2 +/−Cx37 −/− (white bars) mice are shown. Lacteals from Foxc2 +/−Cx37 −/− mice were significantly shorter than WT, Foxc2 +/−, and Cx37 −/− mice. Scale bar: (A–L) 200 µm. Values are presented as means, with error bars indicating standard error of the mean. Asterisks, p < 0.05.

Article Snippet: Antibodies Primary antibodies used for immunostaining were as follows: rabbit antibodies to Cx37 ( Simon et al., 2006 ), Cx43 (C6219, Sigma), Prox1 (11–002, AngioBio; ab11941, Abcam), pHH3 (06–570, Millipore); rat antibodies to CD31 (550274, BD Biosciences); goat antibodies to Foxc2 (ab5060, Abcam), Vefgr3 (AF743, R&D Systems).

Techniques: Immunostaining, Confocal Microscopy

Journal: Developmental biology

Article Title: Combining Foxc2 and Connexin37 deletions in mice leads to severe defects in lymphatic vascular growth and remodeling

doi: 10.1016/j.ydbio.2015.06.004

Figure Lengend Snippet: Gross images of E18.5 mice. Littermates with the following genotypes are shown: Cx37 +/− (A,F), Foxc2 +/− (B,G), Cx37 −/− (C,H), Foxc2 +/−Cx37 +/− (D,I), and Foxc2 +/−Cx37 −/−(E,J). Lateral views of the whole embryo (A–E), with corresponding images of the hindlimb (F–J). Foxc2 +/−Cx37 −/− embryos presented with mild generalized edema (E) that also affected the hindlimbs (J). Flattening of the nasal bridge (E, upper arrow) and micrognathism (E, lower arrow) also occurred. No overt differences in gross embryonic morphology were notable among the other genotypes (A-D, F-I).

Article Snippet: Antibodies Primary antibodies used for immunostaining were as follows: rabbit antibodies to Cx37 ( Simon et al., 2006 ), Cx43 (C6219, Sigma), Prox1 (11–002, AngioBio; ab11941, Abcam), pHH3 (06–570, Millipore); rat antibodies to CD31 (550274, BD Biosciences); goat antibodies to Foxc2 (ab5060, Abcam), Vefgr3 (AF743, R&D Systems).

Techniques:

Journal: Developmental biology

Article Title: Combining Foxc2 and Connexin37 deletions in mice leads to severe defects in lymphatic vascular growth and remodeling

doi: 10.1016/j.ydbio.2015.06.004

Figure Lengend Snippet: Lymph-blood mixing in the lymphatics of the skin (C–E), intestines (G,H), and heart (J,K) of Foxc2 +/−Cx37 −/− mice at E18.5. (A) Evans blue dye (EBD) injection of E18.5 WT embryo showing normal drainage pattern from the inguinal lymph node (arrow) and thoracoepigastric lymphatic vessels. (B) Thoracoepigastric lymph vessels, higher magnification view of the white box from (A); note the fine caliber of WT lymph vessels in this region. (C) Blood within the inguinal lymph node (lower box, arrow denotes same anatomical region as lymph node in panel A) and thoracoepigastric lymph vessels (upper box) of a Foxc2 +/−Cx37 −/−embryo. (D) Higher magnification view of lymph node from (C). (E) Higher magnification view of thoracoepigastric lymph vessels from (C); blood-filled lymph vessels are digitally outlined in white for better contrast. (F) Gross image of the small intestine from an E18.5 Cx37 +/− control embryo, compared to a Foxc2 +/−Cx37 −/− littermate (G). (H) Higher magnification view of white box from (G); blood is present within the serosal lymphatics in the proximal small intestine. (I) Gross image of the heart from E18.5 Cx37 +/− control embryo; right/left auricles and apex are labeled. (J) Blood accumulation in blind-ended vessels of the pericardium in an E18.5 Foxc2 +/−Cx37 −/− embryo. (K) Higher magnification view of white box from (J), arrows highlight blood-filled lymph vessels.

Article Snippet: Antibodies Primary antibodies used for immunostaining were as follows: rabbit antibodies to Cx37 ( Simon et al., 2006 ), Cx43 (C6219, Sigma), Prox1 (11–002, AngioBio; ab11941, Abcam), pHH3 (06–570, Millipore); rat antibodies to CD31 (550274, BD Biosciences); goat antibodies to Foxc2 (ab5060, Abcam), Vefgr3 (AF743, R&D Systems).

Techniques: Injection, Control, Labeling

Journal: Developmental biology

Article Title: Combining Foxc2 and Connexin37 deletions in mice leads to severe defects in lymphatic vascular growth and remodeling

doi: 10.1016/j.ydbio.2015.06.004

Figure Lengend Snippet: Cx37 is expressed in the submucosal lymphatics of the small intestine and colon. Cryosections of the small intestine (A–F) and colon (G–L) of E18.5 WT mice are shown. Vegfr3 immunostaining was used to identify submucosal lymphatic vessels. (A) Vegfr3 and (B) Cx37 immunolabeling of the submucosal lymphatics of the small intestine. (C) Vegfr3 (magenta) and Cx37 (green) signals colocalize in the submucosal lymphatics and lacteal. (D–F) Higher magnification view of the area denoted by the arrow in (C), arrowhead in (E) indicates relevant area of Cx37 expression within the lacteal. Vegfr3 (G) and Cx37 (H) immunolabeling of the submucosal lymphatics of the colon. (I) Vegfr3 (magenta) and Cx37 (green) signals colocalize in the submucosal lymphatics. (J–L) Higher magnification view of the area denoted by the arrow in (I). The bright, saturated Cx37 signal in (E) is from the arterial submucosal vasculature, also in the lower part of panel (H). Scale bars: (A-C, G-I) 50 µm; (D-F, J-L) 25 µm.

Article Snippet: Antibodies Primary antibodies used for immunostaining were as follows: rabbit antibodies to Cx37 ( Simon et al., 2006 ), Cx43 (C6219, Sigma), Prox1 (11–002, AngioBio; ab11941, Abcam), pHH3 (06–570, Millipore); rat antibodies to CD31 (550274, BD Biosciences); goat antibodies to Foxc2 (ab5060, Abcam), Vefgr3 (AF743, R&D Systems).

Techniques: Immunostaining, Immunolabeling, Expressing

Journal: Developmental biology

Article Title: Combining Foxc2 and Connexin37 deletions in mice leads to severe defects in lymphatic vascular growth and remodeling

doi: 10.1016/j.ydbio.2015.06.004

Figure Lengend Snippet: Mesenteric lymph vessels of Foxc2 +/−Cx37 −/− mice lack valves and are dilated at E18.5. Prox1 whole-mount immunostaining highlights lymphatic vessels of the mesentery in WT (A), Foxc2 +/− (B), Cx37 −/− (C), and Foxc2 +/−Cx37 −/− (D) mice at E18.5. Corresponding Prox1 (green) and CD31 (magenta) color composites are shown for WT (E), Foxc2 +/− (F), Cx37 −/−(G), and Foxc2 +/−Cx37 −/− (H) mesenteries at E18.5. (I–L) Prox1 whole-mount immunostaining at P0 for WT (I), Foxc2 +/− (J), Cx37 −/− (K), and Foxc2 +/−Cx37 −/− (L) mesenteries. (M–P) Prox1 (green) and CD31 (magenta) color composites are shown for WT (M), Foxc2 +/− (N), Cx37 −/− (O), and Foxc2 +/−Cx37 −/− (P) mesenteries at P0. Arrows denote lymphatic valve forming areas; note that none are present in Foxc2 +/−Cx37 −/− mice. Scale bar: (A–P) 200 µm.

Article Snippet: Antibodies Primary antibodies used for immunostaining were as follows: rabbit antibodies to Cx37 ( Simon et al., 2006 ), Cx43 (C6219, Sigma), Prox1 (11–002, AngioBio; ab11941, Abcam), pHH3 (06–570, Millipore); rat antibodies to CD31 (550274, BD Biosciences); goat antibodies to Foxc2 (ab5060, Abcam), Vefgr3 (AF743, R&D Systems).

Techniques: Immunostaining

Journal: Developmental biology

Article Title: Combining Foxc2 and Connexin37 deletions in mice leads to severe defects in lymphatic vascular growth and remodeling

doi: 10.1016/j.ydbio.2015.06.004

Figure Lengend Snippet: The dermal lymphatics of Foxc2 +/−Cx37 −/− mice at E18.5 lack valves, are smaller in caliber, and can develop structures resembling cystic lymphangiomas. Prox1 immunolabeling (A, D, G, J, M) and CD31 immunolabeling (B, E, H, K, N) for WT, Foxc2 +/−, Cx37 −/−, and Foxc2 +/−Cx37 −/− dermal lymphatic vessels. (C, F, I, L, O) Color composites of corresponding panels (A, D, G, J, M) for Prox1 (green) and (B, E, H, K, N) for CD31 (magenta). (M–O) Structure resembling a cystic lymphangioma in a Foxc2 +/−Cx37 −/− skin sample. (P–U) Dermal lymphatic vascular network quantitation. (P) Dermal lymph vessel diameter of Foxc2 +/−Cx37 −/− mice was significantly smaller than WT mice. Dermal lymph vessel diameter of Foxc2 +/− mice was also significantly lower than WT. (Q) Dermal lymph vessel area density of Foxc2 +/−Cx37 −/− mice was significantly lower than WT and Cx37 −/− mice. Additionally, lymph vessel area density of Foxc2 +/− mice was significantly lower than WT. (T) There were significantly fewer dermal lymph valves in Foxc2 +/−Cx37 −/− mice compared to WT, Foxc2 +/−, and Cx37 −/− mice. There was also a significant reduction in dermal lymph valves in Foxc2 +/− and Cx37 −/− mice compared to WT. There were no differences in dermal lymph vessel length density (R), branch point density (S), or vessel tortuosity (U) between genotypes. Scale bar: (A–O) 200 µm. Six 10x fields were evaluated per embryo, n = 3 for each genotype. Values are presented as means, with error bars indicating standard error of the mean. Asterisks, p < 0.05.

Article Snippet: Antibodies Primary antibodies used for immunostaining were as follows: rabbit antibodies to Cx37 ( Simon et al., 2006 ), Cx43 (C6219, Sigma), Prox1 (11–002, AngioBio; ab11941, Abcam), pHH3 (06–570, Millipore); rat antibodies to CD31 (550274, BD Biosciences); goat antibodies to Foxc2 (ab5060, Abcam), Vefgr3 (AF743, R&D Systems).

Techniques: Immunolabeling, Quantitation Assay

Journal: Developmental biology

Article Title: Combining Foxc2 and Connexin37 deletions in mice leads to severe defects in lymphatic vascular growth and remodeling

doi: 10.1016/j.ydbio.2015.06.004

Figure Lengend Snippet: Evans blue dye (EBD) lymphangiography of WT, Foxc2 +/−, Foxc2 +/−Cx37 +/−, and Foxc2 +/−Cx43 +/− adult mice. EBD drainage patterns in the ear (A–D), abdominal cavity (E–H), and thoracic cavity (I–L). EBD readily filled the lymphatics of ear (injection site denoted by the asterisk), the lumbar lymph nodes (E-H, labeled “LN”), and thoracic duct (denoted by arrows in I-L) of WT, Foxc2 +/−, Foxc2 +/−Cx37 +/−, and Foxc2 +/−Cx43 +/− mice. No obstructions or reflux of EBD were found in the areas examined.

Article Snippet: Antibodies Primary antibodies used for immunostaining were as follows: rabbit antibodies to Cx37 ( Simon et al., 2006 ), Cx43 (C6219, Sigma), Prox1 (11–002, AngioBio; ab11941, Abcam), pHH3 (06–570, Millipore); rat antibodies to CD31 (550274, BD Biosciences); goat antibodies to Foxc2 (ab5060, Abcam), Vefgr3 (AF743, R&D Systems).

Techniques: Injection, Labeling, Reflux