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First published online 15 April 2003
doi: 10.1242/jcs.00429

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Decreased intercellular dye-transfer and downregulation of non-ablated connexins in aortic endothelium deficient in connexin37 or connexin40

Department of Physiology, University of Arizona, Tucson, AZ 85724, USA

View larger version (50K): [in a new window]   Fig. 1. Deletion of Cx37 or Cx40 reduces the levels of non-ablated connexins in aortic endothelium. (A) Immunoblots of alkaline-extracted endothelial cell membranes isolated from thoracic aortas of 6-7-week-old and 4-6-month-old mice. Pooled membranes from six aortas were loaded in each lane. Densitometric quantification of all blots is presented in Table 1. Connexin levels were normalized to PECAM levels measured on the same blot. Cx37 protein was substantially reduced in aortic endothelium of Cx40–/– mice compared with wild-type levels. The drop in Cx37 was more pronounced in aortas from the younger mice (17-fold) than aortas from 4-6-month-old mice (3.0-fold). Cx40 was reduced in the endothelium of aortas from Cx37–/– mice, but the decrease was not as large (2.6-4.2-fold). VE-cadherin and PECAM levels were not significantly altered, but caveolin-1 levels were slightly reduced in Cx37–/– and Cx40–/– aortic endothelium of 3-7-week-old mice. (B) Anti-Cx37 and anti-Cx40 antibodies reacted only with appropriate GST-connexin fusion proteins. (C) Cx43 was not detected in western blots of alkaline-extracted endothelial cell membranes but was detected in a similar preparation of whole aorta. (D) Chemiluminescent detection of GST-Cx37 fusion protein was linear over a 16-fold dilution range.

View larger version (62K): [in a new window]   Fig. 2. Cx37 and Cx43 levels are elevated in the medial layer of Cx40–/– aorta. (A) Alkaline-extracted membranes from whole aortas of 6-7-week-old mice were immunoblotted. Membranes from the equivalent of 1.5 aortas are loaded in each lane. Cx37 levels slightly increased (1.4-fold) in Cx40–/– aorta, after normalizing to PECAM. Cx43 levels increased fourfold in Cx40–/– aorta. (B) SDS sample buffer lysates of aortic endothelium and alkaline-extracted membranes from medial-layer only preparations were immunoblotted (equivalent of 1.5 aortas per lane). Total endothelial Cx37 declined 8.2-fold in Cx40–/– aorta, whereas medial Cx37 increased 4.4-fold. Cx43 also increased about fourfold in the medial layer of Cx40–/– aorta. Cx40 was detected only in the endothelial fraction, as was PECAM. Cx43 was detected only in the medial layer preparation.

View larger version (126K): [in a new window]   Fig. 3. Endothelial Cx37 immunostaining is markedly reduced in Cx40–/– aorta. Sections of thoracic aortas from E18.5 embryos (A-H) or 7-week-old mice (I-P) were immunostained for Cx37 or Cx40. Differential interference contrast images are shown to the right of each panel. In sections of aorta from E18.5 Cx40–/– embryos (C,D), endothelial Cx37 immunostaining was virtually absent, whereas endothelial Cx37 staining was readily detected in wild-type sections (A,B). Cx40 immunostaining in sections of E18.5 Cx37–/– aorta (G, H) was similar to that observed with sections of E18.5 wild-type aorta (E,F). Sections of aorta from a 7-week-old Cx40–/– animal showed a substantial reduction in endothelial Cx37 immunostaining (K,L) compared with wild-type (I,J). Only rarely were punctate signals observed in the endothelium. Cx40 immunostaining in sections of 7-week-old Cx37–/– aorta was slightly reduced (O,P) compared with wild-type signals (M,N). En face immunostaining of segments of 7-week-old aorta confirmed that endothelial Cx37 immunostaining was greatly reduced in Cx40–/– aortic segments (R) compared with wild-type (Q). For the inset in panel R, the exposure level was adjusted to bring out faint Cx37 immunostaining in the Cx40–/– endothelium. En face Cx40 staining was slightly reduced in Cx37–/– aortic segments (T) compared with wild-type (S). Bars, 20 µm.

View larger version (80K): [in a new window]   Fig. 4. Cx37 immunostaining is detectable in the medial layer of mouse aorta as well as in endothelium. Sections of thoracic aortas from 6-7-week-old wild-type (A), Cx37–/– (B) and Cx40–/– (C,D) mice were immunostained with anti-Cx37 antibody (A,B,C) or with secondary antibody only (D) and photographed at high magnification. The lumen of the aorta is on the right in each panel. (A) In wild-type aorta, very weak punctate staining was observed in the medial layer, as well as strong endothelial staining. (B) The specificity of the medial layer signal was confirmed by the absence of medial layer staining in Cx37–/– aortic sections. (C) Medial layer Cx37 staining was elevated in Cx40–/– sections. (D) Secondary antibody-only control showed no signal in the medial layer of Cx40–/– aorta. (E,F) Sections of wild-type and Cx40–/– aorta from 4-month-old mice were immunostained for Cx37. In addition to endothelial signal, faint punctate Cx37 staining was observed in the medial layer of wild-type sections. Medial layer Cx37 staining was particularly evident in aortas from 4-month-old Cx40–/– mice. Bar, 20 µm.

View larger version (58K): [in a new window]   Fig. 5. Deletion of Cx37 or Cx40 does not alter mRNA levels of non-ablated connexins in aortic endothelium. (A) RT-PCR for Cx37, Cx40, Cx43 and GAPDH mRNA was performed with RNA isolated from aortic endothelium. Amplicons were of the predicted size and depended on reverse transcription (RT). Cx37 and Cx40 amplicons were absent in samples derived from Cx37–/– or Cx40–/– mice, respectively. (B) Semiquantitative RT-PCR was performed. Connexin signals were normalized to GAPDH signals. (C) Quantification of Cx37 signals obtained from groups of wild-type and Cx40–/– animals yielded similar amplification curves. (D) Mean Cx37, Cx40 and Cx43 mRNA levels (relative to wild-type levels) are plotted. Wild-type signals for each connexin mRNA were assigned the value of 1.0. Error bars represent s.e.m. There were no significant changes in the levels of non-ablated connexin mRNAs present in Cx37–/– and Cx40–/– aortic endothelium (P>0.05). (E) Endothelial or whole aorta RNA preparations (equivalent lengths of aorta) from wild-type mice were analyzed by RT-PCR for smooth-muscle actin mRNA to test for contamination of endothelial RNA fractions with medial layer RNA. A 1/500 dilution of whole aorta cDNA yielded a similar amplification curve to that of undiluted endothelial cDNA. Contamination was therefore approximately only 0.2%. Cx40 RNA levels were the same in each preparation, whereas Cx37 RNA levels were slightly higher in the whole aorta sample.

View larger version (96K): [in a new window]   Fig. 6. Biocytin transfer is reduced in aortic endothelium of connexin-deficient mice. Biocytin and FITC-dextran were injected into endothelial cells of wild-type, Cx37–/–, Cx40–/– or Cx37+/–Cx40–/– thoracic aorta isolated from 6-7-week-old animals. Biocytin transfer was reduced in Cx37–/– (C) and Cx40–/– (E) endothelium compared with wild-type endothelium (A). Biocytin-containing cells in panel A extended beyond the photographic field of view. Transfer was further reduced in Cx37+/–Cx40–/– endothelium (G). FITC-dextran marked the injected cell, but did not transfer to adjacent cells. There was more autofluorescence in panels (B,F) than (D,H) because the FITC-dextran was photographed after fixing the tissue. Quantification is presented in Fig. 10 and Table 2. Bar, 50 µm.

View larger version (115K): [in a new window]   Fig. 7. NBD-TMA transfer is reduced in aortic endothelium of connexin-deficient mice. The fluorescent tracer NBD-TMA was injected into endothelial cells of wild-type (A), Cx37–/– (B), Cx40–/– (C) or Cx37+/–Cx40–/– (D) thoracic aorta from 8-12-week-old animals. NBD-TMA transfer was reduced in Cx40–/– endothelium compared with wild-type, but not in Cx37–/– endothelium. NBD-TMA transfer was further reduced in Cx37+/–Cx40–/– endothelium. Injected cells are marked with an asterisk. Quantification is presented in Table 2. Bar, 50 µm.

View larger version (23K): [in a new window]   Fig. 10. Quantification of biocytin transfer in postnatal and E18.5 aortic endothelium. The mean number of biocytin-labeled cells is plotted for postnatal animals (A) or E18.5 embryos (B). In A, the bar for >8-week-old wild-type mice represents pooled data from C57BL/6 and C57BL/6-129/Sv strains, which were not significantly different. Error bars represent s.e.m. *P<0.001 and #P<0.05 versus respective wild-type values.

View larger version (116K): [in a new window]   Fig. 8. Cx37–/–Cx40–/– E18.5 aortas do not show compensatory expression of Cx43 and exhibit normal expression of blood vessel cell markers. Sections of E18.5 aorta from wild-type (A,C,E) and Cx37–/–Cx40–/– (B,D,F) aorta were immunostained for Cx37, Cx40 and Cx43. (B,D) Cx37 and Cx40 were not detected in aortas with the Cx37–/–Cx40–/– genotype. (F) Cx37–/–Cx40–/– aortas did not show compensatory expression of Cx43 in endothelium. Cx37–/–Cx40–/– aortas exhibited normal expression of the endothelial marker, PECAM-1 (G,H) and smooth-muscle actin (SMA) (I,J). (K,L) Silver nitrate staining, which was performed to examine endothelial cell morphology and cell-cell contacts, was similar in Cx37–/–Cx40–/– (L) and wild-type (K) aorta. Bar, 50 µm for A-J, 20 µm for K,L.

View larger version (78K): [in a new window]   Fig. 9. Biocytin transfer is eliminated in Cx37–/–Cx40–/– aortic endothelium from E18.5 embryos and is sharply reduced in E18.5 Cx40–/– endothelium. Biocytin was injected into endothelial cells of thoracic aortas from wild-type (A), Cx37–/– (B), Cx40–/– (C) or Cx37–/–Cx40–/– (D) E18.5 embryos. Biocytin transfer occurred equally well in Cx37–/– and wild-type aortic endothelium. Dye-transfer was reduced by 96% in Cx40–/– aortas and was eliminated in Cx37–/–Cx40–/– endothelium. Quantification is presented in Fig. 10 and Table 2. Bar, 50 µm.

View larger version (14K): [in a new window]   Fig. 11. Survival curves for Cx37+/–Cx40–/– and wild-type mice. Cx37+/–Cx40–/– and wild-type mice were housed for two years and the percentage of surviving animals was plotted every two weeks. Cx37+/–Cx40–/– animals began dying earlier than wild-type mice and showed a decrease in survival after two years (13% versus 42%).