Increased connexin43 expression in human saphenous veins in culture is associated with intimal hyperplasia Sébastien Déglise, MD, David Martin, MS, Hervé.

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Increased connexin43 expression in human saphenous veins in culture is associated with intimal hyperplasia Sébastien Déglise, MD, David Martin, MS, Hervé Probst, MD, François Saucy, MD, Daniel Hayoz, MD, Gérard Waeber, MD, Pascal Nicod, MD, Hans-Beat Ris, MD, Jean-Marc Corpataux, MD, Jacques-Antoine Haefliger, PhD Journal of Vascular Surgery Volume 41, Issue 6, Pages 1043-1052 (June 2005) DOI: 10.1016/j.jvs.2005.02.036 Copyright © 2005 The Society for Vascular Surgery Terms and Conditions

Fig 1 Upper panel, Development of intimal hyperplasia in human saphenous vein segments after 14 days in culture. A, Compared with control vein, (B) intimal and subintimal space thickness, corresponding to intimal hyperplasia (IH) is markedly increased (van Gieson-elastin staining) in human venous segment after 14 days in culture. C and D, Immunolabeling of the sections shown in A and B, with antibodies against α-actin (brown color) revealed the presence of smooth muscle cell proliferation in the neointima (arrows in A, B, C, and D, represent the lamina elastica interna). E and F, Immunolabeling with antibodies against the factor VIII demonstrated the presence of endothelial cells along the vessel. Original magnification, ×400 in A, B, C, D and ×200 for E and F. L, Lumen; I, intima; IH, intimal hyperplasia; M, media. Lower panel, Quantitative assessment of six measurements (one per human vein segment) of intimal and subintimal space thickness, corresponding to intimal hyperplasia, was significantly increased in vein segments incubated in culture for 14 days compared with controls (Native); **P < .01. Journal of Vascular Surgery 2005 41, 1043-1052DOI: (10.1016/j.jvs.2005.02.036) Copyright © 2005 The Society for Vascular Surgery Terms and Conditions

Fig 2 Connexin43 (Cx43) is increased at both mRNA and protein levels in veins after 14 days in culture. A, Multiple connexin transcripts are expressed in the human saphenous vein wall. Reverse-transcribed human saphenous vein RNA was amplified by polymerase chain reaction (PCR) using primers specific for human Cx37, Cx40, Cx43, and Cx45. The amplification of four amplicons corresponding to Cx37, Cx40, Cx43, and Cx45 are shown after ethidium bromide staining. These products were not amplified in samples that were not reverse-transcribed before PCR processing (–). This experiment was performed in triplicate with different human vein segments. B, Expression of Cx43 mRNA analyzed by quantitative real-time PCR is increased in vein that is developing intimal hyperplasia. Quantitative assessment of six to seven measurements (one per human vein segment) showed that Cx43 mRNA was increased sixfold in vein segments cultured during a period of 14 days compared with control veins (Native). Values represent ratios between Cx43 and β2-microglobulin mRNAs expression. Data are expressed as mean ± SEM; ***P < .001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. C and D, Western blot analysis of total proteins showed that the levels of Cx43 were markedly increased (about 10-fold) in vein after 14 days in culture compared with control levels. Data are expressed as mean ± SEM; P < .001. BA, bovine aorta. Under both conditions, Cx43 was detected as two bands of 43–44 kDa. Each lane represents a sample from a different vein. All lanes were loaded with 75 μg total proteins. In the same samples, levels of tubulin were not modified. As positive control, 25 μg of freshly scraped bovine aortic endothelium was used. Journal of Vascular Surgery 2005 41, 1043-1052DOI: (10.1016/j.jvs.2005.02.036) Copyright © 2005 The Society for Vascular Surgery Terms and Conditions

Fig 3 Cx43 protein levels change during the development of intimal hyperplasia in human saphenous vein. Western blot analysis of total proteins content revealed that the expression of connexin43 (Cx43) in venous samples progressively increased during the first 3 days of organ culture. In the same samples, no change in the expression of Cx40 was observed during culture period. All lanes were loaded with 75 μg of total proteins. Each lane represents a sample from a different vein. As positive controls, 25 μg of bovine aorta (BA) endothelial proteins and 40 μg of rat lung (RL) proteins were used. Levels of tubulin proteins were not modified. Journal of Vascular Surgery 2005 41, 1043-1052DOI: (10.1016/j.jvs.2005.02.036) Copyright © 2005 The Society for Vascular Surgery Terms and Conditions

Fig 4 Connexin43 (Cx43) is increased in smooth muscle cells of human saphenous vein in culture. Upper panel, Phase-contrast views of sections of human saphenous vein before (B) and after14 days in culture (D, F). Immunofluorescence labeling with specific antibodies located Cx43 at discrete spots dispersed throughout the media of the vein wall (A). A sizable increase in the number and pattern of these spots was noticed in human vein that was developing hyperplasia after 14 days in culture (C, E). The limited amount of Cx43 expressed by the endothelial cells is visible in panel E. Cx43 expression in smooth muscle located in the intimal hyperplasia cells is visible on panel C and G. Immunofluorescence labeling with Cx40 antibodies reflected the presence of Cx40 only in the endothelial cells (H). L, Lumen; I, intima; IH, intimal hyperplasia; M, media. Original magnification: ×400. Lower panel, Immunofluorescence labeling of serial sections of the human saphenous vein before (A, B) and after 14 days in culture (C, D). Alpha-smooth muscle actin was localized in the smooth muscle cells of the human vein (A, C) and demonstrated that Cx43 is located in the human vein smooth muscle cells (B and D). Cx43 and α-smooth muscle staining revealed that the increase in the number of Cx43 immunofluorescent spots observed after 14 days in culture occurred in the smooth muscle cells (B, D). Arrows indicate the presence of α-smooth muscle actin in A and C and Cx43 in B and D. L, Lumen; M, media. Original magnification, ×400. Journal of Vascular Surgery 2005 41, 1043-1052DOI: (10.1016/j.jvs.2005.02.036) Copyright © 2005 The Society for Vascular Surgery Terms and Conditions

Fig 5 Fluvastatin decreased intimal hyperplasia and connexin43 (Cx43) levels in human saphenous veins in culture. A, Quantitative assessment of six measurements (one per human vein segment) of intimal thickness, corresponding to intimal hyperplasia, was significantly decreased in vein segments after 14 days in culture in the presence of fluvastatin compared with control veins cultured without fluvastatin. **P < .01. B, Western blot revealed that the levels of Cx43 were decreased in vein segments incubated in the presence of fluvastatin compared with controls. C, Quantitative evaluation of six immunoblots (one per vein) revealed that the levels of Cx43 were decreased threefold to fourfold in veins incubated with fluvastatin. **P < .01. Journal of Vascular Surgery 2005 41, 1043-1052DOI: (10.1016/j.jvs.2005.02.036) Copyright © 2005 The Society for Vascular Surgery Terms and Conditions