Knock-down of endothelial connexins impairs angiogenesis

Connexins (Cx) are suggested to play important roles in growth and differentiation. Aim of our study was to investigate the role of endothelial Cx in the angiogenic process. Several parameters of angiogenesis were assessed in 18 h Matrigel in vitro angiogenesis assays with human umbilical vein endothelial cells (HUVEC). Prior to culture on Matrigel cells were treated with nicotine or the gap junction inhibitor palmitoleic acid (PA), or siRNA-knock-down of either Cx37, Cx40 or Cx43 was performed. Changes in Cx expression and their effects on gap-junctional communication were investigated using immunofluorescence microscopy, Western blot and Lucifer Yellow dye transfer. Knock-down of each Cx-isoform significantly reduced the amount of specific Cx protein in HUVEC. Cx-knock-down as well as treatment with PA impaired intercellular communication via gap junctions and diminished significantly the number of capillary branches. Knock-down of Cx43 and Cx40 or treatment with PA reduced complexity pattern in the angiogenesis assay. Nicotine significantly reduced expression of Cx43 and Cx37 as well as average length of capillary branches, number of branches and pattern in the Matrigel assay. We can conclude that connexins are involved in angiogenesis, in particular in branch formation. This can partly explain the changes in angiogenesis seen under nicotine treatment.     

Chronic regulation of the expression of gap junction proteins connexin40, connexin43, and connexin45 in neonatal rat cardiomyocytes

Gap junction channels form the basis of intercellular communication in the heart. In the working myocardium, the connexin43 (Cx43) is most abundantly found, whereas connexin40 (Cx40) is expressed in the atria and in the conduction system [together with low levels of connexin45 (Cx45)]. However, little is known about the differential regulation of the connexins by pathophysiologically stimuli such as tumor necrosis factor alpha (TNFalpha). Inasmuch as TNFalpha may play a contributory role in the concert of factors involved in the pathophysiology of heart failure and because this cardiac disease often leads to ventricular reentrant arrhythmia, the goal of our study was to find out whether TNFalpha may influence the expression of the cardiac connexins connexin43, connexin40, and connexin45. Neonatal rat cardiomyocytes were exposed to TNFalpha (10, 40, 100, 400, and 1000 pg/ml) for 24 h with or without additional treatment with the mitogenic-activated protein kinase (MAP-kinase) inhibitors SB203580 [4-(4-fluorophenyl)-2-(4-methyl-sulfinylphenyl)-5-(4-pyridyl)-1H-imidazole; 10(-5) M, protein38 mitogenic-activated protein kinase (p38 MAP kinase) inhibitor] or the MEK1 (mitogenic-activated protein kinase/extracellular signal-regulated kinase kinase) inhibitor PD98059 [2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; 10(-5) M]. Connexin43, connexin40, and connexin45 expressions were analysed using Western blot analysis, immunohistology, and polymerase chain reaction (PCR) studies (connexin43 and connexin40). TNFalpha induced a concentration-dependent increase in connexin43 (by 2.9+/-0.6, P<0.05, n=5) but not in connexin40 or connexin45 expressions. Both connexins (40 and 45) showed a very low expression near the detection limit. The increases in connexin43 expression could be completely suppressed by SB203580 (0.9+/-0.4, P<0.05, n=5) but not by PD98059. In absence of a stimulating drug, these inhibitors (SB203580 or PD98059) did not affect connexin43 content. Additional PCR experiments revealed increases in connexin43 mRNA under the influence of 100 pg/ml TNFalpha (211+/-38%, P<0.05, n=5), which could be completely suppressed by SB203580. In contrast, the connexin40 expression remained unchanged. From these results, we conclude that TNFalpha can differentially regulate cardiac connexin expression via p38 MAP kinase pathway and thus may alter intercellular communication. This may contribute to the changes observed in heart failure with regard to the formation of an arrhythmogenic substrate.     

Assessment of immunosuppressive drug interactions: inhibition of lymphocyte function in peripheral human blood

Cyclosporin (CsA) or tacrolimus (TRL) is routinely combined with either sirolimus (SRL) or mycophenolate mofetil (MMF) in immunosuppressive regimes in organ transplantation. The aim of our study was to establish a specific human blood assay of lymphocyte function in order to assess interactions of these drug combinations. Different concentrations (10(6)-10(9) nM) of CsA, TRL, SRL or mycophenolic acid (MPA, the active metabolite of MMF) was added to whole blood of five human volunteers. Drug combinations were studied by adding 250, 500 or 1000 nM of MPA to different concentrations of CsA, TRL, or SRL or by adding 1, 10 or 25 nM of SRL to different concentrations of CsA or TRL. After concanavalin-A stimulation, whole blood cultures were analyzed by flow cytometry detecting lymphocyte proliferation and activation by bivariate expression of proliferating cell nuclear antigen (PCNA)/DNA content and T cell-surface activation antigens (e.g. CD25, CD95, and CD154). We found an order of potency inhibiting lymphocyte function with SRL>TRL>CsA>MPA. In addition, we observed enhanced inhibition of PCNA, CD25, CD95 or CD154, if either CsA or TRL was combined with low concentrations of MPA, or SRL alone or if SRL was combined with low concentrations of MPA. Data analysis revealed an independent functional synergism or partial agonism in most combinations. This human blood assay is able to assess lymphocyte function and to monitor immunosuppressive therapy. The assay also permits pharmacological analysis of drug interactions, which will lead to improved safety and therapeutic efficacy in transplanted patients.     

Comparing the ultrastructural effects of two different cardiac preparation- and perfusion-techniques in a porcine model of extracorporal long-term preservation.

OBJECTIVE: In heart transplantation a well-preserved myocardial ultrastructure is an important precondition for functional regeneration. Aim of the study is to optimize the conditions in this new established model of extracorporeal cardiac perfusion. METHODS: (I) In six pigs, hearts were arrested with Bretschneider Histidine-Tryptophan-Ketoglutarate cardioplegia and cold ischemia, explanted and connected to a circulating constant pressure Langendorff system (80-90mmHg) and perfused with leukocyte depleted autologous blood. (II) Beating hearts of seven pigs were explanted and connected immediately to the Langendorff system (40-50mmHg). Myocardial biopsies (n=55) were taken in situ and during the following 12h of reperfusion, and were prepared for electron microscopy. RESULTS: Cardioplegia and hypothermia (group I) induced mitochondrial edema and myofibrillar degeneration in cardiomyocytes and severe endothelial edema. During 4h of reperfusion, mitochondrial edema, myofibrillar, and sarcolemmal damages in cardiomyocytes increased. Moderate endothelial degeneration, interstitial edema, and bleedings appeared. In contrast, in group II after 6h of reperfusion endothelia showed only mild alterations. Cardiomyocytes showed myofibrillary but not mitochondrial degeneration. Interstitial edema and bleedings were mild. CONCLUSION: Avoiding cardioplegia and hypothermia, and using lower perfusion pressure resulted in a better preservation of the ultrastructure in explanted hearts at the Langendorff system.     

Cardiac fibroblasts inhibit β-adrenoceptor-dependent connexin43 expression in neonatal rat cardiomyocytes

Cardiac fibroblasts play an important role in adverse cardiac remodelling. As in many cardiac diseases connexin43 (Cx43) is altered, we wanted to elucidate whether fibroblasts may influence cardiac Cx43 expression. We used four different cell culture systems of neonatal rat cardiomyocytes (CM) and fibroblasts (FB): type 1, pure CM culture; type 2, co-culture of CM/FB; type 3, pure FB culture; type 4, Transwell® system: CM/FB co-cultured but separated by a microporous membrane. Stimulation of types 1–3 cell culture models with isoprenaline significantly enhanced Cx43-protein and Cx43-mRNA expression as well as phosphorylation of ERK and translocation of AP1 and CREB only in the CM cultures; whereas, the CM/FB co-cultures and the FB cultures did not respond to isoprenaline. Similarly, if CM and FB were separated by a microporous membrane (Transwell® system) the isoprenaline-induced increase in CM Cx43 was completely suppressed, suggesting the existence of a soluble factor responsible for the suppressant effect of FB. Angiotensin II determination in types 1 and 2 cell culture supernatants revealed that the CM/FB co-cultures exhibited a significant higher angiotensin II release than the CM cultures. Furthermore, we aimed to inhibit angiotensin II signal transduction pathway: blockade of AT₁ receptors or PKC inhibition restored the responsiveness of CM/FB co-cultures to isoprenaline. Moreover, external addition of angiotensin II to CM cultures also resulted in suppression of isoprenaline-stimulated Cx43 expression in an AT₁-receptor- and PKC-dependent manner. Thus, our study indicates that cardiac fibroblasts inhibit β-adrenoceptor-dependent Cx43 signalling in CM involving angiotensin II.