Autocrine control of angiogenesis by endogenous acetylcholine in an in vitro model using human endothelial cells: evidence for an autocrine cholinergic system in endothelial cells.

We wanted to elucidate whether acetylcholine as the endogenous ligand at cholinoceptors (ChRs) may have effects on angiogenesis and whether they are transduced through muscarinic or nicotinic ChRs. Human umbilical vein endothelial cells were cultured until confluence and thereafter seeded in Matrigel in vitro angiogenesis assays for 18 hours. During the entire cell culture and angiogenesis period, cells were treated with vehicle, eserine (1 µM), in the absence or presence of additional atropine (1 µM) or mecamylamine (1 µM). Finally, the resulting angiogenetic network was investigated histologically. Eserine significantly enhanced acetylcholine formation. When acetylcholine acted through muscarinic ChRs (eserine + mecamylamine), we observed enhanced complexity of the angiogenic network pattern with increased tube length and cell number. In contrast, when acting through nicotinic ChRs (eserine + atropine), we found reduced complexity of pattern with less branches, shorter tubes, and reduced cell number. If acting on both types of ChRs (eserine alone), there were only very small effects. Using α-bungarotoxin, lobeline, and dihydro-ß-erythroidine, we also could show that these effects to various degrees involve α7, α3/ß2, and α4/ß2 n-ChRs. In conclusion, our results support the hypothesis that human umbilical vein endothelial cells possess an autocrine nonneuronal cholinergic system regulating angiogenesic branch formation through the partially opposing effects of n-ChRs and m-ChRs.

Nicotine effects on human endothelial intercellular communication via α4ß2 and α3ß2 nicotinic acetylcholine receptor subtypes.

Since previous in vitro experiments revealed that nicotine can impair endothelial intercellular communication via the downregulation of connexin43 (Cx43), we wanted to find out which nicotinic acetylcholine receptors are involved in the molecular mechanism of communication failure. Cultured human endothelial cells were exposed to 1 µM nicotine for 5 days. Intercellular communication was measured using dye transfer study with/without subtype-specific nicotinic acetylcholine receptor (nAChR) inhibitors. Reverse transcriptase (RT)-PCR was used to further investigate the regulation of nAChR subtypes. Electron microscopy together with MAP LC3-II western blot was used to investigate possible autophagy processes. In cultured human endothelial cells, nicotine decreased the Cx43 protein amount as shown by western blot and immunohistochemistry; however, together with an unaltered mRNA expression as shown by RT-PCR. The nicotine-induced Cx43 downregulation functionally impaired intercellular dye transfer, which could be prevented by mecamylamine, κ-bungarotoxin, lobeline, and dihydro-ß-erythroidine but not α-bungarotoxin, indicating that the nAChR subtypes α4ß2 and α3ß2 but not α7 are involved in signal cascade. RT-PCR analysis revealed that nicotine exposure resulted in the upregulation of α3 and ß4 and the downregulation of α4-nAChR, while α7- and ß2-nAChR-mRNA expressions remained unaltered. Furthermore, nicotine increased total protein ubiquinylation and proteasome activity as was shown by immunohistochemistry and peptide degradation analysis. Evidence of enhanced autophagic processes was assured by the occurrence of autophagic vacuoles in transmission electron microscopy and enhanced formation of MAP LC3-II in western blot. Reduced intercellular endothelial communication together with programmed cell death helps to explain the toxic effect of nicotine leading to endothelial dysfunction. The nAChR involved in the impairment of intercellular communication seem to be α4ß2 and α3ß2 but not α7.