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.

Reno-protective effects of epigallocatechingallate in a small piglet model of extracorporeal circulation.

Cardiopulmonary bypass still often is a necessary tool in cardiac surgery in particular in the correction of congenital heart defects in small infants. Nevertheless, among the complications linked to extracorporeal circulation (ECC) with cardiopulmonary bypass (CPB) in both infants and adults one of the most serious problems is renal impairment. Since this might be caused by ischemia/reperfusion injury and accumulation of free radicals, we used (-)-epigallocatechin-3-gallate (EGCG), a derivate from green tea, which is known to possess antioxidant, antiapoptotic and NO-scavenging properties in order to find out whether EGCG may protect the kidney. 23 four-week-old Angler Sattelschwein-piglets (8-15 kg) were divided into three groups: control-group (n=7), ECC-group (n=10), EGCG-group (n=6). The ECC- and EGCG-group were thoracotomized and underwent CPB for 120 min followed by a 90-min recovery-time. The EGCG-group received 10 mg/kg EGCG before and after CPB. Histology revealed that CPB led to widening of Bowman's capsule, and to vacuolization of proximal tubular cells (p<0.05) which could be prevented by EGCG (p<0.05). Using immunohistology, we found significant nuclear translocation of hypoxia-inducible-factor-1-alpha (HIF-1-alpha) and increased nitrotyrosine formation in the ECC-group. Both were significantly (p<0.05) inhibited by EGCG. ECC-induced loss of energy-rich phosphates was prevented by EGCG. In blood samples we found that CPB resulted in increases in creatinine and urea (in serum) and led to loss of total protein (p<0.05), which all was not present in EGCG-treated animals. We conclude that CPB causes damage in the kidney which can be attenuated by EGCG.

On the different roles of AT1 and AT2 receptors in stretch-induced changes of connexin43 expression and localisation.

Cyclic mechanical stretch (CMS) and angiotensin II (ATII) play an important role in cardiac remodelling. Thus, we aimed to examine how ATII affects CMS-induced changes in localisation and expression of the gap junction protein connexin43 (Cx43). Neonatal rat cardiomyocytes cultured on gelatin-coated Flexcell cell culture plates were kept static or were exposed to CMS (110 % of resting length, 1 Hz) for 24 h with or without additional ATII (0.1 µmol/L). Moreover, inhibitors of ATII receptors (AT-R) were used (for AT(1)-R: losartan 0.1 µmol/L, for AT(2)-R: PD123177 0.1 µmol/L). Thereafter, the cardiomyocytes were investigated by immunohistology, PCR and Western blot. After 24 h of CMS, cardiomyocytes were significantly elongated and orientated 75 ± 1.6° nearly perpendicular to the stretch axis. Furthermore, CMS significantly accentuated Cx43 at the cell poles (ratio Cx43 polar/lateral static: 2.32 ± 0.17; CMS: 10.08 ± 3.2). Additional ATII application significantly reduced Cx43 polarisation (ratio Cx43 polar/lateral ATII: 4.61 ± 0.42). The combined administration of ATII and losartan to CMS further reduced Cx43 polarisation to control levels, whilst the AT(2)-R blocker PD123177 restored polarisation. Moreover, CMS and ATII application resulted in a significant Cx43 protein and Cx43 mRNA up-regulation which could be blocked by losartan but not by PD123177. Thus, CMS results in a self-organisation of the cardiomyocytes leading to elongated cells orientated transversely towards the stretch axis with enhanced Cx43 expression and Cx43 accentuation at the cell poles. ATII enhances total Cx43 mRNA and protein expression probably via AT(1)-R (=inhibitory effect of losartan) and reduces Cx43 polarisation presumably via AT(2)-R, since PD123177 (but not losartan) inhibited the negative effects of ATII on polarisation.

Granular Media Calcinosis in the Aortic Walls of Patients With Bicuspid and Tricuspid Aortic Valves.

BACKGROUND: Bicuspid aortic valve (BAV), the most frequent congenital cardiac abnormality, is associated with a higher risk for ascending aortic aneurysms and aorta-related complications (ie, dissection and rupture). The aim of this study was to quantify granular media calcinosis (GMC) in the ascending aortic wall of patients with BAV. METHODS: We analyzed samples of the ascending aorta from patients with BAV (n = 54) and patients with tricuspid aortic valve (TAV) (n = 33) who underwent aortic repair, regarding medial thickness and diameter expansion. Additionally, the convexity and concavity of the samples were stained for GMC and elastin fragmentation. RESULTS: The quantitative analysis revealed a significantly higher extent of GMC in patients with BAV at the aortic convexity and concavity compared with patients with TAV, independent of aortic diameter. Additionally, GMC increased with enlargement of the aortic diameter in patients with BAV. Furthermore, we found a significantly reduced total medial thickness in patients with BAV compared with patients with TAV. CONCLUSIONS: Our findings highlight GMC as a prominent feature of bicuspid aortopathy.

Protective effects of pulsatile flow during cardiopulmonary bypass.

BACKGROUND: Children with congenital heart disease are often operated at a very young age. Cardiopulmonary bypass (CPB) has become indispensable for these sometimes very complex operations, but one cannot neglect a possible negative impact of CPB on organ function. Traditionally, CPB was developed with non-pulsatile flow but there are clinical observations that pulsatile flow might be superior with improved patient outcomes. Therefore, we attempted to elucidate whether CPB with pulsatile flow preserves organ integrity compared with nonpulsatile flow. METHODS: We studied 27 piglets of 4 weeks age and divided them into 3 experimental groups: control group (no CPB); non-pulsatile and pulsatile-CBP with 90-minutes CPB and 120-minutes recovery and reperfusion. Thereafter, histology of kidney, liver, and hippocampus was performed. Moreover, we measured adenosine triphosphate (ATP) content in these organs. RESULTS: Histologic evaluation revealed that laminar flow produced significant cellular edema in the kidney and hippocampus. Additionally, markers for hypoxia, apoptosis, and nitrosative stress were elevated but predominately in the hippocampus and proximal tubules of the kidney. Most of these alterations were reduced to or near control levels with pulsatile CPB. Moreover, ATP content of all 3 organs examined was higher and kidney and liver enzymes were lower in the pulsatile group compared with the non-pulsatile CPB. With regard to histologic changes, the liver seemed to be a less sensitive organ. CONCLUSIONS: In our study during pulsatile CPB, organ damage was significantly attenuated as compared with non-pulsatile CPB. Therefore, in pediatric patients pulsatile CPB may improve clinical outcomes.

Hippocampal Neuroprotection by Minocycline and Epigallo-Catechin-3-Gallate Against Cardiopulmonary Bypass-Associated Injury.

Surgical correction of congenital cardiac malformations mostly implies the use of cardiopulmonary bypass (CPB). However, a possible negative impact of CPB on cerebral structures like the hippocampus cannot be neglected. Therefore, we investigated the effect of CPB on hippocampus CA1 and CA3 regions without or with the addition of epigallocatechin-3-gallate (EGCG) or minocycline. We studied 42 piglets and divided them into six experimental groups: control without or with EGCG or minocycline, CPB without or with EGCG or minocycline. The piglets underwent 90 minutes CPB and subsequently, a 120-minute recovery and reperfusion phase. Thereafter, histology of the hippocampus was performed and the adenosine triphosphate (ATP) content was measured. Histologic evaluation revealed that CPB produced a significant peri-cellular edema in both CA regions. Moreover, we found an increased number of cells stained with markers for hypoxia, apoptosis and nitrosative stress. Most of these alterations were significantly reduced to or near to control levels by application of EGCG or minocycline. ATP content was significantly reduced within the hippocampus after CPB. This reduction could not be antagonized by EGCG or minocycline. In conclusion, CPB had a significant negative impact on the integrity of hippocampal neural cells. This cellular damage could be significantly attenuated by addition of EGCG or minocycline.

Organ-protective effects on the liver and kidney by minocycline in small piglets undergoing cardiopulonary bypass.

Cardiopulmonary bypass (CPB) often is required for the operative correction of congenital heart defects in small infants. Unfortunately, CPB is associated with injury of inner organs such as the brain, kidney, lung, and liver. Renal failure and increase in liver enzymes are typical side effects observed after CPB. Here, we investigate whether organ protection of the kidney and liver can be achieved with the application of minocycline, which is known-besides its anti-infective effects-to act as a poly-ADP-ribose-polymerase inhibitor. Twenty-nine 4-week-old Angler Sattelschwein-piglets (8-15 kg) were divided into four groups: control group (n = 8), CPB group (n = 9), minocycline-control group (n = 6), and the minocycline-CPB group (n = 6). CPB groups were thoracotomized and underwent CPB for 120 min (cross-clamp, 90 min; reperfusion, 30 min) followed by a 90-min recovery time. The control groups also were thoracotomized but not connected to CPB. The minocycline group received 4 mg/kg minocycline before and 2 mg/kg after CPB. In the kidneys, CPB histologically resulted in widening of Bowman's capsule, and-mainly in tubules-formation of poly-ADP-ribose, nitrosylation of tyrosine-residues, nuclear translocation of hypoxia-induced factor HIF-1α, and of apoptosis-inducing factor (AIF). In addition, we found significantly less ATP in the kidney and significantly increased plasma urea and creatinine. Similar but gradually attenuated changes were found in the liver together with significantly elevated de-Ritis coefficient. These changes in the kidney and liver were significantly diminished by minocycline (except AIF in the liver which was similar in all groups). In conclusion, CPB causes damage in the kidney and-to a lower degree-in the liver, which can be attenuated by minocycline.

Assay validation of phosphorylated S6 ribosomal protein for a pharmacodynamic monitoring of mTOR-inhibitors in peripheral human blood.

BACKGROUND: Therapeutic drug monitoring (TDM) of immunosuppressive drugs after organ transplantation is based on measuring blood levels alone, which often results in under- or over-immunosuppression. Previous studies have shown the potential of measuring pharmacodynamic drug effects for TDM, but assessment of biomarkers for individual drugs is still not clinical routine. Therefore, we validated a specific assay to measure the pharmacodynamic effects of mammalian target of rapamycin (mTOR)-inhibitors on phosphorylated S6 ribosomal protein (p-S6RP), a downstream target of mTOR. METHODS: Clinical relevant concentrations of sirolimus (SRL, 0.9-91.4 µg/L), cyclosporine A (CsA, 75.1-1202 µg/L), mycophenolate acid (MPA, 0.08-3.2 mg/L), or dexamethasone (DEX, 0.5-200 ng/mL) were added to whole-blood from healthy volunteers. Activated whole-blood was analyzed by phospho-flow cytometry to measure p-S6RP in T cells. RESULTS: Phospho-flow analysis revealed that SRL suppressed p-S6RP in human T cells in a dose-dependent manner with a half-maximal inhibitory concentration (IC(50)) at 19.8 nM and a maximal inhibitory effect (I(max) %) at 91.9%. Neither CsA, MPA, nor DEX inhibited mTOR-related S6RP-phosphorylation. Coefficient of variations from 0.03 to 0.05, 0.12 to 0.25, and 0.14 to 0.38 for intra-, interassay, and interindividual variability respectively, showed robustness of our assay. Furthermore, samples can be stored at RT or 4°C up to 2 h after withdrawal. CONCLUSION: We validated a robust whole-blood assay that allows the specific measurement of SRL- and everolimus-induced inhibition of T cells' function through detection of p-S6RP. Future studies in organ transplanted recipients will show if this assay has the potential to enhance a TDM for mTOR-inhibitor drugs in combination therapies.