Gender-specific remodeling in atrial fibrillation?

BACKGROUND: The authors wanted to investigate whether the remodeling process in AF regarding gap junction proteins, collagen I, and amyloid may be gender dependent in humans. METHODS: In total, 123 patients with sinus rhythm (SR, n = 41) or atrial fibrillation (AF, n = 82) suffering from mitral valve disease undergoing cardiac surgery were included. Of the 123 patients, 66 patients (SR: n = 17, AF: n = 49) were investigated biochemically for the expression of the atrial gap junction proteins connexin40 (Cx40), connexin43 (Cx43) and collagen I and 57 patients (SR: n = 24; AF: n = 33) using histochemical methods for possible amyloid depositions. RESULTS: AF led to increased levels of Cx40, Cx43, and collagen I protein. Regarding Cx40 this upregulation was significantly higher in female than in male patients. For AF-induced changes in collagen or Cx43, there were no significant gender-dependent differences. Amyloid depositions were found with increasing age, but were not significantly related to AF or gender. CONCLUSIONS: Remodeling in AF seems to be similar in men and women, with a tendency for women exhibiting somewhat stronger AF-induced changes in Cx40, which is probably a secondary effect because there is nothing known about hormone sensitivity of the Cx40 promoter, and a not significant tendency for higher Cx43 and collagen I.

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.

Arterial flow reduces oxidative stress via an antioxidant response element and Oct-1 binding site within the NADPH oxidase 4 promoter in endothelial cells.

The main sources of oxidative stress in the vessel wall are nicotine adenine dinucleotide phosphate (NADPH) oxidase (Nox) complexes. The endothelium mainly expresses the Nox4-containing complex; however, the mechanism by which shear stress in endothelial cells regulates Nox4 is not well understood. This study demonstrates that long-term application of arterial laminar shear stress using a cone-and-plate viscometer reduces endothelial superoxide anion formation and Nox4 expression. In primary human endothelial cells, we identified a 47 bp 5'-untranslated region of Nox4 mRNA by 5'-rapid amplification of cDNA ends (5'-RACE) PCR. Cloning and functional analysis of human Nox4 promoter revealed a range between -1,490 and -1,310 bp responsible for flow-dependent downregulation. Mutation of an overlapping antioxidative response element (ARE)-like and Oct-1 binding site at -1,376 bp eliminated shear stress-dependent Nox4 downregulation. Consistent with these observations, electrophoretic mobility shift assays (EMSA) demonstrated an enhanced shear stress-dependent binding of Nox4 oligonucleotide containing the ARE-like/Oct-1 binding site, which could be inhibited by specific antibodies against the transcription factors nuclear factor erythroid 2-related factor 2 (Nrf2) and octamer transcription factor 1 (Oct-1). Furthermore, shear stress caused the translocation of Nrf2 and Oct-1 from the cytoplasm to the nucleus. Knockdown of Nrf2 by short hairpin RNA (shRNA) increased Nox4 expression twofold, indicating a direct cross-talk between Nrf2 and Nox4. In conclusion, an ARE-like/Oct-1 binding site was noticed to be essential for shear stress-dependent downregulation of Nox4. This novel mechanism may be involved in the flow-dependent downregulation of endothelial superoxide anion formation.

No upregulation of lectin-like oxidized low-density lipoprotein receptor-1 in serum-deprived EA.hy926 endothelial cells under oxLDL exposure, but increase in autophagy.

The oxidized low-density lipoprotein (oxLDL)-dependent activation of the lectin-like oxLDL receptor-1 (LOX-1) triggers apoptosis in vascular cells and appears to be involved in atherosclerosis. Autophagy might be an alternate to apoptosis in endothelial cells. The EA.hy926 endothelial cell line has been reported to undergo necrosis under oxLDL stimulation. For this reason, we studied the expression of LOX-1 and its oxLDL-dependent function in EA.hy926 cells under serum starvation. Untreated and oxLDL-treated cells expressed the LOX-1 protein at similar levels 6h after starvation. After 24h without oxLDL and with native LDL (nLDL), statistically significant higher levels were found in LOX-1 than in the oxLDL-treated probes. The oxLDL cultures with low LOX-1 expression displayed stronger features of autophagy than those with nLDL as there were remodelling of actin filaments, disrupture of adherens junctions (immunofluorescence staining), and autophagosomes with the characteristic double membrane at the ultrastructural level. For the advanced oxLDL exposure times (18 and 24 h), autophagic vacuoles/autophagolysosomes were morphologically identified accompanied by a decrease in lysosomes. The autophagosome marker protein MAP LC3-II (Western blotting) was significantly augmented 6 and 18 h after oxLDL treatment compared with cultures treated with nLDL and medium alone. Signs of apoptosis were undetectable in cultures under oxLDL exposure, yet present under staurosporin (apoptosis inducer), i.e. presence of apoptotic bodies and cleaved caspase 3. We conclude that serum starvation upregulates LOX-1 in EA.hy926 cells, whereas the additional oxLDL treatment downregulates the receptor and intensifies autophagy probably by increase in oxidative stress.

NO-mediated regulation of NAD(P)H oxidase by laminar shear stress in human endothelial cells.

The flowing blood generates shear stress at the endothelial cell surface. In endothelial cells, NAD(P)H oxidase complexes have been identified as major sources of superoxide anion (.O(2)(-)) formation. In this study, we analysed the effect of laminar shear stress on .O(2)(-) formation by cytochrome c reduction assay and on NAD(P)H oxidase subunit expression by standard calibrated competitive reverse transcription-polymerase chain reaction and Western blot in human endothelial cells. Primary cultures of human umbilical vein endothelial cells were exposed to laminar shear stress in a cone-and-plate viscometer for up to 24 h. Short-term application of shear stress transiently induced .O(2)(-) formation. This was inhibited by NAD(P)H oxidase inhibitor gp91ds-tat, but NAD(P)H oxidase subunit expression was unchanged. Long-term arterial laminar shear stress (30 dyne cm(-2), 24 h) down-regulated .O(2)(-) formation, and mRNA and protein expression of NAD(P)H oxidase subunits Nox2/gp91(phox) and p47(phox). In parallel, endothelial NO formation and eNOS, but not Cu/Zn SOD, protein expression was increased. Down-regulation of .O(2)(-) formation, gp91(phox) and p47(phox) expression by long-term laminar shear stress was blocked by l-NAME. NO donor DETA-NO down-regulates .O(2)(-) formation, gp91(phox) and p47(phox) expression in static cultures. In conclusion, our data suggest a transient activation of .O(2)(-) formation by short-term shear stress, followed by a down-regulation of endothelial NAD(P)H oxidase in response to long-term laminar shear stress. NO-mediated down-regulation by shear stress preferentially affects the gp91(phox)/p47(phox)-containing NAD(P)H oxidase complex. This mechanism might contribute to the regulation of endothelial NO/.O(2)(-) balance and the vasoprotective potential of physiological levels of laminar shear stress.

Lectin-like oxidized low-density lipoprotein receptor-1-mediated autophagy in human granulosa cells as an alternative of programmed cell death.

The LOX-1 receptor, identified on endothelial cells, mediates the uptake of oxidized low-density lipoprotein (oxLDL). The oxLDL-dependent LOX-1 activation causes endothelial cell apoptosis. We here investigated the presence of LOX-1 in granulosa cells from patients under in vitro fertilization therapy. We were interested in the oxLDL-dependent LOX-1 receptor biology, in particular in the induction of apoptosis. In the human ovary, LOX-1 was localized in regressing antral follicles. In granulosa cell cultures, oxLDL-induced mRNA expression of LOX-1 in a time- and dose-dependent manner. The LOX-1 inhibitors (anti-LOX-1 antibody and kappa-carrageenan) abrogated the up-regulation of LOX-1. The oxLDL (100 microg/ml) treatment caused the autophagy form of programmed cell death: 1) reorganization of the actin cytoskeleton at the 6-h time point; 2) uptake of YO-PRO, a marker for the early step of programmed cell death, before propidium iodide staining to signify necrosis; 3) absence of apoptotic bodies and cleaved caspase-3; 4) abundant vacuole formation at the ultrastructural level; and 5) decrease of the autophagosome marker protein MAP LC3-I at the 6-h time point indicative of autophagosome formation. We conclude that follicular atresia is not under the exclusive control of apoptosis. The LOX-1-dependent autophagy represents an alternate form of programmed cell death. Obese women with high blood levels of oxLDL may display an increased rate of autophagic granulosa cell death.

NADPH oxidase in endothelial cells: impact on atherosclerosis.

An elevated vascular superoxide anion formation has been implicated in the initiation and progression of hypertension and atherosclerosis. In this review, we would like to discuss the generation of superoxide anions by an NADPH oxidase complex in vascular cells. Special focus is on the induction of endothelial NADPH oxidase by proatherosclerotic stimuli. We propose a proatherosclerotic vicious cycle of increased NADPH oxidase-dependent superoxide anion formation, augmented generation and uptake of oxidatively modified low-density lipoprotein, and further potentiation of oxidative stress by oxidized low-density lipoprotein itself, angiotensin II, and endothelin-1 in endothelial cells. Furthermore, novel homologues of NADPH oxidase subunit gp91(phox) are summarized. Future directions of research for a better understanding of the role of NADPH oxidase in the pathogenesis of atherosclerosis and clinical implications are discussed.

Augmented endothelial uptake of oxidized low-density lipoprotein in response to endothelin-1.

Endothelin-1 (ET-1) may be involved in the development and progression of atherosclerosis. Furthermore, endothelin receptor blockade was shown to reduce the formation of atherosclerotic lesions in experimental studies. Another potent pro-atherosclerotic risk factor is oxidized low-density lipoprotein (oxLDL). Endothelial cells mediate the uptake of oxLDL by the recently identified lectin-like oxLDL receptor-1 (LOX-1), which accumulates in atherosclerotic lesions. In the present study, we analysed the effects of ET-1 on oxLDL uptake and LOX-1 expression in primary cultures of human umbilical vein endothelial cells (HUVEC). ET-1 stimulated uptake of oxLDL in HUVEC, which reached a maximum after 1 h. In further studies, we found a similar induction of LOX-1 mRNA and protein expression in response to ET-1. The augmented oxLDL uptake and the increased LOX-1 expression in response to ET-1 are mediated by the endothelin receptor B. Our data support a new pathophysiological mechanism by which locally and systemically increased ET-1 levels, e.g. in hypertensive patients, could promote LOX-1-mediated oxLDL uptake in human endothelial cells. This mechanism could promote the development and progression of endothelial dysfunction and atherosclerosis. In addition, endothelin receptor blockade could be considered as a new anti-atherosclerotic therapeutic principle.