A role for 4-hydroxy-2-nonenal in premature placental senescence in preeclampsia and intrauterine growth restriction.

Premature placental senescence is a hallmark of pregnancy-related disorders such as intrauterine growth restriction (IUGR) and preeclampsia (PE), two major cause of maternal and neonatal morbidity and mortality. Oxidative stress and lipid peroxidation are involved in the pathogenesis of PE and IUGR, and may play a role in placental aging. In this study, we investigated whether 4-hydroxy-2-nonenal (HNE), a lipid peroxidation-derived aldehyde present in preeclamptic placentas, may contribute to premature senescence in placenta-related complications. Placentas from PE-affected women, exhibited several senescence patterns, such as an increased expression of phosphorylated (serine-139) histone γH2AX, a sensitive marker of double-stranded DNA breaks, the presence of lipofuscin granules, and an accumulation of high molecular weight cross-linked and ubiquitinated proteins. PE placentas showed an accumulation of acetylated proteins consistent with the presence of HNE-adducts on sirtuin 1 (SIRT1). Likewise, oxidative stress and senescence markers together with SIRT1 modification by HNE, were observed in murine placentas from mice treated with lipopolysaccharide during gestation and used as models of IUGR. The addition of HNE and ONE (4-oxo-2-nonenal), to cultured HTR-8/SVneo human trophoblasts activated the senescence-associated- ß-galactosidase, and generated an accumulation of acetylated proteins, consistent with a modification of SIRT1 by HNE. Altogether, these data emphasize the role of HNE and lipid peroxidation-derived aldehydes in premature placental senescence in PE and IUGR, and more generally in pathological pregnancies.

Sphingomyelinase Disables Inactivation in Endogenous PIEZO1 Channels.

Endogenous PIEZO1 channels of native endothelium lack the hallmark inactivation often seen when these channels are overexpressed in cell lines. Because prior work showed that the force of shear stress activates sphingomyelinase in endothelium, we considered if sphingomyelinase is relevant to endogenous PIEZO1. Patch clamping was used to quantify PIEZO1-mediated signals in freshly isolated murine endothelium exposed to the mechanical forces caused by shear stress and membrane stretch. Neutral sphingomyelinase inhibitors and genetic disruption of sphingomyelin phosphodiesterase 3 (SMPD3) cause PIEZO1 to switch to profoundly inactivating behavior. Ceramide (a key product of SMPD3) rescues non-inactivating channel behavior. Its co-product, phosphoryl choline, has no effect. In contrast to ceramide, sphingomyelin (the SMPD3 substrate) does not affect inactivation but alters channel force sensitivity. The data suggest that sphingomyelinase activity, ceramide, and sphingomyelin are determinants of native PIEZO gating that enable sustained activity.

nSMase2 (Type 2-Neutral Sphingomyelinase) Deficiency or Inhibition by GW4869 Reduces Inflammation and Atherosclerosis in Apoe-/- Mice.

OBJECTIVE: Atherosclerosis is a chronic multifactorial and inflammatory disease of large and medium arteries and the leading cause of cardiovascular diseases worldwide. The aim of this study was to investigate whether and how the nSMase2 (type 2-neutral sphingomyelinase), a key enzyme of sphingolipid metabolism, may contribute to the development of atherosclerotic lesions. APPROACH AND RESULTS: The role of nSMase2 in atherosclerosis was investigated in Apoe-/-;Smpd3fro/fro mice, mutant for nSMase2, and in Apoe-/-;Smpd3+/+ mice intraperitoneally injected with GW4869, a pharmacological nSMase2 inhibitor. The defect or inhibition of nSMase2 resulted in a reduction of atherosclerotic lesions and a decrease in macrophage infiltration and lipid deposition, although cholesterolemia remained unchanged. nSMase2 inhibition decreased the inflammatory response of murine endothelial cells to oxLDL (oxidized low-density lipoprotein), as assessed by the significant reduction of MCP-1 (monocyte chemoattractant protein 1), ICAM-1 (intercellular adhesion molecule-1), and VCAM-1 (vascular cell adhesion molecule-1) mRNA expressions and macrophage recruitment. Likewise, in RAW264.7 or in macrophages isolated from Apoe-/-/Smpd3fro/fro or Apoe-/-/Smpd3+/+ mice stimulated by lipopolysaccharides, nSMase2 inhibition resulted in a decrease in the expression of inflammatory molecules. Mechanistically, the anti-inflammatory response resulting from nSMase2 inhibition involves Nrf2 (nuclear factor [erythroid-derived 2]-like 2 or NF-E2-related factor-2) activation in both endothelial cells and macrophages, as assessed by the lack of protective effect of GW4869 in endothelial cells silenced for Nrf2 by small interfering RNAs, and in lipopolysaccharide-stimulated macrophages issued from Nrf2-KO mice. CONCLUSIONS: The genetic deficiency or inhibition of nSMase2 strongly decreases the development of atherosclerotic lesions in Apoe-/- mice, by reducing inflammatory responses through a mechanism involving the Nrf2 pathway. Inhibitors of nSMase2 may, therefore, constitute a novel approach to slow down atherosclerosis progression.

Proatherogenic effects of 4-hydroxynonenal.

4-hydroxy-2-nonenal (HNE) is a α,ß-unsaturated hydroxyalkenal generated by peroxidation of n-6 polyunsaturated fatty acid. This reactive carbonyl compound exhibits a huge number of biological properties that result mainly from the formation of HNE-adducts on free amino groups and thiol groups in proteins. In the vascular system, HNE adduct accumulation progressively leads to cellular dysfunction and tissue damages that are involved in the progression of atherosclerosis and related diseases. HNE contributes to the atherogenicity of oxidized LDL, by forming HNE-apoB adducts that deviate the LDL metabolism to the scavenger receptor pathway of macrophagic cells, and lead to the formation of foam cells. HNE activates transcription factors (Nrf2, NF-kappaB) that (dys)regulate various cellular responses ranging from hormetic and survival signaling at very low concentrations, to inflammatory and apoptotic effects at higher concentrations. Among a variety of cellular targets, HNE can modify signaling proteins involved in atherosclerotic plaque remodeling, particularly growth factor receptors (PDGFR, EGFR), cell cycle proteins, mitochondrial and endoplasmic reticulum components or extracellular matrix proteins, which progressively alters smooth muscle cell proliferation, angiogenesis and induces apoptosis. HNE adducts accumulate in the lipidic necrotic core of advanced atherosclerotic lesions, and may locally contribute to macrophage and smooth muscle cell apoptosis, which may induce plaque destabilization and rupture, thereby increasing the risk of athero-thrombotic events.

Hyaluronan synthase-2 upregulation protects smpd3-deficient fibroblasts against cell death induced by nutrient deprivation, but not against apoptosis evoked by oxidized LDL.

The neutral type 2 sphingomyelinase (nSMase2) hydrolyzes sphingomyelin and generates ceramide, a major bioactive sphingolipid mediator, involved in growth arrest and apoptosis. The role of nSMase2 in apoptosis is debated, and apparently contradictory results have been observed on fibroblasts isolated from nSMase2-deficient fragilitas ossium (homozygous fro/fro) mice. These mice exhibit a severe neonatal dysplasia, a lack of long bone mineralization and delayed apoptosis patterns of hypertrophic chondrocytes in the growth plate. We hypothesized that apoptosis induced by nutrient deprivation, which mimics the environmental modifications of the growth plate, requires nSMase2 activation. In this study, we have compared the resistance of fro/fro fibroblasts to different death inducers (oxidized LDL, hydrogen peroxide and nutrient starvation). The data show that nSMase2-deficient fro/fro cells resist to apoptosis evoked by nutrient starvation (fetal calf serum/glucose/pyruvate-free DMEM), whereas wt fibroblasts die after 48h incubation in this medium. In contrast, oxidized LDL and hydrogen peroxide are similarly toxic to fro/fro and wt fibroblasts, indicating that nSMase2 is not involved in the mechanism of toxicity evoked by these agents. Interestingly, wt fibroblasts treated with the SMase inhibitor GW4869 were more resistant to starvation-induced apoptosis. The resistance of fro/fro cells to starvation-induced apoptosis is associated with an increased expression of hyaluronan synthase 2 (HAS2) mRNAs and protein, which is inhibited by ceramide. In wt fibroblasts, this HAS2 rise and its protective effect did not occur, but exogenously added HA exhibited a protective effect against starvation-induced apoptosis. The protective mechanism of HAS2 involves an increased expression of the heat-shock protein Hsp72, a chaperone with antiapoptotic activity. Taken together, these results highlight the role of nSMase2 in apoptosis evoked by nutrient starvation that could contribute to the delayed apoptosis of hypertrophic chondrocytes in the growth plate, and emphasize the antiapoptotic properties of HAS2.

Defects in gallbladder emptying and bile Acid homeostasis in mice with cystic fibrosis transmembrane conductance regulator deficiencies.

BACKGROUND & AIMS: Patients with cystic fibrosis (CF) have poorly defined defects in biliary function. We evaluated the effects of cystic fibrosis transmembrane conductance regulator (CFTR) deficiency on the enterohepatic disposition of bile acids (BAs). METHODS: Bile secretion and BA homeostasis were investigated in Cftr(tm1Unc) (Cftr-/-) and CftrΔF508 (ΔF508) mice. RESULTS: Cftr-/- and ΔF508 mice did not grow to normal size, but did not have liver abnormalities. The gallbladders of Cftr-/- mice were enlarged and had defects in emptying, based on (99m)technetium-mebrofenin scintigraphy or post-prandial variations in gallbladder volume; gallbladder contraction in response to cholecystokinin-8 was normal. Cftr-/- mice had abnormal gallbladder bile and duodenal acidity, and overexpressed the vasoactive intestinal peptide-a myorelaxant factor for the gallbladder. The BA pool was larger in Cftr-/- than wild-type mice, although there were no differences in fecal loss of BAs. Amounts of secondary BAs in portal blood, liver, and bile of Cftr-/- mice were much lower than normal. Expression of genes that are induced by BAs, including fibroblast growth factor-15 and BA transporters, was lower in the ileum but higher in the gallbladders of Cftr-/- mice, compared with wild-type mice, whereas enzymes that synthesize BA were down-regulated in livers of Cftr-/- mice. This indicates that BAs underwent a cholecystohepatic shunt, which was confirmed using cholyl-(Ne-NBD)-lysine as a tracer. In Cftr-/- mice, cholecystectomy reversed most changes in gene expression and partially restored circulating levels of secondary BAs. The ΔF508 mice overexpressed vasoactive intestinal peptide and had defects in gallbladder emptying and in levels of secondary BAs, but these features were less severe than in Cftr-/- mice. CONCLUSIONS: Cftr-/- and CftrΔF508 mice have defects in gallbladder emptying that disrupt enterohepatic circulation of BAs. These defects create a shunt pathway that restricts the amount of toxic secondary BAs that enter the liver.

Influence of hydroxyapatite microstructure on human bone cell response.

Microstructure of calcium phosphate ceramics has been shown to influence long-term in vitro cellular events like proliferation and differentiation, and to favor bone integration in vivo. As long-term cellular events are known to be dependent of early cell adhesion events, we decided to study the in vitro influence of the microstructure of a microporous hydroxyapatite (mHA) and a nonmicroporous hydroxyapatite (pHA) ceramic on serum protein adsorption and SaOs-2 human bone cells attachment after 30 min, 1, 4, and 24 h and cell growth after 96 h. Plastic coverslips were used as controls. Hydroxyapatite composition of mHA and pHA was confirmed by X-ray diffraction and Fourier transform infra-red spectroscopy. The surface energies of ceramics were calculated from contact-angle measurements in di-iodomethane, water or complete culture medium. The total surface energy was 44.8 mJ/m(2) for pHA and 48.7 mJ/m(2) for plastic. The contact-angle measurement was impossible on mHA likely because they displayed 12% of open microporosity, pHA ceramic exhibiting only closed pores (2.5%). Moreover, the roughness amplitude was largely higher on mHA (Sa = 4.35 microm) than on pHA (Sa = 0.065 microm) and plastic (Sa = 0.042 microm). Three different techniques were used to evaluate protein adsorption on the ceramics. SDS-PAGE of desorbed proteins demonstrated that more proteins desorbed from mHA (66.02 microg/m(2)) than from pHA (17.2 microg/m(2)) or plastic (0.08 microg/m(2)). A new method was used to evaluate in situ the quantity of adsorbed total proteins: the temperature-programmed desorption (TPD) analysis coupled with mass spectrometry. The TPD analysis confirmed that 10-fold more proteins adsorbed on mHA compared with those on pHA. A direct immunolabeling on ceramics revealed than more fibronectin and serum albumin adsorbed on microporous ceramic than on dense ceramic. The morphology of SaOs-2 cells was the same on all the substrates after 30 min. At later time points, cell morphology on mHA was radically different than on other surfaces, with the particularity of the cytoplasmic edge that appeared undistinguishable from the surface. Only the extremity of the cells and lamellipodia were visible. Cells seemed like "adsorbed" by the mHA surface, whereas on plastic and pHA surfaces the cells displayed classical aspects of polygonal spreading. The cells displayed on mHA the highest initial attachment potential after 30 min, 1, 4, 24 h but the lower proliferation potential after four days. This study confirms that a microporous ceramic surface can modulate the adsorption of proteins and further the adhesion and proliferation of human bone cells.

Quantitative kinetic analysis of gene expression during human osteoblastic adhesion on orthopaedic materials.

Little information was found in the literature about the expression on hydroxyapatite (HA) materials of genes specific of cellular adhesion molecules although more were found on titanium-based substrates. Hence, the goal of this work was to study by a kinetic approach from 30 min to 4 days the adhesion of Saos-2 cells on microporous (mHA) and non-microporous hydroxyapatite (pHA) in comparison to polished titanium. Our strategy associated the visualization of adhesion proteins inside the cells by immunohistochemistry and the quantitative expression of genes at mRNA level by real-time PCR. The cell morphology was assessed using scanning electron microscopy and the number of cells thanks to biochemical techniques. The cellular attachment was the highest on mHA from 30 min to 24 h although the cell growth on mHA was the lowest after 4 days. Generally, the Saos-2 osteoblastic cells morphology on mHA was radically different than on other surfaces with the particularity of the cytoplasmic edge, which appeared un-distinguishable from the surface. The revelation by specific antibodies of proteins of the cytoskeleton (actin) and the focal adhesions (FAK, phosphotyrosine) confirmed that adhesion and spreading were different on the 3 materials. The actin stress fibres were less numerous and shorter on mHA ceramics. Cells had more focal contacts after 4 h on mHA compared to other substrates but less after 24 h. The highest values of total proteins were extracted from mHA at 0.5 and 24 h and from pHA at 1, 4, and 96 h. The alphav and beta1 integrin, actin, FAK, and ERK gene expression were found to be different with adhesion time and with materials. C-jun expression was comparable on mHA, titanium and plastic but was largely higher than on pHA at 0.5 and 1 h. On the contrary, c-fos expression was the highest on pHA after 0.5 h and the lowest after 1h. This difference between c-fos and c-jun expression on pHA after 0.5 h could be related to the fact that these two genes may differ in their signalling pathways. The expression of the alkaline phosphatase gene after 4 days was lower on mHA compared to other materials demonstrating that the microstructure of the mHA ceramic was not favourable to Saos-2 cells differentiation. Finally, it was demonstrated in this study that HA and titanium surfaces influence as well gene expression at early times of adhesion as the synthesis of adhesion proteins but also proliferation and differentiation phases. Indeed, the signal transduction pathways involved in adhesion of Saos-2 cells on HA and titanium were confirmed by the sequential expression of alphav and beta1 integrins, FAK, and ERK genes followed by the expression of c-jun and c-fos genes for proliferation and alkaline phosphatase gene for differentiation.

The influence of culture conditions on extracellular matrix proteins synthesized by osteoblasts derived from rabbit bone marrow.

The influence of culture conditions on the extracellular matrix (ECM) protein expressions of rabbit bone marrow stromal cells has been studied. The focus was on the effects of two kinds of sera, fetal calf serum (FCS) and Ultroser, on cells treated with dexamethasone. The induction of osteoblastic differentiation by dexamethasone addition is confirmed, particularly when cells are cultured in FCS. Bone marrow stromal cells produce alkaline phosphatase positive CFU-F and produce ECM with some mineralized nodules. Analysis by means of two-dimensional gel electrophoresis showed important changes in the composition of ECM proteins after dexamethasone treatment. Overexpression, underexpression, and new synthesized proteins were observed. The most significant modification was linked to the synthesis of four new proteins visible in the acidic area with a low molecular weight of around 17 kDa. These proteins did not correspond to those ECM proteins known to be induced by dexamethasone. Moreover, the effect of dexamethasone on osteoblastic differentiation induction appears very limited when cells are cultured in Ultroser compared to FCS. The protein pattern with Ultroser is different to that obtained with FCS. Cells cultured in Ultroser synthesized no new protein. The different behavior of cells according to the type of medium used is discussed in terms of the osteogenic factors present in the two different sera.