A20 inhibits oxidized low-density lipoprotein-induced apoptosis through negative Fas/Fas ligand-dependent activation of caspase-8 and mitochondrial pathways in murine RAW264.7 macrophages

A20 was originally characterized as a TNF-inducible gene in human umbilical vein endothelial cells. As an NF-kappaB target gene, A20 is also induced in many other cell types by a wide range of stimuli. Expression of A20 has been shown to protect from TNF-induced apoptosis and also functions via a negative-feedback loop to block NF-kappaB activation induced by TNF and other stimuli. To date, there are no reports on whether A20 can protect OxLDL-induced apoptosis in macrophages. For the first time we report that A20 expression blocks OxLDL-mediated cell toxicity and apoptosis. OxLDL induced the expression of Fas and FasL, and the subsequent caspase-8 cleavage and treatment with a neutralizing ZB4 anti-Fas antibody blocked apoptosis induced by OxLDL. Expression of dominant negative FADD efficiently prevented OxLDL-induced apoptosis and caspase-8 activation. A20 expression significantly attenuated the increased expression of Fas and FasL, and Fas-mediated apoptosis. These findings suggest that A20-mediated protection from OxLDL may occur at the level of Fas/FADD-caspase-8 and be FasL dependent. Treatment of RAW264.7 cells with OxLDL induces a series of time-dependent events, including the release of cytochrome c, Smac and Omi from the mitochondria to the cytosol, activation of caspase-9, -6, -2, and -3, which are blocked by A20 expression. No cleaved form of Bid was detected, even treatment with OxLDL for 48 h. Expression of dominant negative FADD also efficiently prevented OxLDL-induced the above apoptotic events. The release of cyto c, Smac and Omi from mitochondria to cytosol, activated by OxLDL treatment, and the activation of caspase-9 may not be a downstream event of caspase-8-mediated Bid cleavage. Therefore, the protective effect of A20 on mitochondrial apoptotic pathway activated by OxLDL may be dependent on FADD. A20 expression reversed OxLDL-mediated G(0)/G(1) stage arrest by maintaining the expression of cyclin B1, cyclin D1, and cyclin E, and p21 and p73. Thus, A20 expression blocks OxLDL-mediated apoptosis in murine RAW264.7 macrophages through disrupting Fas/FasL-dependent activation of caspase-8 and the mitochondria pathway.     

Protective role of glutathione synthesis in response to oxidized low density lipoprotein in human vascular endothelial cells

Impairment of endothelial cells by oxidized low density lipoprotein (OxLDL) is believed to be the first step in atherogenesis. It is also believed that oxidative stress/antioxidant imbalance is involved in the cell damage by OxLDL. However, little is known about the interaction between OxLDL and antioxidants. In this study, we show that treatment of human vascular endothelial cells with OxLDL caused a gradual increase of glutathione (gamma-glutamylcysteinyl glycine, GSH) levels in 24 h. OxLDL increased the intracellular levels of reactive oxygen species (ROS) and stimulated the expression of gamma-glutamylcysteine synthetase (gamma-GCS), the rate-limiting enzyme for the GSH synthesis, the mitogen-activated protein kinase (MAPK) activity, and the AP-1-DNA binding activity. The luciferase activity of gamma-GCS promoter containing AP-1 site was activated by OxLDL. Collectively, OxLDL induces gamma-GCS expression mediated by AP-1 resulting in an increase of GSH levels. The MAPK activity stimulated by ROS may be involved in the activation of AP-1. The increase in GSH by OxLDL may afford cellular protection against OxLDL-induced oxidative stress.     

Angiopoietin-1 inhibits intrinsic apoptotic signaling and vascular hyperpermeability following hemorrhagic shock

Studies from our laboratory demonstrated the involvement of intrinsic apoptotic signaling in hyperpermeability following hemorrhagic shock (HS). Angiopoietin 1 (Ang-1), a potent inhibitor of hyperpermeability, was recently shown to inhibit apoptosis. The purpose of our study was to determine the effectiveness of Ang-1 in attenuating HS-induced hyperpermeability and its relationship to apoptotic signaling. HS was induced in rats by withdrawing blood to reduce the mean arterial pressure to 40 mmHg for 1 h, followed by reperfusion. Mesenteric postcapillary venules were examined for changes in hyperpermeability by intravital microscopy. Mitochondrial release of second mitochondrial derived activator of caspases (smac) and cytochrome c were determined by Western blot and ELISA, respectively. Caspase-3 activity was determined by fluorometric assay. Parallel studies were performed in rat lung microvascular endothelial cell (RLMEC) monolayers, utilizing HS serum and the proapoptotic Bcl-2 homologous antagonist/killer [BAK (BH3)] peptide as inducers of hyperpermeability. In rats, Ang-1 (200 ng/ml) attenuated HS-induced hyperpermeability versus the HS group (P < 0.05). Ang-1 prevented HS-induced collapse of mitochondrial transmembrane potential (DeltaPsi(m)), smac and cytochrome c release, and caspase-3 activity (P < 0.05). In RLMEC monolayers, HS serum and BAK (BH3) peptide both induced hyperpermeability that was inhibited by Ang-1 (P < 0.05). Ang-1 attenuated HS and BAK (BH3) peptide-induced collapse of DeltaPsi(m), smac release, cytochrome c release, activation of caspase-3, and vascular hyperpermeability. In vivo, BAK (BH3) induced vascular hyperpermeability that was attenuated by Ang-1 (P < 0.05). These findings suggest that Ang-1's role in maintaining microvascular endothelial barrier integrity involves the intrinsic apoptotic signaling cascade.     

Low shear stress regulates monocyte adhesion to oxidized lipid-induced endothelial cells via an IkappaBalpha dependent pathway

In regions of a vessel that experience low shear stress and reversing flow patterns, early features in the pathogenesis of atherosclerosis include the accumulation of oxidized LDL (OxLDL) and adhesion of monocytes to endothelial cells (EC). Here we investigated the hypothesis that low shear stress (2 dyn/cm2) and OxLDL are synergistic for enhanced expression of vascular cell adhesion molecule (VCAM-1) and human aortic endothelial cell (HAEC)-monocyte adhesion. This study shows low shear stress can significantly reduce IkappaBalpha levels, activate NF-kappaB, increase the expression of VCAM-1 in HAEC and binding of monocytes. OxLDL itself cannot significantly increase the expression of VCAM-1 in HAEC and binding of monocytes, but through activation of NF-kappaB and degradation of IkappaBalpha induced by low shear stress it can significantly enhance VCAM-1 expression and monocyte adhesion, over that in unmodified LDL or control. These results suggest that low shear stress can regulate monocyte adhesion to oxidized lipid-induced endothelial cells via an IkappaBalpha-dependent pathway, and that low shear stress together with OxLDL may likely play an important role in atherogenesis.     

Cannabinoid (CB2) receptor deficiency reduces the susceptibility of macrophages to oxidized LDL/oxysterol-induced apoptosis

Macrophage apoptosis is an important process in the pathophysiology of atherosclerosis. Oxidized low-density lipoproteins (OxLDL) are a major component of lesions and potently induce macrophage apoptosis. Cannabinoid receptor 2 (CB2), the predominant macrophage cannabinoid receptor, modulates several macrophage processes associated with ongoing atherosclerosis; however, the role of CB2 in macrophage apoptosis is unknown. To determine if CB2 influences a macrophage apoptotic pathway relevant to atherosclerosis, we examined the effect of CB2 deficiency on OxLDL-induced macrophage apoptosis. In situ terminal transferase-mediated dUTP nick end labeling (TUNEL) analysis of resident peritoneal macrophages detected significantly fewer apoptotic CB2(-/-) macrophages than CB2(+/+) macrophages after incubation with OxLDL (27.9 +/- 4.7% vs. 61.9 +/- 8.5%, P < 0.001) or 7-ketocholesterol (7KC) (18.9 +/- 10.5% vs. 54.1 +/- 6.9%, P < 0.001), an oxysterol component of OxLDL. Caspase-3 activity; proteolytic conversion of procaspase-3; and cleavage of a caspase-3 substrate, PARP, were also diminished in 7KC-treated CB2(-/-) macrophages. Furthermore, the deactivation of the prosurvival kinase, Akt, in response to 7KC was impaired in CB2(-/-) macrophages. These results suggest that CB2 expression increases the susceptibility of macrophages to OxLDL-induced apoptosis, in part, by modulating the effect of oxysterols on the Akt survival pathway and that CB2 may influence atherosclerosis by modulating lesional macrophage apoptosis.     

Novel Nox inhibitor of oxLDL-induced reactive oxygen species formation in human endothelial cells

In this study, we investigated effects of a novel NAD(P)H oxidase (Nox)-inhibitor 3-benzyl-7-(2-benzoxazolyl)thio-1,2,3-triazolo[4,5-d]pyrimidine (VAS2870) on oxidized low-density lipoprotein (oxLDL)-mediated reactive oxygen species (ROS) formation in human endothelial cells. Primary cultures of human umbilical vein endothelial cells were cultured to confluence and ROS formation was induced with 50microg/ml oxLDL for 2h. ROS formation was detected by chemiluminescence (CL) using the Diogenes reagent. OxLDL induced ROS formation in human endothelial cells (171+/-12%; n=10, P<0.05 vs. control). This augmented ROS formation in response to oxLDL was completely inhibited by the Nox inhibitor VAS2870 (101+/-9%; n=7, P<0.05 vs. oxLDL). Similar results were obtained with superoxide dismutase (91+/-7%; n=7, P<0.05 vs. oxLDL). However, the Nox4 mRNA expression level was neither changed by oxLDL nor VAS2870. We conclude that VAS2870 could provide a novel strategy to inhibit the augmented endothelial superoxide anion formation in response to cardiovascular risk factors.     

Radiation attenuates physiological angiogenesis by differential expression of VEGF, Ang-1, tie-2 and Ang-2 in rat brain

The etiology of radiation-induced cerebrovascular rarefaction remains unknown. In the present study, we examined the effect of whole-brain irradiation on endothelial cell (EC) proliferation/apoptosis and expression of various angiogenic factors in rat brain. F344 × BN rats received either whole-brain irradiation (a single dose of 10 Gy γ rays) or sham irradiation and were maintained for 4, 8 and 24 h after irradiation. Double immunofluorescence staining was employed to visualize EC proliferation/apoptosis in brain. The mRNA and protein expression levels of vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), endothelial-specific receptor tyrosine kinase (Tie-2), and Ang-2 in brain were determined by real-time RT-PCR and immunofluorescence staining. A significant reduction in CD31-immunoreactive cells was detected in irradiated rat brains compared with sham-irradiated controls. Whole-brain irradiation significantly suppressed EC proliferation and increased EC apoptosis. In addition, a significant decrease in mRNA and protein expression of VEGF, Ang-1 and Tie-2 was observed in irradiated rat brains. In contrast, whole-brain irradiation significantly upregulated Ang-2 expression in rat brains. The present study provides novel evidence that whole-brain irradiation differentially affects mRNA and protein expression of VEGF, Ang-1, Tie-2 and Ang-2. These changes are closely associated with decreased EC proliferation and increased EC apoptosis in brain.     

A redox-sensitive pathway mediates oxidized LDL-induced downregulation of insulin-like growth factor-1 receptor

Oxidized low density lipoprotein (OxLDL) has multiple proatherogenic effects, including induction of apoptosis. We have recently shown that OxLDL markedly downregulates insulin-like growth factor-1 receptor (IGF-1R) in human aortic smooth muscle cells, and that IGF-1R overexpression blocks OxLDL-induced apoptosis. We hypothesized that specific OxLDL-triggered signaling events led to IGF-1R downregulation and apoptosis. We examined OxLDL signaling pathways and found that neither IGF-1R downregulation nor the proapoptotic effect was blocked by inhibition of OxLDL-triggered extracellular signal-regulated kinase, p38 mitogen-activated protein kinase (MAPK), or peroxisome proliferator-activated receptor gamma (PPARgamma) signaling pathways, as assessed using specific inhibitors. However, antioxidants, polyethylene glycol catalase, superoxide dismutase, and Trolox completely blocked OxLDL downregulation of IGF-1R and OxLDL-induced apoptosis. Nordihydroguaiaretic acid, AA-861, and baicalein, which are lipoxygenase inhibitors and also have antioxidant activity, blocked IGF-1R downregulation and apoptosis as well as reactive oxygen species (ROS) production. These results suggest that OxLDL enhances ROS production possibly through lipoxygenase activity, leading to IGF-1R downregulation and apoptosis. Furthermore, anti-CD36 scavenger receptor antibody markedly inhibited OxLDL-induced IGF-1R downregulation and apoptosis as well as ROS production. In conclusion, our data demonstrate that OxLDL downregulates IGF-1R via redox-sensitive pathways that are distinct from OxLDL signaling through MAPK- and PPARgamma-involved pathways but may involve a CD36-dependent mechanism.     

Crystalline silica induces apoptosis in human endothelial cells in vitro

We investigated whether incubation of cultured human aortic endothelial cells (HAEC) with crystalline silica at the concentration 1 cm2/ml (chosen on the basis of a pilot experiment) leads to alterations typical of apoptosis. The binding of annexin V as early, and DNA fragmentation as late events of apoptosis were measured besides the number of cells with depolarized mitochondria. The generation of reactive oxygen species (ROS) by HAEC in presence of silica was determined as well as silica ability to in vitro generate hydroxyl radicals was investigated. After 18 h of silica incubation, about 30% of viable cells bound annexin V. After 24 h of silica treatment, the percentage of cells with fragmented DNA (Tunel positive) was 27% and it increased up to 50% after 48 h, whereas in untreated cells this percentage was 7% and 11% after 24 and 48 h, respectively. The presence of fragmented DNA in cells treated with silica was confirmed by agarose gel electrophoresis. In agreement with these results showing an induction of HAEC apoptosis by silica incubation, the number of cells with depolarized mitochondria was significantly higher after silica treatment as compared to the control. Apoptosis was also obtained with silica added to aliquots of anti-C5a-absorbed-medium. In the cells exposed to silica there was a significant increasing of ROS generation in comparison to the untreated cells. Apoptosis might be due to peroxidative stress since silica can generate hydroxyl radicals.     

Overexpression of angiopoietin-2 impairs myocardial angiogenesis and exacerbates cardiac fibrosis in the diabetic db/db mouse model

The angiopoietins/Tie-2 system is essential for the maintenance of vascular integrity and angiogenesis. The functional role of angiopoietin-2 (Ang-2) in the regulation of angiogenesis is dependent on other growth factors such as VEGF and a given physiopathological conditions. This study investigates the potential role of Ang-2 in myocardial angiogenesis and fibrosis formation in the diabetic db/db mouse. Diabetic db/db mice received intramyocardial administration of either adenovirus Ang-2 (Ad-CMV-Ang-2) or Ad-β-gal. The levels of Tie-2, VEGF, caspase-3, Wnt7b, fibroblast-specific protein-1 (FSP-1), and adhesion molecules (ICAM-1 and VCAM-1) expression were measured. Apoptosis, capillary density, and cardiac fibrosis were also analyzed in the db/db mouse hearts. Overexpression of Ang-2 suppressed Tie-2 and VEGF expression in db/db mouse hearts together with significant upregulation of Wnt7b expression. Overexpression of Ang-2 also sensitizes ICAM-1 and VCAM-1 expression in db/db mouse hearts. Immunohistochemical analysis revealed that overexpression of Ang-2 resulted in a gradual apoptosis as well as interstitial fibrosis formation, these leading to a significant loss of capillary density. Data from these studies were confirmed in cultured mouse heart microvascular endothelial cells (MHMEC) exposed to excessive Ang-2. Exposure of MHMEC to Ang-2 resulted in increased caspase-3 activity and endothelial apoptosis. Knockdown of Ang-2 attenuated high glucose-induced endothelial cell apoptosis. Further, counterbalance of Ang-2 by overexpression of Ang-1 reversed loss of capillary density and fibrosis formation in db/db mouse hearts. Our data demonstrate that Ang-2 increases endothelial apoptosis, sensitizes myocardial microvascular inflammation, and promotes cardiac fibrosis and thus contributes to loss of capillary density in diabetic diseases.     

Signaling and regulation of endothelial cell survival by angiopoietin-2

Angiopoietins are ligands for endothelial cell-specific Tie-2 receptors. Whereas angiopoietin-1 (Ang-1) activates these receptors and promotes cell survival, migration, and sprouting, little information is available regarding how Ang-2 influences these cells. In this study, we evaluated signaling pathways and biological effects of physiological concentrations of Ang-2 in cultured human umbilical vein endothelial cells. Ang-2 at 150 and 300 ng/ml elicited a transient (reaching peak values within 15 min of exposure) increase in the phosphorylation of Tie-2 receptors, protein kinase B (Akt), ERK1/2, and p38 members of the mitogen-activated protein kinases. However, unlike Ang-1, Ang-2 significantly inhibited JNK/SAPK phosphorylation. When vascular endothelial growth factor (VEGF) was present along with Ang-2, ERK1/2 phosphorylation was inhibited, whereas augmentation of Ang-1-induced ERK1/2 phosphorylation was triggered by VEGF. Ang-2 treatment had no effect on cell migration and in vitro wound healing but significantly attenuated serum deprivation-induced apoptosis and promoted survival. These effects were completely reversed by phosphatidylinositol 3 (PI3)-kinase and ERK1/2 inhibitors but were augmented by an inhibitor of the p38 pathway. These results suggest that Ang-2 promotes endothelial cell survival through the ERK1/2 and PI3-kinase pathways and that this angiopoietin is not a strong promoter of endothelial cell migration. We also conclude that the nature of interactions in terms of ERK1/2 activation between Ang-2 and VEGF is different from that of Ang-1 and VEGF.     

Angiotensin-(1-7) Attenuates Kidney Injury Due to Obstructive Nephropathy in Rats

Background

Angiotensin-(1–7) [Ang-(1–7)] counteracts many actions of the renin-angiotensin-aldosterone system. Despite its renoprotective effects, extensive controversy exists regarding the role of Ang-(1–7) in obstructive nephropathy, which is characterized by renal tubulointerstitial fibrosis and apoptosis.

Methods

To examine the effects of Ang-(1–7) in unilateral ureteral obstruction (UUO), male Sprague-Dawley rats were divided into three groups: control, UUO, and Ang-(1–7)-treated UUO rats. Ang-(1–7) was continuously infused (24 μg/[kg·h]) using osmotic pumps. We also treated NRK-52E cells in vitro with Ang II (1 μM) in the presence or absence of Ang-(1–7) (1 μM), Mas receptor antagonist A779 (1 μM), and Mas receptor siRNA (50 nM) to examine the effects of Ang-(1–7) treatment on Ang II-stimulated renal injury via Mas receptor.

Results

Angiotensin II (Ang II) and angiotensin type 1 receptor (AT₁R) protein expression was higher in UUO kidneys than in controls. Ang-(1–7) treatment also decreased proapoptotic protein expression in UUO kidneys. Ang-(1–7) also significantly ameliorated TUNEL positive cells in UUO kidneys. Additionally, Ang-(1–7) reduced profibrotic protein expression and decreased the increased tumor growth factor (TGF)-β1/Smad signaling present in UUO kidneys. In NRK-52E cells, Ang II induced the expression of TGF-β1/Smad signaling effectors and proapoptotic and fibrotic proteins, as well as cell cycle arrest, which were attenuated by Ang-(1–7) pretreatment. However, treatment with A779 and Mas receptor siRNA enhanced Ang II-induced apoptosis and fibrosis. Moreover, Ang II increased tumor necrosis factor-α converting enzyme (TACE) and decreased angiotensin-converting enzyme 2 (ACE2) expression in NRK-52E cells, while pretreatment with Ang-(1–7) or A779 significantly inhibited or enhanced these effects, respectively.

Conclusion

Ang-(1–7) prevents obstructive nephropathy by suppressing renal apoptosis and fibrosis, possibly by regulating TGF-β1/Smad signaling and cell cycle arrest via suppression of AT₁R expression. In addition, Ang-(1–7) increased and decreased ACE2 and TACE expression, respectively, which could potentially mediate a positive feedback mechanism via the Mas receptor.     

Apoptosis induced by oxidized low density lipoprotein in human monocyte-derived macrophages involves CD36 and activation of caspase-3.

Macrophage death may play a crucial role in the progression of atherosclerotic lesions. Here we present evidence that CD36 is involved in oxidized LDL (OxLDL)-induced apoptosis in human monocyte-derived macrophages. Anti-CD36 mAb SMO and OKM-5 reduced the number of apoptotic cells in OxLDL-treated macrophages by more than 94%, but they did not block ceramide-triggered apoptosis. Thrombospondin inhibited the induction of apoptosis by OxLDL in a dose-dependent manner with an IC50 of 10-30 microM. OxLDL did not induce apoptosis in CD36-negative macrophages, demonstrating the essential role of this scavenger receptor in OxLDL-triggered programmed cell death. Neither anti-CD36 Ig nor thrombospondin triggered programmed cell death suggesting that binding to CD36 alone is not sufficient to initiate apoptosis. However, inhibitors of OxLDL-induced apoptosis did not block the uptake of 3H-labeled OxLDL. In contrast, acetylated LDL and polyinosinic acid, ligands of scavenger receptor A (SRA), inhibited uptake of 3H-labeled OxLDL by 65 and 49%, respectively, but did not block OxLDL-induced apoptosis, indicating that SRA is not involved in this process. OxLDL also stimulated caspase-3 activity in human macrophages. Activation of caspase-3 was blocked by anti-CD36 Ig and the caspase-3 inhibitor Z-DEVD-FMK. These results suggest that binding of OxLDL to CD36 initiates a yet unknown OxLDL-specific signaling event, which leads to the rapid activation of caspase-3 resulting in apoptosis of human macrophages. Our data demonstrate a novel role for CD36 in macrophage biology with likely consequences for the development of atherosclerotic lesions.     

A moderate reduction in extracellular pH protects macrophages against apoptosis induced by oxidized low density lipoprotein

We investigated the effect of pH on macrophage apoptosis induced by oxidized low density lipoprotein (OxLDL), as human atherosclerotic lesions have regions of low pH. Hydroperoxide-rich and oxysterol-rich LDL caused 38% and 74% apoptosis of J774 macrophages, respectively, at 24 h, as measured by the externalization of phosphatidylserine. Native LDL, however, did not cause apoptosis. Reducing the pH of the culture medium from 7.4 to 7.0 inhibited apoptosis induced by hydroperoxide-rich or oxysterol-rich OxLDL by 61% and 46%, respectively (P < 0.001). These data were confirmed by semiquantitative analysis of cytochrome c release from mitochondria. Decreasing the extracellular pH to 7.0 reduced the uptake of hydroperoxide-rich and oxysterol-rich (125)I-labeled LDL by 82% and 42%, respectively, and reduced cell surface binding of oxysterol-rich LDL by 31%. This may explain the reduced apoptosis. Additionally, low pH did not affect OxLDL-induced apoptosis of human monocytes, which do not possess scavenger receptors for OxLDL, but reduced apoptosis of human monocyte-derived macrophages, which do possess them. Our investigations suggest that the presence of areas of low pH within atherosclerotic lesions may reduce the uptake of OxLDL and reduce macrophage apoptosis, thus affecting lesion progression.     

Angiopoietin-1 treatment reduces inflammation but does not prevent ventilator-induced lung injury

Background

Loss of integrity of the epithelial and endothelial barriers is thought to be a prominent feature of ventilator-induced lung injury (VILI). Based on its function in vascular integrity, we hypothesize that the angiopoietin (Ang)-Tie2 system plays a role in the development of VILI. The present study was designed to examine the effects of mechanical ventilation on the Ang-Tie2 system in lung tissue. Moreover, we evaluated whether treatment with Ang-1, a Tie2 receptor agonist, protects against inflammation, vascular leakage and impaired gas exchange induced by mechanical ventilation.

Methods

Mice were anesthetized, tracheotomized and mechanically ventilated for 5 hours with either an inspiratory pressure of 10 cmH₂O (‘low’ tidal volume ∼7.5 ml/kg; LV_T) or 18 cmH₂O (‘high’ tidal volume ∼15 ml/kg; HV_T). At initiation of HV_T-ventilation, recombinant human Ang-1 was intravenously administered (1 or 4 µg per animal). Non-ventilated mice served as controls.

Results

HV_T-ventilation influenced the Ang-Tie2 system in lungs of healthy mice since Ang-1, Ang-2 and Tie2 mRNA were decreased. Treatment with Ang-1 increased Akt-phosphorylation indicating Tie2 signaling. Ang-1 treatment reduced infiltration of granulocytes and expression of keratinocyte-derived chemokine (KC), macrophage inflammatory protein (MIP)-2, monocyte chemotactic protein (MCP)-1 and interleukin (IL)-1β caused by HV_T-ventilation. Importantly, Ang-1 treatment did not prevent vascular leakage and impaired gas exchange in HV_T-ventilated mice despite inhibition of inflammation, vascular endothelial growth factor (VEGF) and Ang-2 expression.

Conclusions

Ang-1 treatment downregulates pulmonary inflammation, VEGF and Ang-2 expression but does not protect against vascular leakage and impaired gas exchange induced by HV_T-ventilation.     

Rosiglitazone attenuates NF-κB-mediated Nox4 upregulation in hyperglycemia-activated endothelial cells

Vascular complications, a major cause of morbidity and mortality in diabetic patients, are related to hyperglycemia-induced oxidative stress. Previously, we reported that rosiglitazone (RSG) attenuated vascular expression and activity of NADPH oxidases in diabetic mice. The mechanisms underlying these effects remain to be elucidated. We hypothesized that RSG acts directly on endothelial cells to modulate vascular responses in diabetes. To test this hypothesis, human aortic endothelial cells (HAECs) were exposed to normal glucose (NG; 5.6 mmol/l) or high glucose (HG; 30 mmol/l) concentrations. Select HAEC monolayers were treated with RSG, caffeic acid phenethyl ester (CAPE), diphenyleneiodonium (DPI), small interfering (si)RNA (to NF-κB/p65 or Nox4), or Tempol. HG increased the expression and activity of the NADPH oxidase catalytic subunit Nox4 but not Nox1 or Nox2. RSG attenuated HG-induced NF-κB/p65 phosphorylation, nuclear translocation, and binding to the Nox4 promoter. Inhibiting NF-κB with CAPE or siNF-κB/p65 also reduced HG-induced Nox4 expression and activity. HG-induced H(2)O(2) production was attenuated by siRNA-mediated knockdown of Nox4, and HG-induced HAEC monocyte adhesion was attenuated by treatment with RSG, DPI, CAPE, or Tempol. These results indicate that HG exposure stimulates HAEC NF-κB activation, Nox4 expression, and H(2)O(2) production and that RSG attenuates HG-induced oxidative stress and subsequent monocyte-endothelial interactions by attenuating NF-κB/p65 activation and Nox4 expression. This study provides novel insights into mechanisms by which the thiazolidinedione peroxisome proliferator-activated receptor-γ ligand RSG favorably modulates endothelial responses in the diabetic vasculature.     

Lipoprotein aggregation protects human monocyte-derived macrophages from OxLDL-induced cytotoxicity

Oxidative modifications render low density lipoprotein cytotoxic and enhance its propensity to aggregate and fuse into particles similar to those found in atherosclerotic lesions. We showed previously that aggregation of oxidized LDL (OxLDL) promotes the transformation of human macrophages into lipid-laden foam cells (Asmis, R., and J. Jelk. 2000. Large variations in human foam cell formation in individuals. A fully autologous in vitro assay based on the quantitative analysis of cellular neutral lipids. Atherosclerosis. 148: 243-253). Here, we tested the hypothesis that aggregation of OxLDL enhances its clearance by human macrophages and thus may protect macrophages from OxLDL-induced cytotoxicity. We found that increased aggregation of OxLDL correlated with decreased macrophage injury. Using 3H-labeled and Alexa546-labeled OxLDL, we found that aggregation enhanced OxLDL uptake and increased cholesteryl ester accumulation but did not alter free cholesterol levels in macrophages. Acetylated LDL was a potent competitor of aggregated oxidized LDL (AggOxLDL) uptake, suggesting that scavenger receptor A plays an important role in the clearance of AggOxLDL. Inhibitors of actin polymerization, cytochalasin B, cytochalasin D, and latrunculin A, also prevented AggOxLDL uptake and restored OxLDL-induced cytotoxicity. This suggests that OxLDL-induced macrophage injury does not require OxLDL uptake and may occur on the cell surface. Our data demonstrate that aggregation of cytotoxic OxLDL enhances its clearance by macrophages without damage to the cells, thus allowing macrophages to avoid OxLDL-induced cell injury.