4-HNE Staining Research Articles

Pressure Overload-Induced Transient Oxidative Stress Mediates Perivascular Inflammation and Cardiac Fibrosis through Angiotensin II

Oxidative stress is implicated in the pathogenesis of various cardiovascular diseases. We have shown that in Wistar rats with a suprarenal aortic constriction (AC), pressure overload-induced transient perivascular inflammation (monocyte chemoattractant protein-1 [MCP-1] induction and macrophage accumulation) in the early phase is the determinant of reactive myocardial fibrosis and resultant diastolic dysfunction in the late phase. Thus, we investigated the role of reactive oxygen species production in cardiac remodeling in AC rats. Superoxide production and the footprint of lipid peroxidation were assessed using dihydroethidium staining and immunohistostaining against 4-hydroxy-2-nonenal (4-HNE), respectively. In sham rats, dihydroethidium and 4-HNE signals were scarcely found in the heart. At day 3, AC rats showed dihydroethidium signals mainly in the intramyocardial arterial wall, whereas modest 4-HNE staining was observed diffusely in the myocardium. These signals declined to lower levels by day 14 despite sustained hypertension. Chronic administration of a subdepressor dose of an angiotensin II type 1 receptor blocker candesartan reduced the pressure overload-induced dihydroethidium and 4-HNE signals at day 3. Moreover, candesartan decreased MCP-1 induction and macrophage infiltration at day 3 and prevented myocardial fibrosis at day 14, without affecting left ventricle and myocyte hypertrophy. In conclusion, acute pressure overload induced self-limited superoxide production mainly in the vascular wall. The reactive oxygen species production would contribute to the perivascular inflammation and subsequent myocardial fibrosis. Angiotensin II was suggested to have a pressure-independent effect on the reactive oxygen species production.

Cardiac Metallothionein Synthesis in Streptozotocin-Induced Diabetic Mice, and Its Protection against Diabetes-Induced Cardiac Injury

Oxidative stress is involved in the pathogenesis of diabetes and its cardiovascular complications. Metallothionein (MT), a stress-response protein, is significantly increased in the liver and kidney of diabetic animals. We examined whether diabetes also induces cardiac MT synthesis through oxidative damage and whether MT overexpression protects the heart from injury. Diabetes was induced in mice by single injection of streptozotocin (STZ), and cardiac MT mRNA and protein levels were measured 2 weeks and 2 months after STZ treatment. Diabetes significantly increased cardiac MT synthesis 2 weeks and 2 months after STZ treatment, with no change in cardiac metals including zinc, copper, and iron. Serum and cardiac vasopeptide endothelin and inflammatory cytokine tumor necrosis factor-alpha were also significantly increased in diabetic hearts, as were the ratio of oxidized to reduced glutathione and the immunohistochemical staining of 3-nitrotyrosine and 4-hydroxynonenal. To explore the biological importance of increased MT synthesis in the heart, MT-overexpressing transgenic mice were treated with STZ and then examined 2 months later. A loss of inotropic reserve, uncovered during beta-adrenergic stimulation, and the presence of cardiac fibrosis, shown by increased Sirius red staining of collagen, were evident in the wild-type diabetic mice but not in the MT-overexpressing transgenic diabetic mice. These results suggest that diabetes-induced cardiac MT expression likely associates with systemic increases in endothelin-1 and tumor necrosis factor-alpha and the resulting cardiac oxidative stress. Overexpressing cardiac MT significantly protects the heart from diabetes-induced injury.

Histochemical accumulation of oxidative damage products is associated with Alzheimer-like pathology in the canine

An important lesion in Alzheimer's disease (AD) patient brains is the neurofibrillary tangle (NFT). Hyperphosphorylated tau is its major component. In a former paper we described some NFT in the canine brain. During aging, moreover, advanced glycation end products (AGE) might accumulate. Glycated tau induces lipid peroxidation in vivo and tau and AGE antigens have been mentioned to co-localize in NFT. This indicates that AGE may play an important role in Alzheimer disease (AD) by oxidation of tau. The aim of the present study was to investigate amyloid, neurofibrillary tangles, Abeta precursor protein, Abeta, tau, ubiquitin, advanced glycation end products, 4-hyroxynonenal protein and lipofuscin in a series of dogs of varying ages. The results showed a significant positive correlation between age and amyloid quantity (Congo red staining), HNE staining and lipofuscin (LF), and between amyloid quantity and HNE staining and LF. Staining for AβPP seemed to have a tendency to increase with age, whereas staining for tau, ubiquitin and AGE each only gave limited positive results in a proportion of the older dogs. Preliminary studies including loss of cognitive capabilities in the older dogs and chemical measurement of lipofuscin-like pigment (LFP) accumulation in brain extracts revealed an increase with old age and dementia. The Congo red, HNE and LF results suggest that deposition of amyloid with aging might be associated with formation of end products of lipid peroxidation. The finding of the limited positive signals for tau, ubiquitin and AGE in some old cases might indicate that the spontaneous brain pathology of the aged dog reveals similarities to early stages observed in AD in humans especially those with Down syndrome.

NADPH oxidase-derived oxidant stress is critical for neutrophil cytotoxicity during endotoxemia.

Neutrophils can cause liver injury during endotoxemia through generation of reactive oxygen species. However, the enzymatic source of the oxidant stress and the nature of the oxidants generated remain unclear. Therefore, we investigated the involvement of NADPH oxidase in the pathophysiology by using the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI) in the galactosamine/endotoxin (700 mg/kg Gal:100 microg/kg ET) model of liver injury. In addition, we measured chlorotyrosine as indicator for hypochlorous acid formation by myeloperoxidase. Gal/ET treatment of male C3HeB/FeJ mice resulted in sinusoidal neutrophil accumulation and parenchymal cell apoptosis (14 +/- 3% of cells) at 6 h. At 7 h, 35% of neutrophils had transmigrated. The number of apoptotic cells increased to 25 +/- 2%, and the overall number of dead cells was 48 +/- 3%; many of them showed the characteristic morphology of necrosis. Hepatocytes, which colocalized with extravasated neutrophils, stained positive for chlorotyrosine and 4-hydroxynonenal (4-HNE) protein adducts. In contrast, animals pretreated with DPI (2.5 mg/kg) were protected against liver injury at 7 h (necrosis = 20 +/- 2%). These livers showed little chlorotyrosine or 4-HNE staining, but apoptosis and neutrophil accumulation and extravasation remained unaffected. However, DPI-treated animals showed serious liver injury at 9 h due to sustained apoptosis. The results indicate that NADPH oxidase is responsible for the neutrophil-derived oxidant stress, which includes formation of hypochlorous acid by myeloperoxidase. Thus NADPH oxidase could be a promising therapeutic target to prevent neutrophil-mediated liver injury. However, the long-term benefit of this approach needs to be investigated in models relevant for human liver disease.

α-Phenyl- n- tert-butyl-nitrone attenuates hypoxic–ischemic white matter injury in the neonatal rat brain

White matter of the neonatal brain is highly sensitive to hypoxic–ischemic insult. The susceptibility of premature oligodendrocytes (OLs) to free radicals (FRs) produced during hypoxia–ischemia (HI) has been proposed as one of the mechanisms involved. To test this hypothesis, and to further investigate if the FR scavenger α-phenyl- N- tert-butyl-nitrone (PBN) attenuates hypoxic–ischemic white matter damage (WMD), postnatal day 4 (P4) SD rats were subjected to bilateral common carotid artery ligation (BCAL), followed by 8% oxygen exposure for 20 min. Pathological changes were evaluated on P6 and P9, 2 and 5 days after the HI insult. HI caused severe WMD including rarefaction, necrosis and cavity formation in the corpus callosum, external and internal capsule areas. OL injury was evidenced by degeneration of O4 positive OLs on P6. Disrupted myelination was verified by decreased immunostaining of myelin basic protein (MBP) on P9. Axonal injury was demonstrated by increased amyloid precursor protein (APP) immunostaining on both P6 and P9. Two lipid peroxidation end products, malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), showed a one-fold elevation within 1–24 h following HI. 4-HNE immunostaining was found to specifically localize in the white matter area. Furthermore, pyknotic O4+ OLs were double-labeled with 4-HNE. These findings suggest that FRs are involved in the pathogenesis of neonatal WMD. PBN (100 mg/kg, i.p.) treatment alleviated the pathological changes of WMD following HI. It improved the survival of O4 positive OLs, attenuated hypomyelination and reduced axonal damage. PBN treatment also decreased the brain concentration of MDA/4-HNE and positive 4-HNE staining in the white matter area. These findings indicate that in the current WMD model, PBN protects both OLs and axons, the two main components in the white matter, from neonatal HI insult. FR scavenging appears to be the primary mechanism underlying its neuroprotective effect.

January 22 Highlights

Kastenbauer et al. observed increased tyrosine nitration (a marker for peroxynitrite) 4-HNE staining in the leptomeninges, and inflammatory cells of a bacterial meningitis patient. in the brain and CSF of patients with acute bacterial meningitis. Increased CSF nitrotyrosine concentrations were associated with a depletion of ascorbate, an increased oxidation of the natural peroxynitrite scavenger uric acid, and an adverse outcome of meningitis. see page 186 The accompanying editorial by Simon and Beckman points out that the cause of the encephalopathy of meningitis remains unclear. CSF and brain pathology does not reflect the severity of the clinical picture. A diffusable toxin is likely and the Kastenbauer …

4-hydroxy-2-nonenal (4-HNE) staining by anti-HNE antibody.

In almost all aerobic cells, the oxygen metabolism generates reactive oxygen species (ROS), such as superoxide, hydroxyl radicals, and hydrogen peroxide. These ROS can peroxidize membrane lipids of a cell and its organelles, and can also attack DNA or protein (). During the process of lipid peroxidation, polyunsaturated fatty acids (PUFA), especially linoliec acid, arachidonic acid, and docosahexaenoic acid, in biomembranes are degraded to a great variety of water-soluble, short-chain carbonyl compounds (). Malonaldehyde and other aldehydes, such as alkaneals, 2-alkenals, hydroxyalkenals (), and phospholipid-bound aldehydes () are generated in the lipid peroxidation process. The major representative of 4-hydroxyalkenals, 4-hydroxynonenal (4-HNE), is the main product formed from omega 6-PUFA (). 4-HNE, a highly toxic aldehyde product of lipid peroxidation (), is a sensitive marker of oxidative damage and lipid peroxidation and can be evaluated by immunohistochemical staining using an anti-4-HNE monoclonal antibody (MAb) (, , ) and labeled goat anti-mouse IgG antibody ().

Lipid peroxidation in the postnatal rat brain. Formation of 4-hydroxynonenal in the supraventricular corpus callosum of postnatal rats.

Lipid peroxidation is known to be associated with many neurodegenerative diseases and with traumatic brain injury, but its occurrence in the normal developing brain has not been reported. The present study was carried out using a specific antibody that recognises proteins modified by the end-product of lipid peroxide decomposition, 4-hydroxynonenal (HNE), to evaluate evaluate possible lipid peroxidation products in the brains of developing rats by immunocytochemistry and electron microscopy. Moderately dense labelling was observed in the supraventricular corpus callosum in the 7- and 8-day-old rats, whilst very dense labelling was observed in the same region, in the 9- and 10-day-old rats. Very little immunoreactivity was observed at 14 days, and no staining was observed in the corpus callosum in adult rats. HNE staining was not observed in neuronal cell bodies that give rise to callosal axons in the overlying cerebral cortex. Electron microscopy showed dense HNE staining on the basal laminae of blood vessels and on the plasma membranes of unmyelinated axons. Large numbers of rounded cells with features of oligodendrocyte precursor cells were labelled by Perl's stain in the supraventricular corpus callosum at postnatal day 7 and postnatal day 10, i.e. at times corresponding to high levels of HNE immunoreactivity. In contrast, very few such cells were observed in the adult brain, corresponding to the very little or no Perl's staining in the adult. These results suggest that lipid peroxidation observed in the supraventricular corpus callosum at postnatal day 10 could result from an accumulation of iron in this region, at this time.

Distribution of hydroxynonenal-modified proteins in the kainate-lesioned rat hippocampus: evidence that hydroxynonenal formation precedes neuronal cell death

Decomposition of lipid peroxides gives rise to a wide range of aldehydes. 4-Hydroxyalkenals and in particular 4-hydroxynonenal (HNE) are often the most toxic products. Frequently, it is unclear at which stage in the tissue injury process HNE is formed, i.e., is it a late stage or an early stage in which HNE contributes to subsequent cell death? The present study was carried out using an antibody to HNE-modified proteins to elucidate the time course and distribution of HNE in the lesioned hippocampus after kainate injections. HNE was absent from normal neurons, but dense staining to HNE was observed in degenerating neurons after kainate injection. The increase in HNE staining occurred as early as 1 d postinjection, at a time when there was no histological evidence of cell death. HNE immunoreactivity was observed in the degenerating CA1 and CA3 fields at 3 d and 1 week postinjection, but was confined to a cluster of neurons at the edge of the degenerating CA fields, at 2 and 3 weeks postinjection. These observations suggest that HNE formation is an early event after this tissue injury, and may contribute to later cell death.

Collagen type I synthesized by pancreatic periacinar stellate cells (PSC) co-localizes with lipid peroxidation-derived aldehydes in chronic alcoholic pancreatitis.

Chronic alcoholic pancreatitis (CAP) is characterized by progressive pancreatic fibrosis and loss of the acinar cell mass, but the pathogenesis of pancreatic fibrosis in the human is poorly understood. It has been recently suggested that lipid peroxidation-derived aldehydes such as 4-hydroxynonenal (HNE) are involved in tissue damage and fibrosis in other organs. The aim of this study was to evaluate the role of oxidative stress in the development of alcohol-induced pancreatic fibrosis in humans, and to assess the contribution of pancreatic periacinar stellate cells (PSC) in the in vivo synthesis of extracellular matrix components during CAP. Lipid peroxidation was evaluated in tissue specimens obtained from patients with CAP who underwent surgical procedures, by immunohistochemistry using a monoclonal antibody directed against HNE-protein adducts. Immunohistochemical determination of collagen type I, alpha-smooth muscle actin (alpha-SMA), and the beta subunit of human platelet-derived growth factor (PDGF-Rbeta) was also performed. In addition, the tissue mRNA expression of procollagen I, PDGF-Rbeta, and transforming growth factor-beta1 (TGF-beta1) was evaluated by in situ hybridization. In CAP, increased formation of HNE-protein adducts was evident in acinar cells adjacent to the interlobular connective tissue that stained positively for collagen type I. HNE staining was absent in normal pancreas. Several non-parenchymal periacinar cells (PSC) underlay the HNE-stained acinar cells. Those PSC stained positively for alpha-SMA and PDGF-Rbeta and showed active synthesis of procollagen type I by in situ expression of the specific mRNAs. The pattern of expression of PDGF-Rbeta mRNA reflected that observed in immunostaining, showing increased amounts of transcripts in PSC. TGF-beta1 mRNA expression was increased in CAP, but transcripts were found in several cell types including PSC, acinar, and ductal cells. These results indicate that significant lipid peroxidation phenomena occur in CAP and that they are associated with active synthesis of collagen by PSC.