Michael J. Duryee

Association of malondialdehyde-acetaldehyde (MAA) adducted proteins with atherosclerotic-induced vascular inflammatory injury

Atherosclerosis is a vascular injury characterized by elevated tissue levels of tumor necrosis factor-alpha (TNF-alpha), increased expression of endothelial cell adhesion molecules, and vascular wall inflammatory cell infiltration. Foam cells are associated with atherosclerotic plaque material, and low density lipoprotein (LDL) is a lipid component of foam cells. Malondialdehyde (MDA) is an oxidative product of unsaturated fatty acids and is also present in atherosclerotic lesions. MDA-modified (adducted) proteins, including MDA-modified LDL, are present in atherosclerotic human vascular tissue. Acetaldehyde (AA) is the major metabolic product of ethanol oxidation. Both MDA and AA are highly reactive aldehydes and will combine with proteins to produce an antigenically distinct protein adduct, termed the MAA adduct. This study demonstrates that proteins modified in the presence of high concentrations of MDA can produce MAA-modified proteins in vitro. In addition, MAA adducted proteins are capable of inducing rat heart endothelial cell cultures (rHEC) to produce and release TNF-alpha, and cause rHEC upregulation of endothelial adhesion molecule expression, including ICAM-1. These adhesion molecules are required for circulating inflammatory cells to adhere to endothelium which allows inflammatory cell tissue infiltration. Additionally, MAA modified proteins were defected in human atherosclerotic aortic vascular tissue but not in normal aortic tissue. Since atherosclerosis is associated with an inflammatory vascular injury characterized by elevated tissue TNF-alpha concentrations and inflammatory cell infiltration, these data suggest that MAA-adducted proteins may be formed in atherosclerotic plaque material and may be involved in the inflammatory reaction that occurs in atherosclerosis. These data further suggest that previous studies demonstrating MDA modified protein in atherosclerotic plaque may in fact have MAA modified proteins associated with them.

Aldehyde dehydrogenase 2 deficiency ameliorates alcoholic fatty liver but worsens liver inflammation and fibrosis in mice

Aldehyde dehydrogenase 2 (ALDH2) is the major enzyme that metabolizes acetaldehyde produced from alcohol metabolism. Approximately 40‐50% of East Asians carry an inactive ALDH2 gene and exhibit acetaldehyde accumulation after alcohol consumption. However, the role of ALDH2 deficiency in the pathogenesis of alcoholic liver injury remains obscure. In the present study, wild‐type and ALDH2−/− mice were subjected to ethanol feeding and/or carbon tetrachloride (CCl4) treatment, and liver injury was assessed. Compared with wild‐type mice, ethanol‐fed ALDH2−/− mice had higher levels of malondialdehyde‐acetaldehyde (MAA) adduct and greater hepatic inflammation, with higher hepatic interleukin (IL)‐6 expression but surprisingly lower levels of steatosis and serum alanine aminotransferase (ALT). Higher IL‐6 levels were also detected in ethanol‐treated precision‐cut liver slices from ALDH2−/− mice and in Kupffer cells isolated from ethanol‐fed ALDH2−/− mice than those levels in wild‐type mice. In vitro incubation with MAA enhanced the lipopolysaccharide (LPS)‐mediated stimulation of IL‐6 production in Kupffer cells. In agreement with these findings, hepatic activation of the major IL‐6 downstream signaling molecule signal transducer and activator of transcription 3 (STAT3) was higher in ethanol‐fed ALDH2−/− mice than in wild‐type mice. An additional deletion of hepatic STAT3 increased steatosis and hepatocellular damage in ALDH2−/− mice. Finally, ethanol‐fed ALDH2−/− mice were more prone to CCl4‐induced liver inflammation and fibrosis than ethanol‐fed wild‐type mice. Conclusion: ALDH2−/− mice are resistant to ethanol‐induced steatosis but prone to inflammation and fibrosis by way of MAA‐mediated paracrine activation of IL‐6 in Kupffer cells. These findings suggest that alcohol, by way of acetaldehyde and its associated adducts, stimulates hepatic inflammation and fibrosis independent from causing hepatocyte death, and that ALDH2‐deficient individuals may be resistant to steatosis and blood ALT elevation, but are prone to liver inflammation and fibrosis following alcohol consumption. (Hepatology 2014;60:146–157)

Lipopolysaccharide is a cofactor for malondialdehyde-acetaldehyde adduct-mediated cytokine/chemokine release by rat sinusoidal liver endothelial and kupffer cells

BACKGROUND The nonparenchymal cells of the liver have been suggested to play a significant role in the inflammatory processes observed in the development and/or progression of alcoholic liver disease. Our laboratories have shown that malondialdehyde-acetaldehyde (MAA)-modified proteins can induce immune responses, cytokine/chemokine secretion, and antigen processing and presentation by liver sinusoidal endothelial cells (SECs). Another molecule that has been shown to induce similar types of responses in Kupffer cells (KCs) is lipopolysaccharide (LPS). Because these materials induce similar responses, it was the purpose of this study to investigate the relationship between LPS and MAA-modified proteins in the development of proinflammatory responses by SECs and KCs. METHODS For these studies, SECs and KCs were isolated from chow-fed, pair-fed, and ethanol-fed rats. Cells were stimulated with media alone, bovine serum albumin (Alb), or MAA-modified Alb (MAA-Alb) in the presence or absence of LPS 1 ng/ml, and the supernatants were assayed by enzyme-linked immunosorbent assay for tumor necrosis factor alpha, macrophage chemotactic protein 1, and macrophage inhibitory protein. RESULTS All three cytokines/chemokines were 3 to 5 times higher when SECs or KCs were stimulated by MAA-Alb in the presence of LPS, in contrast to cells stimulated with Alb or media in the presence of LPS. Chronic ethanol consumption (6 weeks) had variable effects on the secretion of these cytokines/chemokines but in general did not alter the increased secretion in response to MAA-Alb in the presence of LPS. CONCLUSIONS These studies strongly suggest that the sensitization of SECs and KCs by LPS plays a significant role in the development and/or progression of alcoholic liver disease, and the subsequent activation by MAA-modified proteins may be a mechanism by which proinflammatory processes are initiated.

Malondialdehyde–acetaldehyde adduct is the dominant epitope after MDA modification of proteins in atherosclerosis

Antibodies to malondialdehyde (MDA)-modified macromolecules (adducts) have been detected in the serum of patients with atherosclerosis and correlate with the progression of this disease. However, the epitope and its formation have not been characterized. Studies have shown that excess MDA can be degraded to acetaldehyde, which combines with proteins to from a stable dihydropyridine adduct. To investigate, mice were immunized with MDA adducts in the absence of adjuvant and showed an increase in antibodies to MDA adducts and the carrier protein as the concentration of MDA was increased. In fact, a number of the commercially available antibodies to MDA-modified proteins were able to be inhibited by a chemical analogue, hexyl-MAA. Also, MDA-MAA adducts were detected in the serum and aortic tissue of JCR diabetic/atherosclerotic rats. These studies determined that commercially available antibodies to MDA predominantly react with the MAA adduct and are present in the JCR model of atherosclerosis in both the serum and the aortic tissue. Therefore, the immune response to MDA-modified proteins is most probably to the dihydropyridine structure (predominant epitope in MAA), which suggests that MAA adducts may play a role in the development and/or progression of atherosclerosis.

Immune responses to methamphetamine by active immunization with peptide-based, molecular adjuvant-containing vaccines.

Vaccines to methamphetamine (meth) were designed by covalently attaching a meth hapten (METH) to peptide constructs that contained a conformationally biased, response-selective molecular adjuvant, YSFKPMPLaR (EP54). Rats immunized with EP54-containing meth vaccines generated serum antibody titers to authentic meth, an immune outcome that altered meth self-administration. Immunization increased meth self-administration suggesting pharmacokinetic antagonism. The ability of immune sera to bind a METH-modified target protein dramatically decreased during and shortly after the meth self-administration assay, suggesting effective sequestration of free meth. However, the binding ability of immune sera to the METH-modified target protein was recovered 34 days after meth-free clearance time.

Malondialdehyde-Acetaldehyde Adducts and Anti–Malondialdehyde-Acetaldehyde Antibodies in Rheumatoid Arthritis

Malondialdehyde‐acetaldehyde (MAA) adducts are a product of oxidative stress associated with tolerance loss in several disease states. This study was undertaken to investigate the presence of MAA adducts and circulating anti‐MAA antibodies in patients with rheumatoid arthritis (RA).

IL-6 and its receptors in coronary artery disease and acute myocardial infarction.

Biomarkers such as interleukin-6 (IL-6), soluble interleukin-6 receptor (sIL-6R), and high sensitive C-reactive protein (hsCRP) have been reported to be elevated in acute myocardial infarction (AMI). The aim of this study is to determine the relationship between these markers during AMI, as well as their relationship to clinical parameters in an effort to discern their predictive potential in cardiac events. Serum was collected from 73 patients with; AMI, stable coronary artery disease (CAD), and controls during cardiac catheterization. Biomarker levels were determined and correlated with clinical data. IL-6 (11.75pg/ml, P<0.05) and sIL-6R (41,340pg/ml, P=0.05) were elevated in AMI compared with CAD and controls. At presentation, hsCRP was elevated in AMI patients (4.69mg/L) compared to controls (2.69mg/L, P<0.05); however, there was a significant decrease in hsCRP between AMI (4.69mg/L) and CAD patients (7.4mg/L, P<0.05). After 24h post-AMI hsCRP levels were increased compared to stable CAD (60.46mg/L, P<0.05) and were preceded by increased IL-6 at presentation. Soluble Gp130 (sGp130) showed no significant change between AMI, CAD, and control patients. However, sGp130 positively correlated with peak troponin in AMI (R=0.587, P<0.01), and negatively correlated with previous AMI (R=-0.382, P<0.05). Circulating monocyte mRNA expression isolated from selected AMI patients showed an increase in IL-6 mRNA (5.28-fold, P<0.01) and a decrease in both IL-6R (0.374-fold, P<0.01) and sGp130 mRNA (0.38-fold, P<0.01) as compared to CAD and controls. Results demonstrate that IL-6 and sIL-6R are associated with AMI and cardiac injury. These data support the hypothesis that trans-IL-6 receptor binding may alter intracellular signaling, and blocking of IL-6 receptor binding may be pathogenic in AMI. These data may be predictive of mechanism(s) by which plaques become unstable and rupture.

High-Pressure Distention of the Saphenous Vein During Preparation Results in Increased Markers of Inflammation: A Potential Mechanism for Graft Failure

BACKGROUND Coronary artery disease is the single leading cause of death in the United States. Commonly it is treated with coronary bypass grafting using the saphenous vein (SV) or internal mammary artery (IMA) as a conduit. Unfortunately, the SV has much lower patency rates compared with the IMA. Several hypotheses exist as to why occlusion occurs more commonly in SV grafts than in IMA grafts. However detailed studies in this area have been limited. This study investigates the effects of pressure distention on inflammation in SV conduit used in coronary artery bypass grafting (CABG). METHODS Saphenous vein distention pressure was measured intraoperatively during 48 CABG procedures. A segment of SV was excised from the conduit before distention. Because the vein was used for coronary artery grafting, sequential pieces were archived for evaluation. Real-time polymerase chain reaction (RT-PCR) and immunohistochemical analyses were performed to investigate a change in the expression of biomarkers. RESULTS Upregulation of various biomarkers occurred. These biomarkers included scavenger receptors A and B (SR-A, SR-B), toll-like receptors 2 and 4 (TLR2, TLR4), platelet endothelial cell adhesion molecule (PECAM), vascular cell adhesion molecule (VCAM), and intercellular cell adhesion molecule (ICAM) in segments of SV that were subjected to distention. Immunohistochemical results mirrored RT-PCR findings. A significant correlation was observed between biomarkers and pressure values. CONCLUSIONS These studies demonstrate that markers of inflammation are upregulated in response to SV distention. The data suggest that the pressure used in graft preparation procedures should be regulated to avoid inflammation and its potential to induce graft failure.

An in vitro method of alcoholic liver injury using precision-cut liver slices from rats

Alcohol abuse results in liver injury, but investigations into the mechanism(s) for this injury have been hampered by the lack of appropriate in vitro culture models in which to conduct in depth and specific studies. In order to overcome these shortcomings, we have developed the use of precision-cut liver slices (PCLS) as an in vitro culture model in which to investigate how ethanol causes alcohol-induced liver injury. In these studies, it was shown that the PCLS retained excellent viability as determined by lactate dehydrogenase and adenosine triphosphate (ATP) levels over a 96-h period of incubation. More importantly, the major enzymes of ethanol detoxification; alcohol dehydrogenase, aldehyde dehydrogenase, and cytochrome P4502E1, remained active and PCLS readily metabolized ethanol and produced acetaldehyde. Within 24 h and continuing up to 96h the PCLS developed fatty livers and demonstrated an increase in the redox state. These PCLS secreted albumin, and albumin secretion was decreased by ethanol treatment. All of these impairments were reversed following the addition of 4-methylpyrazole, which is an inhibitor of ethanol metabolism. Therefore, this model system appears to mimic the ethanol-induced changes in the liver that have been previously reported in human and animal studies, and may be a useful model for the study of alcoholic liver disease.

Malondialdehyde-acetaldehyde (MAA) modified proteins induce pro-inflammatory and pro-fibrotic responses by liver endothelial cells.

Recent reports have implicated liver non-parenchymal cells in liver injury as they can secrete; pro-inflammatory cytokines, excessive matrix proteins during fibrosis, and have been shown to initiate local immune responses [1-3]. Of the non-parenchymal cells, sinusoidal liver endothelial cells (SLECs) have been suggested to be a major contributor to the inflammatory processes observed in alcoholic liver disease (ALD) [4]. This is because SLECs are involved in the recruitment of leukocytes into the liver following the activation of an immune response, such as that observed in alcoholic hepatitis [4]. This process involves the release of cytokines TNF-alpha and IL-1beta, which increases the expression of adhesion molecules on the surface of SLECs and the release of chemokines (MIP-2 or MCP-1), which recruit leukocytes into the liver from the circulation [5,6]. These chemokines cause increased binding of leukocytes to the vessel walls and promote the trans-migration of these immune cells across the SLECs by the process of diapedesis [7,8]. Recent studies have shown that proteins can be modified by the metabolites of chronic ethanol consumption. Two of these metabolites, malondialdehyde (MDA) and acetaldehyde (AA), have been shown to synergistically bind to proteins (adduct) to form a product termed MAA [9]. This results in the binding and degradation of the adducted protein. However, chronic ethanol consumption decreases their degradation, but not the binding of these MAA modified proteins. Thus, the extended presence and binding of the MAA-modified proteins to their appropriate receptors could result in the induction of some biological response. It is thought that since these adducts are chronically present, a potential inflammatory response may be initiated similar to that observed in chronic viral infections. Additionally, observation of SLECs when exposed to MAA-Alb showed morphological changes in these cells that was suggestive of cell death (personal observation). Preliminary studies suggested that these changes may be due to TNF-alpha secretion by SLECs. Thus, it was the purpose of these experiments to begin assessing whether MAA-modified proteins can activate SLECs.