Brian D. Lamon

Inflammation at the molecular interface of atherogenesis: an anthropological journey.

Despite the multifactorial nature of atherosclerosis, substantial evidence has established inflammation as an often surreptitious, yet critical and unifying driving force which promotes disease progression. To this end, research has defined molecular networks initiated by cytokines, growth factors and other pro-inflammatory molecules which promote hallmarks of atherosclerosis such as endothelial dysfunction, macrophage infiltration, LDL oxidation, cell proliferation and thrombosis. Although commonly associated with risk factors such as dyslipidemia, diabetes and hypertension, the global etiology of atherosclerosis may be alternatively attributed to underlying anthropological pressures. The agricultural, industrial and technological revolutions produced alterations in dietary, social and economic factors which have collectively exaggerated the exposure of the human genome to environmental stimuli. Furthermore, advances in sanitation, nutrition, and medicine have increased the lifespan of humans, effectively prolonging blood vessel exposure to these factors. As a result, the vasculature has become conditioned to respond to injury with what is arguably an overzealous immunological response; thus setting the stage for the prevalence of cardiovascular disease, including atherosclerotic plaque development in Western populations. Evidence suggests that each of these alterations can be linked to specific mediators in the inflammatory process. Integration of these factors with an inflammation-based hypothesis of atherosclerosis has yet to be extrapolated to observations in the realms of basic and clinical sciences and is the focus of this review.

Maintaining Equilibrium by Selective Targeting of Cyclooxygenase Pathways Promising Offensives Against Vascular Injury

Cardiovascular disease is the leading cause of death in most Western countries. Vascular abnormalities associated with cardiovascular disease are attributable to a variety of risk factors, including hypercholesterolemia, hypertension, hyperglycemia, lipotoxicity, obesity, aging, and tobacco smoke.1,2 Importantly, altered ·NO bioavailability is a major underlying mechanism linking each of these risk factors. Indeed, the endothelium is recognized as a pivotal regulator of vascular function by maintaining homeostatic levels of ·NO and prostanoids derived from arachidonic acid (AA) metabolism.3 Eicosanoids, synthesized from unsaturated fatty acids, are biologically active molecules that play a wide range of regulatory roles in the cardiovascular, renal, immune, and gastrointestinal systems.4 Alterations in their biosyntheses can promote hypertension, diabetes, and, in particular, atherosclerosis, an inflammatory disease characterized by the accretion of fat-laden plaques in the arterial wall that can lead to vasoocclusive events. During atherogenesis, eicosanoid production (from the cyclooxygenases [COXs], lipoxygenases [LOXs], and cytochrome P450s pathways) is altered by mechanisms that are not yet well understood. The dichotomous nature of eicosanoids requires that their balance is maintained, and, as such, these pathways are a relevant therapeutic target against cardiovascular disease.5 Thus, it is now appreciated that cardiovascular disease can be triggered by an absolute deficiency of ·NO and/or an imbalance between “beneficial” and “harmful” eicosanoids in the vasculature and that these pathways are mutually interactive. In this review we consider pharmacological therapies possessing the potential for greater safety and efficacy than nonsteroidal antiinflammatory drugs (NSAIDs) in the treatment of chronic vasoinflammatory conditions. Fundamental physiological distinctions are now recognized for the structurally similar enzymes, COX-1 and COX-2. Constitutively expressed COX-1 is found in almost all of the tissues where it subserves diverse physiological functions. Basal expression of COX-2 is limited to the kidney, brain, and reproductive system4 but can be …

Characterization of a cellular denitrase activity that reverses nitration of cyclooxygenase

Protein 3-nitrotyrosine (3-NT) formation is frequently regarded as a simple biomarker of disease, an irreversible posttranslational modification that can disrupt protein structure and function. Nevertheless, evidence that protein 3-NT modifications may be site selective and reversible, thus allowing for physiological regulation of protein activity, has begun to emerge. We have previously reported that cyclooxygenase (COX)-1 undergoes heme-dependent nitration of Tyr(385), an internal and catalytically essential residue. In the present study, we demonstrate that nitrated COX-1 undergoes a rapid reversal of nitration by substrate-selective and biologically regulated denitrase activity. Using nitrated COX-1 as a substrate, denitrase activity was validated and quantified by analytic HPLC with electrochemical detection and determined to be constitutively active in murine and human endothelial cells, macrophages, and a variety of tissue samples. Smooth muscle cells, however, contained little denitrase activity. Further characterizing this denitrase activity, we found that it was inhibited by free 3-NT and may be enhanced by endogenous nitric oxide and exogenously administered carbon monoxide. Finally, we describe a purification protocol that results in significant enrichment of a discrete denitrase-containing fraction, which maintains activity throughout the purification process. These findings reveal that nitrated COX-1 is a substrate for a denitrase in cells and tissues, implying that the reciprocal processes of nitration and denitration may modulate bioactive lipid synthesis in the setting of inflammation. In addition, our data reveal that denitration is a controlled process that may have broad importance for regulating cell signaling events in nitric oxide-generating systems during oxidative/nitrosative stress.

Inducible nitric oxide synthase gene deletion exaggerates MAPK-mediated cyclooxygenase-2 induction by inflammatory stimuli

Cyclooxygenase (COX)-2 and inducible nitric oxide (NO) synthase (iNOS) are responsive to a wide array of inflammatory stimuli, have been localized to vascular smooth muscle cells (SMCs), and are intimately linked to the progression of vascular disease, including atherosclerotic lesion formation. We and others have shown that the production and subsequent impact of COX products appear to be correlative with the status of NO synthesis. This study examined the impact of inflammation-driven NO production on COX-2 expression in SMCs. Concurrent stimulation of quiescent rat aortic SMCs with lipopolysaccharide (LPS) and interferon (IFN)-gamma increased COX-2, iNOS, and nitrite production. Pharmacological inhibition of NO synthase (N(G)-monomethyl-l-arginine) concentration- and time-dependently magnified LPS + IFN-gamma-mediated COX-2 mRNA and protein induction in a cGMP-independent manner. COX-2 induction was associated with activation of the ERK, p38, and JNK mitogen-activated protein kinase (MAPK) pathways. Interestingly, NO synthase inhibition enhanced ERK, p38, and to a lesser extent JNK phosphorylation but suppressed MAPK phosphatase (MKP)-1 induction in response to LPS + IFN-gamma. Similarly, the exposure of SMCs from iNOS(-/-) mice to LPS + IFN-gamma produced an enhancement of COX-2 induction, p38, and JNK phosphorylation and an attenuated upregulation of MKP-1 versus their wild-type counterparts. Taken together, our data indicate that NO, in part derived from iNOS, negatively regulates the immediate early induction of COX-2 in response to inflammatory stimuli.

Physical evidence for substrate binding in preventing cyclooxygenase inactivation under nitrative stress

Prostaglandin biosynthesis is catalyzed by two spatially and functionally distinct active sites in cyclooxygenase (COX) enzymes. Despite the crucial role of COXs in biology, molecular details regarding the function and regulation of these enzymes are incompletely defined. Reactive nitrogen species, formed during oxidative stress, produce modifications that alter COX functionalities and prostaglandin biosynthesis. We previously established that COX-1 undergoes selective nitration on Tyr385 via a mechanism that requires the presence of bound heme cofactor. As this is a critical residue for COX-1 catalysis, nitration at this site results in enzyme inactivation. We now show that occupancy of the COX-1 active site with substrate protects against Tyr385 nitration and redirects nitration to alternative Tyr residues on COX-1, preserving catalytic activity. This study reveals a novel role for the substrate in protecting COX-1 from inactivation by nitration in pathophysiological settings.

Inducible nitric oxide synthase provides protection against injury-induced thrombosis in female mice

Nitric oxide (NO) is an important vasoactive molecule produced by three NO synthase (NOS) enzymes: neuronal (nNOS), inducible (iNOS), and endothelial NOS (eNOS). While eNOS contributes to blood vessel dilation that protects against the development of hypertension, iNOS has been primarily implicated as a disease-promoting isoform during atherogenesis. Despite this, iNOS may play a physiological role via the modulation of cyclooxygenase and thromboregulatory eicosanoid production. Herein, we examined the role of iNOS in a murine model of thrombosis. Blood flow was measured in carotid arteries of male and female wild-type (WT) and iNOS-deficient mice following ferric chloride-induced thrombosis. Female WT mice were more resistant to thrombotic occlusion than male counterparts but became more susceptible upon iNOS deletion. In contrast, male mice (with and without iNOS deletion) were equally susceptible to thrombosis. Deletion of iNOS was not associated with a change in the balance of thromboxane A(2) (TxA(2)) or antithrombotic prostacyclin (PGI(2)). Compared with male counterparts, female WT mice exhibited increased urinary nitrite and nitrate levels and enhanced ex vivo induction of iNOS in hearts and aortas. Our findings suggest that iNOS-derived NO in female WT mice may attenuate the effects of vascular injury. Thus, although iNOS is detrimental during atherogenesis, physiological iNOS levels may contribute to providing protection against thrombotic occlusion, a phenomenon that may be enhanced in female mice.

Pitavastatin suppresses mitogen activated protein kinase-mediated Erg-1 induction in human vascular smooth muscle cells.

Statins have been demonstrated to elicit a broad range of cellular events resulting in an attenuation of the inflammatory response and enhanced protection to the components of the vessel wall. The present study was designed to examine the effect of pitavastatin on pathways associated with the proinflammatory gene, early growth response (Egr)-1, in human vascular smooth muscle cells. Pretreatment with pitavastatin resulted in a dose-dependent reduction in Egr-1 protein and suppressed Egr-1 mRNA expression in response to phorbol 12-myristate 13-acetate (PMA). A reduction in Egr-1 expression reduced the activation of NGFI-A binding protein (NAB)-2, an Egr-1-dependent gene. Furthermore, these events appeared to be dependent on the ability of pitavastatin to attenuate signaling cascades associated with extracellular regulated kinase (ERK) 1/2, but not p38 and c-Jun N-terminal kinase (JNK).

Silent Partner in Blood Vessel Homeostasis? Pervasive Role of Nitric Oxide in Vascular Disease.

The endothelium generates powerful mediators that regulate blood flow, temper inflammation and maintain a homeostatic environment to prevent both the initiation and progression of vascular disease. Nitric oxide (NO) is arguably the single most influential molecule in terms of dictating blood vessel homeostasis. In addition to direct effects associated with altered NO production (e.g. vasoconstriction, excessive inflammation, endothelial dysfunction), NO is a critical modulator of vaso-relevant pathways including cyclooxygenase (COX)-derived prostaglandin production and angiotensin II generation by the renin-angiotensin system. Furthermore, NO may influence the selectivity of COX-2 inhibitors and ultimately contribute to controversies associated with the use of these drugs. Consistent with a central role for NO in vascular disease, disruptions in the production and bioavailability of NO have been linked to hypertension, diabetes, hypercholesterolemia, obesity, aging, and smoking. The ability of the vessel wall to control disease-associated oxidative stress may be the most critical determinant in maintaining homeostatic levels of NO and subsequently the prospect of stroke, myocardial infarction and other CV abnormalities. To this end, investigation of mechanisms that alter the balance of protective mediators, including pathways that are indirectly modified by NO, is critical to the development of effective therapy in the treatment of CV disease.

Pitavastatin Differentially Modulates MicroRNA-Associated Cholesterol Transport Proteins in Macrophages.

There is emerging evidence identifying microRNAs (miRNAs) as mediators of statin-induced cholesterol efflux, notably through the ATP-binding cassette transporter A1 (ABCA1) in macrophages. The objective of this study was to assess the impact of an HMG-CoA reductase inhibitor, pitavastatin, on macrophage miRNAs in the presence and absence of oxidized-LDL, a hallmark of a pro-atherogenic milieu. Treatment of human THP-1 cells with pitavastatin prevented the oxLDL-mediated suppression of miR-33a, -33b and -758 mRNA in these cells, an effect which was not uniquely attributable to induction of SREBP2. Induction of ABCA1 mRNA and protein by oxLDL was inhibited (30%) by pitavastatin, while oxLDL or pitavastatin alone significantly induced and repressed ABCA1 expression, respectively. These findings are consistent with previous reports in macrophages. miRNA profiling was also performed using a miRNA array. We identified specific miRNAs which were up-regulated (122) and down-regulated (107) in THP-1 cells treated with oxLDL plus pitavastatin versus oxLDL alone, indicating distinct regulatory networks in these cells. Moreover, several of the differentially expressed miRNAs identified are functionally associated with cholesterol trafficking (six miRNAs in cells treated with oxLDL versus oxLDL plus pitavastatin). Our findings indicate that pitavastatin can differentially modulate miRNA in the presence of oxLDL; and, our results provide evidence that the net effect on cholesterol homeostasis is mediated by a network of miRNAs.