Lung lymphatic thrombosis and dysfunction caused by cigarette smoke exposure precedes emphysema in mice.

The lymphatic vasculature is critical for lung function, but defects in lymphatic function in the pathogenesis of lung disease is understudied. In mice, lymphatic dysfunction alone is sufficient to cause lung injury that resembles human emphysema. Whether lymphatic function is disrupted in cigarette smoke (CS)-induced emphysema is unknown. In this study, we investigated the effect of CS on lung lymphatic function. Analysis of human lung tissue revealed significant lung lymphatic thrombosis in patients with emphysema compared to control smokers that increased with disease severity. In a mouse model, CS exposure led to lung lymphatic thrombosis, decreased lymphatic drainage, and impaired leukocyte trafficking that all preceded the development of emphysema. Proteomic analysis demonstrated an increased abundance of coagulation factors in the lymph draining from the lungs of CS-exposed mice compared to control mice. In addition, in vitro assays demonstrated a direct effect of CS on lymphatic endothelial cell integrity. These data show that CS exposure results in lung lymphatic dysfunction and a shift in thoracic lymph towards a prothrombic state. Furthermore, our data suggest that lymphatic dysfunction is due to effects of CS on the lymphatic vasculature that precede emphysema. These studies demonstrate a novel component of CS-induced lung injury that occurs early in the pathogenesis of emphysema.

Atherosclerosis in LDLR-knockout mice is inhibited, but not reversed, by the PPARgamma ligand pioglitazone.

Thiazolidinediones, a class of drugs for the treatment of type-2 diabetes, are synthetic ligands for peroxisome proliferator-activated receptor-gamma. They have been demonstrated to possess cardioprotective effects in humans and anti-atherogenic properties in animal models. However, the question remains whether a peroxisome proliferator-activated receptor-gamma ligand can reverse the development of atherosclerosis. In this study, we tested the effects of pioglitazone on the development of established atherosclerosis in low-density lipoprotein receptor-null mice. We observed that atherosclerosis in low-density lipoprotein receptor-null mice progressed when mice were fed a high-fat diet. Pioglitazone treatment of atherogenic mice prevented this progression of atherosclerosis from its middle stages of disease, but was not able to reverse it. Withdrawal of the high-fat diet from mice with advanced atherosclerosis did not result in a reduction in lesion sizes. Pioglitazone treatment also had no effect on advanced atherosclerosis. Levels of high density lipoprotein cholesterol correlated inversely with lesion development when pioglitazone was given during lesion progression. However, pioglitazone had no effect on circulating high density lipoprotein levels in mice in which treatment was initiated following 14 weeks on the high-fat diet. These findings have implications for the analysis of therapeutic agents in murine models of atherosclerosis and the use of pioglitazone in patients with established atherosclerosis.

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).

Inducible nitric oxide synthase mediates prostaglandin h2 synthase nitration and suppresses eicosanoid production.

Nitric oxide (NO) modulates the biological levels of arachidonate-derived cell signaling molecules by either enhancing or suppressing the activity of prostaglandin H(2) isoforms (PGHS-1 and PGHS-2). Whether NO activates or suppresses PGHS activity is determined by alternative protein modifications mediated by NO and NO-derived species. Here, we show that inducible NO synthase (iNOS) and PGHS-1 co-localize in atherosclerotic lesions of ApoE(-/-) mouse aortae. Immunoblotting and immunohistochemistry revealed Tyr nitration in PGHS-1 in aortic lesions but markedly less in adjacent nonlesion tissue. PGHS-2 was also found in lesions, but 3-nitrotyrosine incorporation was not detected. 3-Nitrotyrosine formation in proteins is considered a hallmark reaction of peroxynitrite, which can form via NO-superoxide reactions in an inflammatory setting. That iNOS-derived NO is essential for 3-nitrotyrosine modification of PGHS-1 was confirmed by the absence of 3-nitrotyrosine in lesions from ApoE(-/-)iNOS(-/-) mice. Mass spectrometric studies specifically identified the active site residue Tyr385 as a 3-nitrotyrosine modification site in purified PGHS-1 exposed to peroxynitrite. PGHS-mediated eicosanoid (PGE(2)) synthesis was more than fivefold accelerated in cultured iNOS(-/-) versus iNOS-expressing mouse aortic smooth muscle cells, suggesting that iNOS-derived NO markedly suppresses PGHS activity in vascular cells. These results further suggest a regulatory role of iNOS in eicosanoid biosynthesis in human atherosclerotic lesions.

Pitavastatin alters the expression of thrombotic and fibrinolytic proteins in human vascular cells.

In addition to lowering blood lipids, clinical benefits of 3-hydroxy-3-methylglutaryl coenzyme A (HMG Co-A; EC 1.1.1.34) reductase inhibitors may derive from altered vascular function favoring fibrinolysis over thrombosis. We examined effects of pitavastatin (NK-104), a relatively novel and long acting statin, on expression of tissue factor (TF) in human monocytes (U-937), plasminogen activator inhibitor-1 (PAI-1), and tissue-type plasminogen activator (t-PA) in human aortic smooth muscle cells (SMC) and human umbilical vein endothelial cells (HUVEC). In monocytes, pitavastatin reduced expression of TF protein induced by lipopolysaccharide (LPS) and oxidized low-density lipoprotein (OxLDL). Similarly, pitavastatin also reduced expression of TF mRNA induced by LPS. Pitavastatin reduced PAI-1 antigen released from HUVEC under basal, OxLDL-, or tumor necrosis factor-alpha (TNF-alpha)-stimulated conditions. Reductions of PAI-1 mRNA expression correlated with decreased PAI-1 antigen secretion and PAI-1 activity as assessed by fibrin-agarose zymography. In addition, pitavastatin decreased PAI-1 antigen released from OxLDL-treated and untreated SMC. Conversely, pitavastatin enhanced t-PA mRNA expression and t-PA antigen secretion in untreated OxLDL-, and TNF-alpha-treated HUVEC and untreated SMC. Finally, pitavastatin increased t-PA activity as assessed by fibrin-agarose zymography. Our findings demonstrate that pitavastatin may alter arterial homeostasis favoring fibrinolysis over thrombosis, thereby reducing risk for thrombi at sites of unstable plaques.