Fluid-phase pinocytosis of native low density lipoprotein promotes murine M-CSF differentiated macrophage foam cell formation

During atherosclerosis, low-density lipoprotein (LDL)-derived cholesterol accumulates in macrophages to form foam cells. Macrophage uptake of LDL promotes foam cell formation but the mechanism mediating this process is not clear. The present study investigates the mechanism of LDL uptake for macrophage colony-stimulating factor (M-CSF)-differentiated murine bone marrow-derived macrophages. LDL receptor-null (LDLR-/-) macrophages incubated with LDL showed non-saturable accumulation of cholesterol that did not down-regulate for the 24 h examined. Incubation of LDLR-/- macrophages with increasing concentrations of (125)I-LDL showed non-saturable macrophage LDL uptake. A 20-fold excess of unlabeled LDL had no effect on (125)I-LDL uptake by wild-type macrophages and genetic deletion of the macrophage scavenger receptors CD36 and SRA did not affect (125)I-LDL uptake, showing that LDL uptake occurred by fluid-phase pinocytosis independently of receptors. Cholesterol accumulation was inhibited approximately 50% in wild-type and LDLR-/- mice treated with LY294002 or wortmannin, inhibitors of all classes of phosphoinositide 3-kinases (PI3K). Time-lapse, phase-contrast microscopy showed that macropinocytosis, an important fluid-phase uptake pathway in macrophages, was blocked almost completely by PI3K inhibition with wortmannin. Pharmacological inhibition of the class I PI3K isoforms alpha, beta, gamma or delta did not affect macrophage LDL-derived cholesterol accumulation or macropinocytosis. Furthermore, macrophages from mice expressing kinase-dead class I PI3K beta, gamma or delta isoforms showed no decrease in cholesterol accumulation or macropinocytosis when compared with wild-type macrophages. Thus, non-class I PI3K isoforms mediated macropinocytosis in these macrophages. Further characterization of the components necessary for LDL uptake, cholesterol accumulation, and macropinocytosis identified dynamin, microtubules, actin, and vacuolar type H(+)-ATPase as contributing to uptake. However, Pak1, Rac1, and Src-family kinases, which mediate fluid-phase pinocytosis in certain other cell types, were unnecessary. In conclusion, our findings provide evidence that targeting those components mediating macrophage macropinocytosis with inhibitors may be an effective strategy to limit macrophage accumulation of LDL-derived cholesterol in arteries

Macropinosome formation is M-CSF dependent.

<p>A and B. Wild-type macrophages were differentiated with M-CSF for 7 days and visualized by phase-contrast microscopy following the treatments described below. Macrophages differentiated with M-CSF were pretreated 30 min with DMSO drug vehicle (A), or 5 µM of cFMS (i.e., M-CSF receptor) tyrosine kinase inhibitor, GW2580 (B). Pretreatment was carried out without either serum or M-CSF. Withdrawal of M-CSF caused disappearance of the macrophage vacuoles. Subsequently, these macrophage cultures were treated 30 min with fresh serum-free medium containing M-CSF (50 ng/ml) without (A) or with GW2580 (B). Macrophages treated with M-CSF without GW2580 showed numerous vacuoles shown to be macropinosomes in Video S1. In contrast, there was complete inhibition of macropinosome formation when macrophage cultures were treated with GW2580 (also see Video S2). Scale bar in B = 75 µm and also applies to A. (C) Wild-type macrophages were incubated 24 h with 1 mg/ml LDL without or with 5 µM GW2580, and then cholesterol accumulation was assessed. Macrophages incubated without LDL had 111±3 nmol cholesterol/mg protein. ** = <i>p</i><0.01.</p

Role of active nitrogen molecules in progression of septic shock

Introduction: Active nitrogen molecules are formed as a result of cell metabolism. They are essential for cell metabolism, but when produced in excess, they contribute to the pathogenesis of several disease processes. These nitrogen molecules play an important role in vascular instability of septic shock. This study was planned to detect the role of active nitrogen molecules in the progression of septic shock. Materials and methods: Blood samples were collected from 118 critically ill patients admitted in ICU and from 95 healthy relatives accompanying the patients. Patients were categorized into three groups: systemic inflammatory response syndrome (n=54), sepsis (n=35) and septic shock (n=29). Plasma total nitrite (nitrites and nitrates), cytokines like tumour necrosis factor-α (TNF-α) and plasma lactate were measured to assess inflammatory activity and severity of septic shock. Results: High plasma levels of nitrite and nitrate (No<SUB>2−</SUB>/No<SUB>3−</SUB>) were observed in critically ill patients (mean level 78.92 µmol/l in sepsis and 97.20 µmol/l in septic shock). Mean plasma TNF- level in sepsis was 213.50 pg/ml and septic shock was 227.38 pg/ml. Conclusion : Plasma No<SUB>2−</SUB>/No<SUB>3−</SUB> and TNF-α levels were high in patients with sepsis and septic shock, which increased with severity of sepsis