Victoria P. Noffsinger

HDL remodeling during the acute phase response.

Objective—The purpose of this study was to examine the interactive action of serum amyloid A (SAA), group IIA secretory phospholipase A2 (sPLA2-IIA), and cholesteryl ester transfer protein (CETP) on HDL remodeling and cholesterol efflux during the acute phase (AP) response elicited in humans after cardiac surgery. Methods and Results—Plasma was collected from patients before (pre-AP), 24 hours after (AP-1 d), and 5 days after cardiac surgery (AP-5 d). SAA levels were increased 16-fold in AP-1 d samples. The activity of sPLA2-IIA was increased from 77.7±38.3 U/mL (pre-AP) to 281.4±57.1 U/mL (AP-1 d; P<0.001). CETP mass and activity reduction was commensurate to the reduction of HDL cholesterol levels. The combined action of SAA, sPLA2-IIA, and CETP in vitro markedly remodeled HDL with the generation of lipid-poor apoA-I from both pre-AP and AP-1 d HDL. The net result of this remodeling was a relative preservation of ABCA1- and ABCG1-dependent cholesterol efflux during the acute phase response. Conclusions—Our results show that the many and complex changes in plasma proteins during the acute phase response markedly remodel HDL with functional implications, particularly the relative retention of cholesterol efflux capacity.

Impact of serum amyloid A on high density lipoprotein composition and levels

Serum amyloid A (SAA) is an acute-phase protein mainly associated with HDL. To study the role of SAA in mediating changes in HDL composition and metabolism during inflammation, we generated mice in which the two major acute-phase SAA isoforms, SAA1.1 and SAA2.1, were deleted [SAA knockout (SAAKO) mice], and induced an acute phase to compare lipid and apolipoprotein parameters between wild-type (WT) and SAAKO mice. Our data indicate that SAA does not affect apolipoprotein A-I (apoA-I) levels or clearance under steady-state conditions. HDL and plasma triglyceride levels following lipopolysaccharide administration, as well as the decline in liver expression of apoA-I and apoA-II, did not differ between both groups of mice. The expected size increase of WT acute-phase HDL was surprisingly also seen in SAAKO acute-phase HDL despite the absence of SAA. HDLs from both mice showed increased phospholipid and unesterified cholesterol content during the acute phase. We therefore conclude that in the mouse, SAA does not impact HDL levels, apoA-I clearance, or HDL size during the acute phase and that the increased size of acute-phase HDL in mice is associated with an increased content of surface lipids, particularly phospholipids, and not surface proteins. These data need to be transferred to humans with caution due to differences in apoA-I structure and remodeling functions.

Deficiency of Endogenous Acute Phase Serum Amyloid A Does Not Affect Atherosclerotic Lesions in Apolipoprotein E–Deficient Mice

Objective—Although elevated plasma concentrations of serum amyloid A (SAA) are associated strongly with increased risk for atherosclerotic cardiovascular disease in humans, the role of SAA in the pathogenesis of lesion formation remains obscure. Our goal was to determine the impact of SAA deficiency on atherosclerosis in hypercholesterolemic mice. Approach and Results—Apolipoprotein E–deficient (apoE−/−) mice, either wild type or deficient in both major acute phase SAA isoforms, SAA1.1 and SAA2.1, were fed a normal rodent diet for 50 weeks. Female mice, but not male apoE−/− mice deficient in SAA1.1 and SAA2.1, had a modest increase (22%; P⩽0.05) in plasma cholesterol concentrations and a 53% increase in adipose mass compared with apoE−/− mice expressing SAA1.1 and SAA2.1 that did not affect the plasma cytokine levels or the expression of adipose tissue inflammatory markers. SAA deficiency did not affect lipoprotein cholesterol distributions or plasma triglyceride concentrations in either male or female mice. Atherosclerotic lesion areas measured on the intimal surfaces of the arch, thoracic, and abdominal regions were not significantly different between apoE−/− mice deficient in SAA1.1 and SAA2.1 and apoE−/− mice expressing SAA1.1 and SAA2.1 in either sex. To accelerate lesion formation, mice were fed a Western diet for 12 weeks. SAA deficiency had effect neither on diet-induced alterations in plasma cholesterol, triglyceride, or cytokine concentrations nor on aortic atherosclerotic lesion areas in either male or female mice. In addition, SAA deficiency in male mice had no effect on lesion areas or macrophage accumulation in the aortic roots. Conclusions—The absence of endogenous SAA1.1 and 2.1 does not affect atherosclerotic lipid deposition in apolipoprotein E–deficient mice fed either normal or Western diets.

The Impairment of Macrophage-to-Feces Reverse Cholesterol Transport during Inflammation Does Not Depend on Serum Amyloid A

Studies suggest that inflammation impairs reverse cholesterol transport (RCT). We investigated whether serum amyloid A (SAA) contributes to this impairment using an established macrophage-to-feces RCT model. Wild-type (WT) mice and mice deficient in SAA1.1 and SAA2.1 (SAAKO) were injected intraperitoneally with 3H-cholesterol-labeled J774 macrophages 4 hr after administration of LPS or buffered saline. 3H-cholesterol in plasma 4 hr after macrophage injection was significantly reduced in both WT and SAAKO mice injected with LPS, but this was not associated with a reduced capacity of serum from LPS-injected mice to promote macrophage cholesterol efflux in vitro. Hepatic accumulation of 3H-cholesterol was unaltered in either WT or SAAKO mice by LPS treatment. Radioactivity present in bile and feces of LPS-injected WT mice 24 hr after macrophage injection was reduced by 36% (P < 0.05) and 80% (P < 0.001), respectively. In contrast, in SAAKO mice, LPS did not significantly reduce macrophage-derived 3H-cholesterol in bile, and fecal excretion was reduced by only 45% (P < 0.05). Injection of cholesterol-loaded allogeneic J774 cells, but not syngeneic bone-marrow-derived macrophages, transiently induced SAA in C57BL/6 mice. Our study confirms reports that acute inflammation impairs steps in the RCT pathway and establishes that SAA plays only a minor role in this impairment.

Deficiency of Endogenous Acute-Phase Serum Amyloid A Protects apoE−/− Mice From Angiotensin II–Induced Abdominal Aortic Aneurysm Formation

Objective— Rupture of abdominal aortic aneurysm (AAA), a major cause of death in the aged population, is characterized by vascular inflammation and matrix degradation. Serum amyloid A (SAA), an acute-phase reactant linked to inflammation and matrix metalloproteinase induction, correlates with aortic dimensions before aneurysm formation in humans. We investigated whether SAA deficiency in mice affects AAA formation during angiotensin II (Ang II) infusion. Approach and Results— Plasma SAA increased ≈60-fold in apoE−/− mice 24 hours after intraperitoneal Ang II injection (100 &mgr;g/kg; n=4) and ≈15-fold after chronic 28-day Ang II infusion (1000 ng/kg per minute; n=9). AAA incidence and severity after 28-day Ang II infusion was significantly reduced in apoE−/− mice lacking both acute-phase SAA isoforms (SAAKO; n=20) compared with apoE−/− mice (SAAWT; n=20) as assessed by in vivo ultrasound and ex vivo morphometric analyses, despite a significant increase in systolic blood pressure in SAAKO mice compared with SAAWT mice after Ang II infusion. Atherosclerotic lesion area of the aortic arch was similar in SAAKO and SAAWT mice after 28-day Ang II infusion. Immunostaining detected SAA in AAA tissues of Ang II–infused SAAWT mice that colocalized with macrophages, elastin breaks, and enhanced matrix metalloproteinase activity. Matrix metalloproteinase-2 activity was significantly lower in aortas of SAAKO mice compared with SAAWT mice after 10-day Ang II infusion. Conclusions— Lack of endogenous acute-phase SAA protects against experimental AAA through a mechanism that may involve reduced matrix metalloproteinase-2 activity.

Serum Amyloid A3 is a High-Density Lipoprotein-associated Acute Phase Protein

Serum amyloid A (SAA) is a family of acute-phase reactants. Plasma levels of human SAA1/SAA2 (mouse SAA1.1/2.1) can increase ≥1,000-fold during an acute-phase response. Mice, but not humans, express a third relatively understudied SAA isoform, SAA3. We investigated whether mouse SAA3 is an HDL-associated acute-phase SAA. Quantitative RT-PCR with isoform-specific primers indicated that SAA3 and SAA1.1/2.1 are induced similarly in livers (∼2,500-fold vs. ∼6,000-fold, respectively) and fat (∼400-fold vs. ∼100-fold, respectively) of lipopolysaccharide (LPS)-injected mice. In situ hybridization demonstrated that all three SAAs are produced by hepatocytes. All three SAA isoforms were detected in plasma of LPS-injected mice, although SAA3 levels were ∼20% of SAA1.1/2.1 levels. Fast protein LC analyses indicated that virtually all of SAA1.1/2.1 eluted with HDL, whereas ∼15% of SAA3 was lipid poor/free. After density gradient ultracentrifugation, isoelectric focusing demonstrated that ∼100% of plasma SAA1.1 was recovered in HDL compared with only ∼50% of SAA2.1 and ∼10% of SAA3. Thus, SAA3 appears to be more loosely associated with HDL, resulting in lipid-poor/free SAA3. We conclude that SAA3 is a major hepatic acute-phase SAA in mice that may produce systemic effects during inflammation.

The dual role of group V secretory phospholipase A 2 in pancreatic β-cells

PurposeGroup X (GX) and group V (GV) secretory phospholipase A2 (sPLA2) potently release arachidonic acid (AA) from the plasma membrane of intact cells. We previously demonstrated that GX sPLA2 negatively regulates glucose-stimulated insulin secretion (GSIS) by a prostaglandin E2 (PGE2)-dependent mechanism. In this study we investigated whether GV sPLA2 similarly regulates GSIS.MethodsGSIS and pancreatic islet-size were assessed in wild-type (WT) and GV sPLA2-knock out (GV KO) mice. GSIS was also assessed ex vivo in isolated islets and in vitro using MIN6 pancreatic beta cell lines with or without GV sPLA2 overexpression or silencing.ResultsGSIS was significantly decreased in islets isolated from GV KO mice compared to WT mice and in MIN6 cells with siRNA-mediated GV sPLA2 suppression. MIN6 cells overexpressing GV sPLA2 (MIN6-GV) showed a significant increase in GSIS compared to control cells. Though the amount of AA released into the media by MIN6-GV cells was significantly higher, PGE2 production was not enhanced or cAMP content decreased compared to control MIN6 cells. Surprisingly, GV KO mice exhibited a significant increase in plasma insulin levels following i.p. injection of glucose compared to WT mice. This increase in GSIS in GV KO mice was associated with a significant increase in pancreatic islet size and number of proliferating cells in β-islets compared to WT mice.ConclusionsDeficiency of GV sPLA2 results in diminished GSIS in isolated pancreatic beta-cells. However, the reduced GSIS in islets lacking GV sPLA2 appears to be compensated by increased islet mass in GV KO mice.