Ratio determination of plasma wild-type and L159R apoA-I using mass spectrometry: tools for studying apoA-IFin

Owen, John S.; Bharadwaj, Manish S.; Thomas, Michael J.; Bhat, Sujata V.; Samuel, Michael P.; Sorci‐Thomas, Mary G.

doi:10.1194/jlr.d600031-jlr200

Cited by 11 publications

(16 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Compared to control vector treated mice, diet-fed DKO mice expressing apoA-I had normal levels of LN cholesterol, similar to TgWT ApoA-I mice that have plasma apoA-I concentrations of ∼110 mg/dl. DKO mice expressing the mutant apoA-I, Tg L159R, have plasma levels of a mutant form of human apoA-I at ∼10 mg/dl23. In these mice LN cholesterol levels were intermediate between control and HD Ad ApoA-I mice, but this low level was sufficient to reduce LN cell expansion.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Apolipoprotein A-I and Its Role in Lymphocyte Cholesterol Homeostasis and Autoimmunity

Wilhelm

Zabalawi²,

Grayson³

et al. 2009

ATVB

Self Cite

114

122

View full text Add to dashboard Cite

Objective-The purpose of this study was to determine the effects of an atherogenic diet on immune function in LDLr Ϫ/Ϫ , ApoA-I Ϫ/Ϫ mice. Methods and Results-When LDLrϪ/Ϫ , ApoA-I Ϫ/Ϫ (DKO), and LDLr Ϫ/Ϫ (SKO) mice were fed an atherogenic diet, DKO had larger peripheral lymph nodes (LNs) and spleens compared to SKO mice. LNs were enriched in cholesterol and contain expanded populations of T, B, dendritic cells, and macrophages. Expansion of all classes of LN cells was accompanied by a Ϸ1.5-fold increase in T cell proliferation and activation. Plasma antibodies to dsDNA, ␤2-glycoprotein I, and oxidized LDL were increased in DKO, similar to levels in diet-fed Fas lpr/lpr mice, suggesting the development of an autoimmune phenotype. Both LN enlargement and cellular cholesterol expansion were "prevented" when diet-fed DKO mice were treated with helper dependent adenovirus expressing apoA-I. Independent of the amount of dietary cholesterol, DKO mice consistently showed lower plasma cholesterol than SKO mice, yet greater aortic cholesterol deposition and inflammation. Conclusions-ApoA-I prevented cholesterol-associated lymphocyte activation and proliferation in peripheral LN of diet-fed DKO mice. A Ϸ1.5-fold increase in T cell activation and proliferation was associated with a Ϸ3-fold increase in concentrations of circulating autoantibodies and Ϸ2-fold increase in the severity of atherosclerosis suggesting a common link between plasma apoA-I, inflammation, and atherosclerosis.

show abstract

Section: Resultsmentioning

confidence: 99%

“…Unlike letters indicate statistical significance at p<0.05. TgWT ApoA-I in DKO mice have apoA-I plasma concentrations ∼110 mg/dl while DKO mice expressing L159R ApoA-I have plasma apoA-I concentrations ∼10 mg/dl23.…”

Section: Figuresmentioning

confidence: 97%

Apolipoprotein A-I and Its Role in Lymphocyte Cholesterol Homeostasis and Autoimmunity

Wilhelm

Zabalawi²,

Grayson³

et al. 2009

ATVB

Self Cite

114

122

View full text Add to dashboard Cite

show abstract

“…Plasma concentrations of human apoA-I WT and apoA-I L159R were determined using a combination of Enzyme-Linked Immunosorbent Assay (ELISA) and mass spectrometry [39], as previously described. Plasma mouse apoE was determined by ELISA, in which a 96 well plate was coated with WUE4 purified anti-apoE that was kindly provided by Dr. Holtzman.…”

Section: Methodsmentioning

confidence: 99%

“…Standards for the ratio determination of L159R to wild-type apoA-I were prepared from pure stocks of the two proteins as previously described [39]. Serial dilutions were used to generate five standards, each containing 1 µg of total protein.…”

Section: Methodsmentioning

confidence: 99%

Dysfunctional HDL containing L159R ApoA-I leads to exacerbation of atherosclerosis in hyperlipidemic mice

Sorci‐Thomas

Zabalawi

Bharadwaj

et al. 2012

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biolog

Self Cite

View full text Add to dashboard Cite

The mutation L159R apoA-I or apoA-IL159R (FIN) is a single amino acid substitution within the sixth helical repeat of apoA-I. It is associated with a dominant negative phenotype, displaying hypoalphaproteinemia and an increased risk for atherosclerosis in humans. Mice lacking both mouse apoA-I and LDL receptor (LDL−/−, apoA-I−/−) (double knockout or DKO) were crossed > 9 generations with mice transgenic for human FIN to obtain L159R apoA-I, LDLr−/−, ApoA-I−/− (FIN-DKO) mice. A similar cross was also performed with human wild-type (WT) apoA-I (WT-DKO). In addition, FIN-DKO and WT-DKO were crossed to obtain WT/FIN-DKO mice. To determine the effects of the apoA-I mutations on atherosclerosis, groups of each genotype were fed either chow or an atherogenic diet for 12 weeks. Interestingly, the production of dysfunctional HDL-like particles occurred in DKO and FIN-DKO mice. These particles were distinct with respect to size, and their enrichment in apoE and cholesterol esters. Two-dimensional gel electrophoresis indicated that particles found in the plasma of FIN-DKO mice migrated as large α3-HDL. Atherosclerosis analysis showed that FIN-DKO mice developed the greatest extent of aortic cholesterol accumulation compared to all other genotypes, including DKO mice which lack any apoA-I. Taken together these data suggest that the presence of large apoE enriched HDL particles containing apoA-I L159R lack the normal cholesterol efflux promoting properties of HDL, rendering them dysfunctional and pro-atherogenic. In conclusion, large HDL-like particles containing apoE and apoA-IL159R contribute rather than protect against atherosclerosis, possibly through defective efflux properties and their potential for aggregation at their site of interaction in the aorta.

show abstract

“…Both the concentrated BSA and apoA-I (4 -5 mg/ml) were filtered through a 0.45-m SpinX column (Costar) prior to subcutaneous injection. The human apoA-I concentration in plasma samples from mice bled at timed intervals following injection of apoA-I or BSA was determined by ELISA, as described previously (13,14).…”

mentioning

confidence: 99%

Apolipoprotein A-I Modulates Regulatory T Cells in Autoimmune LDLr−/−, ApoA-I−/− Mice

Wilhelm

Zabalawi

Owen

et al. 2010

Journal of Biological Chemistry

Self Cite

123

141

View full text Add to dashboard Cite

The immune system is complex, with multiple layers of regulation that serve to prevent the production of self-antigens. One layer of regulation involves regulatory T cells (Tregs) that play an essential role in maintaining peripheral self-tolerance. Patients with autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis have decreased levels of HDL, suggesting that apoA-I concentrations may be important in preventing autoimmunity and the loss of self-tolerance. In published studies, hypercholesterolemic mice lacking HDL apoA-I or LDLr ؊/؊ , apoA-I ؊/؊ (DKO), exhibit characteristics of autoimmunity in response to an atherogenic diet. This phenotype is characterized by enlarged cholesterol-enriched lymph nodes (LNs), as well as increased T cell activation, proliferation, and the production of autoantibodies in plasma. In this study, we investigated whether treatment of mice with lipid-free apoA-I could attenuate the autoimmune phenotype. To do this, DKO mice were first fed an atherogenic diet containing 0.1% cholesterol, 10% fat for 6 weeks, after which treatment with apoA-I was begun. Subcutaneous injections of 500 g of lipid-free apoA-I was administered every 48 h during the treatment phase. These and control mice were maintained for an additional 6 weeks on the diet. At the end of the 12-week study, DKO mice showed decreased numbers of LN immune cells, whereas Tregs were proportionately increased. Accompanying this increase in Tregs was a decrease in the percentage of effector/effector memory T cells. Furthermore, lipid accumulation in LN and skin was reduced. These results suggest that treatment with apoA-I reduces inflammation in DKO mice by augmenting the effectiveness of the LN Treg response.

show abstract

Ratio determination of plasma wild-type and L159R apoA-I using mass spectrometry: tools for studying apoA-IFin

Cited by 11 publications

References 32 publications

Apolipoprotein A-I and Its Role in Lymphocyte Cholesterol Homeostasis and Autoimmunity

Apolipoprotein A-I and Its Role in Lymphocyte Cholesterol Homeostasis and Autoimmunity

Dysfunctional HDL containing L159R ApoA-I leads to exacerbation of atherosclerosis in hyperlipidemic mice

Apolipoprotein A-I Modulates Regulatory T Cells in Autoimmune LDLr−/−, ApoA-I−/− Mice

Contact Info

Product

Resources

About