Lymphocyte activation gene 3 and coronary artery disease
Diana Golden, Antonina Kolmakova, Sunitha Sura, Anthony T. Vella, Ani Manichaikul, Xin-Qun Wang, Suzette J. Bielinski, Kent D. Taylor, Yii-Der Ida Chen, Stephen S. Rich, Annabelle Rodriguez
Diana Golden, Antonina Kolmakova, Sunitha Sura, Anthony T. Vella, Ani Manichaikul, Xin-Qun Wang, Suzette J. Bielinski, Kent D. Taylor, Yii-Der Ida Chen, Stephen S. Rich, Annabelle Rodriguez
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Clinical Research and Public Health Cardiology Inflammation

Lymphocyte activation gene 3 and coronary artery disease

Abstract

BACKGROUND: The lipoprotein scavenger receptor BI (SCARB1) rs10846744 noncoding variant is significantly associated with atherosclerotic disease independently of traditional cardiovascular risk factors. We identified a potentially novel connection between rs10846744, the immune checkpoint inhibitor lymphocyte activation gene 3 (LAG3), and atherosclerosis.

METHODS: In vitro approaches included flow cytometry, lipid raft isolation, phosphosignaling, cytokine measurements, and overexpressing and silencing LAG3 protein. Fasting plasma LAG3 protein was measured in hyperalphalipoproteinemic (HALP) and Multi-Ethnic Study of Atherosclerosis (MESA) participants.

RESULTS: In comparison with rs10846744 reference (GG homozygous) cells, LAG3 protein levels by flow cytometry (P < 0.001), in lipid rafts stimulated and unstimulated (P = 0.03), and phosphosignaling downstream of B cell receptor engagement of CD79A (P = 0.04), CD19 (P = 0.04), and LYN (P = 0.001) were lower in rs10846744 risk (CC homozygous) cells. Overexpressing LAG3 protein in risk cells and silencing LAG3 in reference cells confirmed its importance in phosphosignaling. Secretion of TNF-α was higher (P = 0.04) and IL-10 was lower (P = 0.04) in risk cells. Plasma LAG3 levels were lower in HALP carriers of the CC allele (P < 0.0001) and by race (P = 0.004). In MESA, race (P = 0.0005), age (P = 0.003), lipid medications (P = 0.03), smoking history (P < 0.0001), and rs10846744 genotype (P = 0.002) were independent predictors of plasma LAG3. In multivariable regression models, plasma LAG3 was significantly associated with HDL-cholesterol (HDL-C) (P = 0.007), plasma IL-10 (P < 0.0001), and provided additional predictive value above the Framingham risk score (P = 0.04). In MESA, when stratified by high HDL-C, plasma LAG3 was associated with coronary heart disease (CHD) (odds ratio 1.45, P = 0.004).

CONCLUSION: Plasma LAG3 is a potentially novel independent predictor of HDL-C levels and CHD risk.

FUNDING: This work was supported by an NIH RO1 grant (HL075646), the endowed Linda and David Roth Chair for Cardiovascular Research, and the Harold S. Geneen Charitable Trust Coronary Heart Disease Research award to Annabelle Rodriguez. MESA is conducted and supported by the National Heart, Lung, and Blood Institute (NHLBI) in collaboration with MESA investigators. Support for MESA is provided by contracts HHSN268201500003I, N01-HC-95159, N01-HC-95160, N01-HC-95161, N01-HC-95162, N01-HC-95163, N01-HC-95164, N01-HC-95165, N01-HC-95166, N01-HC-95167, N01-HC-95168, N01-HC-95169, UL1-TR-001079, UL1-TR-000040, and DK063491. Cardiometabochip genotyping data for the MESA samples was supported in part by grants and contracts R01HL98077, N02-HL-64278, HL071205, UL1TR000124, DK063491, RD831697, and P50 ES015915.

Authors

Diana Golden, Antonina Kolmakova, Sunitha Sura, Anthony T. Vella, Ani Manichaikul, Xin-Qun Wang, Suzette J. Bielinski, Kent D. Taylor, Yii-Der Ida Chen, Stephen S. Rich, Annabelle Rodriguez

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Figure 4

LAG3 protein is crucial in B cell receptor (BCR) signaling.

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LAG3 protein is crucial in B cell receptor (BCR) signaling. Protein was ...
Protein was isolated from EBV-transformed B cells expressing the reference (GG) or risk (CC) allele under basal or cocktail-stimulated (PMA/ionomycin+IL-4) conditions for 2 hours. (A) Ramos cell line as control B cells, reference (GG-003) cell line or risk (CC-008) cell line. As compared with basal conditions, p-CD79A (P = 0.04), p-CD19 (P = 0.04), p-SYK (P = 0.005), p-LYN (P = 0.001), p-PLCγ2 (P = 0.004) and p-PKCβ (P = 0.003) were increased in stimulated reference cells. Similar experiments were performed for additional cell lines derived from subjects expressing the reference or risk allele for a total of 6 independent cell lines (n = 3 for the reference cells and n = 3 for the risk-expressing cells) (Supplemental Figure 4). (B) The effect of overexpression of lentiviral LAG3-GFP or short-hairpin, shRNA-LAG3 silencing on BCR phosphosignaling in unstimulated cells. Lanes: 1, mock transduction in risk cells; 2, transduction of lentiviral LAG3-GFP in risk cells; 3, transduction of lentiviral LAG3-GFP in reference cells; 4, mock transduction in reference cells; 5, transduction of scrambled shRNA in reference cells; and 6, transduction of shRNA-LAG3 in reference cells. As compared with cells transduced with the mock vector, p-LYN (P = 0.04) and p-PKCβ (P = 0.03) were increased in risk cells overexpressing LAG3 protein (lane 2), while p-LYN (P = 0.04) and p-PKCβ (P = 0.01) were increased in reference cells overexpressing LAG3 protein (lane 3) as compared with cells expressing the mock vector. (C) The effect of overexpression of lentiviral LAG3-GFP or shRNA-LAG3 silencing on BCR phosphosignaling in stimulated cells. Lanes: 1, mock transduction in risk cells; 2, transduction of lentiviral LAG3-GFP in risk cells; 3, transduction of lentiviral LAG3-GFP in reference cells; 4, mock transduction in reference cells; 5, transduction of scrambled shRNA in reference cells; and 6, transduction of shRNA-LAG3 in reference cells. As compared with cells transduced with mock vector, p-LYN (P = 0.01) and p-PKCβ (P = 0.01) were increased in stimulated risk and reference cells overexpressing LAG3 protein (lanes 2 and 3, respectively). As compared with mock-transduced reference cells, LAG3 silencing was associated with reduced p-LYN (P = 0.002) and p-PKCβ (P = 0.009) (lane 6). In lanes 4 and 5, reference cells showed the expected increase in p-LYN and p-PKCβ following stimulation. The results were analyzed using a 2-sided Student’s t test; n = 3, P < 0.05 was considered significant.