Antibodies against Oxidized Phospholipids and Osteoporosis

Central Arkansas Veterans Hlthcare Sys, North Little Rock AR

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Abstract

Phosphatidylcholine is the most common phospholipid in cell membranes, microvesicles, and lipoproteins; it contains the phosphocholine head group (PC) and polyunsaturated fatty acids (PUFA) that are susceptible to lipid peroxidation by reactive oxygen species. Lipid peroxidation of PUFA generates oxidized phospholipids (OxPLs) that are highly reactive and cause extensive cell damage. Oxidized phospholipids containing phosphocholine (PC-OxPLs) are pathogenic in multiple conditions, including osteoporosis, cardiovascular diseases, and steatohepatitis. Following the peroxidation of PUFA, the phosphocholine headgroup undergoes a conformational change and becomes an epitope that is recognized and bound by the natural antibody E06 IgM which blocks the deleterious effects of OxPLs. Transgenic mice overexpressing a single chain (scFv) form of the antigen-binding domain of the E06 IgM (E06-scFv) have increased bone mass at 6 months of age and are protected against age-induced bone loss. E06-scFv increases bone formation and osteoblast number at both cancellous and cortical sites. OxPLs decrease Wnt10b and Wnt target genes in osteoblastic cells. Consistent with this, with unbiased approaches such as bulk RNA sequencing of vertebrae and single cell RNA sequencing of osteoblastic cells we found that Wnt10b and Wnt target genes are upregulated in E06-scFv transgenic mice compared to WT. Increasing the levels of antibodies against PC-OxPLs, by either overexpressing E06-scFv or injecting a monoclonal E06 IgG antibody, is protective against multiple diseases where PC-OxPLs play a pathogenic role such as atherosclerosis, ischemia reperfusion injury, vascular inflammation, and steatohepatitis. Importantly, short term administration of a monoclonal form of E06 IgG increases mineral apposition rate, indicating that the injectable antibody increases osteoblast function. We have investigated the relationship between the endogenous levels of anti-PC antibodies and bone mineral density (BMD) in humans in a cross-sectional study of 247 Veterans and found that the levels of anti-PC IgM negatively correlate with both the T- and Z-scores at the femur. This correlation is maintained after adjusting for age, race, and gender. Consistent with this, patients with osteopenia and osteoporosis at the femoral sites had higher levels of anti-PC IgM compared to those with normal BMD. These results suggest that higher levels of endogenous anti-PC IgM in patients with osteopenia and osteoporosis reflect higher exposure to PC-OxPLs, although insufficient to prevent their adverse effects. Based on these findings, we hypothesize that PC-OxPLs inhibit osteoblast differentiation and function by affecting Wnt signaling and their neutralization with exogenous administration of a monoclonal E06 IgG antibody is an effective therapeutic intervention to increase bone mass in mice. In humans, anti-PC IgM are a marker of for increased exposure to PC-OxPLs and may predict the loss of bone with time. Studies proposed in this application will establish whether E06-scFv increases bone mass by increasing Wnt10b by characterizing the bone phenotype of E06-scFv transgenic mice crossed with mice in which Wnt10b has been globally deleted. We will also determine whether weekly injections of murine monoclonal E06 IgG increase bone mass and reproduce the anabolic effects of the E06-scFv transgene in young and old mice. Additionally, we will expand the clinical studies to determine whether the levels of the anti- PC IgM correlate with baseline BMD, with longitudinal BMD changes in the elderly and with fractures. This will be accomplished by measuring the levels of anti-PC IgM in a large cohort of patients from two epidemiological studies: the Osteoporotic Fractures in Men (MrOS) and the Study of Osteoporotic Fractures (SOF) in women. Successful completion of these preclinical and clinical studies may lead to a novel therapy to simultaneously prevent or ameliorate osteoporosis together with multiple age-related disease that are very common in the aging veteran population.