Antibodies against Oxidized Phospholipids and Osteoporosis

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.

Grant Number: 5I01BX003901-06
NIH Institute/Center: VA
Principal Investigator: Elena Ambrogini