Treatment with L-4F, in the absence or presence of pravastatin, effectively reduced manifestations of lupus-like autoantibody production, glomerulonephritis, and osteopenia in our apoE-/-Fas-/- B6 murine model of accelerated atherosclerosis in SLE. Only mice treated with L-4F, with or without pravastatin, had significantly reduced glomerular tuft size, IgG anti-PGPC and IgG anti-POVPC antibodies, lower plasma proinflammatory cytokine/chemokine levels, and increased total and vertebral BMD compared to vehicle controls. Furthermore, mice treated with L-4F alone also had significantly lower levels of IgG anti-dsDNA and IgG anti-cardiolipin autoantibodies. Although larger aortic lesions were consistently present in all the treatment groups, lesion characteristics of the combination treatment group indicate decreased macrophage infiltration and inflammation, potentially suggestive of plaque remodeling. Despite the reported success of the immunomodulatory effects of statins in mouse models, no increased effects were appreciated in mice treated with the combination treatment compared to those receiving L-4F alone. To our knowledge, our L-4F treatment regimen has not been previously used in murine models of atherosclerosis in SLE.
Statins in SLE patients and murine models have shown varying degrees of success in recent trials [7
]. Pravastatin was successful in reducing total cholesterol and LDL at both 10 mg/day and 40 mg/day doses, but failed to exhibit anti-inflammatory properties in rheumatoid arthritis patients [38
]. Conversely, atorvastatin showed positive results in the prevention of endothelial-dependant vasodilation and reduction in disease activity in SLE patients at 20 mg/day in a controlled trial, but failed as a mono-therapy in a NZB/NZW murine lupus model to control anti-dsDNA antibodies, proteinuria, and kidney disease [39
]. Nachtigal et al. mentions that compared to human studies, higher doses of statins are normally required in mouse models; this is potentially a result of the inactivation of HMG-CoA reductase inhibitors by P450 enzyme induction and the elevation of HMG-CoA reductase levels [42
]. These studies suggest that the efficacy of statins as treatment for systemic inflammation, characteristic of SLE, is dependent on the study protocol, dosage, and/or inclusion/exclusion criteria for study participation. In our attempt to achieve synergistic effects between our statin regimen and our administered novel peptide, our suboptimal dose of pravastatin alone did not significantly control the progression of either renal deterioration, production of IgG autoantibodies to dsDNA or oxPLs, or formation of atherosclerotic lesions in our model.
Since statin regimens have had such varied results among different studies, we added an apolipoprotein mimetic peptide to potentially contribute pleiotropic effects as seen in other murine models of atherosclerosis [23
]. Recent studies have shown the effectiveness of piHDL as a predictor of subclinical atherosclerosis in SLE patients [45
]. Since L-4F effectively reduced the proinflammatory effects of LDL in preliminary studies (Figure ), we believed L-4F could potentially be utilized to target inflammatory lipids and as a result, limit the progression of inflammation, including atherosclerotic manifestations, in our lupus model.
Renal involvement and glomerulonephritis are serious complications that can present in patients diagnosed with SLE. Elevated plasma levels of VCAM-1, which also plays a role in perpetuating atherosclerotic plaque formation, are associated with nephritis and increased disease activity in SLE patients [37
]. Similarly, Yao et al. proposed a correlation between increased renal lesions, elevated levels of VCAM-1, and degree of symptom severity in patients with lupus nephritis [47
]. In our study, lower circulating VCAM-1 levels were consistent with 11% and 19% smaller mean glomerular tuft areas seen in L-4F or combination treatment mice, respectively, compared to vehicle controls after 27 weeks of treatment.
L-4F treatment in the presence or absence of pravastatin also significantly prevented overall bone loss and additional osteopenic manifestations within the lumbar spine, as reflected in significantly higher total BMD and vertebral BMD in treatment mice, compared to vehicle controls. Feng et al. showed that five-month-old female apoE-/-
mice experienced a greater decrease in vertebral BMD than in femoral BMD by the time they reached nine months [16
]; this could account for the minimal difference seen among the femoral BMD values of the different treatment groups. Okamatsu et al. previously demonstrated, in a series of neutralization studies, that RANKL, a stimulator of osteoclastogenesis, activation, and survival, triggers CCL9, which further stimulates osteoclast activation for bone resorption [48
]. Mice receiving L-4F had significantly lower plasma levels of CCL9 than control mice, which correspond with improved trabecular bone characteristics observed in L-4F-treated mice compared to vehicle controls. Furthermore, Graham et al. demonstrated that the production of RANKL, by T lymphocytes could be induced by circulating oxPLs [49
], indicating that osteopenic manifestations could be linked to atheroma formation as a result of elevated levels of circulating oxPLs.
OxPLs, such as POVPC and PGPC, are commonly found in oxidized LDL and aid in the development of fatty streaks, which may contribute to accelerated atherosclerosis in SLE [50
]. Mice with L-4F or combination treatment showed significantly decreased levels of IgG autoantibodies to both POVPC and PGPC without significant alteration in plasma lipid levels (Figure ). In addition, L-4F successfully improved the anti-inflammatory function of HDL and reduced the proinflammatory nature of LDL, as determined in cultures of human aortic endothelial cells. Increased levels of circulating CCL19 has been correlated with unstable plaques in patients with CVD compared to patients with stable plaques [36
]. Significantly decreased levels of circulating CCL19 and VCAM-1, both linked to plaque formation and instability, are consistent with possibly improved lesion characteristics in both L-4F and combination treatment mice.
Despite reduced inflammation, as indicated by lower levels of circulating plasma proinflammatory chemokines and reduced lipoprotein inflammatory activity in cultures of human aortic endothelial cells, all treatment groups presented enlarged aortic lesions compared to vehicle controls. In response, we investigated the composition of the local plaque environment at the aortic root to determine the relationship between size and stability of the atheromas in our model. In humans, advanced plaques, which are more vulnerable to rupture, are characterized by large populations of infiltrated macrophages, lower concentrations of smooth muscle cells, and lower collagen content with thinner fibers [51
]. Aortic lesions from mice treated with L-4F plus pravastatin had 37% less mean macrophage area and 59% more mean smooth muscle cell area compared to vehicle controls (Figure ). Although these characteristics indicate improved plaque composition, tissue levels of VCAM-1 did not reflect the significantly decreased levels seen in circulation in L-4F-treated mice and showed minimal deviation across the four treatment groups. Similarly, collagen content of lesions from the different groups did not vary significantly. Despite this, the improved changes in atheroma cellular composition of both infiltrating macrophages and SMC content, in combination with circulating levels of IgG anti-oxPLs and atherogenic chemokines, possibly suggest improved stability and potential remodeling of atherosclerotic lesions in L-4F-treated mice compared to vehicle controls.
The histopathologic composition of the lesions indicated that oil red O and α-actin stained areas predominantly contributed to the increased lesion size in the three treatment groups compared to the control group. This may be the result of elevated neutral triglycerides and lipids (as reflected by oil red O staining) and increased smooth muscle (as reflected by α-actin staining). However, there was no apparent increase in volume of infiltrated CD68+ macrophages, collagen content, and t-cell concentration in any of the treatment groups compared to the control group that could have contributed to the increased lesion size. In a previous study, daily treatment with oral pravastatin (50 μg/mouse) and subcutaneously injected L-4F (200 μg/mouse) did not show improvement of aortic plaques in a small cohort of nine-month-old female ApoE-/-
B6 mice compared to baseline studies [53
]. Similarly, daily suboptimal doses of oral atorvastatin (10 mg/kg) have been shown to mildly increase plaque size, albeit not significantly, in two-month-old female apoE-/-
mice when compared to controls [54
Circulating proinflammatory cytokines and chemokines trended lower in mice receiving L-4F with or without pravastatin. The presence of circulating markers in the control mice, such as IL-10, a Th2 cytokine involved in B cell activity upregulation and linked to increased IgG anti-dsDNA autoantibodies [55
], CCL19, and VCAM-1, indicate increased autoimmune response and increased risk for unstable atherosclerotic plaques due to their role in humoral immunity or monocyte recruitment to plaque sites [36
]. Reduction of these circulating cytokines by L-4F or combination treatment may have contributed to limiting inflammation-induced glomerulonephritis by dampening autoimmune responses in our mice. Biomarkers such as these could potentially be developed into a chemokine score to monitor and assess disease activity in patients with SLE and atherosclerosis. Similarly, Bauer et al. proposed that monitoring CCL19 and other interferon-regulated chemokines would be beneficial for the assessment of current disease activity and prediction of future flares in patients with SLE [57