The present study has multiple findings that may have important implications for the treatment of human atherosclerosis using LA, a commonly used dietary supplement, at low doses. 1) LA decreases atherosclerotic plaque burden within a 12 week period with changes seen as early as 6 weeks; 2) Decreased plaque burden was paralleled by marked improvements in vascular function: 3) LA reduces NADPH oxidase dependent O2•- and NF-κB-mediated inflammatory responses, offering a molecular basis for the anti-atherosclerotic effects of LA; 4) LA anti-inflamatory effects involves reduction in key adhesion and chemokine molecules involved in T cell trafficking to atherosclerotic plaque; effects that were confirmed by dose dependent effects of LA in reducing T cell migration across CCL5/SDF-1 gradients and prevention of T cells to the endothelium.
MRI scanning demonstrated that 6 weeks of dietary supplementation with LA was sufficient to inhibit atherosclerotic lesion development in WHHL rabbits. We induced rapid progression of plaque by high cholesterol feeding for 6 weeks prior to randomization. LA treatment inhibited progression of plaque in the absence of notable changes in plasma lipoprotein levels. Our data are consistent with the effects noted by Zhang et al but differ in dosing and timing of LA treatment (Zhang et al fed mice with 0.2% (wt/wt) LA for a period of 10 weeks.). In contrast, our studies were in established atherosclerosis with much lower doses (Zhang, et al., 2008
). Zhang et al demonstrated an effect of LA in reducing TG concentrations in the ApoE-/-
mouse model while total cholesterol increased in the ApoE-/-
strain. These disparate effects on lipoprotein metabolism raise the question of context dependent effects of LA that may relate to degree and genesis of lipoprotein abnormalities in the animal model studied. In our studies, high fat diet administration in the setting of additional genetic predisposition lead to extremely high values of most lipid parameters and precluded detection of any changes that may have been induced by LA.
An additional important observation in this study pertained to the weight loss noted with LA. LA has been described to have appetite suppressant effect through an AMP kinase mechanism in the hypothalamus (Kim, et al., 2004
). The only definitive way to discount an effect on food intake is to perform pair-feeding experiments. Our experiments were very similar to a pair fed experimental situation, in that the LA and control groups consumed equal amount of pre-apportioned diet. Thus the weight loss effects are unlikely to relate to reduced food intake. While it may be difficult to dissociate the effects on weight loss from its direct effects in reducing plaque progression, we believe that latter effects are distinct and dominant. In the study by Zhang et al that did employ pair feeding, the attenuation of lesion area was not accounted by weight loss alone (Zhang, et al., 2008
). Studies by Kim et al in rats, demonstrated weight loss at doses that ranged from 0.25, 0.5 and 1%, wt/wt (Kim, et al., 2004
). Our studies were designed cognizant of these earlier studies and therefore employed far lower doses (20 mg/d) than that employed by either study to limit the weight loss effects. Nevertheless it appears that weight loss is seen in the rabbit model and may potentially explain the effects on insulin sensitivity. The effects of LA in potentiating insulin mediated dilation are important as LA has been suggested to be an insulin sensitizing agent through PI3 kinase/Akt dependent pathways (Jacob, et al., 1999
; Yaworsky, et al., 2000
). LA has been shown to enhance IRS-1 expression and insulin-stimulated IRS-1 association with PI3K (Saengsirisuwan, et al., 2004
). Our results indeed do confirm a marked insulin sensitizing effect of LA in the vasculature although. Steady state, levels (non-insulin stimulated) of phosphorylated Akt and eNOS in the aorta were no different. The lack of difference in phosphorylation states by westerns does not by itself argue against the in-vivo functional data as the phosphorylation data was obtained in the absence of agonist stimulation. Thus there could still be important differences in the presence of insulin or acetylcholine. Additionally eNOS and Akt is regulated by phosphorylation at multiple sites and the lack of difference at one site does not preclude differences at other site.
Our results demonstrating a favorable effect on endothelial function is consistent with our prior observations suggesting an effect of LA even in the context of multiple risk factors in diseases such as metabolic syndrome and Type II diabetes (Sola, et al., 2005
). Notably, although LA treatment did not affect the vasoconstriction to PE and endothelin-1, it reduced the constriction to angiotensin II through down-regulation of AT1 mRNA, similar to its effects in the renal vasculature (Mervaala, et al., 2003
). These effects are consistent with therapeutic synergism seen in our human studies evaluating the effects of AT-1 receptor blockade in conjunction with LA supplementation (Sola, et al., 2005
). The renin-angiotensin-aldosterone system, a crucial regulator of vascular homeostasis, has consistently been shown to play a prominent role in atherogenesis (Daugherty, et al., 2000
; Munzel, et al., 2008
; Weiss, et al., 2001
). Conversely, blockade of AT1 receptors in atherosclerosis normalizes NADPH oxidase activity, improves endothelial function, and reduces plaque area and macrophage infiltration (Hornig, et al., 2001
; Sorescu, et al., 2001
; Warnholtz, et al., 1999
). Thus LA may potentially exert additional synergistic effects with drugs that target the RAAS system in atherosclerosis, a finding that may be worthy of exploration in the future.
A novel finding was the pronounced effects of LA on T cell transmigration. Our results show that LA markedly reduced T cell infiltration in the atherosclerotic lesion. This may be attributed to reduced migration or “homing” of T cells presumably through its effects on reducing adhesion molecules such as CD62L (L-Selectin) and ICAM and/or chemotactic influences such as RANTES and SDF-1, factors critically important in the migration of T cell populations to the plaque. It is interesting to note that RANTES is regulated by NFkB and the effects of LA on RANTES expression may relate to these effects (Lee, et al., 2000
; Munzel, et al., 2000
). These observations are similar to the effect of LA in modulating neural inflammation and reducing the migration of T cells to the central nervous system in experimental encephalomyelitis, through effects on cyclic AMP pathways (Chaudhary, et al., 2006
; Schillace, et al., 2007
). Thus treatment with LA has broad anti-inflammatory and metabolic effects that may be beneficial in inflammatory diseases.
In conclusion, LA exerts important metabolic and vascular protective effects in models of established atherosclerosis. These findings provide an important impetus to design appropriate human trials with LA.