This study investigated in a murine model the efficacy of probucol to suppress cerebral capillary dysfunction and heightened neurovascular inflammation that occurs with aging and long-term ingestion of SFA enriched diets.
Previous studies have equivocally demonstrated the presence of plasma-derived proteins within brain parenchyme in mice maintained on SFA enriched diets for 3 to 12 months [4, 5, 8–10]. Diets enriched in SFA also promote secretion from epithelial cells of the small intestine of Aβ associated with chylomicrons [7, 11, 12] and these findings previously led us to propose that exaggerated exposure to SFA and/or chylomicron-Aβ compromise capillary permeability. Whatever the mechanism, plasma derived apoB lipoprotein-Aβ extravasation to brain parenchyme and exaggerated retention upon extracellular matrices has been demonstrated in mice fed an SFA-enriched diet for 6 months . In more recent studies, synergistic effects of aging and western styled diets on cerebral capillary dysfunction were demonstrated . Notably however, marked capillary dysfunction was also shown in aged (12 month) mice maintained on a LF diet that was free of saturated fats and cholesterol.
Several lines of evidence suggest that probucol may be an agent of therapeutic value for individuals with AD . In addition to positive capillary function regulatory effects in mice maintained for relatively short periods of time on SFA/cholesterol diets, probucol has other potentially useful metabolic effects including synaptic potentiation ; inhibition of Aβ biogenesis ; promotion of Aβ-chaperoning (reduction in formation of toxic oligomers) ; vasodilatory effects ; and anti-oxidant/-inflammatory properties [4, 13]. However, further consideration of probucol in a therapeutic context for prevention of non-familial AD would be strengthened by longitudinal in vivo studies in an aging context.
This study used an established model of BBB dysfunction induced by aging and exacerbated by the provision of dietary SFA to explore the effects of probucol on cerebral capillary function. The dietary intervention was physiologically relevant with approximately 40% of energy derived as fats. The SFA diet was well tolerated and mice randomized to this treatment were found to be normolipidemic and had similar body weight to LF fed controls at the conclusion of treatment. Probucol predictably lowered plasma cholesterol but this was not associated with either parenchymal extravasation of plasma proteins, or measures of neurovascular inflammation and so was not considered further.
In this study, we firstly confirm that parenchymal extravasation of plasma derived IgG is increased as a consequence of aging and exacerbated by SFA feeding consistent with our previous study . We extend those findings and now show that parenchymal retention of apoB lipoproteins is markedly increased in mice maintained for 12 months on an otherwise healthy LF diet. A synergistic effect of aging with SFA resulted essentially in a doubling of the age-induced effect. The co-provision of probucol with SFA was found to completely abolish the long-term SFA induced effect, a phenomenon previously reported in mice maintained on an SFA diet for just 3 months . However, probucol had no marked effect on the age-associated increased abundance of plasma-derived proteins within brain parenchyme, only showing parenchymal IgG and apoB reduction to the 12 months LF level but not to the 3 months LF mice without the ageing effect. Some regional differences in effectiveness were also noted. Probucol effectively abolished the SFA induced accumulation of IgG and apoB within the CTX following 12 months of intervention. However, within the HPF probucol had only modest but not significant effect on apoB retention at 12 months of intervention.
GFAP and COX-2 are widely used measures of neurovascular inflammation. Both measures were increased principally within the cortex in mice maintained on a LF diet for 12 months compared to those following 3 months of intervention. However, GFAP and COX-2 were markedly elevated as a consequence of SFA in both CTX and HPF. The co-provision of probucol had a remarkable suppressive effect on these measures of neurovascular inflammation essentially completely abolishing the SFA-induced effect that persisted for the duration of intervention. Collectively, probucol appears to protect BBB integrity through the suppression of neurovascular inflammation in aged mice maintained on an SFA diet.
Another possible mechanism by which probucol prevents the long-term high SFA induced BBB dysfunction may attribute to the reduced BBB exposure to circulating Aβ that are associated with apoB lipoproteins. Indirect evidence comes from studies in amyloid transgenic mice, where it was reported that onset and progression of amyloidosis was positively associated with the secretion into blood of Aβ that was associated with nascent chylomicron . The SFA diet used in this study was previously demonstrated to increase the enterocytic association of Aβ with nascent chylomicrons [11, 12]. In other studies; probucol was shown to strongly suppress the chylomicron-Aβ in mice fed an SFA enriched diet for 3 months . Hence the proposition that postprandial-Aβ is associated with age-induced capillary dysfunction is worthy of consideration.
Enterocytic apoB (an obligatory structural element of chylomicrons) is a useful surrogate marker of chylomicron biosynthetic rates . The abundance of Aβ that is colocalized with nascent chylomicrons is therefore a direct measure of protein abundance per lipoprotein particle assembled.
This study reports that mice maintained on a LF diet for 12 months had substantially elevated Aβ relative to nascent chylomicrons within the absorptive epithelial cells of the small intestine compared to 3 months LF control mice. However, enterocytic apoB production remained comparable. Long-term ingestion of SFA enriched diets doubled the enterocytic postprandial Aβ production concomitant with increased enterocytic apoB lipoprotein, suggesting increased release of lipoproteins and Aβ into the circulation. However, the enterocytic production of Aβ was completely abolished by the co-provision of probucol with the SFA diet. In mice, it is not feasible and perhaps not relevant to measure postprandial chylomicron-Aβ in plasma because of the limited sampling volume; separation of plasma lipoprotein classes; the effects of anesthesia and mimicking the ‘chronic’ (12 month) dietary intervention in the context of a singular acute fat challenge. Nonetheless, our data showed a significant increase in the plasma Aβ by the long-term ingestion of SFA enriched diet. The study also provided evidence of a quite strong positive association between chylomicron-Aβ, cerebral-capillary permeability and parenchymal apoB lipoprotein abundance. Consistent with the notion that chylomicron-Aβ per se, rather than chylomicron concentration is more relevant to capillary integrity, there was a relatively poor positive association of permeability with enterocytic apoB. Furthermore, probucol had a substantial effect on enterocytic Aβ, but a weaker and non-significant effect on apoB abundance. These data collectively suggest that probucol may protect the BBB integrity through reduced BBB exposure to postprandial lipoprotein-Aβ by attenuating the enterocytic production of postprandial Aβ.
This is the first long-term intervention study showing potential beneficial effects of probucol on cerebral capillary integrity in the context of reducing risk for Alzheimer’s disease. This study demonstrates persistent suppression by probucol of neurovascular inflammation and differential effects of capillary permeability in aged mice that had been maintained on an SFA-enriched diet. The beneficial effects of probucol on SFA-induced capillary dysfunction may in part be a consequence of suppression of postprandial lipoprotein-Aβ secretion and decreased vascular exposure, and of its anti-oxidative/-inflammatory properties.