This study is the first to demonstrate the capacity of whole mushrooms, delivered as DMSO extracts, to reduce adhesion molecule expression with subsequent reductions in human monocyte binding to human aortic endothelial cells. Specifically, we have demonstrated that dietary mushrooms significantly reduced cell surface expression of VCAM-1, ICAM-1, and E-selectin. Furthermore, there were significant reductions in adhesion of pre-loaded human monocytes to both unstimulated, or quiescent, and pro-inflammatory, cytokine-stimulated HAEC as summarized in Table . The health implications are that diverse mushrooms, including common and specialty mushrooms can protect against cardiovascular disease by interfering with events that contribute to atherogenesis.
Summary of effects by mushrooms on adhesion molecule expression and monocyte binding
Mushrooms have received marginal attention regarding their potential role in protecting against cardiovascular disease although studies have suggested a protective effect against CVD. For example, powders (5% dry weight) of shiitake, maitake, and Agaricus bisporus
significantly decreased plasma cholesterol levels and blood pressure in rats presumably due to the bioactive agent eritadenine [20
]. Furthermore, dietary oyster mushrooms (P. ostreatum
) also elicited a hypocholesterolemic effect and inhibited lipid peroxidation, a process linked to oxidative stress, in rabbits [23
]. This is in agreement with our results in that white button and crimini (A. bisporus
), maitake and oyster significantly reduced AM expression and monocyte binding, which would counter a hypercholesterolemic, or hyperlipidemic, effect. That is, even with elevated plasma levels of lipids, the reduced expression of AM and subsequent inhibition of binding of monocytes would interrupt the atherogenic process. This is further supported by the observation that a 10% (w/w) oyster mushroom diet significantly reduced both the incidence and size of aortic atherosclerotic plaques in rabbits suggesting interaction with physiological and molecular processes of atherogenesis [23
]. Percario et al. also showed that a natural antioxidant-rich mushroom, Agaricus sylvaticus
, could prevent the development of atherosclerosis in an environment of hypercholesterolemia using a rabbit model [25
]. Collectively, several studies suggest that dietary mushrooms can inhibit or slow CVD although more research is needed.
In this report, we have demonstrated that both common and specialty mushrooms can modulate processes at the cellular level where AM expression is attenuated and binding of monocytes blunted. Others have identified alternative mechanisms for a potential CVD protective effect by dietary mushrooms. For example, oyster mushrooms (10% of diet) reduced significantly the incidence of atherosclerotic plaques in rabbits as well as plaque size by 26% compared to the control group and prevented formation of atheromas, reduced foam cell number, and reduced coronary arterial injury [24
]. Other dietary antioxidants (vitamins C, E, and beta carotene) have been shown to reduce lesion area by reducing concentrations of soluble adhesion molecules by 36-61% in mice [26
]. This observation is important because soluble AM have been proposed as a critical assessment tool for asymptomatic CVD in high-risk patients [27
]. While we did not measure soluble AM in this study, the capacity of the test mushrooms to reduce cell surface expression in HAEC is equally compelling as a protective mechanism. Other proposed mechanisms of cardioprotection in rodents consuming Grifola frondosa
, Inonotus obliquus
, Antrodia xantha
, and Rigidoporus ulmarius
include inhibition of COX-1 and COX-2 activities, inhibition of iNOS via reduced NF-kB binding, and potent antiangiogenesis activity [28
]. Collectively, the data support the capacity of both exotic and commonly consumed mushrooms to beneficially modulate numerous processes associated with atherogenesis.
We also noted in this study that the three most frequently measured AM including VCAM-1, ICAM-1 and E-selectin, were reduced singly, collectively, or in various combinations of two after incubation with mushrooms yet binding of monocytes was consistently reduced after incubation with each mushroom extract. Others have shown similar results with different dietary agents using the HAEC cell model, which is a non-transformed primary endothelial cell line. For example, pretreatment of HAEC with a red wine polyphenol extract significantly inhibited oxysterol-induced cell surface expression of adhesion molecules and subsequent adherence of monocytes to monolayers by inhibiting primarily VCAM-1 [31
]. Subsequent studies by Naito et al. showed that tocotrienols exerted a similar effect [32
]. Dietary consumption of antioxidant polyphenols from avenathramide-enriched mixtures (AEM) derived from oats significantly reduced IL-1β-stimulated VCAM-1, ICAM-1, and E-selectin expression in HAEC by 50, 20, and 20% respectively, and reduced U937 binding at concentrations as low as 20 μg/mL [33
]. Ginkgo biloba extracts reduced both VCAM-1 and ICAM-1 and adhesion of monocytes to HAEC [34
]. In a study using structurally related dietary carotenoids, Martin et al. found that pre-incubation of HAEC with beta-carotene, lutein and lycopene significantly reduced VCAM-1 expression, beta-carotene and lutein significantly reduced E-selectin expression, and beta-carotene, lutein and lycopene significantly reduced the expression of ICAM-1, yet lycopene was the only carotenoid to significantly reduce binding of U937 to HAEC [19
]. The antioxidant pyrrolidine dithiocarbamate selectively inhibited in HUVEC cells VCAM-1 induction, but not ICAM-1, after stimulation with TNF-α and LPS. E-selectin was only partially inhibited after TNF-α incubation [35
]. Pretreatment of HUVECs with vitamin E and probucol significantly reduced the expression of VCAM-1 induced by oxidized LDL, but did not reduce expression of ICAM-1 [36
]. In contrast, the well recognized antioxidants N-acetyl-L-cysteine and troglitazone, an antidiabetic agent, reduced or completely abolished VCAM-1, E-selectin, and ICAM-1 expression in endothelial cells after pro-oxidant signals including oxidized LDL and TNF-α [37
]. Thus, each of the three commonly tested AM can be modulated either singly or in combination by diverse dietary agents and typically at least one, but often more, are inhibited. This suggests that attenuation of at least one AM, without specificity as to which one, may be adequate for inhibition of binding of monocytes but also that there are many other contributors to this process that may not be measured.
It is noteworthy that mushrooms also contain high levels of bioactive agents including polyphenols and the novel antioxidant ergothioneine, which is produced exclusively in mushrooms and some bacteria [38
]. Dietary polyphenols such as catechin and quercetin have been shown to significantly reduce VCAM-1 expression and binding of monocytes to HAEC and ergothioneine exerts antioxidant activity that may be protective in a pro-inflammatory environment [40
]. The test mushrooms used in this study have been analyzed and found to contain considerable amounts of both ergothioneine and total polyphenols [43
]. Moreover, all test mushrooms exhibited significant antioxidant capacities using assays for oxygen radical absorbance capacity (ORAC), and hydroxyl (HORAC), peroxynitrite (NORAC), and superoxide (SORAC) radical averting capacity. Mushrooms also contain high levels of selenium and copper, essential micronutrients, needed for proper function of antioxidant enzymes, which can reduce oxidative stress and inflammation often found in CVD [45
]. It is possible that varying levels of these bioactive agents alone or in combination contributed to the beneficial effects observed.