The principal observation in this study is that quercetin and two of its metabolites, quercetin 3′-sulphate and quercetin 3-glucuronide, inhibit key inflammatory changes in the porcine isolated coronary artery induced by prolonged exposure to LPS. The basis of this potentially beneficial effect of the flavonoid can be largely attributed to prevention of the induction of NOS.
Lipopolysaccharide-induced changes in the porcine coronary artery
Prolonged exposure of the coronary artery to LPS induced a significant reduction in contractile responses to both KCl and U46619, with a slightly greater effect against the former, and an impairment of endothelium-dependent relaxations to substance P. The effect of LPS on contractile responses is qualitatively similar to that noted in other studies (Gibreal et al., 2000; Piepot et al., 2000), but was not accompanied by a reduction in the potency of either agonist. In the case of substance P-induced relaxations, suppression of endothelium-dependent responses by LPS has been previously reported for the porcine coronary artery (Qi et al., 2007). The inhibitory effects of LPS on contractile responses was also observed in endothelium-denuded segments, which suggests that these changes are a function of independent actions on endothelial and non-endothelial cells in the blood vessel.
These changes in vascular responsiveness were accompanied by increased production of nitrite ions (as determined by the Griess reaction) and immunohistochemical evidence for induction of NOS in the blood vessel. The adventitial location for iNOS in the coronary artery is similar to that reported for the rat aorta (Kleschyov et al., 1998), where exposure to LPS caused a 4- to 10-fold greater activity for nitric oxide production in the tunica adventitia compared with the tunica media. A strong pharmacological link between biochemical and functional events was provided by the finding that exposure to 1400 W, a selective inhibitor of iNOS (Garvey et al., 1997), prevented LPS-induced changes in contractile responses and nitric oxide formation in the porcine coronary artery. As the effect of 1400 W on LPS-induced changes in the coronary artery were mimicked by inhibition of NFκB (with Bay 11-7082) it would appear that activation of this pathway precedes induction of NOS, as described in macrophages (Chen et al., 2005).
The effect of quercetin and quercetin metabolites
Quercetin, quercetin 3′-sulphate and quercetin 3-glucuronide suppressed the LPS-induced changes in endothelial and vascular responses, and the elevation in nitrite ion production, with the aglycone exhibiting activity at concentrations as low as 0.1 µM. As the effect of quercetin on LPS-induced changes was also noted in endothelium-denuded segments of the coronary artery, it would appear that the endothelium is not the primary site of action for this flavonoid. A direct action of quercetin on iNOS appears unlikely as immunohistochemical evidence clearly implicates suppression of the induction of the enzyme in the tunica adventitia, in a manner comparable with that reported for the anti-inflammatory agent dexamethasone (Korhonen et al., 2002). Quercetin has been reported to inhibit LPS-induced nitric oxide production in various non-vascular cells, including RAW 264.7 macrophages (Chen et al., 2001), J774.1 macrophages (Raso et al., 2001; Hamalainen et al., 2007)) and mouse BV microglia (Chen et al., 2005). Crucially, however, the potency of quercetin in the porcine isolated coronary artery is approximately 10- to 30-fold greater than that noted in cultured cells, where it is typically >3 µM. The efficacy of quercetin and its metabolites in this vascular model stands in marked contrast to that of myricetin, which failed to modify LPS-induced suppression of contractile responses.
Quercetin-induced changes of nitric oxide production superficially mirrors the effects observed on LPS-induced suppression of contractile response. While this point is reinforced by the lack of effect of myricetin on LPS-induced nitrite production, the precise relationship between biochemical events and functional changes is clearly complex. The highest concentration of quercetin examined (10 µM) significantly impaired vasoconstrictor responses and substance P-mediated relaxations per se (see Table 1). Thus, the overall effect of 10 µM quercetin on LPS-induced changes in contractile responses is the sum of two opposing actions and less than that observed with 1 µM quercetin – but comparable with that noted for the overall effect of 0.1 µM quercetin. Also, it remains a possibility that the overall effect of LPS on contractile responses is product of the induction of several inflammatory mediators rather than just nitric oxide (Qi et al., 2007). Overall, it is noteworthy that quercetin modified the coronary actions of LPS in a similar manner to that reported for eritoran (a Toll-like receptor 4 antagonist) against endotoxin in rat aortic segments (Ehrentraut et al., 2007), which suggests that this flavonoid may be beneficial in inflammatory conditions.
It is well recognized that a key mediator of inflammatory responses in cells is the translocation of nuclear factor-κB (NF-κB) from the cytoplasm to the nucleus and activation of numerous genes, including those for NOS and pro-inflammatory cytokines (Liu and Malik, 2006). In the case of endothelial and vascular smooth muscle cells NFκB has been linked to increased expression of cell adhesion molecules (Read et al., 1994), the induction of NOS (Hattori et al., 2003) and associated with development of early atherosclerotic lesions (Hajra et al., 2000). Thus, stabilization of the NF-κB/IκB complex in the coronary artery could explain the protective effect of quercetin against LPS-induced changes in contractile responses, nitric oxide production and the expression of PECAM-1 (CD31) found in this study. This possibility is reinforced by the observation that a combination of quercetin and Bay K 11-7082, a known inhibitor of NFκB (Pierce et al., 1997) was no more effective against LPS-induced changes in the coronary artery than quercetin alone (see Figure 4), yet neither condition completely prevented the inhibitory effect of LPS. While the precise molecular target for this beneficial effect of quercetin was not investigated, the failure of myricetin to mimic the effect of quercetin on LPS-induced nitric oxide production, and to also ‘antagonise’ the effect of quercetin (see Figure 7), indicates that these structurally related flavonoids may be useful in future studies. It is noteworthy that the lack of effect of myricetin on inflammatory responses in the coronary artery is not a selective effect for the vasculature. Blonska et al. (2003) noted that quercetin and kaempferol were capable of inhibiting LPS-induced production of IL-1β in RAW 264.7 cells but myricetin was inactive.
Although the potency of the flavonoids against inflammatory events in the coronary artery is greater than that reported in macrophages, suggesting a selective action on the vasculature, the physiological relevance remains unclear. Quercetin is extensively metabolized in man and the concentrations used in this study exceeded the peak plasma levels of the aglycone (0.03 µM) and the metabolites (3 µM) detected following dietary ingestion (Kroon et al., 2004; Wang and Morris, 2005). However, two recent studies raise the possibility that flavanoids can be either generated in situ from metabolites accumulated in activated cells (Kawai et al., 2008a) or even preferentially concentrated in cells (Kawai et al., 2008b), potentially the threshold for physiological significance. Nonetheless, further studies on human blood vessels with lower concentrations of quercetin and the metabolites are warranted. In light of the striking difference between the effect of quercetin and myricetin on the porcine coronary artery, there is also a need to establish whether myricetin can inhibit the effect of quercetin in human vascular cells, effectively behaving as an ‘antagonist’.
The finding that quercetin can oppose the proinflammatory effect of LPS on the vasculature may also hold therapeutic significance. Recently, quercetin has been shown to attenuate both the release of pro-inflammatory cytokines in response to LPS in mice and the associated lethality (Teng et al., 2009). Significantly, this effect of quercetin was manifest even when administered several hours after exposure to LPS, raising the possibility that a similar mechanism may occur in the vasculature.
In conclusion, we have demonstrated that one of the major dietary flavonoids, quercetin and its principal human metabolites oppose pro-inflammatory events in both endothelial cells and vascular smooth muscle cells. These effects of quercetin are evident at lower concentrations than previously reported in studies using other cell types and suggests a selective action on the vasculature, particularly against the induction of NOS. Further studies on human blood vessels are warranted to establish whether these observations are relevant to the well-documented beneficial effects of dietary flavonoids (Halliwell, 2007; Boots et al., 2008).