The data presented in this study showed that the impaired contractile responses to both depolarisation with high K+
and the receptor-dependent agonist 5-HT were early vascular pathological changes in mCIA animals. This contractile dysfunction was not associated with a change in BP or affected by ex vivo
iNOS and eNOS/nNOS inhibition. Furthermore, plasma NOx
levels were similar in both groups, which suggests that excess NO production did not mediate the aberrant vascular contractility observed in this model of inflammatory arthritis. The fact that this vascular dysfunction was unaffected by ex vivo
nNOS and COX inhibition suggests that H2
(Capettini et al., 2008
) and vasodilatory prostanoids were not involved.
Interestingly, contractile dysfunction was not accompanied by overt endothelial dysfunction given that endothelium-dependent relaxation responses to ACh were similar to those seen in tissues from non-immunized control mice. Moreover, relaxation responses to exogenously donated NO were unaffected. Together, these observations suggest that there is ample endothelium-derived NO bioavailability during mCIA, and that the VSM functionally responds to both endogenous and exogenous NO to produce vasodilation. As such, this finding is contrary to what is expected from data already published, given the widely described endothelial dysfunction in patients with established RA (Khan et al., 2010
Endothelial dysfunction is characterized by reduced NO bioavailability and impaired endothelium-dependent relaxation responses. With regard to causative mechanisms in CVD, such changes constitute well-described primary events and the presence of endothelial dysfunction predicts the risk of future adverse cardiovascular events in older high-risk and younger populations alike (Martin and Anderson, 2009
). However, it would seem that, in the mCIA model, pathological changes at the VSM rather than the endothelial level are the first to present, at least functionally.
It is highly likely that the observed departure from the traditional endothelial dysfunction model of CVD development is related to the fact that mCIA represents a first presentation autoinflammatory insult and not the relapsing/remitting condition seen in well-established human RA. Interestingly, recent independent studies also describe the absence of measurable endothelial dysfunction in both new onset inflammatory arthritis (Foster et al., 2012
) and newly diagnosed RA (Sodergren et al., 2010
; van Eijk et al., 2011
) patients. Moreover, in a sub-group of RA patients 18 months after the initial evaluation, a significant increase in common carotid artery intima-media thickening (IMT) was observed in the absence of any change in endothelial function (Sodergren et al., 2010
). Given that increased IMT is a result of phenotypical/structural changes at a VSM level, these findings tend to complement those described in the present study. The question remains as to the underlying mechanism(s) responsible for the VSM dysfunction and impaired constrictor responses.
Our data rule out a role for increased NO production or prostanoids in the contractile dysfunction observed. This finding is surprising given the major role of the inflammatory cytokine TNF-α in both RA and iNOS induction. In fact, previous studies using the same model have reported increases in iNOS mRNA (Juarranz et al., 2005
) and plasma NOx
(Sakaguchi et al., 2004
). However, in those studies, animals were killed 14 days (Juarranz et al., 2005
) and 12 weeks (Sakaguchi et al., 2004
) after the onset of arthritis, and the increases observed were relatively minor. Moreover, the animals in the present study were used well within the times of those described above. Again, this indicates that early non-endothelium/non-NO-dependent events may underlie the observed contractile dysfunction. Any possible role for inflammation-associated excess production of NO may be involved in much later pathological changes in RA.
Whilst it is possible that the systemic inflammatory milieu associated with arthritis could have a negative effect on receptor-mediated VSM contraction, the fact that both depolarization and agonist-induced responses are impaired would argue against this concept. Consequently, the fact that similar contractile dysfunction in an unrelated disease model was accompanied by increases in MMP-9 (Chung et al., 2007
) leads us to hypothesise that this gelatinase may play a role in the vascular impairment associated with the mCIA model (see below).
As indicated, the present study demonstrates that levels of MMP-9 protein in plasma samples and aorta homogenates are elevated following the development of arthritis. The much greater change in aortic levels, combined with the significant increase in the active form of MMP-9 in the aortae, strongly suggests a possible role for tissue-derived MMP-9 in the impaired contractile responses described above. Furthermore, the finding that incubation of aortic tissues from naïve control animals with exogenous MMP-9 (at a concentration similar to that seen in mCIA-derived aortic homogenates) produced a detrimental effect on vessel contractility also implies a possible role for circulating MMP-9 in this process. Interestingly, such incubations were without negative effects on subsequent endothelium-dependent relaxation responses, a fact that emphasises the significance of the earliest changes in contractile function with regard to subsequent vascular responsiveness.
The observation of increased levels of aortic MMP-9 is a novel finding in this model and perhaps represents the earliest pathological change in the vasculature in response to systemic inflammation. A role for MMP-9 in the inhibition of vasoconstriction has been described previously (Chew et al., 2004
; Chung et al., 2007
), with possible mechanisms related to the inhibition of extracellular calcium entry into the VSM cells having been identified (Raffetto and Khalil, 2008
). Importantly, it is suggested that this effect is both concentration and time-dependent and is reversible (Chew et al., 2004
). Although the role of concentration and time cannot be refuted, data from the present study would argue against the reversibility. In the previous study (Chew et al., 2004
), only short 1 h incubations with MMP-9 were used, and such short incubation periods might allow the consequent acute effects to be reversed following removal of MMP-9. However, in our experiments, following 24 h incubations with MMP-9, a significant contractile dysfunction remained, even though the enzyme was completely washed away. While dysfunctional mechanisms of extracellular calcium entry into the VSM cells cannot be ruled out as an underlying cause, no such evidence has been published. Therefore, given the irreversible nature of the MMP-9-induced effect, it is likely that more permanent damage is responsible. Interestingly such a hypothesis is supported by preliminary experiments in which aortic tissues from mCIA animals were incubated ex vivo
with a MMP-9 inhibitor and contractile dysfunction was still observed (unpubl. obs.).
Despite the fact that IL-1β has been intrinsically linked to both RA and MMP-9 secretion (Goldbach-Mansky, 2009
), it would seem that this pyrogen does not play a role in the contractile dysfunction observed in this study. Whilst plasma levels of IL-1β were significantly elevated in mCIA over the course of disease progression, the contractile dysfunction produced following ex vivo
incubations of aortic tissues from naïve animals with IL-1β was not associated with increased MMP-9 production. Indeed given that l
-NAME and 1400W, and to a certain extent indomethacin, prevented the IL-1β-induced impairment of contraction suggests the well-described up-regulation of NO production, and to a lesser extent COX products, in these tissues in response to this inflammatory mediator. Such an action in the mCIA model has already been ruled out by experiments described above.
MMP-9 would seem to be a common denominator in the pathogenesis of RA and cardiovascular-related diseases (Ram et al., 2006
). Importantly, this gelatinase is associated with destructive roles resulting in compromised blood vessel function. For instance, in an animal model of Kawasaki disease, a disease characterized by vasculitis or inflammation of middle-sized arteries, such arterial inflammation was associated with MMP-9 up-regulation and the breakdown of elastin (Lau et al., 2008
). Similarly in a mouse model of Marfan syndrome, a disease characterized by increased arterial stiffness and life-threatening aortic aneurysm formation (Kingwell and Boutouyrie, 2007
), the degeneration of elastic fibre integrity and the deterioration in vascular compliance is associated with an increased presence of gelatinases (Chung et al., 2007
). Because MMP-9 has been implicated in the development of arterial stiffness in human studies (Yasmin et al., 2005
; Vlachopoulos et al., 2007
), an important question with regard to RA is what consequences could such MMP-9-associated effects have on the subsequent vascular health of affected individuals?
A change in VSM phenotype from contractile to synthetic is important in the development and progression of diseases such as hypertension and atherosclerosis. The consequence of such ‘phenotypic switching’ is the promotion of VSM proliferation and extracellular matrix remodelling leading to a thickened arterial wall and narrowing of the vessel lumen. It has been suggested that VSM cell-derived MMP-9 activity mediates this process (Cho and Reidy, 2002
) via its basement-membrane and elastin degrading capabilities (Senior et al., 1991
). Such a notion is substantiated by the observation that MMP-9 inhibition or gene deletion prevents VSM cell proliferation and migration (Galis et al., 2002
). Importantly, IMT (Tanasescu et al., 2009
) and arterial stiffness (loss of vascular elasticity and compliance) (Yildiz, 2010
) are commonplace in patients with RA and correlate directly with cumulative inflammatory burden and disease severity independently of established cardiovascular risk factors (Crilly et al., 2009
). As such, it would seem that a MMP-9/IMT/arterial stiffness pathological triangle may exist in RA.
In conclusion, the data presented here demonstrate an early impairment of vascular contractile responses associated with disease activity in the mCIA model of inflammatory arthritis in the absence of overt endothelial dysfunction. Because the progression of RA is not linear, the health of sufferers deteriorates quickly following its development. As such, there is now significant interest in an ‘early window of opportunity’ with regard to treatment of the disease. Nonetheless, despite changes in the course of RA in recent years, the increased risk of CVD has not altered. Perhaps the window of opportunity should also be concentrated on the development of vascular disease. Structural dysregulation in the vasculature associated with increased MMP-9 would seem a potential target for novel early intervention.