We confirmed here our prior finding that in CRPtg, the neointima that develops in response to vascular ligation is increased ~twofold compared to NTG that do not express human CRP.1, 2
The major new finding of this study is that mouse C3 is required for this action of human CRP. Thus in CRPtg/C3-/-
mice that lack expression of complement C3 (the activation of which is the converging point of all three complement pathways and the source of the effector functions of complement), we observed that the neointimal response to injury was significantly less than that of CRPtg mice that have a fully functional complement system. In fact, neointima formation in CRPtg/C3-/-
was no more robust than that seen in mice that did not express human CRP but did express mouse C3.
While the liver is the primary source for the synthesis of C3,24
many other specialized cells - including macrophages25, 26
- can synthesize C3. The results generated here indicate that the requirement of mouse C3 for the effect of human CRP in the vascular injury response is related to human CRP mediated induction of expression of C3 by mouse macrophages residing in the periadventitia. Given that CRP also activates complement, this sets the stage for a feed-forward cycle in the injured blood vessel, wherein CRP both induces expression of complement and activates it.
Similar to CRPtg mice, it has been demonstrated that complement components27
are present in the atherosclerotic intima, and that CRP colocalizes with C5b-9 in early atherosclerotic lesions in humans.14
The effect of complement activation on the development of the neointima in experimental animals has been controversial, with some studies showing that interruption of the complement cascade reduces the extent of the lesion,29-31
while others showed no effect,32
or even an enhancement of neointima formation.33, 34
Although there is no consensus on the interpretation of these apparently conflicting findings, these differences could be dependent on the models utilized to induce neointima formation. While studies showed that blocking complement led to a reduction in neointima formation in animals with diet-induced disease,29, 30
other studies that were carried out in animals with genetically-induced disease; i.e. APOE-/-
mice suggested an increase in neointima formation or no effect.32-34
Caution is warranted however, because the vascular remodeling process associated with carotid artery ligation versus atherogenesis is not the same. A report by Shagdarsuren et al., the only study so far to address the effect of complement on neointima formation in the setting of mechanical vascular injury, suggested that blocking the complement cascade at the level of C1q retarded neointima formation.31
Our findings are consistent with this view in that the neointimal response to carotid artery ligation, a model of vascular injury that is similar (not identical) to the one used by Shagdarsuren et al., was significantly reduced in the absence of the C3
Importantly, complement activation by CRP is distinguished by selective activation of early components with little or no formation of the distal C5-convertase or C5b-9 membrane attack complex.reviewed in 11
CRP also recruits factor H, inhibiting the alternative complement pathway amplification loop and the C5 convertases.35
In accordance with these observations, the deposition of C3, but not C5, in the lesion was increased in our CRPtg compared to NTG mice, and CRP induced macrophages in culture to increase their expression of C3 but not C5.
It is now generally accepted that inflammation plays an integral role in the pathogenesis of vascular injury.36, 37
In parallel with evolution of the inflammatory model of vascular disease, epidemiological data emerged that point to CRP as a strong predictor of vascular events.38
Recently, a large prospective randomized study demonstrated the additive value of serum levels of CRP to that of cholesterol in directing therapy in a population with no other indication for cholesterol lowering therapy.39
The evidence that CRP is present in vascular lesions and that it stimulates and activates inflammatory cells, endothelial cells and vascular smooth muscle cells, has sparked a debate on whether it is directly involved in the pathogenesis of vascular disease or is simply a marker of its ongoing activity.5
Although there are well known genetic determinants of serum CRP levels,40
two large studies have failed to show an association between these genetic determinants and cardiovascular disease outcomes, raising suspicion that elevated serum levels could be an effect rather than a cause of vascular disease.41, 42
These indirect studies are inherently incapable of establishing or refuting a direct cause-and-effect relationship linking CRP to vascular injury. Ultimately, human studies in which CRP is selectively lowered in randomized controlled trials will be needed to answer this question.43
Meanwhile, studies that explore the actions of human CRP in vivo, in a system that expresses human CRP in a fashion that parallels the human condition, can provide very useful information. In this study we showed that by decreasing circulating levels of human CRP in CRPtg with a human CRP-specific ASO drug, both circulating human CRP levels and C3 expression/deposition in injured arteries were reduced. Both outcomes would be predicted to be of clinical benefit in patients.
Our new results need to be interpreted in the context of our previous finding that FcγRI is essential for CRP to mediate vascular remodeling, i.e. in the absence of expression of the FcγRI ligand-binding α-chain or the cell-signaling γ-chain, human CRP does not enhance the neointimal response to injury in CRPtg.1
Based on those earlier findings and the new ones reported here we are now testing the following model: In the context of vascular injury CRP activates resident/recruited periadventitial macrophages, likely by interacting with FcγRI,1, 44
thus stimulating them to increase expression of C3. Locally expressed C3, perhaps in conjunction with circulating (hepatically derived) C3, itself an acute phase protein,45
accumulates and is readily available for activation by CRP. Ultimately this generates anaphylotoxic and chemotactic fragments of C3, which recruit more macrophages to the site of injury, so the process feeds forward. This model accounts for why both FcγRI deletion and C3 deletion eliminate the CRP-mediated exacerbation of the neointimal response to injury in CRPtg: in the absence of FcγRI CRP is unable to stimulate macrophage expression of C3, and in the absence of C3 the feed-forward loop is broken. Although smooth muscle cells are known to be intimately involved in the development of the neointima after carotid artery ligation, we found no evidence that human CRP colocalizes with smooth muscle actin.1
Rather, human CRP colocalized with the macrophage marker F4/80. Efforts are currently underway to validate our CRP→macrophage FcγRI→macrophage C3 model in CRPtg. If these are fruitful, and the model is confirmed in humans, these findings could have important therapeutic implications.