Physicians' Academy for Cardiovascular Education

Finding a biomarker for atherosclerosis-related inflammation to test the inflammation hypothesis of atherosclerosis

Passacquale G et al., Cardiovasc Res 2015

The role of inflammatory biomarkers in developing targeted cardiovascular therapies: lessons from the cardiovascular inflammation reduction trials
Passacquale G,  Di Giosia P, and Ferro A
Cardiovasc Res 2015 109: 9-23
Recent research efforts have been dedicated to the potential of anti-inflammatory add-on therapy to conventional cardiovascular (CV) therapy to reduce residual CV risk. Several novel molecules targeting selected inflammatory pathways have been evaluated in phase III secondary prevention trials, such as lipoprotein-associated phospholipase A2 (Lp-PLA2) inhibitors, soluble (s)PLA2-inhibitors and antioxidants. Although their anti-inflammatory effect seemed promising in phase II studies, these agents did not reduce CV event rates in large population-based studies.
The anti-inflammatory rationale originated from the observations that the efficacy of statins to prevent CV events can partly be attributed to their ability to lower C-reactive protein (CRP). This has raised the question whether reduction of inflammation per se is protective against atherosclerotic progression, thereby positively affecting clinical outcome.

The available evidence to date may seem to contradict the hypothesis that residual CV risk implies residual inflammation as a causal factor. These studies, however, did not characterise the inflammatory profile of the participants. Due to likely heterogeneity of the study population, effectiveness of anti-inflammatory therapy may be expected to vary considerably. This may have diluted a possible effect in subgroups of patients with ‘residual inflammation’ despite conventional prophylactic treatment.
Quantifying inflammation is most commonly performed by measuring the biomarker CRP. The specificity of CRP, however, is limited for atherosclerosis-related inflammation, and its prognostic value for future CV events in optimally treated patients is particularly weak in secondary prevention. Thus, modulation of CRP may not reflect an effect on atherosclerosis, and lack of an effect may not necessarily imply absence of an effect on arterial inflammation.

This review article considers the use and limitations of available and novel biomarkers to reflect the inflammatory status of the arterial wall. According to the authors, the circulating monocytes phenotype, namely a change towards a pro-atherogenic profile, deserves particular attention.

Inflammatory biomarkers, plaque vulnerability and cardiovascular events: usefulness and limitations of C-reactive protein

The availability of a relatively inexpensive standardised methodology to measure CRP makes it the widest applied test in CV inflammation reduction trials, but its value as indicator of atherosclerosis-related inflammation is controversial.  
Its predictive value for future CV events in primary prevention trials, and added value in addition to Framingham risk calculation were not consistently shown. Also, in asymptomatic patients, CRP does not predict atherosclerotic burden well, thus limiting its use in clinical decision-making.
Even more so in the context of secondary prevention, CRP did not predict recurrence of CV events in stable, prophylactically treated ACS patients. Nor was a correlation seen between CRP-reduction and changes in plaque size or composition in response to tested anti-inflammatory therapies.
Phase II trials have yielded inconsistent results with regard to the effects of anti-inflammatory agents on CRP. CRP is a measure of systemic inflammation, and its link with plaque composition and vulnerability and CV event rates needs to be further elucidated. Imaging studies could help advance understanding, but their application in large studies is hardly feasible due to costs and technical reasons.
CRP was shown to be a more reliable biomarker of disease progression and future events in statin trials in which the baseline CRP level was generally above 2 mg/L. This level has been identified as cut-off value to distinguish between sub-optimal and optimal anti-inflammatory effects in response to statins. These in turn predicted more favourable CV outcomes independently of the LDL target achieved. Thus, recruiting patients with CRP persistently > 2mg/L despite conventional therapy may yield a more relevant population to test the efficacy of anti-inflammatory agents in secondary CV prevention. This strategy is adopted in the ongoing CANTOS study.

Emerging inflammatory biomarkers: focus on monocyte phenotype

1. Prognostic value for CV events

Monocytes play a primary role in atherogenesis and plaque progression towards vulnerability. Monocytes have varying levels of pro-inflammatory cytokine production and infiltration of the arterial wall. Functionally distinct subsets can be identified through their pattern recognition receptors: e.g. intermediate CD14++CD16+ monocytes seem to have a more pro-inflammatory profile than the CD14++CD16- subset. Studies have described associations between predominance of certain subsets over others and CHD, but monocyte phenotype did not always correlate with CRP. Level of CD16+ monocytes correlated with plaque vulnerability, while CRP did not in a direct comparison. Indeed, prevalence of CD16+ monocytes has been described to be an independent risk factor in population studies. And a shift towards a pro-atherogenic monocyte phenotype appears to accompany atherosclerosis progression. Also, in asymptomatic, at risk patients, the level of this subset was found to correlate strongly with presence of subclinical atherosclerosis.

2. Pro-atherogenic implications

The different functional atherogenic effects of monocyte subsets is attributed to the pro-inflammatory cytokines they synthesise as well as the ability to infiltrate the arterial wall. It should be noted, however, that these insights are based on in vitro experiments. Also the interaction of monocyte subsets with the arterial wall is extrapolated from in vitro experiments, which suggests that the stage of disease likely affects preferential recruitment of one monocyte subtype over another.
By lack of a diagnostic technique applicable to the clinical setting to monitor different circulating monocytes into plaques, it remains unclear how experimental findings translate into the more complex situation of human disease. The phenotype of resident macrophages further complicates the situation. Two main subtypes are distinguished: the classical activated M1 lipid-triggered foam cells and the anti-inflammatory M2 involved in healing and repair.

3. Relationship between the phenotype of circulating monocytes and degree of plaque inflammation

Studies in mouse models of atherosclerosis have shown different colonisation of arterial lesions by M1 or M2 macrophages, depending on disease state or in response to treatment, with M2 predominating in advanced lesions. In histopathological analysis of human plaques, mostly the spatial distribution of M1 and M2 subtypes appears to differ during disease progression, rather than their abundance. Much remains to be elucidated on the role of systemic inflammation in determining arterial inflammation in human disease.
Differential expression of various molecules on the extracellular surface of intermediate monocytes likely give rise to the different biological functions of subsets. The specific properties of intermediate monocytes suggests a central role for CD14++CD16+ monocytes in activation of the innate immune system, as seen in atherosclerosis. Several lines of evidence suggest a dynamic process leading to more CD16+ cells and fewer classical monocytes, in response to specific inflammatory stimuli. It is postulated that acquisition of a CD16+ phenotype by circulating cells could act as a bridge between the innate and adaptive immune systems, and might give rise to a specific immune response against pro-atherogenic stimuli that ultimately leads to disease progression.
This hypothesis needs to be tested, to evaluate the effect of immunomodulatory interventions on monocyte phenotype in vivo and whether this affects clinical outcomes. Also, prospective studies are now indicated to examine whether monocyte phenotype determination is useful in predicting the extent of atherosclerotic disease, and response to therapy.

4. Inflammation and classical cardiovascular risk factors

The effect of immunomodulatory agents on classical CV risk factors should also be considered, although the extent to which classical therapies act via reducing inflammation is unclear.
Three classes of anti-inflammatory drugs are discussed that have entered phase III trials, to address the relevance of immune deregulation in the pathogenesis of atherosclerosis, assuming that no effects of the tested immunomodulatory drugs are seen on classical CV factors.

* Anti-cytokines

TNF-α antagonists have been shown to reduce CRP levels in patients with rheumatoid arthritis (RA), but in these studies the biomarker could merely reflect RA disease control. Both TNF-α-antagonists and IL-6 blockers have been associated with an adverse impact on lipid profile. Possible CV protective effects of these antagonists are currently evaluated in a phase iV trial in patients with moderate and severe RA.
IL-1β antagonists exert a hypoglycaemic effect through stimulation of β-cell secretory function. Evidence suggest that antagonising IL-1 or its receptor also lowers inflammatory biomarkers. Study results on their effect on inflammation in patients with ACS are awaited, including the CANTOS trial that evaluates the IL-1β-targeted humanised antibody canakinumab.

* Anti-inflammatory drugs targeting oxidised LDL

Uptake of oxidised LDL (ox-LDL) cholesterol particles by monocytes stimulates pro-inflammatory cytokine release. Internalisation by macrophages within plaques promotes their apoptosis, thereby contributing to plaque necrotic core formation and growth.
Conflicting data were obtained with the antioxidant succinobucol with regard to prevention of coronary disease. In the phase III ARISE trial, no efficacy was shown on primary endpoints, and an unfavourable impact on lipid profile was seen.
Inhibitors of PLA2 can also lower ox-LDL. Again, studies yielded an unclear picture on their benefit in reducing inflammation and endpoints and potential harm of these agents.

Future perspectives

Another immunomodulatory approach under development is vaccination against targets involved in plaque development. Experiments are performed with immunisation with LDL particles or cholesterol-related antigens. Precise mechanisms underlying the anti-atherogenic effect remain to be elucidated, but may involve facilitated clearance of lipoproteins from the circulation before they accumulate within the arterial wall.
Anti-CETP vaccine reduced plaque formation in the aorta of an atherosclerosis rabbit model. Induction of neutralising CETP-antibodies in humans was below expectation to date.
Other, cholesterol-unrelated targets have also been explored, and research into vaccination against VEGFR2, expressed by proliferating endothelial cells involved in neo-angiogenesis, is ongoing.


Characterisation of the inflammatory profile should also be performed in phase III trials, to determine the relationship between residual inflammation, immunomodulation and CV protection. Limitations of CRP should be appreciated, and additional and more specific biomarkers of atherosclerosis-related inflammation and plaque vulnerability should be considered. Monocyte phenotype may prove useful in this context.
Find the article online at Cardiovasc Res