Search for Reliable Circulating Biomarkers to Predict Carotid Plaque Vulnerability

Round 1

Reviewer 1 Report

It is a very interesting review regarding circulating biomarkers to predict carotid plaque vulnerability. The authors describe a lot of different candidates including inflammatory, lipid-related, and mi-RNA biomarkers.

This subject is very difficult and still remains unclear.

However,  there is still al lack of significant marker associated with the risk of rapture of atherosclerotic plaque that could be routinely performed...

The authors only highlight biomarkers- candidates ,but which of them is in Your opinion the best? is there a chance to choose one of them and suggest its assessment in clinical practice?

It is not difficult to list the markers one by one and recall the references, you should approach them critically and confront them with each other. The conclusions are very general, banal and shallow. It brings nothing new to our current knowledge. Please try to improve it and give appropriate indications for practitioners.

In general, I did find important flaws or issues to discuss.

In manuscript I found only some minor drawbacks to correct:

Line 35-  percent- should be wright together, not "per cent",

Line -36- You should correct and wright  "20%  of them are caused by  large artery or large-vessel atherosclerosis...." because there is also a group of ischemic stroke due to small-vessel disease  and here is small artery atherosclerosis account for 20-25% of all strokes.

Author Response

It is not difficult to list the markers one by one and recall the references, you should approach them critically and confront them with each other. The conclusions are very general, banal and shallow. It brings nothing new to our current knowledge. Please try to improve it and give appropriate indications for practitioners.

The scope of our review was to highlight the need for finding circulating biomarkers related to carotid plaque vulnerability that, in conjunction with imaging features of the plaque, help to predict high risk patients of suffering an atherothrombotic stroke. We aimed to collect the putative biomarkers reported in this regard, particularly focusing our search in those related with inflammation and with lipoproteins, because of their involvement in the development of atherosclerosis. Unfortunately, although several candidates have been proposed, as explained throughout the review, a reliable biomarker to predict carotid plaque vulnerability remains to be established. For this reason, further studies with big cohorts are essential to find suitable biomarkers (with high sensitivity and specificity) for atherothrombotic stroke. In clinical practice, the establishment of a biomarker or, more likely, a combination of biomarkers, which corroborate and/or provide additional information to that found by imaging, would be very useful and would allow the selection of patients at high risk. As illustrated in Figure 3 in the manuscript, the measurement of reliable biomarkers allow to select those patients candidate to other tests, such as 18-FDG-PET, to better predict the patients’ outcomes and helping to make clinical decisions. 

We kindly thank the reviewer’s comments and the suggestion of improving the Conclusions. This section has been re-written according to the reviewer’s opinion and we have tried to summarize the concepts explained above and to discuss some pros and cons of the use of inflammatory and lipoprotein-related biomarkers in the context of atherothrombotic stroke in order to provide a more critical point of view. We have also included a new section “9. Plasma biomarkers in the clinical practice and future directions”, to give our opinion about the hypothetically future steps in the search for circulating biomarkers in atherothrombotic stroke and their implementation in the clinical practice. We think that this issue may be of interest for practitioners as well as for researchers.

The authors only highlight biomarkers- candidates ,but which of them is in Your opinion the best? is there a chance to choose one of them and suggest its assessment in clinical practice?

We agree with the reviewer that we do not take part for a specific biomarker. It is difficult to choose one, because, in spite of the reported studies, none of them is being used routinely in the clinical practice. It is more feasible the combination of several putative biomarkers, because atherothrombotic stroke is a complex disease in which several pathways are involved. Our hypothesis is that some circulating inflammatory biomarkers in combination with some modified form of LDL, or even some components found increased in modified LDL (i.e. specific apolipoproteins), may be predictors of plaque vulnerability.

Among inflammatory molecules, FABP4 is a promising candidate, because it seems to be more specific than other putative markers, which increase in overall inflammatory processes. This hypothesis is suggested by some previous bibliography and preliminary unpublished data from our group of investigation. FABP4 is highly expressed and released by activated macrophages, and it is related to lipid accumulation and to inflammation. Interestingly, FABP4 concentration is increased in plasma and plaque from atherothrombotic stroke patients, and its expression correlates with carotid plaque instability. In addition, it is well-known that modified forms of LDL can induce the expression of several inflammatory mediators in macrophages, including FABP4 (reference 90 in the manuscript, Puig et al. 2020), and that LDL play an essential role in the origin and progression atherosclerosis, as suggested in Figure 2. According to these observations, we think that the combination of circulating FABP4 and electronegative LDL or oxLDL may be a good option in the prediction of plaque vulnerability. However, this is our personal opinion and we consider that there is not enough evidence to state it in the review.

In manuscript I found only some minor drawbacks to correct:

Line 35-  percent- should be wright together, not "per cent",

Line -36- You should correct and wright  "20%  of them are caused by  large artery or large-vessel atherosclerosis...." because there is also a group of ischemic stroke due to small-vessel disease  and here is small artery atherosclerosis account for 20-25% of all strokes.

Drawbacks in lines 35 and 37 are corrected. Thank you.

Reviewer 2 Report

The authors present an interesting paper in the form of a simple review with an acceptable recent bibliography.

However, in view of the epidemiological and pathophysiological importance of the subject, further elements and modifications are necessary in order to make the manuscript complete.

• Authors should specify why they did not perform a systematic review in order to make the literature review more specific and interesting.

• Introduction:

line 42: the authors should specify if there are further theories in the literature on the progression of the atheromatous lesion related to the mechanisms of share stress and alteration of laminar flow, both in the sense of increased flow and in the sense of reduction of flow.

• Atherosclerosis and plaque vulnerability:

line 99: the authors should better illustrate the temporal path and the related inflammatory changes that characterize the natural history of atheromatous plaque. What were the main inflammatory pathways that led to the initial thickening, the subsequent intimal hyperplasia and finally the late atherosclerotic lesion?

• Carotid plaque vulnerability and ischemic stroke:

line 140: in recent years, the role of CEUS in the diagnosis and follow up of unstable and vulnerable carotid plaques has grown significantly. The authors should illustrate the recent evidence on the role of CEUS and its possible advantages over second level and invasive examinations such as CTA of the supra-aortic trunks. Furthermore, it is essential that the authors illustrate the recent evidence of the applications of dedicated softwares for 3D reconstruction of the plaque and the relationship of statistical and clinical significance with respect to the anatomical-pathological characteristics of the plaque.

• Search for circulating biomarkers in atherothrombotic ischemic stroke:

line 171: if possible, the authors should report data and scientific evidences confirming the eventual non-linear statistical association between the cerebral ischemic event and common cardiovascular risk factors, such as Franingham's score items. Furthermore, the authors should specify whether in the literature there are examples of linear statistical association between failure of revascularization procedures and plasma inflammatory biomarkers or any other scientific evidence that allows to identify inflammatory biomarkers as a possible expression of the pathophysiology of the disease that they intend to examine.

• Modified LDL:

Line 317: the authors should illustrate the possible role of statins in reducing the risk of cardiovascular and neurological events in patients with carotid stenosis. Authors should specify if recent evidences on the role of particular statins with adequate pharmacological dosages in reducing ischemic risk and atheromatous plaque vulnerability are showed

• Enzymes, adhesion molecules and cell receptors:

line 378: in order to make the role of MMP-9 more complete, the authors should illustrate the possible role of enhancing the action of gelatinases by NGAL, its role in the pathogenesis of severe vascular pathology and in particular its potential role in the evolution of plaque instability and rupture and ischemic stroke.

• Other stroke biomarkers

Line 470: the authors should specify whether the biomarkers illustrated in the chapter have been evaluated through in vitro, in vivo or experimental studies on subjects suffering from atheromatous disease.

It is also important to evaluate not only the statistical significance deriving from the results of the various studies but also the clinical feasibility and costs deriving from these biomolecular diagnostic procedures, if possible

• Novel therapies

Line 472: the authors should specify the role of some antibiotic drugs administered at sub-antimicrobial dosages which resulted in a significant reduction in the plasma levels of the main inflammatory biomarkers with important clinical effects. Another fundamental element is the evaluation of the role of systemic anti-inflammation as a first level of therapy

Author Response

Authors should specify why they did not perform a systematic review in order to make the literature review more specific and interesting.

We performed an extensive review in Pubmed by combining the following search terms: ischemic stroke; carotid atherosclerosis; biomarkers; inflammation; lipids; lipoproteins. In addition, we looked for other specific molecules that were related with the topic of the review, such as miRNA, modified LDL, chemokines, adhesion molecules, adipokines,… For selecting studies suitable for the present review, we pre-specified the following criteria: putative biomarkers for atherothrombotic stroke, retrospective and prospective studies, biomarkers in circulation and in carotid plaque, symptomatic and asymptomatic human patients.

Introduction:

line 42: the authors should specify if there are further theories in the literature on the progression of the atheromatous lesion related to the mechanisms of share stress and alteration of laminar flow, both in the sense of increased flow and in the sense of reduction of flow.

We agree with the reviewer that alterations in blood flow contribute to the progression of atherosclerosis. Low shear stress zones with non-laminar flow (turbulent), for example in curvatures or bifurcation (branches), are exposed to endothelial damage and are thus more prone to plaque development and rupture. On the other hand, maintenance of a physiologic laminar blood flow is atheroprotective.  In human carotid arteries, it has been demonstrated that wall shear stress is lower in carotids with plaques than in plaque-free carotid arteries (Gnasso et al. In vivo association between low wall shearstress and plaque in subjects with asymmetrical carotid atherosclerosis, Stroke 1997, 28:993-998). A sentence about the role of shear stress in atherosclerosis has been added: “Particularly, low shear stress zones, with non-laminar flow, are more prone to plaque development and rupture” (lines 69-70)

Atherosclerosis and plaque vulnerability:

line 99: the authors should better illustrate the temporal path and the related inflammatory changes that characterize the natural history of atheromatous plaque. What were the main inflammatory pathways that led to the initial thickening, the subsequent intimal hyperplasia and finally the late atherosclerotic lesion?

According to the reviewer’s suggestion, we have tried to improve the explanation in order to clarify the temporal path and the inflammatory pathways involved in the different stages of the atherosclerotic lesion (please, see lines 91-118 in the new version of the manuscript).

Carotid plaque vulnerability and ischemic stroke:

line 140: in recent years, the role of CEUS in the diagnosis and follow up of unstable and vulnerable carotid plaques has grown significantly. The authors should illustrate the recent evidence on the role of CEUS and its possible advantages over second level and invasive examinations such as CTA of the supra-aortic trunks. Furthermore, it is essential that the authors illustrate the recent evidence of the applications of dedicated softwares for 3D reconstruction of the plaque and the relationship of statistical and clinical significance with respect to the anatomical-pathological characteristics of the plaque.

We agree with the reviewer on the importance of CEUS and other carotid ultrasound techniques (such as 3D carotid imaging) in the diagnosis of unstable and vulnerable plaques. Indeed, two of the authors of the present review published an article this year about the clinical significance of CEUS (Camps-Renom P. et al. Eur J Neurol. 2020). Therefore, in this new version of the manuscript we have added some new information related to the ultrasound imaging techniques allowing the study of carotid plaque vulnerability.

“Doppler ultrasound provides some interesting information related to plaque vulnerability, including plaque echolucency that is associated with increased lipid and inflammatory molecule content and microemboli detection that is related to the risk of stroke recurrence [12], among others. In addition, some new ultrasound techniques have been developed in recent years providing interesting information about plaque vulnerability. For example, Contrast-Enhanced Ultrasound (CEUS) allows for the detection of intraplaque neovascularization and it has been related to the risk of stroke recurrence independently of the degree of stenosis [23]. Furthermore, there has been a significant increase in the use of three-dimensional carotid ultrasound reconstruction software for estimating carotid plaque burden, which allows a fast and reproducible volumetric study of the plaque, and a better risk stratification of individuals [25]” (lines 147-155).

Search for circulating biomarkers in atherothrombotic ischemic stroke:

line 171: if possible, the authors should report data and scientific evidences confirming the eventual non-linear statistical association between the cerebral ischemic event and common cardiovascular risk factors, such as Franingham's score items. Furthermore, the authors should specify whether in the literature there are examples of linear statistical association between failure of revascularization procedures and plasma inflammatory biomarkers or any other scientific evidence that allows to identify inflammatory biomarkers as a possible expression of the pathophysiology of the disease that they intend to examine.

As the reviewer suggests, cardiovascular risk factors, such as the ones included in the Framingham score, are associated with cardiovascular events, including ischemic stroke. This score combines clinical characteristics of the patients and laboratory findings. However, except for age, none of the items included in the Framingham score allow an accurate selection of patients for carotid revascularization. In fact, this score is designed essentially for primary cardiovascular prevention not for identifying patients with carotid atherosclerosis at high risk of stroke. Currently, the selection of patients for carotid revascularization is based on age, prior symptoms of stroke, and carotid stenosis. These features are often insufficient to decide on the best treatment in some frequent clinical situations and this is the focus of the review. Our main objective is to define putative plasmatic biomarkers that may help in the future in selecting patients with vulnerable carotid plaques in the clinical practice. Thus, we honestly think that reporting evidence concerning the associations described between age, hypertension or diabetes, among other classical vascular risk factors, and the risk of stroke globally, is out of the scope of this review.

Regarding reviewers’ suggestion about inflammatory biomarkers, stroke per se generates a systemic inflammatory reaction as early as in the acute stage and this is directly linked to the infarct size and to the recanalization status. Not recanalized patients have greater infarct volumes, and some biomarkers, such as IL-6 (inversely), TNF-alpha, MMP9 and ICAM-1 were associated with greater infarct volumes on CT (Sotgiu et al. Inflammatory biomarkers in blood of patients with acute brain ischemia. European Journal of Neurology 2006). This is important, because it may confound some results when analyzing the association between inflammatory biomarkers and vulnerable carotid plaques. We thank the reviewer for this observation. We have added the following sentence in the manuscript: “In addition, some inflammatory biomarkers are associated with stroke per se and directly related to infarct volume" (In “6-Inflammatory Biomarkers”, lines 356-357).

Modified LDL:

Line 317: the authors should illustrate the possible role of statins in reducing the risk of cardiovascular and neurological events in patients with carotid stenosis. Authors should specify if recent evidences on the role of particular statins with adequate pharmacological dosages in reducing ischemic risk and atheromatous plaque vulnerability are showed

We agree with the reviewer that it is interesting to comment the role of statins in reducing the risk of cardiovascular events, including stroke recurrence. Statins are the most widely used lipid-lowering treatment and it has been demonstrated that their use in patients with non-cardioembolic strokes (including small-vessel disease and atherothrombotic strokes) reduces the incidence of recurrence and other cardiovascular events. As commented in the manuscript, statins reduce carotid atherosclerosis by lowering the plasmatic levels of LDL (references 49-51 in the manuscript), but they also have pleiotropic effects including attenuation of chronic inflammation.  In the field of stroke, the main evidence exists for atorvastatin 80 mg, rosuvastatin 40 mg and simvastatin 20 mg. The studies and trials are numerous. However, we think that this information is out of the scope of the review.

Following this suggestion we added the following information: “Statins are the most widely used lipid-lowering treatment and it has been demonstrated that their use in patients with non-cardioembolic strokes (including small-vessel disease and atherothrombotic strokes) reduces the incidence of stroke recurrence and other cardiovascular events. Statins reduce inflammation of the atherosclerotic plaques by lowering the plasmatic levels of LDL, but they also have pleiotropic effects including attenuation of chronic inflammation.” (lines 263-268)

Enzymes, adhesion molecules and cell receptors:

line 378: in order to make the role of MMP-9 more complete, the authors should illustrate the possible role of enhancing the action of gelatinases by NGAL, its role in the pathogenesis of severe vascular pathology and in particular its potential role in the evolution of plaque instability and rupture and ischemic stroke.

We agree with the reviewer that to include NGAL provide more complete information in this section. We have decided to include a new reference in the manuscript (reference 118, Eilenberg et al. Eur J Vasc Endovasc Surg 2019) that proposes NGAL (and NGAL-MMP9 complex) as a biomarker for the detection of unstable carotid plaques in asymptomatic patients. This sentence has been added in the text: “Asymptomatic patients with vulnerable plaques also display high levels of serum neutrophil gelatinase-associated lipocalin (NGAL), which modulates the activity of MMP-9, and MMP-9/NGAL complexes”

Other stroke biomarkers

Line 470: the authors should specify whether the biomarkers illustrated in the chapter have been evaluated through in vitro, in vivo or experimental studies on subjects suffering from atheromatous disease.

It is also important to evaluate not only the statistical significance deriving from the results of the various studies but also the clinical feasibility and costs deriving from these biomolecular diagnostic procedures, if possible

Biomarkers illustrated in this section and polymorphisms of other putative biomarkers previously discussed in the review have been evaluated in vivo in stroke patients from the atherothrombotic subtype when possible, or in both stroke patients and asymptomatic patients, as stated in the first question raised by the reviewer about review criteria.

Only biomarkers related to neuronal damage have not been attributed specifically to the atherothrombotic stroke subtype. However, they have been mentioned in the present review as they might be interesting to be evaluated in the context of a putative “predictive scores“as an indirect measure of the brain affectation to evaluate for example risk of recurrence or functional disability of the patient.

Regarding miRNA profile analysis, as their levels change dynamically regarding pathology, they might be valuable in the detection and monitoring of certain critical steps in the atherosclerotic process. This technology is actually at the bench, and mainly associated to pilot clinical studies. These studies raise a lot of valuable information but lack of replication in large clinical cohorts and thus clinical significance is still poor.  The miRNAs mentioned in the review are the ones that we consider are in the track to be selected for a future validation. As the relevant role of miRNAs in pathology is being evidenced, it may jump soon to be investigated in large clinical trials, however, their implementation in clinical routine laboratories will probably not be immediate.

According to the reviewer we have slightly modified this section to include some of the above-mentioned observations.

Lines 457-458: “Therefore, genetic analysis may add complementary relevant information to analyze protein levels, or may be informative by itself”.

Line 472-473: “however, it may be interesting to take them into account when several molecules are analyzed together to generate a predictive score”.

Lines 475-476: “however, at the moment, lack of research in large clinical trials may delay its implementation in the clinical setting”

Novel therapies. Line 472: the authors should specify the role of some antibiotic drugs administered at sub-antimicrobial dosages which resulted in a significant reduction in the plasma levels of the main inflammatory biomarkers with important clinical effects. Another fundamental element is the evaluation of the role of systemic anti-inflammation as a first level of therapy

It is well-established that chronic bacterial infection is significantly associated with the development of atherosclerosis and its clinical complications, such as myocardial infarction, and stroke. Bacterial infection seems to play a role in the initiation, the progression, or the destabilization of atherosclerotic plaques. In some cases, bacteria may act directly on vessel wall by promoting endothelial dysfunction and inflammation, but, in other cases, can be an indirect effect of enhancing the chronic inflammatory response. Therefore, as commented by the reviewer, sub-antimicrobial dosages of antibiotic could be beneficial. It is also suggested by some research regarding antibiotic drugs and atherosclerosis. For example, a study from Sander et al. (new reference 196 in the manuscript) showed a protective effect of roxithromycin in the progression of atherosclerosis in patients serologically tested for Chlamydia pneumonia. In this new version of the manuscript we have added this sentence in the Novel therapies section.

“It has also been hypothesized that some chronic bacterial infections may be associated with the development of atherosclerosis and the risk of complications, such as myocardial infarction or stroke. These bacterial infections seem to play a role in the initiation, the progression, and the destabilization of atherosclerotic plaques. In this regard, sub-antimicrobial dosages of antibiotic could be beneficial in inhibiting inflammation. This is suggested by some studies demostrating the benefits of antibiotics in selected patients. For example, a study from Sander et al. showed a protective effect of roxithromycin treatment in the progression of arteriosclerosis arteriosclerosis [196]. Once again, a better knowledge of plasmatic biomarkers associated with chronic-infection-immune response will probably help in selecting patients who are candidates to receive antibiotic drugs” (lines 537-545).

As commented by the reviewer, a first level of therapy to inhibit immune response and excessive inflammation may be the use of anti-inflammatory and other immunomodulation drugs. From lines 511 to 533, we review the current evidence of immunomodulation in the setting of atherosclerosis. Although there are promising studies regarding the beneficial role of anti-inflammatory molecules, such as IL-10 and colchicine, in atherosclerosis, these drugs are still not clinically used in the treatment of patients with atherosclerosis. We have added a sentence in this regard: “In addition, a first line of therapy would also be the use of anti-inflammatory drugs. In this regard, promising studies show the beneficial role of anti-inflammatory molecules, such as IL-10 or colchicine, in atherosclerosis, but they need to be further studied and they are still not used in the treatment of patients” (lines 534-537).

Reviewer 3 Report

The authors presented an excellent review of the possibility of using different biological markers to predict the instability of carotid atherosclerotic plaques. I propose to publish the review in the present form.

Author Response

The authors presented an excellent review of the possibility of using different biological markers to predict the instability of carotid atherosclerotic plaques. I propose to publish the review in the present form.

Thank you for your kind comments

Round 2

Reviewer 2 Report

Thank you for your revision

In this way, tha manuscript appears to be better and it can published in this form