Mast Cells in Carotid Plaques and Cardiovascular Events
Mast Cells in Carotid Plaques and Cardiovascular Events
Atherosclerosis is a chronic inflammatory disorder where numerous inflammatory cell types are known to be involved. Such cells can enter the plaque from the circulation through the endothelium and create an inflammatory environment in the vessel wall. A characteristic of a rupture-prone plaque that has gained attention of the scientific community is the presence of intraplaque vessels. In this study, we show that MCs associate with microvessel density in the plaque. It is known that not only MCs, but also other inflammatory cells, are capable of inducing new vessel formation, suggesting that an overall inflammatory environment is responsible for the formation of the neovessels in the plaque. We also observed that, besides MCs, macrophages, and neutrophils are associated with microvessel density. However, this study provides a sufficient sample size to show that the association between microvessel density and MCs is significant and independent of the presence of other inflammatory cell types. Particularly interesting is the finding that high MC numbers in the plaque were associated with a higher average microvessel density independent of the amount of macrophages and neutrophils. These data suggest that MCs might be important for plaque neovascularization. Nevertheless, when all cell types are present an even higher microvessel density is observed. A possible explanation might be that, as the plaque progresses, the number of inflammatory cells increases due to increased extravasation of the cells into the plaque via the newly formed microvessels. Of note, in this association study it remains an open question whether the MC numbers are a cause or a consequence of vessel formation, i.e. whether MCs are responsible for the induction of the new vessels or whether the MCs and other inflammatory cells enter the plaque via the vessels after their formation. Probably both mechanisms occur, as a low number of MCs are already present in the normal arterial wall and increase with plaque progression. In this study, we did not observe a correlation between known MC chemoattractants and MC numbers within the plaque, while for monocytes and their attractors a positive association was observed. This could be explained by the life span of MCs, which is long compared with other inflammatory cell types. Subsequently, chemokine levels at the moment of plaque excision may not reflect earlier MC recruitment. Additional research is needed to elucidate the underlying mechanism of MC migration towards the atherosclerotic plaque.
Necrotic areas in atherosclerotic plaques did not contain MCs. Large plaques with avascular fatty areas would result in an undervalued number of MCs when expressed per mm. Therefore, in the histological analyses we expressed the number of the MCs/plaque. Here, we show that plaques with high MC numbers show signs of increased plaque vulnerability. Previous studies demonstrated that this observation is also reflected in the association with the clinical outcome, since higher MC numbers were observed in the plaques of patients with unstable angina or symptomatic carotid artery disease. We confirm these observations in our patient cohort where we also noticed more MC in the plaques of symptomatic patients compared with asymptomatic patients.
The impact of MCs on atherosclerotic plaque progression has long been underestimated, as it was considered that MCs were only present in low numbers. Therefore, another important finding in this study is that MCs are highly prevalent in the atherosclerotic lesion: in some of the plaques total MC numbers of ~800 cells per section were detected, with an overall median of just >100 cells per section. Plaques often showed confluency of CD68-positive foam cells. The percentage of macrophages is, therefore, depicted as percentage of the covered area, because it was not always possible to quantify the individual cells. This makes comparison of absolute macrophage and MC numbers difficult in our cohort. It is though acknowledged that macrophages by far outnumber all other cell types in the advanced atherosclerotic plaques. Nevertheless, together with experimental data in the literature, our data suggest that the MC is a prominent inflammatory cell type accumulating in the atherosclerotic plaque during plaque progression.
We considered that particularly degranulating MCs would be responsible for inducing intraplaque neovascularization as they are frequently observed near the microvessels. However, we could not observe an association between degranulating MC numbers and microvessel density. Also, no association was observed between degranulating MCs and any of the other rupture-prone characteristics or future events. This might suggest that the induction of new vessel formation is more related to a regulated non-exocytotic release of pro-angiogenic growth factors rather than to the extent of an exocytotic release of countable granules, i.e. degranulation of activated MCs.
In this study, we show that MC presence is associated with thrombus formation and IPH. It was hypothesized that MC components can induce hyperpermeability or erosion of endothelial cells of the microvessels in the plaques eventually leading to IPH or thrombus formation. This is in accordance with the most important finding in our study, that plaque MC numbers associated independently with future cardiovascular events. In contrast, in the study of Hellings et al. showing that microvessel density is also predictive for future events, neither macrophage nor neutrophil numbers were found to associate with the occurrence of secondary manifestations.
Interestingly, we also observed higher plasma tryptase levels in patients that experienced a secondary event. The tryptase levels correlated positively with the number of (degranulating) MCs in the plaque, implying that the presence of degranulating, i.e. tryptase-secreting MCs in a plaque is not only responsible for the local matrix degradation, but also representative for the systemic changes that might be responsible for the secondary manifestations. The correlation of intraplaque MC numbers with future events has never been reported previously; however, MC tryptase plasma levels have been tested as a possible biomarker for cardiovascular disease. Thus, in agreement with our data, elevated tryptase levels were observed in patients with substantial coronary heart disease with the highest levels in the subgroup with acute myocardial infarction. In addition, higher MC tryptase levels were observed in patients with significant coronary artery disease (CAD) defined by stenosis of over 50%. Moreover, in another study of the group of Deliargyris, tryptase levels were elevated in patients with CAD. Conversely, no differences in MC tryptase were observed in patients with acute coronary syndrome. Besides tryptase, several other MC-derived components have also been associated with disease severity, and, in general, most studies underline the importance of MC mediators in inflammatory diseases.
Taken together, we show here for the first time that intraplaque MC numbers and plasma MC tryptase associate with future cardiovascular events. However, the results of this clinical proof-of-concept study are not sufficient to suggest utilization of the above observations in a regular clinical setting for prediction studies. Nevertheless, the data do strengthen the hypothesis that the presence of MCs in advanced carotid plaques increases risk for secondary cardiovascular manifestations, possibly by inducing intraplaque neovascularization, and via matrix degradation, which may together increase the incidence of IPH and thrombus formation. However, extensive research is necessary before MC stabilizing agents can be considered as a possible therapeutic opportunity preventing clinical manifestations by plaque stabilization in the future. For example, animal experiments inhibiting MC activation by stabilizing agents should be performed to proof causality between plaque MCs and neovascularization.
In conclusion, we show that MC numbers in the carotid atherosclerotic plaque associate with future cardiovascular events. These data correspond with the strong association found between MC numbers and intraplaque neovascularisation, now evolving as an important characteristic of rupture-prone atherosclerotic lesions, which may trigger acute atherothrombotic complications in the vulnerable patients.
Discussion
Atherosclerosis is a chronic inflammatory disorder where numerous inflammatory cell types are known to be involved. Such cells can enter the plaque from the circulation through the endothelium and create an inflammatory environment in the vessel wall. A characteristic of a rupture-prone plaque that has gained attention of the scientific community is the presence of intraplaque vessels. In this study, we show that MCs associate with microvessel density in the plaque. It is known that not only MCs, but also other inflammatory cells, are capable of inducing new vessel formation, suggesting that an overall inflammatory environment is responsible for the formation of the neovessels in the plaque. We also observed that, besides MCs, macrophages, and neutrophils are associated with microvessel density. However, this study provides a sufficient sample size to show that the association between microvessel density and MCs is significant and independent of the presence of other inflammatory cell types. Particularly interesting is the finding that high MC numbers in the plaque were associated with a higher average microvessel density independent of the amount of macrophages and neutrophils. These data suggest that MCs might be important for plaque neovascularization. Nevertheless, when all cell types are present an even higher microvessel density is observed. A possible explanation might be that, as the plaque progresses, the number of inflammatory cells increases due to increased extravasation of the cells into the plaque via the newly formed microvessels. Of note, in this association study it remains an open question whether the MC numbers are a cause or a consequence of vessel formation, i.e. whether MCs are responsible for the induction of the new vessels or whether the MCs and other inflammatory cells enter the plaque via the vessels after their formation. Probably both mechanisms occur, as a low number of MCs are already present in the normal arterial wall and increase with plaque progression. In this study, we did not observe a correlation between known MC chemoattractants and MC numbers within the plaque, while for monocytes and their attractors a positive association was observed. This could be explained by the life span of MCs, which is long compared with other inflammatory cell types. Subsequently, chemokine levels at the moment of plaque excision may not reflect earlier MC recruitment. Additional research is needed to elucidate the underlying mechanism of MC migration towards the atherosclerotic plaque.
Necrotic areas in atherosclerotic plaques did not contain MCs. Large plaques with avascular fatty areas would result in an undervalued number of MCs when expressed per mm. Therefore, in the histological analyses we expressed the number of the MCs/plaque. Here, we show that plaques with high MC numbers show signs of increased plaque vulnerability. Previous studies demonstrated that this observation is also reflected in the association with the clinical outcome, since higher MC numbers were observed in the plaques of patients with unstable angina or symptomatic carotid artery disease. We confirm these observations in our patient cohort where we also noticed more MC in the plaques of symptomatic patients compared with asymptomatic patients.
The impact of MCs on atherosclerotic plaque progression has long been underestimated, as it was considered that MCs were only present in low numbers. Therefore, another important finding in this study is that MCs are highly prevalent in the atherosclerotic lesion: in some of the plaques total MC numbers of ~800 cells per section were detected, with an overall median of just >100 cells per section. Plaques often showed confluency of CD68-positive foam cells. The percentage of macrophages is, therefore, depicted as percentage of the covered area, because it was not always possible to quantify the individual cells. This makes comparison of absolute macrophage and MC numbers difficult in our cohort. It is though acknowledged that macrophages by far outnumber all other cell types in the advanced atherosclerotic plaques. Nevertheless, together with experimental data in the literature, our data suggest that the MC is a prominent inflammatory cell type accumulating in the atherosclerotic plaque during plaque progression.
We considered that particularly degranulating MCs would be responsible for inducing intraplaque neovascularization as they are frequently observed near the microvessels. However, we could not observe an association between degranulating MC numbers and microvessel density. Also, no association was observed between degranulating MCs and any of the other rupture-prone characteristics or future events. This might suggest that the induction of new vessel formation is more related to a regulated non-exocytotic release of pro-angiogenic growth factors rather than to the extent of an exocytotic release of countable granules, i.e. degranulation of activated MCs.
In this study, we show that MC presence is associated with thrombus formation and IPH. It was hypothesized that MC components can induce hyperpermeability or erosion of endothelial cells of the microvessels in the plaques eventually leading to IPH or thrombus formation. This is in accordance with the most important finding in our study, that plaque MC numbers associated independently with future cardiovascular events. In contrast, in the study of Hellings et al. showing that microvessel density is also predictive for future events, neither macrophage nor neutrophil numbers were found to associate with the occurrence of secondary manifestations.
Interestingly, we also observed higher plasma tryptase levels in patients that experienced a secondary event. The tryptase levels correlated positively with the number of (degranulating) MCs in the plaque, implying that the presence of degranulating, i.e. tryptase-secreting MCs in a plaque is not only responsible for the local matrix degradation, but also representative for the systemic changes that might be responsible for the secondary manifestations. The correlation of intraplaque MC numbers with future events has never been reported previously; however, MC tryptase plasma levels have been tested as a possible biomarker for cardiovascular disease. Thus, in agreement with our data, elevated tryptase levels were observed in patients with substantial coronary heart disease with the highest levels in the subgroup with acute myocardial infarction. In addition, higher MC tryptase levels were observed in patients with significant coronary artery disease (CAD) defined by stenosis of over 50%. Moreover, in another study of the group of Deliargyris, tryptase levels were elevated in patients with CAD. Conversely, no differences in MC tryptase were observed in patients with acute coronary syndrome. Besides tryptase, several other MC-derived components have also been associated with disease severity, and, in general, most studies underline the importance of MC mediators in inflammatory diseases.
Taken together, we show here for the first time that intraplaque MC numbers and plasma MC tryptase associate with future cardiovascular events. However, the results of this clinical proof-of-concept study are not sufficient to suggest utilization of the above observations in a regular clinical setting for prediction studies. Nevertheless, the data do strengthen the hypothesis that the presence of MCs in advanced carotid plaques increases risk for secondary cardiovascular manifestations, possibly by inducing intraplaque neovascularization, and via matrix degradation, which may together increase the incidence of IPH and thrombus formation. However, extensive research is necessary before MC stabilizing agents can be considered as a possible therapeutic opportunity preventing clinical manifestations by plaque stabilization in the future. For example, animal experiments inhibiting MC activation by stabilizing agents should be performed to proof causality between plaque MCs and neovascularization.
In conclusion, we show that MC numbers in the carotid atherosclerotic plaque associate with future cardiovascular events. These data correspond with the strong association found between MC numbers and intraplaque neovascularisation, now evolving as an important characteristic of rupture-prone atherosclerotic lesions, which may trigger acute atherothrombotic complications in the vulnerable patients.
