Panoramic imaged carotid atheromas are associated with increased neutrophil count: both validated, independent predictors of near-term myocardial infarction

Abstract

Objectives:

Panoramic images (PXs) demonstrating calcified carotid artery atheromas (CCAAs) are associated with heightened risk of near-term myocardial infarction (MI). Elevated neutrophil counts (NC) within normal range 2,500–6,000 per mm³ are likewise associated with future MI signaling the role neutrophils play in the chronic inflammation process underlying coronary artery atherogenesis. We determined if CCAAs on PXs are associated with increased NC.

Methods:

Investigators implemented a retrospective study of PXs and accompanying medical records of white males ≥ 65 years treated by a VA dental service. Two groups (N = 60 each) were constituted, one with atheromas (CCAA+) and one without (CCAA-). Predictor variable was CCAA + and outcome variable was NC. Bootstrapping analysis determined the difference in mean NCs between two groups, significance set at ≤0.05.

Results:

The study group of (CCAA+) (mean age 75.9; range 69–91 years) demonstrated a mean NC of 4,843 per mm³ and control group (CCAA-) (mean age 75.3; range; 66–94) a mean NC of 4,108 per mm³. The difference between the groups was significant (p = 0.0008) (95% CI of difference of mean: −432, 431; observed effect size 736).

Conclusions:

CCAAs on PXs of elderly white males are associated with elevated NC; amplifying need for medical consultation prior to invasive dental procedures.

Introduction

Atherosclerosis in the coronary vessels arising from chronic, low-grade inflammation, is the number one cause of death among older white males in the Americas and Western Europe.^1–3 The inflammatory process is believed initiated as a response to endothelial injury from lipid deposition, and its severity mirrored by an increase (although within the normal reference range) in the neutrophil count (NC) and a concomitant increase in circulating levels of pro-inflammatory cytokines.⁴ The NC is configurated by multiplying the white blood cell count reference range 4500–10 000 per mm^3,5 by the sum of the percentage of neutrophils (usual range 50–70%).

The clinical correlate of this concept is exemplified by the results of the Welsh, Caerphilly Study, which enrolled more than 2100 white males aged 45–59 and followed them for approximately 10 years. After adjustments of pre-existing heart disease and classic atherogenic risk factors including smoking, those with a mean NC of 4720 per mm³ versus 4,200 per mm³ were significantly more likely to evidence incident ischaemic heart disease (i.e., fatal or non-fatal MI).⁶

Early detection of preclinical atherosclerotic lesions and institution of preventive therapeutics is critical because almost half of those dying from a first MI had an occult form of the disease and did not manifest prodromal symptoms and traditional risk factors.^7,8 Given this imperative and the fact that the atherosclerotic process is systemic in nature involving both the coronary and carotid vasculature, it is critical to note that we and our Swedish colleagues^9,10 have previously demonstrated that older white males having panoramic imaged CCAA (Figure 1) are significantly more likely to suffer a near-term MI than similarly age-matched controls devoid of such lesions. Therefore, we were intrigued by the results of a recent Austrian study. Specifically, a group of clinician-scientists in Vienna conducted a prospective study among more than 500 neurologically asymptomatic white males (mean age 69) having ultrasound confirmed carotid artery atherosclerotic lesions.¹¹ During the 6-year follow-up period, patients who died from cardiovascular diseases had significantly higher median baseline NC (4700 per mm³ vs 4,200 per mm³.

Figure 1.

Panoramic image of a 71-year-old non-Hispanic white male with a neutrophil count of 5404 per mm³. The cropped image, which was digitally enhanced with the manufacturer’s provided software, shows calcified carotid artery plaques (arrow heads) below the angle of the right side of the mandible and anterior to both the preverterbral fascia and the cervical vertebrae.

In order to further clarify the association between systemic inflammation and atherogenesis, we designed a retrospective study conducted among older, white, American military veterans evidencing CCAAs, on PX in order to determine if the presence of such lesions was associated with an elevated NC; a known risk factor/indicator for a future MI. We hypothesized that the study would demonstrate that the mean NC among those patients evidencing carotid atheromas would be significantly greater than among those whose imaging studies were devoid of atheromas. The specific aim of our study was to determine if a group of older males, free of acute dental infection, and having CCAA on their PX would evidence greater levels of systemic inflammation as gauged by higher NC than those without atheromas. The predictor variable was CCAA and the outcome variable the mean NC.

Methods and materials

The investigators designed a retrospective cross-sectional study to address the research purpose. Specifically, the study cohort was comprised of white males ≥ 65 years evaluated by the Dental Service between January 1, 2017 and December 31, 2017. Study inclusion criteria mandated access to electronic medical records (EMR) having technically satisfactory PXs, which visualized the diagnostic region localized by an area 2.5 cm posterior and 2.5 cm inferior to the angle of the mandible while simultaneously capturing the interspace between C₃ and C₄. Similarly, the EMR had to have recorded an NC, which had been determined using standard Coulter counter techniques (Coulter Gen. S Hematology Analyzer, Beckman Coulter Corp Hialeah, Florida) from blood samples obtained within 24 months of the PX (i.e. up to 24 months before and 24 months after obtaining the image). Project enlistment was completed upon development of two cohorts of 60 persons each; 60 who were CCAA+, and 60 who were CCAA-. To ensure selection of an unbiased control sample, individuals in this cohort had to have their PX obtained on the same day as that of an individual enrolled in the study sample as well being closest in age. Furthermore, the clinicians interpreting the PXs for evidence of CCAA were masked as to the individual patient’s NC. Excluded from study enrollment were persons having chronic inflammatory/autoimmune diseases (e.g. rheumatoid arthritis, lupus, Crohn’s disease, ulcerative colitis), malignant haematologic disorders affecting white blood cells (e.g. myeloproliferative syndromes, chronic myeloid leukemia), oncologic diseases, active fungal or bacterial infections (including dental infection), recently traumatized persons, and those receiving medications known to affect the white blood cell count including glucocorticoids. Non-Caucasian males and Caucasian males under age 65 were excluded from study enrollment because of age and ethnic influence on NC.¹² Females were similarly excluded because of potential differences in NC between the genders and the scarcity of females attending the clinic.¹³ The project’s design and implementation were performed in accordance with the Declaration of Helsinki guidelines. The Medical Center’s Institutional Review Board approved the project’s protocol and given the study’s retrospective nature waived the need for informed consent from each patient.

The primary predictor variable was the demonstration of CCAA on a PX. The diagnosis of the presence or absence of CCAA was jointly arrived at by the first (TIC) and senior authors (AHF) utilizing previously published peer review criteria.^14,15 Consistent with these guidelines, nodular radiopaque masses or two opaque vertical lines inferior and/or posterior to the angle of the mandible at the level of the third and fourth cervical vertebrae were deemed to be CCAA. Excluded were cofounding calcified structures frequently imaged in the area such as the triticeous cartilage, tonsilloliths, salivary calculi, phleboliths, styloid ligaments, stylomandibular ligaments, superior cornu of the thyroid cartilage, epiglottis, and hyoid bone.^14,15

The primary outcome variable was the difference in the mean NC between patients with (CCAA+) and without (CCAA-) atheromas plaques on their PXs.

The differences in the mean NC between the CCAA + and CCAA- cohorts were appraised by bootstrapping analysis. Recorded data, in a deidentified fashion, were entered into an electronic database, and imported Stata/IC 15.1 for Mac (64-bit Intel) Revision 27 Jun 2018. P-value level of significance was set at 0.05.

Results

The Dental Service during the enrollment process treated 1006 study eligible White males over the age 65. Of these, 60 (6%) (mean age 75.9; range 69–91 years) were CCAA+ (study cohort) and they evidenced a mean NC of 4843 per mm³. The control cohort of 60 patients (mean age 75.3; range 66–94 years) whose PXs were devoid of atheromas (CCAA-) demonstrated a mean NC of 4108 per mm³. Bootstrapping analysis comparing the difference of the NC means as independent variables substantiated that the NC was significantly (p = 0.0008) higher (95% CI of difference of mean −432, 431; observed effect size 736:) in CCAA + individuals than in those that were CCAA- (Table 1). Analysis of variance in age between members of the study and control groups failed to delineate a statistically meaningful difference.

Table 1.

NC Among CCAA + vs CCAA-

	NC ± SD Mean	p-value
CCAA+	4843 ± 1303	p = 0.0008
CCAA-	4108 ± 1116

CCAA+, Positive for calcified carotid artery atheroma on panoramic imaging; CCAA-, Negative for calcified carotid artery atheroma on panoramic imaging; NC, Neutrophil count per mm³; SD, Standard deviation.

National Health Nutrition Survey, NC Reference Range: mean 4600 per mm³ for white males aged 70–74.¹⁶

Discussion

The purpose of this study was to compare the extent of systemic inflammation measured by NC between groups of older non-Hispanic white males with and without CCAA formation on their PXs (Figure), because both the atheromatous plaques and heightened numbers of neutrophils (NC) are validated “risk indicators/risk factors” associated with a future MI. The authors hypothesized that the CCAA +group would have a higher mean NC than the CCAA- group. The specific aim of the study was to determine whether a group of males with atheromas on their PXs would have greater systemic inflammation than those without atheromas as gauged by the mean NC. The results of our study evidenced that older (mean age almost 76 years) white male military veterans with CCAA on their PX evidenced significantly (p = 0.0008) greater NC levels (4843 per mm³ vs 4108 per mm³ than a cohort of similar patients with PXs devoid of carotid atheromas. Confirmation that our study group’s NC was heightened is borne out by a National Health and Nutrition Survey (NHANES),¹⁶ which established that the reference value for white males between the ages of 71 and 74 is 4,600 per mm³. This finding is clinically important finding because heightened NC and CCAA on PXs are both independent, validated “risk indicators” of near-term MI. The results of our study are also in accord with those of a carotid artery ultrasound study conducted in Korea among a cohort (N = 139; mean age 70) of neurologically asymptomatic individuals having stenotic (50–70%) lesions. Those individuals evidencing calcified carotid plaques had an elevated mean NC of 4,800 per mm³ (range 3,800–5,800 per mm.)³¹⁷

The findings from our study are consistent with the accumulating evidence that a heightened number of neutrophils and their mediators (elaborated cytokines) are integral to initiation and propagation of the chronic, low-grade systemic inflammation underlying the atherogenic process in both the carotid and coronary vasculature, and as shown by other investigators, a strong predictor of future adverse cardiovascular events.^7,18,19 Specifically, neutrophils are activated and recruited by platelet-derived chemokines to sites of endothelial cell injury (dysfunction) resulting from deposition of serum lipids in the sub endothelial space within the arterial wall.^20–28 The then “activated” endothelial cells interact with these neutrophils with consequent expression of cytokines, which attract additional neutrophils and monocytes to the site.²⁹ The activated endothelial cells also elaborate adhesion molecules, which facilitate the capture and transmigration of those neutrophils as well as monocytes and low density lipoprotein (LDL) through the endothelial layer, which has now been rendered more permeable to passage.^30,31 The neutrophils within the vascular wall elaborate additional products, which facilitate monocyte to macrophage differentiation as well as macrophage phagocytosis (uptake) and accumulation of oxidized LDL (resulting from neutrophil release of reactive oxygen species) with consequent macrophage transformation into foam cells. The neutrophils within the wall likewise enhance smooth muscle cell (SMC) responses, that is their recruitment from the tunica media to the subendothelial space where they proliferate and synthetize collagens, fibronectin, and elastin. Furthermore, as the atherosclerotic plaque matures, SMCs and the foam cells are subject to apoptosis resulting in a necrotic lipid core. Simultaneously, calcification forms within the atherosclerotic lesion, often towards its base, near the vascular media. When this occurs in carotid lesions, these can be detected on PXs^{10,11,22,32–34} The clinical correlate of this pathologic process within the coronary vascular tree is often an MI resulting from plaque destabilization and rupture or erosion of the fibrous cap overlying the atherosclerotic lesion triggering formation of an intraluminal thrombus.

Finally as we attempt to put our study results into perspective, it is humbling to note that a number of studies attempting to validate PX diagnoses of CCAA by carotid ultrasound have resulted in somewhat disparate findings. For example in one Turkish study of 93 patients (43 male; 50 female, age range 45–84 years), the accuracy of PX diagnoses was determined to be 63.4%.³⁵ Whereas in a second Turkish study conducted among a mixed gender cohort (N = 70) over age 40, the accuracy of PX diagnoses confirmed by carotid ultrasound was 80.5% with a sensitivity of 79.8% and a specificity of 81.1%.³⁶ These variations in study results most likely arose because the readers misinterpreted the images mistakenly classifying other calcified structures in the region (false positives) as carotid atheromas.³⁷ False-negative interpretations may have also arisen because the calcified carotid plaque was situated inferior to the area encompassed by the panoramic image or because the lesion was insufficiently calcified to be discerned by radiography.

Our study had a number of strengths and weakness. As for strengths, it is to the best of our knowledge, the first to report the association between PXs demonstrating CCAA and increased NC, both of which are validated “risk indicators/risk factors” in development of a future MI. This is a uniquely consequential finding given that 50% of males die of sudden adverse cardiovascular events without having had prior classical symptoms or traditional risk factors, which would have identified them as being a member of a high-risk group. However, our study does have certain limitations. Generalization of our findings to populations other than older white males would be inappropriate given our understanding as to the influences of ethnicity, gender and age upon the NC.^6,13,38 Secondly, our study evaluated only a single neutrophil value among a small patient sample, in a single center. Furthermore, a positive smoking history, shown in a few, but not all studies to affect the NC was often not available in the cohorts’ EMR and, therefore, could not be factored into the results.^13,39

Conclusion

In conclusion, we have demonstrated for the first time in the medical/dental literature an association between the presence of CCAA on PXs and an elevated NC value among older white males; two validated cardiovascular risk factors previously deemed independent. Therefore, we suggest that the comorbid presence of both panoramic imaged vascular plaque and a heightened (≥5000 per mm³) NC, when encountered in an older white male patient, strongly suggests the need for medical consultation prior to performing invasive dental procedures.