Systemic Infections Cause Exaggerated Local Inflammation in Atherosclerotic Coronary Arteries

Abstract

Systemic infections can trigger heart attacks. We conducted an autopsy study to investigate the pathologic effect of systemic infections on coronary artery inflammation.

We studied 14 atherosclerotic patients diagnosed with an acute systemic infection. Our control group (n=13) had atherosclerosis without infection. The groups were similar in luminal stenosis and age. Coronary artery sections were stained with H&E and markers for macrophages (CD68), T cells (CD3), and dendritic cells (S100).

On pathologic examination, 5 infected patients had acute myocardial infarction with thrombosis. Macrophage density in plaques and in periadventitial fat was higher in the infected group (NS). The infected patients' adventitia had significantly more macrophages (1,577 ± 1,872 vs 265 ± 185 per mm²; P=0.047). The macrophage density, similar in the control group's adventitia and plaque, was significantly greater in the infected group's adventitia than in the plaque. The adventitia and periadventitial fat of the infected group had more T cells than did samples from the control group (48.4 ± 45.0 vs 14.1 ± 6.3 per mm²; P=0.002). The groups exhibited similar plaque T-cell density. The infected patients' plaques, but not the adventitia and periadventitial fat, had more dendritic cells than did the controls' (3.2 ± 2.5 vs 0.3 ± 0.5 per mm²; P=0.022).

To our knowledge, this is the 1st report to establish a connection between acute systemic infections and significant increases in inflammatory cells in the atherosclerotic coronary arteries of human beings. This offers a new therapeutic target for preventing heart attacks in high-risk patients.

Inflammation plays a major part in the initiation and progression of atherosclerosis and in the development of its acute clinical manifestations.^1,2 Acute infections, with their consequent inflammation, may affect atherosclerotic disease. This relationship was first proposed by William Osler at the beginning of the 20th century.

The following infectious agents have been linked to atherosclerosis: cytomegalovirus, Chlamydia pneumoniae, herpes simplex viruses 1 and 2, Helicobacter pylori, Mycoplasma pneumoniae, Porphyromonas gingivalis, enterovirus, and, more recently, the influenza virus.^3–5 A series of acute and chronic infections, occurring alone or in combination, may lead to the development and progression of atherosclerosis. By rapidly increasing inflammation in the coronary arteries, acute infections may trigger destabilization and possible rupture of vulnerable plaques.⁶ Given the central role of inflammation in atherosclerosis, we investigated whether a wide range of systemic infections might exacerbate local inflammation in coronary arteries.

Materials and Methods

Study Population

We reviewed the pathology files of a large teaching hospital from 1991 through 2002. The study protocols and procedures were approved by the institutional review boards, and approvals from the treating physicians were obtained before the medical records were released. After excluding patients who might have had an inadequate or altered inflammatory response to infections (for example, persons with human immunodeficiency virus or cancer, or those who were on a regimen involving immunosuppressive or corticosteroid drugs), we identified 14 patients who had clinical and pathologic evidence of coronary artery disease (atherosclerosis) at autopsy and an acute systemic infection within 2 weeks of death. The study group comprised 11 men and 3 women, aged 64 ± 14 yr. This group was compared with 13 control patients (8 men and 5 women; mean age, 65 ± 11 yr) who had died with coronary artery disease but without infection. There were no significant differences between the groups in age (P = 0.82) or sex (P = 0.41). Table I lists the acute infectious agents in the study group.

TABLE I. Acute Infections in the Study Group

Sepsis was defined as the presence of systemic infection as determined by the treating physician or from laboratory data. Twelve study-group patients had upper or lower respiratory infections; 2 had urinary tract infections. Most of the control patients had died abruptly due to pulmonary embolism (2 cases), postoperative decompensation or complications (3), aortic aneurysm (3), acute myocardial infarction (AMI) (1), aortic dissection and AMI (1), asthma (1), airway obstruction (1), and acute respiratory distress syndrome (1).

Histopathologic Examination

The coronary arteries obtained at autopsy were formalin-fixed, paraffin-embedded, and cut into 4-μm-thick serial sections. One to 4 sections per patient (mean, 1.2 sections) were immunohistochemically stained with all 3 of these antibodies: the macrophage marker CD68 (all markers were from DAKO; Carpenteria, Calif), the CD3 marker for T cells, and the S100 protein for dendritic cells. Quantitative morphometric evaluation(blinded) was performed by 2 separate observers who used the Olympus MicroSuite Software™ B3SV on an Olympus BX61 microscope (Olympus America Inc.;Center Valley, Pa). Cell counts were performed in the intimal plaque, adventitia, and periadventitial fat at ×200 magnification for the entire circumference of each coronary artery. The results were presented as the number of cells per mm².

Histologic Definitions

The plaque area was defined as the area inside the internal elastic lamina (IEL). The adventitia was defined as the region extending from the external elastic lamina to the beginning of the periadventitial fat. The periadventitial fat was considered to extend from the adventitia to 250 μm beyond. Macrophage density was the number of macrophages per mm². Stenosis denoted the (IEL area – lumen area)/IEL area, expressed as a percentage. We quantified all the slides that were available.

Statistical Tests

Results are expressed as mean ± standard deviation. Because of the small sample size and nonnormal distribution of the data, we used the Mann-Whitney U test and the Wilcoxon signed rank test to study the significance of our findings. An α level of 0.05 was considered the threshold for statistical significance. The Fisher exact test was used to evaluate categorical variables, such as the sex of the patients. The software used for statistical analysis was the Statistical Package for the Social Sciences, version 9 (SPSS Inc.; Chicago, Ill).

Results

The 2 groups had similar percentages of luminal stenosis (67% ± 14% vs 55% ± 25%; P = 0.23). Subocclusive luminal thrombi were seen in 4 infected patients, and a small luminal thrombus was present in a 5th; all 5 had histologic evidence of AMI. Only 1 organized thrombus was seen in the control group. Of the 5 infected patients who had AMI on pathologic examination, only 2 had been diagnosed clinically to have AMI.

In the plaques, the macrophage density showed a nonsignificant trend toward higher levels in the patients with a systemic infection (582 ± 774 vs 281 ± 321 per mm²; P = 0.41) (Table II). However, in the adventitia of the infected patients, there was a significantly greater number of macrophages than in the adventitia of the control patients (1,577 ± 1,872 vs 265 ± 185 per mm²; P = 0.047) (Figs. 1 and 2).

TABLE II. Densities of Inflammatory Cells in the Plaque and Adventitia

Fig. 1 Section of coronary artery from an infected patient. A) Low-power view (H&E, orig. ×4). B) CD68 staining shows a substantial presence of macrophages that heavily infiltrate the plaque, adventitia, and periadventitial fat, mostly sparing the media. C, D) Increased magnification (×10) of the respective stained sections.

Fig. 2 Section of coronary artery from a control patient. A) Low-power view (H&E, orig. ×4). B) CD68 staining shows an absence of macrophages in comparison with the Figure 1 images of the infected patient's artery. C, D) Increased magnification (orig. ×10) of the respective stained sections.

The macrophage density in the periadventitial fat showed a nonsignificant trend toward higher levels in the infected patients (776 ± 821 vs 212 ± 219 per mm²; P = 0.085). In the control patients, the macrophage density was similar in the adventitia and the plaque (281 ± 321 vs 265 ± 185 per mm²; P = 0.85); however, in the infected patients, the density was significantly higher in the adventitia than in the plaque (1,577 ± 1,872 vs 582 ± 774 per mm²; P = 0.047).

Significantly more T cells were observed in the adventitia and periadventitial fat of the infected patients than in the control patients (48.4 ± 45.0 vs 14.1 ± 6.3 per mm²; P = 0.002). In the intima, the number of T cells did not differ significantly (infected, 14.0 ± 13.2 per mm²; control, 14.3 ± 20.5 per mm²; P = 0.49).

Similarly, the dendritic cell counts were not significantly different in the adventitia and periadventitial fat (infected, 34.1 ± 53.7 per mm²; control, 21.8 ± 15.3 per mm²; P = 0.87). On the other hand, significantly more dendritic cells were seen in the plaque of the infected patients (3.2 ± 2.5 per mm²; control, 0.3 ± 0.5 per mm²; P = 0.022) (Table II; Figs. 3 and 4).

Fig. 3 Section of coronary artery from an infected patient. A) Calcified plaque with substantial neovascularization (H&E, orig. ×20). B) S100-positive dendritic cells are seen mainly around neovascularization (CD68, orig. ×20).

Fig. 4 Higher numbers of macrophages (upper graph) and dendritic cells and T cells (lower graph) were observed in different layers of the atherosclerotic coronary lesions of infected patients versus control patients.

In summary, we found a significantly higher number of macrophages in the coronary adventitia of the infected patients than of the control patients, who were atherosclerotic without infection. The macrophage density tended to be higher in the plaque and periadventitial fat of the infected patients, but this difference was not significant. The infected patients also had more T cells in their adventitia and periadventitial fat, and more dendritic cells in their intima and media. The higher number of inflammatory cells was associated with an increase in the incidence of myocardial infarctions and luminal thrombosis. Interestingly, the percentage of stenosis was not significantly different between the 2 groups.

Discussion

To our knowledge, this is the 1st report to establish a connection between acute systemic infections and significant increases in inflammatory cells—known to play a major role in AMI—in the atherosclerotic coronary arteries of human beings. This discovery suggests a mechanism for the triggering of AMIs after acute infections, and it offers a new therapeutic target for the prevention of heart attacks.

How Might Acute Infections Trigger Acute Myocardial Infarction?

Over several decades, scattered clinical reports have noted that up to one third of myocardial infarctions are preceded by an upper respiratory infection.^6–12 The occurrence of AMI undergoes seasonal variation, having its highest incidence in the winter months, when the incidence of upper respiratory infections is also highest.^13–15 Influenza epidemics have been associated with a significant increase in overall cardiovascular deaths,¹⁶ a spike usually attributed to deaths of patients with known heart failure. In 2000, we described an association between influenza vaccination and a reduced incidence of winter myocardial infarctions,^6,17 a finding later confirmed by others.^18–21 In addition, a study of 75 patients who had severe acute respiratory syndrome (SARS) found that 2 out of 5 deaths were due to AMI²²—an occurrence still overlooked by many.

Infectious agents have several potential effects on the pathophysiology of atherosclerosis and its clinical complications.^5,23 Whereas most suspected infectious agents initiate or aggravate a chronic vascular or systemic inflammatory process, acute systemic infections may, instead, destabilize existing vulnerable plaques. For example, we have shown that inoculating atherosclerotic, apolipoprotein-E–deficient mice with the influenza A virus leads to a marked increase in inflammation and thrombosis in murine atherosclerotic plaques but not in normal regions of the aorta.²⁴ Pro-atherosclerotic changes in mice have also been reported after acute infection with cytomegalovirus and P. gingivalis.²⁵

Systemic infections can exert acute and chronic influence on vascular walls. The effects are either direct (through seeding of the microbe in the vascular wall) or indirect (through release of inflammatory cytokines and other systemic effects) (Fig. 5).5,6,26,27

Fig. 5 Various mechanisms by which acute infections may affect atherosclerotic plaques.

Adventitial Inflammation in Atherosclerosis

Evidence increasingly suggests an important role of adventitia in the inflammation that is present in atherosclerotic plaques. In 2004, we reported that human atherosclerotic coronary plaques with large lipid cores have a significantly greater number of macrophages in their periadventitial fat than do fibrocalcific and nonatherosclerotic arterial segments, which suggests the involvement of adventitial inflammatory cells in plaque vulnerability.²⁸ Our previous findings suggested that the adventitia and periadventitial fat may function as a unit.²⁸ The lack of a functional border between the adventitia and the periadventitial fat has been noted by others after balloon angioplasty.²⁹

Dendritic Cells and Atherosclerosis

An immune reaction against certain antigens may play an important role in the development of atherosclerosis.^30,31 Dendritic cells—antigen-presenting cells that have important functions in immune responses—have been observed in atherosclerotic plaques. In advanced atherosclerotic lesions, clusters of dendritic cells have been described as part of the inflammatory infiltrates that resemble mucosa-associated lymphoid tissue, where they intermingle closely with B lymphocytes.³² Dendritic cells also interact closely with CD4 helper and CD8 cytotoxic cells.³³ In our study, dendritic cells were more numerous in the plaques of the infected group than in those of the control group. We infer that acute systemic infections, by increasing the number of dendritic cells, may enhance the immunologic reactions in the plaques and exacerbate the atherosclerotic process. Further studies are needed to evaluate this hypothesis.

Limitations

Although our study's low sample size might have decreased our power to detect some of the associations at an α level of 0.05, our main findings were statistically significant. At a higher sample size, our significant results would still be valid, and the observed trends—which are already biologically relevant—might also reach levels of significance.

Unfortunately, our autopsy study, by its very nature, limited the number of cases that could be studied. The small sample size did not enable us to differentiate between pathologic responses to different infections, such as gram-negative versus gram-positive infections and those at different loci. Nevertheless, given the long list of infectious agents linked to atherosclerosis and the central role of inflammation as a common final pathway for these agents' effects, we conclude that most acute systemic infections have similar nonspecific, injurious effects on the coronary arteries. In fact, the burden of infection itself may increase the risk of AMI.⁴ More severe infections, such as influenza and SARS, may have a more profound effect on atherosclerotic disease and could warrant separate studies.⁶

Recent reports have suggested that statins can decrease morbidity and mortality rates in atherosclerotic patients who have sepsis.^34,35 Most of our patients had died in the early 1990s, when statin use was very low. In fact, of the patients in our study, only 1 had received lovastatin. Future studies should seek to determine whether statin use can decrease the recruitment of inflammatory cells to the coronary arteries. Statins may be especially useful in lowering the morbidity and mortality rates among people with and without atherosclerotic disease during a future influenza pandemic. In the event of such a disaster, we foresee a large rise in cardiovascular deaths because of the triggering effect of influenza on AMI.^36,37

Implications

If further studies confirm that sepsis promotes the infiltration or survival of inflammatory cells in atherosclerotic arteries, many questions meriting further investigation will be suggested: Why are T cells and dendritic cells drawn to plaque, and macrophages to the adventitia? Is this also seen in atherosclerotic carotid arteries and the aorta? Are some microbes more likely than others to increase plaque inflammation? Does increased plaque inflammation increase the risk of rupture, erosion, or thrombosis? Does adventitial inflammation accelerate the progression of aneurysms? Are some arteries themselves infected? If so, is the inflammatory response beneficial or harmful? Although our study falls short of proving a causal relationship between the preceding systemic infection and the coronary inflammation observed at autopsy, our findings are supported by the growing body of evidence concerning the pivotal role of inflammatory cells in coronary events,³⁸ and these findings deserve further evaluation.

We noted 5 cases of AMI in the infected group, compared with only 1 old myocardial infarction in the control group. However, only 2 of these 5 AMIs had been detected clinically before the patients died—raising the possibility that, in patients with a systemic infection, AMI may easily be missed despite the increased chance of plaque destabilization. Our report highlights the need for clinicians to consider possible myocardial infarction when caring for septic patients.

Inflammatory mediators are central to the pathogenesis of septic shock and multiorgan failure, because sepsis results from an exaggerated systemic inflammatory host response induced by infectious organisms.³⁹ Clearly, the use of anti-inflammatory drugs and statins should be evaluated in patients who have coronary artery disease. These drugs may attenuate the dangerous effects of acute infections on atherosclerotic plaques. The severity of sepsis may be decreased by the administration of statin therapy before the onset of an acute bacterial infection.⁴⁰

Aggressive anti-inflammatory treatment may be indicated for patients who develop an acute infection anywhere in the body. Our findings may also help explain why the risk of myocardial infarction is increased after surgical interventions (which may be associated with bacteremia), and why—if tooth-brushing leads to bacteremia—patients with periodontal disease have an increased rate of coronary and cerebral events.⁴¹

Therapeutic and prophylactic implications for clinicians and patients include oral hygiene; hand-washing; avoidance of infected persons; vaccinations (for example, for influenza, Streptococcus pneumoniae, and Haemophilus influenzae); new indications for antimicrobial, anti-inflammatory, and antithrombotic medications; and closer monitoring of at-risk patients.

Acknowledgments

The authors wish to thank Tomas Klima, MD; Tommy Reese, HTL; and Mr. John Ryan for their help in conducting this study; and Ms Virginia D. Fairchild for preparing this manuscript.