ABSTRACT: Background. Gender differences that exist in patients with acute coronary disease (ACD) are unexplained. We sought to determine if these differences could be related to differences in the pathologic substrate found in the coronary arteries at the time of death. Methods. The hearts of 83 patients (64 men and 19 women) who died of ACD were obtained fresh and uncut at the autopsy table. The coronary arteries were injected with a colored barium gelatin mass. After formalin fixation, the epicardial arteries were dissected intact, decalcified and cut at 2–3 mm intervals, with all segments mounted for histologic study. The severity of luminal stenosis and the frequency of adventitial inflammation, intimal calcification and atheromas were determined microscopically for each segment. Plaque burden was determined histologically by assessing the severity of luminal stenosis for each coronary segment. The number of plaque ruptures (PRs), with and without luminal thrombosis, were tabulated for each heart in the study. These results were compared with 22 control patients who died of noncoronary disease. Results. There are gender similarities as well as significant differences in the pathologic substrate of patients who die of ACD. Active, inflammatory atherosclerosis and associated ACDs develop earlier in life in men than in women, and are associated with death at an earlier age, producing a “gender gap.” There were no significant gender differences in the frequency of PRs. The women were significantly older than the men and had more extensive active disease, but had the same overall plaque burden as men, suggesting women may be resistant to plaque growth, particularly atheroma growth. Conclusions. Gender gap appears to be related to factors peculiar to women who resist atheroma growth, delaying PR and the onset of ACD.
J INVASIVE CARDIOL 2009;21:270–277
The gender differences that exist in patients with acute coronary disease (ACD) remain largely unexplained and may reflect anatomic, biologic or pathophysiologic differences.1,2 Women who develop ACD are often older than men, typically die at an older age and have a worse prognosis following acute myocardial infarction (AMI), or after interventions such as coronary bypass graft surgery (CABG) or percutaneous coronary intervention (PCI).2–6 These differences, often referred to as “gender gap”, are consistently present in all countries and cultures where coronary artery disease exists.1 The low frequency of ACD in premenopausal women suggests they are “protected” or resistant in some way to developing atherosclerosis and subsequent ACD,1, 2, 7–9 but the mechanism of such resistance is unknown. The protective factor(s) appear to be powerful and are not found to operate through lipid-related mechanisms.9 Additionally, levels of known common risk factors do not explain gender gap.1,9 Even women with heterozygous familial hyperlipidemia appear to be protected.10 A sudden increase in ACD does not occur at the time of menopause,1 as might be expected if this resistance were related to female hormones. In fact, the administration of female hormones to the postmenopausal woman is associated with increased risk of ACD.11 The presence or absence of female hormones does not explain gender gap.1,9,12
The aim of this study was to perform an extensive histologic study of the coronary arteries in a large number of men and women who died of ACD to determine whether any structural differences existed in the coronary arteries, particularly in regard to plaque burden, adventitial inflammation, calcification and atheroma formation that could contribute to our understanding of gender gap. This study moves forward from previous quantitative studies13,14 by showing significant gender differences in the age of onset and rate of acceleration of active, progressive, inflammatory atherosclerotic disease that must be considered in evaluating gender gap.
Methods
Study population. The study group consisted of 83 patients: 64 men, age range 32–86 years, mean age 56, and 19 women, age range 49–93, mean age 71, who died of ACD. The hearts of these patients were obtained from the Department of Pathology at Mercy General Hospital, Sacramento, California, and from the Sacramento County Coroner’s office. No hearts were obtained from long-term care facilities and none were involved in forensic studies. The study population is considered to be representative of an unselected population of patients with ACD, men predominating at a ratio of 3:1 over women, similarly found in most studies.2,3 Risk factor data were incomplete for these patients. Twenty-two patients, 3 women, age range 15–67 years, mean age 47 years, and 19 men, age range 19–74 years, mean age 43 years, served as controls. Five control patients died as a result of accidents or trauma, 5 of noncoronary cardiomyopathy, 3 of cancer, 2 of suicide, 2 of stroke and 5 of miscellaneous causes.
Acute coronary syndromes (ACS). The clinical syndromes associated with death included cardiogenic shock (CS), sudden cardiac death (SCD) with AMI, SCD without AMI and cardiac rupture (CR) associated with AMI. CS was considered to be the cause of death in patients with AMI who exhibited progressive deterioration in cardiovascular status including, but not restricted to, progressive hypotension, congestive heart failure, pulmonary edema and renal insufficiency. All CS patients were in intensive care units at Mercy General Hospital. Terminal arrhythmias in these patients were considered to be secondary to CS, not a primary contributor to these deaths. Hospital deaths in patients with AMI were considered arrhythmic in origin if documented by telemetry monitoring or, if SCD occurred in an unmonitored hospital patient with AMI, were considered to be stable. Sudden death was defined as any patient’s death occurring within 6 hours of being in good health or stable condition.
Out-of-hospital SCDs were considered to be arrhythmic in origin. Information is incomplete regarding whether these out-of-hospital SCDs were witnessed. CR was deemed to be present if the pericardial sac was filled with blood at autopsy, associated with a transmural AMI with ventricular perforation. Twelve patients (5 women and 7 men) died following PCI and/or the administration of thrombolytic drugs. No patients received a coronary stent, and no cardiac surgery patients were included in this study. A diagnosis of AMI was made on histologic examination including coagulation necrosis of myocytes (cytoplasmic eosinophilia and nuclear pyknosis) and the presence of leukocyte infiltrates.
Study technique. Hearts were obtained fresh and uncut at the autopsy table. The coronary arteries were injected with a colored barium gelatin mass, red color in the left and blue in the right coronary artery, distending the arteries to physiologic limits before formalin fixation. The coloring material withstands histologic processing, which allows the injection mass to be identified and followed visually and microscopically. After formalin fixation, the epicardial coronary arteries were dissected intact, decalcified, cut at 2–3 mm intervals, photographed as a group and then in groups of 4–6 contiguous segments. Individual segments were photographed when gross abnormalities such as PR were noted. The photographed surface of each segment was then marked with a drop of colored injection mass, the segment numbered and processed in its own cassette and embedded, thus the marked surface was the surface cut first for histologic study. This allowed close correlation between the gross and microscopic appearance of each segment. Subserial sections, usually 3–5 sections, were made from each segment.15–17 The microscopic results reported here represent only the findings in the left main, left anterior descending, circumflex and right coronary arteries. They do not include any marginal, intermediate or diagonal branches of these arteries.
A total of 7,143 coronary segments, average 86 per heart, were examined histologically, approximately 30,000 separate coronary sections when the subserial sections are counted. X-rays were taken of the intact heart before and after injection and X-rays were also taken of the dissected coronary arteries before and after decalcification. Hematoxylin and Eosin (H&E) and Martius Scarlet Blue (MSB) were the primary stains used in this study.
Microscopic examination. All coronary segments were examined microscopically to determine the presence or absence of the following features: First, the severity of luminal stenosis was determined by projecting the microscopic slide and using a planimeter to measure the area of the lumen and the area inside the internal elastic lamina. The luminal area was divided by the area inside the internal elastic lamina area to determine the percentage of luminal stenosis. Initially, all segments were measured using this method, but after experience with 10–15 hearts, the degree of stenosis was estimated solely by observing the microscopic slide. Segments were divided into three groups based on the severity of luminal stenosis in order to quantify the plaque burden. These groups were the segments with 80% luminal stenosis. Second, adventitial inflammation, intimal calcification and atheromas were identified microscopically and tabulated for each coronary segment. These three lesions were selected because they are easily identified (Figures 1–3) and quantified microscopically, are associated with advanced atherosclerotic disease,18 and are consistently present at the site of PRs.16,17,19 They were not necessarily present together in a given segment and could occur singly, doubly or with all three present in a single segment. Tabulating these three components by age and gender, segment-by-segment, provides an overall view of the extent of the atherosclerotic process and of the different plaque components in men and women.
Plaque ulcerations, erosions and ruptures. Any significant breaks or breaches of endothelial integrity, with penetration of colored injection mass into the underlying plaque,16,17 are referred to collectively as PRs. PRs, with and without associated luminal thrombosis, were tabulated for each heart. Any PR extending to adjacent, contiguous segments was counted as one PR.
Adventitial inflammation. Adventitial inflammation was determined to be present by finding dense foci of lymphocytes in the adventitia, as shown in Figures 1–3. The term “adventitial inflammation” in this report refers specifically to these foci of adventitial lymphocytes. In approximately 15% of segments, the adventitial lymphocytes were more scattered. At least 20 lymphocytes per high power (40x) field were considered to represent an inflammatory response. No attempt was made to determine the identity, type, or extent of lymphocytes or other types of inflammatory cells in the intima, but adventitial inflammation was deemed to reflect intimal injury and inflammatory response.20–22 The majority of these lymphocytes are T lymphocytes.21
We interpret these lymphocytic infiltrates to be a reliable histologic marker of active, progressive atherosclerotic disease involving the underlying plaque based on the following observations: First, these lymphocytic infiltrates are found only over underlying intimal plaques, not over normal wall.23 Second, the infiltrates are often present over the plaque shoulder (Figure 3), the frequent site of active disease.24 Third, a good correlation between adventitial infiltrates and underlying intimal inflammation has been reported.20 Fourth, the majority of lymphocytes in the region of atherosclerotic plaques are in an activated state, and although not yet proved, it is reasonable to believe that at least some of the adventitial T cells have also been activated by intimal antigens migrating to the adventitia.25–27 Fifth, T lymphocytes are immune cells, and immune mechanisms interact with metabolic risk factors to initiate, propagate and activate lesions in the arterial tree.25 Sixth, virtually all PRs and/or erosions are associated with inflammation, including adventitial lymphocytic infiltrates,16,17,19,28 and these PRs are considered to be the site of active disease.29 We expand the histologic definition of currently active disease to include any plaque, regardless of size, with or without PR, that has an overlying infiltrate of lymphocytes in the adventitia (Figures 1–3). The overall extent of active disease in the hearts in this study is based on the number of segments showing these adventitial lymphocytic infiltrates. Active, progressive atherosclerotic disease is characterized by plaque growth, inflammation, calcification and atheroma formation, often occurring at multiple sites in the epicardial coronary tree.
Calcification and atheromas. Calcification was identified by finding the characteristic staining reaction on H&E stain (Figure 2) in the intima. Atheromas were identified as plaques with a lipid-rich core containing necrotic pultaceous debris, foam cells, cholesterol crystals and tissue fragments in varying amounts (Figures 1–3). Although calcification and atheromas are formed as a component of active, progressive disease, they are not sensitive indicators of currently active disease and are not consistent predictors of future events.30
Statistical analysis. Statistical analysis of data presented in this report utilized chi-square and Fisher’s exact probability tests; p-values Results
Table 1 presents the frequency distribution, by gender, in patients with ACD. There were no significant gender differences in the frequency of these syndromes, allowing the histologic findings in the different syndromes to be compared.
Table 2 compares the total number of PRs found in these syndromes, as well as the number of PRs per heart found in men and women in this study. There was no significant gender difference in the frequency of PRs in any of these different clinical syndromes. Multiple PRs were the rule in these patients.16 Fifty percent of PRs were associated with luminal thrombosis,16 with no significant gender difference in the frequency of thrombosis in the different clinical syndromes.
Table 3 compares gender differences in age, overall plaque burden and the frequency of adventitial inflammation, calcification, atheroma formation and luminal stenosis in all coronary segments in this study. The women were significantly older than the men. Over half of all segments, 54% in men and 56% in women, showed > 50% luminal stenosis, illustrating that many patients with ACD have diffuse atherosclerotic disease.13,14 Similarly, over 50% of all segments also showed adventitial inflammation, showing that a relationship exists between the extent of adventitial inflammation and the extent of atherosclerosis.
Table 3 shows that plaque growth is associated with increasing inflammation, calcification and atheroma formation in both men and women. However, women had significantly more extensive overall involvement of the coronary tree, particularly with inflammation (p 50% luminal stenosis. A close relationship clearly exists between active disease and plaque burden. Importantly, even though women were older and had more extensive active disease and significantly more atheromas, they did not have a greater plaque burden.
Table 3 also provides a unique overall view of the atherosclerotic process and plaque development in these patients. Adventitial inflammation is the most common lesion, occurring in 50% of all segments in men and 55% of all segments in women, followed by calcification in 35–43%, respectively, and by atheromas in 30–35% of segments. Adventitial inflammation, being the most common, is presumed to be the first to develop, followed by calcification, then by atheroma formation. The descending frequency of these three lesions is similar in both men and women.
Figure 4 compares the frequency of these three components in men and women by decade of life. In the men, active disease begins in the fourth decade and expands to involve more of the coronary tree with each passing decade, reaching a maximum in the sixth decade, when approximately 25–30% of all segments show active disease. Most of the men died in the sixth decade at an average age of 56 years, showing a relationship between the extent of active disease and death from ACD. In men beyond the sixth decade, the rates of expansion and the extent of active disease appeared to stabilize without further progression.1 The extent of active disease appeared to remain relatively constant. Approximately 10–15% of all segments showed active disease, and this remained relatively constant from the seventh through the ninth decades.
In women, active disease was present in the 4 women who died in the fifth and sixth decades, at an average of 51 years, but it was much less extensive than in men at that age. These results show that women have similar, but less extensive active disease than men at this age. After menopause, the disease process expands to involve more of the coronary tree, reaching a maximum in the eighth and ninth decades, when approximately 35% of all segments show active disease. Death in women, occurring at an average of 71 years, is associated with the maximum extent of active disease, similar to men. These results show that there are gender differences in the pathologic substrate, in the age of onset, in the rate of progression, and in the extent of active disease in patients with ACD.
Table 4 compares the gender frequency of PRs by decade of life. Younger men and older women show significantly more acute lesions than their gender counterparts in those age groups. A relationship exists between the extent of active disease and the frequency of PRs in both men and women.
Table 5 compares the plaque burden, adventitial inflammation, calcification and atheroma formation in the 22 control patients. The women were significantly older than the men, but men had the greater plaque burden. These three components of active disease are present in both men and women who do not have ACD, but the extent of involvement of the coronary tree is much less than in patients with ACD. Women again had more extensive inflammation and calcification, but there were no differences in the frequency of atheroma formation. One PR was found in the control hearts.
Discussion
The results presented here show that adventitial lymphocytic infiltrates are a component of active, inflammatory atherosclerosis, and are present in over 50% of all segments examined. Michel reports that the adventitia, including the adventitial inflammatory responses, plays an important role in the vascular “response to injury” by reacting to antigens and/or other mediators that originate within the plaque and pass centrifugally from the intima to the adventitia.22 These adventitial lymphocytes may play a role in the activation of intimal inflammatory cells and in the overall immune response.25,26
This is the first report, to our knowledge, to quantify and compare, by gender, the extent of adventitial lymphocytic infiltrates in patients with ACD. Ridker has reported that high levels of inflammatory biomarkers are associated with increased risk of acute coronary events.31 The results presented here, showing a close correlation between the extent of inflammation and death from ACD, provide histologic support for the clinical findings of Ridker.
Gender similarities. The results show gender similarities as well as significant differences in the pathologic substrate in patients who die of ACD. The same clinical syndromes, the same frequency and type of acute lesions in the different syndromes, the same overall plaque burden, the same components of active disease with similar descending frequency, as well as a similar relationship between active disease and luminal stenosis, are strong evidence that the pathogenesis of atherosclerosis is the same in men and women. They have the same — not a different — form of atherosclerosis, presumably caused by the same injurious agent(s) or processes. If the pathogenesis is the same, why do we have a gender gap?
Gender differences and atheroma growth. Gender gap may be related to gender differences either in susceptibility to or resistance to the development of active disease. Men develop active disease and associated acute lesions early in life; women develop active disease and acute lesions late in life. Young men may be more susceptible to injury and active disease; women, particularly premenopausal women, may be resistant to active disease. The prevailing opinion of most investigators is that women possess inherent, unknown factors that enable them to resist the onset of the disease.8,9 It is not clear whether this resistance is an inherent resistance to atherosclerotic injury, per se, or a resistance to the plaque growth that follows injury, or both. The result of this resistance is minimal atherosclerosis and infrequent ACD in premenopausal women,32,33 a major contributor to gender gap. The role of various risk factors in the pathogenesis of these gender differences was not considered in this study.
However, this gender difference in resistance to active disease does not appear to end abruptly at menopause, but is lost gradually until approximately age 70 years in women, when the frequency of ACD approximates that of men. At this point, women appear to become particularly vulnerable to atherosclerotic injury and the development of active disease. The extensive involvement in women in their eighth and ninth decades (Figure 4) is dramatic and exceeds the maximum observed in men in their sixth decade, showing just how vulnerable women have become. The frequency of acute lesions exceeds that of men and is associated with death from ACD.
We would expect women with this pathologic substrate to have a greater overall plaque burden because plaque burden, in patients with active disease, is related primarily to the growth and enlargement of atheromas.34 The failure to find a greater plaque burden in women, even though they are older, have more extensive active disease, with more frequent atheromas, suggests that the atheromas in women did not grow sufficiently to contribute to a greater overall plaque burden. Women do not appear to be resistant to atheroma formation, per se, because they have more atheromas than men, but appear to be resistant to atheroma growth and expansion. This view is supported by the findings in the control hearts that show younger women, mean age 47 years, have more extensive inflammation, but do not have more frequent atheromas. It is young men who have the greater plaque burden, presumably due to atheroma growth. In terms of atheroma growth, women appear to lag behind men. They appear to be resistant, not only to atherosclerotic injury and to expansion of active disease during their premenopausal years, but continue to be resistant to atheroma growth well beyond menopause. If factors peculiar to women enable them to resist atheroma growth, this could be expected to delay PR in both pre- and post-menopausal women, also delaying the onset of death from ACD, and producing gender gap.
Possible mechanism of resistance. What mechanism, based on this pathologic evidence, can be proposed that might explain resistance to growth of the atheroma in women? Oxidation of low-density lipoprotein (LDL) is considered to be a major factor in the pathogenesis of atherosclerosis and progressive growth of the atheroma.35 If this theory is correct, reducing or modifying the oxidation of LDL may be expected to delay growth of the atheroma, the development of luminal stenosis and, ultimately, the onset of AD. Therefore, the resistance to growth of the atheroma in women may be related to factors peculiar to women that prevent or delay the oxidation of LDL.
Recent reports concerning glutathione peroxidase, a powerful antioxidant enzyme, show premenopausal women have much higher levels of this enzyme than men of the same age.36 Further studies of glutathione peroxidase show that the risk of cardiovascular events, in both men and women, is inversely associated with increased levels of this enzyme.37 These gender differences in the level of glutathione peroxidase continue after menopause, but the blood levels gradually fall in women until they approximate those of men near age 70.36 The steady increase in frequency of ACD beginning with menopause1 coincides with the drop in the level of glutathione peroxidase, suggesting a possible relationship.36 If this, or a similar unknown mechanism, is responsible for the resistance to growth of atheroma in women, it could explain the resistance of premenopausal women to ACD and why postmenopausal women have smaller atheromas.38
Influence of age. This study, like virtually all clinical studies, shows that women are significantly older than men at the time of onset and death from ACD,2,3 raising the question of the role of age in the pathologic changes observed in this study. Age is frequently mentioned as a risk factor for atherosclerosis and ACD in women, but no histologic evidence has been presented to show specific pathologic changes associated with the plaque that can be attributed directly to age. Atherosclerotic plaques tend to become more atheromatous with age,39 but this does not prove age to be a causative factor. The presence of more comorbid conditions in older women may explain their deaths, but does not explain gender gap. If age, per se, were a crucial or important factor in pathogenesis, we would expect all elderly people to have extensive atherosclerosis and ACD, particularly older women.
Atherosclerosis is not a degenerative disease,40 or an age-dependent disease, even though the amount and extent of atherosclerosis tend to increase with age. Atherosclerosis is related to vascular injury caused by an injurious agent or process that attacks the vascular system of persons of any age. Many young people have severe and extensive atherosclerotic disease, equivalent to or greater than many older people. Other investigators have noted neither age nor sex to be a significant factor in the development of plaque burden13,14 or calcification.41 Age, per se, does not appear to play a direct role in the pathogenesis of atherosclerosis or the onset of ACD.
Poor prognosis. Why do women have a poor prognosis following an acute coronary event compared to men? Why do they respond differently to acute coronary interventions and bypass surgery?3 What mechanisms can be considered? Multiple acute lesions,16,17,42–44 with or without luminal thrombosis, frequently unrecognized on angiography, are one factor to be considered. These unrecognized lesions are consistently associated with active disease.16,17 These acute lesions often exist as chronic, open lesions and represent unstable lesions that have the potential to progress rapidly to new or recurrent events, adversely affecting prognosis. The importance of angiographically insignificant lesions that subsequently progress rapidly to produce acute events is now well-recognized.45 These seemingly innocent lesions may actually be unrecognized acute lesions, such as chronic ulcerated plaques16,17 (Figure 2).
Study limitations. This study is limited by a lack of premenopausal women who died of ACD. We used the 4 women who died in the fifth and sixth decades, at an average age of 51 years, as an indication of what we could reasonably expect to find in younger women. The absence of age and/or sex-matched controls is a further limitation. This study does not address the possible role of risk factors and other clinical and laboratory factors in the growth and expansion of atheromas because of incomplete data on all of the patients, particularly those from the coroner’s office.
Conclusion
In summary, the gender gap observed clinically in patients with ACD may be explained, in part, by the gender differences in the pathologic substrate. Premenopausal women appear to be resistant to the development of active, inflammatory atherosclerosis and to other factors responsible for atheroma growth. As a result, plaque rupture and ACD are delayed until later in life, producing a gender gap. Postmenopausal women have more extensive, active, inflammatory disease of the coronary tree than men, which may restrict their ability to respond to acute events and interventions, resulting in a poor prognosis.
_______________________
From the Heart Research Foundation of Sacramento, Sacramento, California.
The author reports no conflicts of interest regarding the content herein.
Manuscript submitted November 20, 2008, provisional acceptance given January 5, 2009, final version accepted February 13, 2009.
Address for correspondence: Richard J. Frink, BS, MS, MD, Heart Research Foundation of Sacramento, 1007 39th Street, Sacramento, CA 95816-5502. E-mail: rjfrink@hrfsac.org
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