The Probable Role of Chlamydia pneumoniae Infection in Acute Stroke

Abstract

Cardiovascular diseases are the most leading cause of worldwide mortality. According to USA statistics, about 1 of 6 cardiovascular deaths is due to stroke. Stroke is the second most common cause of death and a chief cause of disability due to EU data. Treatment, care providing, rehabilitation costs and with the labor loss, the overall cost in EU due to stroke was estimated about €45 billion in year 2017. Acute stroke due to infectious diseases via several possible mechanisms with various clinical presentations were previously reported in the literature. Chlamydia pneumoniae is an obligate intracellular bacteria and extremely common in adult individuals. Besides it being a major cause of pneumonia in adults, association between atherosclerosis and vascular diseases was demonstrated by several sero-epidemiological studies and by direct detection of organism in atherosclerotic lesions by electron microscopy, immunohistochemistry, polymerase chain reaction. Also, several sero-epidemiological studies have demonstrated a link between Chlamydia pneumoniae infection and acute stroke. In this chapter, we will summarize the data in literature regarding the association between Chlamydia pneumoniae infection and acute stroke and we will try to explain the possible mechanisms that could be responsible in pathophysiology of stroke in these patients.

1. Introduction

Several previous studies have demonstrated a possible association between the infection by Chlamydia pneumoniae and atherosclerosis and stroke. The demonstration of the presence of the pathogen in the atherosclerotic parts of the infarct-related arteries and the increased Chlamydia pneumonia antibody titers in stroke patients raises suspicions about the role of Chlamydia pneumonia infections in development of stroke. Besides these, several animal and in vitro studies support the hypothesis that Chlamydia pneumonia has role in atherosclerosis development, and its complications resulting in the stroke. In this chapter, we will summarize the epidemiology, pathophysiology, and its associations with the infections and the possible pathophysiological mechanisms of the Chlamydia pneumonia in the development of atherosclerosis and atherosclerosis-related complications resulting in the cerebral infarcts.

2. Acute stroke

2.1 Definition and epidemiology

Abrupt disturbance of cerebral circulation due to thrombosis or embolism which results in focal neurologic deficit that persist for at least 24 hours is termed as stroke. Cerebral ischemia due to arterial occlusion results in an irreversibly damaged core area with a surrounding reversibly dysfunctional area, which is called penumbra. Cerebral ischemia owing to occluded arterial supply produces focal symptoms and signs that correlate with the influenced cerebral territory. Diagnosis is made by history and detailed neurological examination and confirmed by imaging studies (computerized tomography and magnetic resonance imaging). Further investigations are carried out in order to determine a specific cause responsible for the cerebral ischemia [1]. Yearly more than 13.7 million people have stroke, and 5.8 million people die as a result of stroke in the world [2]. Ischemic stroke comprises about 70% of stroke cases, and this proportion is estimated at about 85–87% in USA [3]. Stroke is the second most common cause of death and adult disability and it affects about 1.1 million people every year in European Union (EU) countries. According to EU cardiovascular disease statistics, treatment and care costs and costs due to labor loss because of stroke were estimated about €45 billion in year 2017 [4, 5].

2.2 Pathology, etiology, and pathophysiology

Oxygen and glucose insufficiency due to ischemia result in depletion of energy stores of neural cells to maintain membrane potentials and transmembrane ion gradients. Leak of potassium and increased calcium entry due to depolarization initiate series of events resulting in activation of calcium-dependent enzymes. This enhanced enzymatic activity of catabolic enzymes and their metabolic products with the oxygen-free radicals result in cell death. Prolonged ischemia leads to irreversible injury results with infarctions and persistent neurologic sequella [1, 6]. Pathological examinations defines two types of cerebral infarctions: First, the infarctions in a major cerebral artery distribution; both the gray and white matter are influenced and acute ischemic changes in neurons, destruction of glial cells, disruption of axons and myelins, and interstitial edema are visible changes in pathology. The second is the lacunar infarctions that are usually seen in chronic hypertensive people; small infarction cavities in size from 0.5 to 1.5 diameter due to occlusion of small cerebral arterioles are the characteristic of pathological finding [1].

The thrombosis comprises about two thirds of ischemic stroke cases. Thrombotic strokes are produced by occlusion of large cerebral arteries. Embolic strokes are produced from thrombi from heart, aortic arch, and large cerebral arteries. The distinction between them is very difficult and the establishment of the source necessitates detailed clinical examination [1].

Vascular disorders as a result of atherosclerosis of the large extracranial arteries in the neck and at the base of the brain are responsible for the cerebral ischemia in the great majority of cases. Atherosclerosis affects large and medium-sized elastic and muscular arteries of cerebral circulation. This complex chronic inflammation initiates in endothelial injury, and followed by the migration of monocytes with T-lymphocytes, transforming of these monocytes into lipid-laden macrophages and in continuation with the vascular smooth muscle cell proliferation; finally, the lesion proceeds into atheroma. The process of atherosclerosis is prevalent in patients with hypertension, diabetes mellitus, hypercholesterolemia/ hypertriglyceridemia, homocystinuria, and cigarette smokers. The enlargement and/or ulcerations of the atheroma would result in cerebral circulation disturbances. The severity of the circulatory problem and the localization of the threatened cerebral territory present with various clinical/neurologic scenarios. The detailed explanation of the arise and evolution of the atherosclerotic lesions and the probable role of Chlamydia pneumonia (CP) will be discussed in detail in this chapter [1, 7].

Inflammatory disorders other than atherosclerosis with arterial involvement can also result in cerebral ischemic syndromes: Giant cell arteritis, systemic lupus erythematous, polyarteritis nodosa, granulomatous angiitis, syphilitic arteritis are some examples of these diseases with central nervous system involvement due to vasculitis of arteries/arterioles. Fibromuscular dysplasia, carotid or vertebral artery dissection, lacunar infarctions in hypertensive individuals, vasculitis and vasopasms in drug abusers (cocaine, amphetamines, etc.), and multiple progressive intracranial arterial occlusions (Moyamoya disease) are other vascular causes of cerebral strokes [8, 9, 10].

Cardiac diseases comprises about one quarter of ischemic strokes [11]. Mural thrombus formation especially after first weeks of myocardial infarction is a well-documented source of cardiac embolism. Atrial fibrillation is a very common and preventable cause of cardiac embolism especially in patients with risk factors for hypercoagulable state. Infective and marantic endocarditis, atrial myxoma, and paradoxical embolus from venous system through a patent foramen ovale (PFO) are other rare causes of cardiac embolism [12].

Chronic myeloproliferative disorders such as thrombocytosis and polycythemia vera, sickle cell anemia, hypercoagulable states due to paraproteinemias, hereditary thrombophilia syndromes are the mostly recognized hematological disorders that cause cerebral strokes chiefly due to increased blood viscosity and thrombophilia [1].

2.3 Acute stroke and infectious diseases

Various infections may cause cerebrovascular complications, mainly due to involvement of the CNS vasculature by the pathogen itself usually with an inflammatory reaction of the immune system. The activation of pro-thrombotic mechanisms as a result of infection, and cardiac embolization in case of an infective endocarditis are other pathogenic mechanisms in the development of stroke [13]. Cerebral infarctions can be seen in viral infections such as HIV and VZV. The underlying mechanism in HIV infection was thought to be direct vasculopathy and hypercoagulable state [14, 15]. Syphilitic arteritis and inflammatory vasculopathy in Lyme’s disease, which is caused by Borrelia burgdorferi, are good examples for spirochetal infections that cause cerebral infarctions [16, 17]. Hemophilus influenzae, Neisseria meningitidis, and Streptococcus pneumoniae—the pathogens causing pyogenic meningitis may lead to cerebral infarctions as a result of disturbance of larger arteries at the skull base by a purulent exudate, and arterial spasm in response to inflammation [18]. Infective endocarditis is a well-documented cause of strokes due to occlusion of intracranial arteries by an embolic material derived from vegetations [19].

3. Chlamydia pneumoniae (CP) and atherosclerosis

3.1 Is CP just a respiratory tract infection agent?

Chlamydia species are gram-negative obligate intracellular bacteria [20]. They can cause infections in both humans and animals. The Chlamydia pneumonia (CP) and the Chlamydia trachomatis (CT) are the most common species responsible for the human infections. Particularly, the CT has a serious socioeconomical burden due to its infections are sexually transmitted [21]. The CT is the most important cause of nongonococcic urethritis in males and also, it is an important cause of cervicitis in females [22]. The control of the CT infection is challenging because the half of the infected patients are asymptomatic [23].

CP commonly causes respiratory disease such as pneumonia, bronchitis, sinusitis, and pharyngitis. According to literature data, it is estimated about the CP is the cause of 10% of community acquired pneumonia. Most of the population were infected during childhood period, and the vast majority of the adult people have serologic evidences of past infection [24]. Biological evidences of CP in atheromatous plaques, and even in influenced areas of Alzheimer’s disease patients brain, cogitate about that agent could not be a just respiratory tract infection agent [25, 26].

The infectious form of CP, the elementary body (EB) which is metabolically inactive, enters the body with inhalation and attaches to mucosal surface. After receptor mediated endocytosis enters into the cells and differentiates into metabolic active form, which is called the reticulate body (RB). The RB is the replicating form of CP and it is capable of modifying host cell pathways. The final EBs are released by cell lysis after 48 to 72 hours to infect other cells. The CP can infect respiratory epithelium, vascular endothelial cells, smooth muscle cells, monocytes, and macrophages. The CP can remain persistent in the host cell by escaping cellular lysosomal pathways. Resistance of CP to the host defense mechanisms determines the chronic infectious state [27, 28]. Intracellular survival of CP in cells of pulmonary alveolar membrane may be a reservoir for the persistent infectious state [29]. CP persistence is described as long-term survival of these metabolic inactive forms in the host cells. These patients are culture negative and the infection is antibiotic resistant due to reduced ribosomal activities [30].

CP can able to spread systemically via bacteremia during severe pulmonary infection and could be carried from recirculating monocytes/macrophages derived from alveolar membrane [31].

3.2 The Chlamydia pneumoniae (CP); virulence factors, the host cells of CP, and cytokine responses and target cell activation mechanisms triggered by the CP infection

3.2.1 Virulence factors of CP

1. CP interacts with the cellular secretory pathway via putative receptor-mediated specific signaling and enters into cell by endocytosis. However, the exact structures of the chlamydial surface (proteins, glycolipids) which initiate and mediate contact to target cells still are not well known [32].

2. CP creates itself an adjusted intracellular environment by releasing specific proteins into cellular cytoplasm. The type III secretion apparatus is a specific CP protein, which translocates some proteins that modulate cellular response [33]. The reactive oxygen species production in CP-infected macrophages are limited by upregulating of antioxidant enzyme systems such as superoxide dismutase (SOD) and γ-glutamylcysteine synthase (γ-GCS) thereby limits the bacteria killing ability of macrophages [34].

3. CP inhibits host cell apoptosis via signaling specific cascades in monocytes; pro-apoptotic proteins that stimulate apoptosis can be degraded by CP and release of apoptotic trigger cytochrome-c can be inhibited by CP and the caspase-3 activity is reduced [33, 35, 36]

All of these virulence factors of CP create an appropriate environment for replication and survival [32].

3.2.2 Host for CP is not only the respiratory epithelial cells

CP can invade various cell types other than respiratory epithelium (vascular endothelial cells, smooth muscle cells, monocytes, granulocytes, even neural glial cells) and lead to enhanced expression of numerous chemokines [37].

Infection of all these cells leads to an enhanced cytokine expression, which results in a series of events resulting with proinflammatory and proproliferating environment. The ICAM-1 (intracellular adhesion molecule), VCAM-1 (vascular cell adhesion molecule), IL-6, IL-8 cytokine and protein expressions from the endothelial cells, the IL-6, MCP-1 (monocyte chemoattractant protein) from the vascular smooth muscle cells, IL-1ß, IL-6, IL-8, MCP-1, TNFα (tumor necrosis factor α), and MMPs (matrix metalloproteinases) from monocytes are some examples for the increase expression of cytokines and proteins in CP infection. Aggravation of inflammation and vascular cell proliferation induction by these cytokines may have an effect on atherosclerosis development [32, 38, 39, 40, 41, 42].

3.2.3 Target cell stimulation by CP and activation of signal transduction

Several receptor systems and signaling pathways are thought to be involved in activation of host cells by CP infection. Stimulation by IL-8, ICAM-1, causes phosphorylation of various kinases, which in turn causes a proinflammatory phenotype in vascular cells [43]. The transcription factor NF-κB that mediates the pro-inflammatory cascades in cells can be activated by CP [44].

Two systems are known to be involved to activation of target cells by CP: Toll-like receptors are the extracellular receptors and Nod proteins are the intracellular receptors.

Toll-like receptor (TLR) 2 and 4 were found to be the essential mediators of CP host cell activation. TLRs are receptors of the immune system, which are responsible for recognition of pathogens. Secretion of cytokines and translocation of NF-κB in dendritic cells are dependent on TLR stimulation. Nod protein system is the intracellular part of the immune system that could be responsible for the cytokine production in chronic infections. Through several kinases finally the NF-κB is activated and immune response is mediated. Various protein kinase systems are activated after contact of CP with the endothelial cells. Mitogen-activated protein kinase family (MAPK) members that are the key elements of proinflammatory, prothrombotic and pro-proliferative responses are the most activated kinases after CP contact. The final activation of NF-κB is followed by expression of pro-inflammatory mediators: ICAM, VCAM, IL-8, MCP-1, RANTES [30].

These all responses coincide shortly after acute contact by CP. The primary infection of monocytes and the vascular smooth muscle cells resemble persistent infection rather than active infection. The less is known about the changes in the persistent CP infection in which the pro-atherosclerotic signaling cascades supervene [45, 46].

3.3 Atherosclerosis and Chlamydia pneumoniae

3.3.1 Endothelial dysfunction to atherosclerosis

The atherosclerosis is characterized by deposition of lipids in the artery wall and infiltration of immune cells such as macrophages, T-cell, and mast cells with a surrounding fibrous cap consisting of mainly collagen, which is formed by the vascular smooth muscle cells.

Rudolph Virchow was the first scientist who recognized the inflammatory nature of the atherosclerotic lesions in history; however, his concept of atherosclerosis consisting of the inflammatory process was complicated to comprehend at that century. Eventually, the atherosclerosis was remained to be a concept of a just an arterial cholesterol and thrombotic debris deposition disease in the last century. Thereafter with the discovery of the smooth muscle cell proliferation with the inflammatory cells in the atherosclerotic plaque in 1960s and 1970s, the inflammation was begun to be considered to be a cause of the atherogenesis [47].

Earliest change of atherosclerosis is the dysfunction of the endothelial lining of the lesion-prone areas in the vascular system. The focal permeation, entrapment, and the modification of the circulating lipoprotein particles in the subendothelial space trigger a series of immune reaction including recruitment of circulating monocytes from the circulation. The monocytes differentiate into macrophages and they become foam cells as they resume to phagocyte the modified lipoproteins. These foam cells are the hallmark of early fatty streak lesions. Initiating event in the atherogenic process was assumed to be an injury to the endothelial lining by noxious substance such as oxidized LDL, cigarette smoking contents, hyperhomocysteinemia, altered hemodynamic forces generated in hypertension. However, this type of endothelial injury was failed to be demonstrated in the animal models of natural atherosclerosis. The uncertainty of the evidences of the direct endothelial cell injury in animal studies of natural atherosclerosis and the recent findings that demonstrate the functions and phenotypic modulation of the endothelial cells raised the term endothelial dysfunction in the development of atherosclerosis [48].

The term “proinflammatory endothelial phenotype” confers to enhanced expression of various effector proteins and cytokines that are responsible for acute and chronic inflammatory responses and disease processes in endothelial cells. In the lesion-prone regions of the arterial vascular tree as a result of endothelial cell activation by the actions of pro-inflammatory cytokines and noxious stimuli, genetic regulation modifications supervene in the endothelial cells primarily driven by the transcription factor NfκB. These include enhanced expression of adhesion molecules such as VCAM-1, ICAM, increased secretion of chemokines, and prothrombotic mediators. The circulating monocytes and T-cells respond to these signals and they migrate into subendothelial space. As a result, the paracrine milieu of cytokines, growth hormones, and reactive oxygen species, which were created by the actions of all activated endothelial cells, smooth muscle cells, monocytes and T-cells all together, eventuate as a vicious cycle of chronic inflammation. This chronic inflammation establishes the pathophysiological basis of the atherosclerosis [48, 49].

Lesion-prone areas for atherosclerosis are the sites which have and disturbed laminar flow patterns. The sites with low oscillatory endothelial shear stress located near branch point of arteries are most susceptible. The abdominal aorta, coronary arteries, iliofemoral arteries, and the carotid bifurcations are the most affected sites. These predilection sites are characterized by the presence of subendothelial macrophages. Modifications of gene expression in endothelial cells are present in these sites [50].

The most of the lesion-prone areas are the bifurcation sites and the other regions with altered hemodynamics. The absence of an disrupted endothelial lining at these branch points in the detailed morphological studies undermines the arterial injury hypothesis to explain this phenomenon. Similar to the changes in lesion-prone areas in vivo, the enhanced endothelial cell turnover, oxidative stress, and the alterations in endothelial cell shape, and changes in cytoskeletal and junctional proteins were demonstrated in vitro studies. These findings suggest the hemodynamic forces might have an effect on endothelial cell dysfunction in atherogenesis [48, 51]. The association between hemodynamic forces and the various genes that are important in development of atherogenesis such as hemostasis, thrombosis, growth regulation, and proinflammatory activation was demonstrated in previous studies [52, 53]. These results suggest a presence of a system of biomechanical endothelial gene regulation [48].

3.3.2 Progression of atherosclerosis

American Heart Association (AHA) defined six lesion types according to atherosclerosis progression.

1. Type I lesion: initial lesions. Intimal thickening and fatty streak lesions are frequent in infants and children. The earliest vascular change is intimal thickening consisting of layers of smooth muscle cells and extracellular matrix with small isolated groups of macrophage foam cells.

2. Type II lesions: include fatty streaks, which are visible as yellow-colored streaks on the intimal surface of arteries. Macrophage foam cells are abundant scattered in smooth muscle cells and proteoglycan-rich intima. T cells are identified in these lesions but they are less numerous than macrophages. Foam cells, easily recognizable by light microscopy, are signs of lipoprotein-driven inflammation occurring in the vascular wall. Xanthomas are harmless and reversible in case of disappearance of the factor that caused their formation. Probably due to maternal risk factors, they are visible in some fetal aortas and infants in the first 6 months of life, but their number decreases in following years. They reappear in lesion-prone areas in adolescence period.

3. Type III lesions: Pathologic intimal thickening. The earliest progressive lesions are primarily composed of layers of smooth muscle cells in a proteoglycan-collagen matrix with an underlying acellular lipid pool rich in hyaluronan and proteoglycans. There is a variable accumulation of macrophages outside the lipid pool. These lesions are found in young adults.

4. Type IV lesions: Atheroma. The lipid core is evident with foam cells.

5. Type V lesions: Fibroatheroma. The lipid core is covered by a fibrous capsule. Necrotic core is present that is made up of cellular debris and this core is covered by a thick fibrous cap consisting of smooth muscle cells in a proteoglycan and collagen matrix. The fibrous cap is critical for the maintenance of the lesion.

6. Type VI lesions: Complicated lesions; intraplaque hemorrhage, fissures, erosions, or thrombosis.

Update to these lesions: type VII lesions, if calcification predominates; type VIII lesions if fibrosis predominates. The type IV lesions (atheroma) can evolve to any of the further stages. The progression does not need to be in a sequential manner [54]. The fate of plaque is determined by the following mechanisms: lipid retention rate, macrophage phenotype, inflammation, apoptosis and necrosis, smooth muscle cell proliferation, arterial remodeling, and stability of fibrous cap. Most of the plaques remain asymptomatic, and some become obstructive, while some of them due to complications of the plaque may elicit acute thrombosis, which present as acute coronary syndromes and stroke [55].

The higher LDL levels induce more progressive disease due to increased amount of lipid retention in the plaque. The modified and oxidized LDL exerts chronic stimulation of the immune system [56].

The phenotype of the recruited macrophages is important for the plaque progression. Macrophages with the M1-like phenotype, possibly via binding of modified LDL to the Toll-like receptors, secrete proinflammatory cytokines such as interleukin-1β and tumor necrosis factor-α and enzymes and reactive oxygen products, which promote further modification of LDLs. This type of proinflammatory phenotype also secretes the mediators that were demonstrated to have a role in atherosclerosis. In contrast, the macrophages with M2 phenotype secrete factors such as transforming growth factor and proresolving lipids, which cease the severity of inflammation [55, 57].

Apoptosis and secondary necrosis of foam cells and smooth muscle cells and impaired removal of the apoptotic remnants cause the formation of necrotic core in the atheroma. The enlargement of the necrotic core induces further plaque inflammation [55, 58].

New vessels can develop in the atherosclerotic lesion mainly originating from adventitial vasa vasorum. They provide an alternative entry way for the immunocytes. Intraplaque hemorrhages from these fragile vessels promote inflammation and lead to expansion of the necrotic core [59].

Smooth muscle cells of the plaque are characterized by presence of abundant secretory organelles. The contractile smooth muscle cells of the tunica media can migrate to intima and phenotypic modifications supervene in these cells. These synthetic phenotypes of smooth muscle cells increase in number with the lesion progression. The collagen, elastin, and proteoglycans of the plaque matrix are produced by these cells. Collagen-rich tissue becomes a dominant component of the plaque as the plaque expands [55].

The involved arterial segment tends to remodel in a way that does not allow the compromisation of the luminal area until plaque volume enlarges. This type of expansive remodeling is seen in fibroatheromas, and the extent of the enlargement is correlated with the plaque inflammation and necrotic core. Continued plaque growth with the shrinkage of the local vessel segment results in the stenosis of the vessel segment. This type of contrictive remodeled arterial segments contains lesions rich in fibrous tissue [55, 60].

3.3.3 Acute clinical presentations of atherosclerosis: The vulnerable plaque

Acute coronary syndromes and the vast majority of strokes are cases caused by luminal thrombi due to plaque rupture or a sudden plaque hemorrhage with or without vasopasms [61, 62]. The atherosclerotic plaque rupture occurs from the site where the cap is thinnest and most infiltrated by the foam cells. Plaque rupture is the most frequent cause of luminal thrombosis [61].

Ruptured plaques contain fewer smooth muscles cells and less collagen when compared with the intact plaques. And these lesions are demonstrated to be heavily infiltrated with macrophages rich in proteolytic activity suggesting the enhanced degradation of extracellular matrix elements. These two concurrent mechanisms leading to loss of supporting elements of the plaque are thought to explain the plaque rupture [63, 64].

Another mechanism leading to the intraluminal thrombus formation is the plaque erosion. The endothelial coating in these lesions is absent; however unlike the ruptured plaque the internal and external elastic lamina and contractile smooth muscle cells are present. The vasospasm of the involved arterial segment was suggested to be as a cause of endothelial damage and resulting thrombosis [65].

These two mechanisms whether the plaque rupture or the plaque erosion result in intraluminal thrombosis are comprised in a concept of a dynamic-active plaque that results in an acute clinical presentation: the vulnerable plaque. This term is used for the plaques to describe a group of histological features that are associated with plaque rupture and subsequent intraluminal thrombosis. The typical rupture-prone vulnerable plaque is the plaque with a thin fibrous cap containing a large necrotic core and infiltrated with abundant macrophages in the cap. Other features of this plaque include neovascularization, plaque hemorrhage, and adventitial inflammation [55, 66].

3.3.4 Infections and atherosclerosis

The traditional risk factors merely are not adequate to explain the development of atherosclerosis in all patients. Additional risk factors that predispose to atherosclerosis development are yet undetected. The triggers of the arterial injury, which result as an atherosclerotic plaque, have not been clearly identified. The oxidized LDL and heat shock proteins are identified factors, which elicit an inflammatory response through a complex autoimmune response [67, 68, 69].

The demonstration of the infectious agents in the atherosclerotic lesions by polymerase chain reaction (PCR) and immunohistochemistry (IHC), and the seroepidemiological studies suggests a presence of an inflammatory trigger pathway initiating with an infection, which results in atherosclerosis and even possibly with its complications [21, 70, 71, 72].

Infectious agents can promote atherosclerosis through direct effect of the agents on cellular components of the vessel. The Chlamydia pneumonia and cytomegalovirus infections are the mostly concerned infectious agents demonstrated to have these effects. These mechanisms include smooth muscle cell proliferation and inhibition of their apoptosis, enhancement of smooth muscle cell migration, enhanced foamy cell formation, and increased expression of cytokines and cellular adhesion molecules, which lead to endothelial cell dysfunction. Autoimmune reaction through molecular mimicry of the heat shock proteins could be other possible mechanism [69].

3.3.5 Chlamydia pneumoniae in the atherosclerotic plaque

Many of previous studies including immunohistochemistry, polymerase chain reaction, and cultures demonstrated the presence of CP in various stages of human atheromatous plaques [21, 73, 74]. The existence of these bacteria in the atherosclerotic plaque can be explained by either direct infection of the vessel and/or transportation via circulating infected monocytes [69].

Although the exact mechanism with the atherosclerosis development is unclear, previous in vitro and animal studies have demonstrated the atherosclerosis relevant alterations triggered by the CP infections; the upregulation of the atherosclerosis-related gene expression products in cultured cells such as the heat shock proteins 60 (HSP60), macrophage scavenger receptor, cytochrome p450, and VEGF165R; smooth muscle cell proliferation enhancement (mediated through platelet-derived growth factor); increased macrophage foam cell formation are some demonstrated examples of these modifications [71, 75, 76, 77].

3.3.6 The vulnerable atherosclerotic plaque, plaque complications, and Chlamydia pneumoniae

As we mentioned previously, the plaque vulnerability depends on plaque volume, both the collagen content and the macrophage population of the fibrous cap, and the cap fatigue caused by mechanical stresses on the plaque (flexion, shear, pressure alterations, etc.). The role of CP in atherosclerosis development through macrophages was demonstrated in previous studies. Cytokines produced by CP-infected macrophages (such as TNF-α, IL-1β, and IL-6) may aggravate inflammatory response resulting with fibrous cap degradation and increase necrotic core volume and thereby increase the plaque size. The activated T lymphocytes by the infected vascular cells (endothelial cells, smooth muscle cells, and macrophages) can aggravate inflammatory activation via the NF-κB pathway, resulting in enhanced VEGF-1 (vascular cell adhesion molecule−1) and the additional inflammatory cell recruitment. Also, these cytokines can promote thrombin generation resulting in a thrombophilic intravascular milieu [78]. All these alterations might be responsible mechanisms for the plaque vulnerability and resulting in acute events. The data are scarce regarding the association of the CP infection and the plaque vulnerability and acute events. In one study, the intimal presence of Chlamydial heat shock proteins (HSP) was demonstrated to be associated with major adverse cardiac events in 6 months following coronary intervention. Also in this study, the C reactive protein (CRP) levels as an indirect measure of the inflammation severity was demonstrated to be correlated with the major cardiac adverse events [79].

Serological studies in patients with acute coronary events demonstrated a possible association with CP infections and acute vascular events. However, this serological finding could be an indirect association and does not establish an exact causality. Moreover, this serological response may also be just a part of a nonspecific humoral response to inflammation. Further studies with using direct detection tools in the vulnerable lesions—if possible—might provide further information [80, 81, 82].

Protective effects of statins on acute cardiac events, stroke, and mortality were demonstrated in previous studies. Beyond cholesterol lowering, their effect on the modulation of the immune response was revealed by clinical studies. The reduction of the CRP (C-reactive protein) levels with statin treatment indicates their immunomodulatory actions. Either the future cardiovascular events or CRP levels—in other words the inflammation—decrease with the statin treatment. Moreover in a recent study, the reduction in major adverse cardiac events with rosuvastatin treatment and especially its efficacy in patients with increased high sensitive CRP (hs-CRP) levels were demonstrated. This study demonstrated that with statin treatment, the lower primary endpoint rate was achieved (acute coronary syndrome, stroke, confirmed cardiac death, etc.) in patients with high hs-CRP despite their low cholesterol levels. These all findings shifted the idea of preventing future vulnerable plaque-mediated events by reducing the cholesterol with statins, to the idea of prevention of these events by reducing the inflammation with statin treatment [83, 84, 85, 86].

Statins inhibit cholesterol production by inhibition of mevalonate pathway. This inhibition also decreases the production of other downstream metabolites such as isoprenoids. Prenylation of certain proteins by certain isoprenoid compounds is essential for their function, such as post-translational modification of membrane GTPases—the members of Ras, Rho, and Rab families. These GTPases are important in various cell signaling pathways, which regulate cell growth, proliferation, and inflammation. Disturbance of these prenylation pathways by statins exert the immune modulatory effect especially through the function of macrophages. Inflammatory responses of M1 macrophages are diminished by modulation of TLR (Toll-like receptors) signaling pathways via inhibition of NF-кB, and modulation of the IFN-γ receptor signaling system. Inhibition of M1 response results in decreased level of cytokine expression such as IL-1ß, IL-6, IL-12, TNFα [87, 88, 89].

In vitro studies demonstrated the modification of immune response by statins in CP-infected human macrophages and endothelial cells. The increased NF-кB expression in CP-infected cells was demonstrated to be inhibited with statins. Also, it was demonstrated that in the vascular smooth muscle cells infected with CP, the reactive oxygen species production, the activity of RhoA and Rac1, and the expression of NF-кB, MCP-1, and RANTES were reduced with cerivastatin [90, 91]. The interruption of the CP-activated signal transduction cascade by statins inhibits the inflammation response in the infected vascular cells. Thereby, inhibition of the inflammation by statins in the possibly CP infected atherosclerotic plaque results in plaque stability. Despite the absence of an direct evidence, these all findings could support the hypothesis that CP infection may have a role in atherosclerosis development and plaque vulnerability via aggravating the immune responses in the plaque.

4. Chlamydia pneumoniae and stroke

Despite the presence of serologic and biologic evidences, and the information from in vitro and animal studies which demonstrated the possible mechanism of the CP role in atherosclerosis development, definite human atherosclerosis evidences are not present yet. Solely, from the human atherosclerosis studies we have the information of serological traces and the PCR and IHC evidences of CP. Despite their biological evidences in human atherosclerotic plaques, the definitive role in the human atherosclerotic disease is not clear. We derive the information in which the possible role of CP in atherosclerosis development from animal and in vitro studies. Moreover, the in-human trials with the antibiotic treatment were failed to demonstrate any atherosclerosis protective result [92, 93].

Serologic traces and PCR/IHC evidences of CP are demonstrated in stroke patients. Many studies demonstrated the increased IgG and IgA antibody titers in stroke patients. In one meta-analysis of these studies demonstrated, CP infection was significantly associated with increased risk of cerebral infarctions and the immunoglobulin A was more effective to predict the risk of stroke. However, the value of these serological studies is limited due to uncertainty in the titers of CP antibodies to diagnose acute, chronic, and chronic active infection. There are also discrepancies between the serological tests and the PCR tests, and also the inconsistency among the PCR studies [94, 95, 96, 97].

The detection of circulating CP DNA from peripheral blood monocytes is more reliable tool to diagnose an active infection; however, this method should be performed cautiously to prevent false-positive and false-negative results. One study demonstrated the higher prevalence of CP DNA in peripheral blood monocytes in symptomatic patients with carotid artery disease. Despite controversy between similar studies, one meta-analysis demonstrated an increased risk for cerebrovascular disease. These finding of evidences of an active infection might lead a speculation of the CP inflammation-associated atherosclerotic plaque vulnerability and resulting stroke [98, 99, 100].

Controversy exits between the PCR studies from atheromatous plaques in patients with carotid artery disease. There can be problems with sensitivity and specify due to PCR testing resulting with discrepant results [101, 102].

Previous studies mainly focused on the large artery atherosclerosis etiology comprising for the stroke. In all these studies, the exact etiological evaluation is not clearly defined. Cardioembolic stroke especially due to atrial fibrillation could also be a possible cause of the stroke in these patients. Animal studies demonstrated the involvement of cardiac muscle involvement by the CP infection. Either by direct atrial tissue involvement and/or via the increased cytokine levels atrial fibrillation could be triggered by the CP infection. The thrombophilic milieu propensity via the increased cytokine levels due to infection could be another potential contributory factor for the development of stroke. Moreover, most of the patients with atrial fibrillation have also coincident widespread atherosclerosis development, which could mean the possible high burden of chronic CP infection. Further well-designed studies with a clear definition of stroke etiology might answer this question [103, 104].

There are two problems arising with the studies, which investigate the possible role of CP infection in stroke patients: First, there is not any clear diagnostic criteria in the serological evaluation of a CP infection and there are controversies between PCR tests and serology; second problem is the etiologic diagnosis of the stroke; the etiology is a vulnerable plaque complication result or a cardioembolism, should be clearly defined [102, 104].

The studies that investigate the CP role in stroke that clearly defined the clinical presentation and etiology (symptomatic carotid artery stenosis and/or stroke) have conflicting results. The PCR studies could not demonstrate the CP presence in the carotid plaques of symptomatic patients. However in a study that used the IHC method, it is demonstrated that the CP in carotid plaques is significantly associated with the cerebrovascular events [102, 105, 106].

Another interesting common finding of these studies is that of similar to finding by Elkind et al., the high serum anti chlamydial Ig-A presence in symptomatic patients. This finding could indicate the possible role of the acute and/or chronic infections of CP anywhere in the body could play a role in atherosclerotic plaque activation and plaque vulnerability [98, 102, 106].

However, it is unclear whether the possible pathophysiologic chain of the events ongoing with increased cytokine levels result in the plaque vulnerability and stroke is unique to CP infection. The epidemiological studies demonstrated the increased risk of stroke after certain infections. The pathophysiological scenario ongoing with increased cytokine levels which ends with the plaque vulnerability related events may not unique to infection by CP. Increased risk of stroke was also demonstrated after certain upper respiratory tract infections. Also, the etiology could also be an atrial fibrillation-related cardioembolism, which also shares similar pathophysiological basis due to increased cytokines. However, there is necessity for large-scale epidemiological studies in which the etiology of stroke is well defined [107, 108, 109].

The levels of proinflammatory cytokines, and the inflammatory markers such as the CRP and ESR (erythrocyte sedimentation rate) are found to be increased in symptomatic carotid stenosis patients. The cerebral ischemia/infarct, and/or the inflammation of the plaque itself could be either an explanation for the increased cytokine levels and inflammatory markers. Also, the possible aggravating etiologies which triggered this chain of events might have a role in this finding [110, 111].

5. Conclusions

The CP infection may be the relevant cause of cerebral strokes as an agent that causes and/or exacerbates atherosclerosis, or it could be a just one of the complicating agents of atherosclerosis through mechanisms of exacerbating the atherosclerotic plaque inflammation.

Further large-scale and prospective studies could find answers to this puzzle, on the condition that the exact diagnosis of recent or ongoing CP diagnosis could be established.