**4.** *Chlamydia pneumoniae* **and atherosclerosis**

The first findings regarding the association of *C. pneumoniae* and coronary artery disease were presented by Shor et al. [84] in 1992. Their study examined coronary artery atherosclerosis and vascular fatty streaks in seven autopsy studies and demonstrated the presence of TWAR-like *C. pneumoniae*. In a later, extended autopsy study, Kuo et al. [21] demonstrated the presence of *C. pneumoniae* in atherosclerotic lesions by PCR and culture studies.

In a 1998 study by Saiku et al. including 40 patients with acute myocardial infarction, 30 patients with coronary artery disease and 41 controls, chlamydial IgA and IgG antibodies were detected in 68% and 50% by micro immunofluorescence, respectively. Both frequencies were significantly higher than in the controls (7.17%). In 68% of patients with acute myocardial infarction, a significant seroconversion was demonstrated in enzyme immunoassay with LPS antigen; this response was absent in patients with coronary heart disease and in all but one of the controls [85].

The presence of *C. pneumoniae* organisms in atherosclerotic lesions was later demonstrated using immunohistochemistry, PCR, in situ DNA hybridizations, and electron microscopy [21, 86–88].

*Chlamydia* has been detected not only in plaques in the coronary arteries, but also in the aortic tissue, aortic aneurysms, and plaques in the carotid and peripheral arteries [22, 24, 89–92].

#### **4.1** *Chlamydia pneumoniae* **and the immune response**

In studies demonstrating the relationship between *C. pneumoniae* and atherosclerosis, circulating cholesterol-containing immune complexes were shown to be present in 50–70% of patients with acute myocardial infarction [93, 94]. These immune complexes (comprising IgG and apolipoprotein (a)) have a proatherogenic effect [95]. Patients with immunocomplexes containing *C. pneumoniae*-specific IgG and apolipoprotein (a) were found to have a 3.8 times higher risk of developing acute myocardial infarction than the control group [96].

Several mechanisms have been proposed to explain the formation of immunocomplexes containing *C. pneumoniae*-specific IgG and apolipoprotein (a). According to one mechanism, structurally similar elements in *C. pneumoniae* and apolipoprotein (a), which is found in lipoprotein (a), causes anti-*C. pneumoniae* antibodies to form an immune complex with apolipoprotein (a) [34, 35, 97].

Another mechanism involves the formation of antibodies against apolipoprotein (a) in association with HLA tissue groups, which is facilitated by *C. pneumoniae* infection. It was reported that HLA class II DR genotypes were more common in patients with high lipoprotein (a) levels and early coronary artery disease compared to the healthy control group. This finding indicates that an immune response to apolipoprotein (a) may occur in connection with the HLA system [95, 98].

Many of the properties of the single-cell-layer endothelium that forms the innermost layer of the arteries are mediated by NO. NO is synthesized from L-arginine by NO synthetase, an endothelial enzyme. NO is a potent inhibitor of platelet aggregation on endothelial cells and a potent vasodilator that acts by reducing vascular tone. In addition, it inhibits atherosclerosis at every stage through its anti-inflammatory properties, which it exerts by preventing the expression of genes that synthesize molecules that cause inflammation, such as ICAM-1, VCAM-1, MCP-1, and P selectin. Conditions known to predispose to atherosclerosis, such as hypertension, diabetes mellitus, smoking, or increased super oxide levels, have been associated with reduced endothelial production or increased destruction of NO [99]. Chlamydial infection in the endothelial layer results in the disruption of these endothelial properties. Stimulation of the release of endothelin 1, which is an especially powerful vasoconstrictor, causes endothelial cells to revert to their proliferative form, initiating the atherosclerosis process [100]. Chlamydial infection also stimulates the release of ELAM-1, VCAM-1, and ICAM-1 from the endothelium. Studies have shown that *C. pneumoniae* is associated with damage to the endothelium, which forms the intima layer of the arteries, in the early stages of the atherosclerosis process [84].

Host monocytes infected with *C. pneumoniae* begin secreting the adhesion molecules E-selectin, ICAM-1, and VCAM-1. These molecules allow adhesion of monocytes from the endothelium to the subendothelium. There the monocytes turn into macrophages and start to increase their cytokine production. Macrophages enlarged from the phagocytosis of oxidized LDL rupture, releasing the bacteria within them into the atherosclerotic plaque to infect neighboring cells [92].

In addition, *C. pneumoniae* proliferating within monocytes and macrophages stimulates the release of IL-6, TNF-ɑ, monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein 1-a (MIP1-a) [84].

#### *A Hidden Organism, Chlamydia in the Age of Atherosclerosis DOI: http://dx.doi.org/10.5772/intechopen.109745*

Another mechanism that may explain the relationship between *C. pneumoniae* infection and atherosclerosis is based on the similarities in structure between the heat shock protein (HSP-60) produced by nearly all bacteria and the HSP produced by humans. Antibodies against the HSP-60 in bacteria may cross-react with human HSP [40]. The immune response to *C. pneumoniae* and/or human HSP in the vascular wall is thought to activate atherosclerosis. Wong et al. showed that *C. pneumoniae* and human HSPs coexisted in atherosclerotic lesions, and incubating mouse macrophages with this HSP caused an increase in matrix-degrading metalloproteinases and TNF-α activity. *Chlamydia*-infected endothelial cells trigger smooth muscle cell proliferation by stimulating the synthesis of endogenous HSP-60 and PDGF [40].

In addition, *C. pneumoniae* HSP has been shown to increase the secretion of lectinlike oxidized LDL receptor 1 (LOX-1) in hypercholesterolemic rabbit endothelial cells. Increased LOX-1 disrupts the LDL regulation system in the host and induces oxidized-LDL-mediated atherosclerosis. LOX-1 forces macrophages to phagocytose oxidized LDL, thereby turning macrophages into foam cells through the phagocytosis of high amounts of oxidized LDL, and foam cells are known to be one of the main players in the atherosclerotic process [74].

*Chlamydia* has been shown to stimulate the toll-like receptor system in host tissue. Toll-like receptor stimulation is considered one of the factors that initiates and promotes atherosclerosis by triggering the cytokine and inflammatory cascade. Thus, smooth muscle cell migration into the media layer is stimulated and the macrophage/ foam cell diapedesis process that causes subintimal thickening progresses [101].

An experimental study by Justin et al. [102] showed that after contaminating porcine coronary arteries with *C. pneumonia* in culture medium, *Chlamydia* proliferating in the arterial wall quickly stimulated smooth muscle cell proliferation in the medial layer of the artery and caused atherosclerosis and significant narrowing in the lumen.

#### **4.2** *Chlamydia pneumoniae* **and lipid metabolism**

*C. pneumoniae* has been shown to adversely affect the regulation of lipid metabolism in host tissue. *C. pneumoniae* continues to live in immune cells after undergoing phagocytosis and thus can survive even in chronic inflammation environments [103].

The unmodified, natural level of LDL is controlled by LDL receptors and does not normally lead to the formation of macrophage-derived foam cells. However, the oxidation of LDL due to chlamydial infection disrupts this balance. It has been shown that *C. pneumoniae*-infected macrophages incubated with LDL turn into foam cells within 22 hours [40]. This effect occurs mostly through the induction of LDL oxidation, phagocytosis of oxidated LDL, and induction of lipid accumulation within cells and in the atherosclerotic plaque. Liu et al. [104] reported that both active and inactive *Chlamydia* trigger lipid accumulation and induce foam cell formation.

A study conducted by Zhao et al. [105] showed that *C. pneumoniae* negatively affects lipid metabolism by decreasing ATP-binding cassette transporter A1 (ABCA1) level, which has an important role in cholesterol transport in macrophages. In a study conducted by Tumurkhuu et al. [106] on the same system, it was shown that *C. pneumoniae* infection affected the lipid reuptake system by stimulating extracellular IL-1β and caused intracellular cholesterol accumulation by reducing the synthesis of ABCA1 and G protein-coupled receptor 109A (GPR109a), which are involved in the niacin and ketone receptor system.

In a study on the effects of *Chlamydia* on lipid metabolism in humans, it was found that chronic inflammation associated with *C. pneumoniae* infection caused a significant increase in cardiovascular risk in individuals with familial hypercholesterolemia [104].

In experimental studies, rabbits intranasally infected with *C. pneumoniae* exhibited findings consistent with early atherosclerosis characterized by aortic inflammation when fed a high-fat diet but not in those fed a normal diet [107, 108]. The combination of hyperlipidemia and *C. pneumoniae* infection has been shown to significantly increase the development of atherosclerosis.

Blessing et al. [109, 110] demonstrated in their study that *C. pneumoniae* inoculation causes inflammation in the heart and aorta in normolipidemic C57BL/6J mice. In the same model, atherosclerosis was shown to accelerate and become widespread when the animals were fed a high-cholesterol diet.

Lantos et al. [111] showed that hyperlipidemic diet-induced atherosclerosis in ApoB100only/LDLR−/− mice accelerated threefold in the presence of *C. pneumoniae* infection.

Apolipoprotein E (apoE) is involved in chylomicron and very-low-density lipoprotein (VLDL) metabolism and has a key role in LDL and cholesterol metabolism. ApoE deficiency leads to dyslipidemia and increases susceptibility to atherosclerosis. In mice with apoE enzyme deficiency, even a single dose of *Chlamydia* inoculation significantly increased atherosclerosis compared to uninfected subjects [112].

New Zealand rabbits do not develop atherosclerosis unless they are fed a hyperlipidemic diet. However, when infected with *C. pneumoniae*, atherosclerosis was observed in these animals within 2 weeks despite being fed a normal diet [113]. These results suggest that *C. pneumoniae* also triggers atherosclerosis independently of lipid levels and acts as an independent factor in the development of atherosclerosis.

In their study on C57BL/6J mice fed a high-cholesterol diet, Zafiratos et al. [114] concluded that the coexistence of *Chlamydia* infection and hyperlipidemia significantly increased levels of TNF receptors 1 and 2 and caused inflammation when compared with hyperlipidemia or *Chlamydia* infection alone. Similarly, a study conducted on IL-17A-deficient mice showed that a hyperlipidemic diet did not result in a significant difference in inflammation or atherosclerosis compared to the control group. However, after infection with *C. pneumoniae*, the control group exhibited significant elevation in inflammation markers in the blood (IL-12p40 and IFN-γ) and increased macrophage accumulation in atherosclerotic plaques compared to the IL-17A-deficient group. This showed that IL-17A plays a role in the *Chlamydia*-induced atherosclerosis process in hyperlipidemic subjects [115].

These studies demonstrate the important role of *Chlamydia*, both independent of lipid physiology and as a cofactor of hyperlipidemia, in the different stages of initiating and advancing atherosclerosis.
