биохимия атеросклероза

422 Paul K.M. Cheung and Grant N. Pierce

coronary heart disease [115] and Cpn can be found in 52% of atheromatous lesions but in only 5% of control samples. Thus, the available observations suggest a statistically significant role for Cpn infection in atherosclerosis. To further support the importance of Cpn in atherosclerotic lesions, recent in vivo and in vitro studies have provided insights into the biochemical pathways by which the pathogen may directly stimulate plaque formation.

Findings from in vitro studies have confirmed the ability of Cpn to infect vascular endothelial cells, smooth muscle cells, and macrophages [116, 117]. Persistent infection of human endothelial cells have been reported, and the replication of Cpn in endothelial cells can induce upregulation of vascular and cellular adhesion molecules, including ICAM-1, VCAM-1, and ELAM-1, all of which signify endothelial cell injury [118, 119]. In addition, inflammatory markers such as IL-6, IL-8, MCP-1, NF-κB, and plasminogen- activator–inhibitor-1 are upregulated in Cpn-infected vascular endothelial and smooth muscle cells [120, 121]. The presence of Cpn lipopolysaccharide (LPS) can be a strong stimulator of inflammatory responses in vascular endothelial cells via toll-like receptor-4 (TLR-4). At the same time, LPS can stimulate TNF-α, IL-1, and TF secretion, leading to a 2–20-fold increase in adhesion of monocytes and polymorphonuclear leukocytes to the endothelial surface [122, 123]. These molecular events are accompanied by an apparent increased efficiency at which neutrophils and monocytes can transmigrate through the Cpn-infected endothelial layer [124] into subendothelial space where monocytes can transform to foam cells and take up residence. in vitro experiments have verified that Cpn can transform the macrophage into a foam cell phenotype [125–127]. At the same time, the proximity of monocytes at the endothelial surface can in turn enhance the susceptibility of endothelial cells to Cpn infection [128].

Cpn also affect vascular smooth muscle function. Besides the proadhesive responses, Cpn infection of endothelial cells can also increase the proliferation of smooth muscle cells in the intimal layer [129]. Cpn can stimulate this proliferation via upregulation of endogenous heat shock protein 60 [96]. Of note, heat shock protein 60 is an important target of the autoimmune response that leads to enhanced atherosclerosis [130]. It has also been suggested that the Cpn-hsp60 can upregulate the expression of macrophage TNF-α and matrix metalloproteinases [131]. These data provide important cellular biochemical evidence of the pathways involved in the proatherogenic effects of Cpn. This lends further strength to the argument that Cpn is an important atherogenic agent. A schematic representation of the pathways that can be employed by Cpn infection to induce atherogenesis is presented in Fig. 19.3.

One of the limitations of these in vitro cellular studies is that the significance of these infection-mediated events in the context of the overall longterm in vivo atherogenesis remains unknown. To address this limitation, investigations using animal models of Cpn infection have provided some important data. The causative role of Cpn in human atherosclerosis is often

Chronic-inflammatory &

autoimmune responses

Direct Injury to Endothelium

Cpn Infection macrophage activation Atherogenesis

SMC Proliferation

Synergism with LDL/oxidized LDL

In foam cell formation

FIGURE 19.3. The three major pathways that Chlamydia pneumonia can contribute to atherogenesis: stimulation of immune and autoimmune responses (against molecules such as hsp60 and α-myosin), direct infection of the endothelial and smooth muscle cells at the lesion, and synergism with LDL and oxidized LDL in the formation of foam cells and the migration of monocytes into the lesion.

studied in tandem with low-density lipoprotein (LDL). LDL is a primary determinant in the atherogenic process. The development of fatty streaks and plaques involves the uptake of LDL by smooth muscle cells and monocytes. It is well known that hyperlipidemia is an important risk factor for human atherosclerosis [106]. In transgenic mouse models that are defective in LDL receptor expression and generate a high level of circulating LDL, Cpn infection can significantly enlarge the lesion area in the presence of a high-choles- terol diet [132]. The atherogenic effect appears to be Cpn dependent as heat-inactivated Cpn does not produce the same effect [133]. In addition, infection with Cpn induced significant atherosclerotic lesions in mice only when they ingested a cholesterol-enriched diet [134]. However, in rabbits, the Cpn infection alone was sufficient to stimulate significant atherosclerosis [135]. In those experiments, Fong et al. [135] noted that lesions in animals fed with low-cholesterol diets were not prominent to the naked eye but microscopically as significant as in animals fed with much higher cholesterol diet. They also demonstrated that atherosclerosis is amplified in the rabbit aorta with Cpn inoculations but not with Mycoplasma pneumoniae. This is consistent with other experiments that have shown Chlamydia trachomatis does not induce atherosclerotic lesions in the same setting that Cpn does [134]. All in all, these findings suggest that Cpn functions strongly as an atherogenic agent when in synergism with LDL but it may also induce initial atherosclerotic lesions on its own. Currently, there is no definitive biochemical knowledge regarding the molecular pathways that LDL and Cpn can interact to enhance plaque formation in human. While Cpn infection does enhance LDL uptake in macrophages in vitro, smooth muscle cells are also capable of transforming into foam cells. To date, there is no data that indicates Cpn infection can stimulate accumulation of LDL molecules within the subendothelial space.

424 Paul K.M. Cheung and Grant N. Pierce

Not all in vivo investigations identify Cpn as a causative agent in atherosclerosis. There are reports that fail to find any atherogenic effects of Cpn infection in mouse models [136, 137]. The presence of conflicting observations in animal models is likely due to different conditions in animal handling, efficiency of infection, and genetic factors that affect the overall process of lesion development. This brings up the possibility that components of the immune system from animal models can react differently to the LDL/Cpn infection (e.g., mouse and rabbits) and thus the development of plaques (or the lack of) in certain animals does not necessarily fully resemble atherogenesis in humans. Several thorough reviews on the strengths and weaknesses of various animal models used in the study of atherosclerosis and Cpn infection have been published recently [96, 138, 139]. In summary, although some differences have been identified in the literature, the in vivo data generally provides strong support for the role of Cpn and circulating lipids as strong risk factors for atherosclerosis.

The amount of in vitro and in vivo data on Cpn infection has provided significant impetus for the development of several clinical trials that investigated the potential for using antibiotics to treat preexisting atherosclerotic diseases. In the late 1990s, the effectiveness of azithromycin and roxithromycin in reducing cardiovascular events of Cpn-induced patients [140–142] was successfully demonstrated. Cardiovascular events were defined as nonfatal myocardial infarction, cardiovascular death, and recurrent angina. However, recent reports from trials involving larger patient population do not demonstrate observable differences between treated and nontreated groups over coronary events and cerebral events [143, 144]. These reports have cast some doubts on the role of Cpn as an important atherogenic agent. On the other hand, the effectiveness of administering antibiotics in eradicating persistent Cpn from patients may be questionable. Frequent reinfection of the pathogen from other humans is likely considering the presence of this pathogen in the general population. There have been reports that Cpn can be found in lesions of patients who are serologically negative of infection [145, 146], and yet serology is the main end point utilized in these clinical trials. In the mouse model, Cpn antibodies can recur within 3 weeks of apparent eradication [147]. Another trial identified protective effects of antibiotic therapy against clinical cardiovascular disease but only in subgroups with one or more additional risk factor present [148]. This would agree well with the Cpn/LDL synergism observations demonstrated in animal work [132, 134].

In summary, there are legitimate questions concerning the association of Cpn infection with atherosclerosis. Is it possible that the in vitro proliferative and proinflammatory events mediated by Cpn infection may not be significant compared to other factors during the clinical development of in vivo atherosclerosis over long period of time? Is it also possible that the immune responses in animals (whether transgenic or not) to Cpn infection can be different from those in the human arterial environment, leading to potentially

misleading observations of Cpn-induced atherosclerosis? Is it possible or justified to develop a novel Cpn-specific treatment for atherosclerotic patients and coronary heart disease?

Thus, at the present time, a causative role for Cpn in atherosclerosis remains highly suggestive but not ultimately proven. As in the case with other potential atherogenic pathogens, more experiments are needed to generate a definitive verdict. To begin to settle the controversy, it may be necessary to employ an ex vivo organ vessel culture environment to study the biochemistry of Cpn infection under controlled conditions to more effectively understand the complex interactions among different cell types and the consequences in structural and morphological changes. Further, it may also be critical to isolate the host immune components (T cell, monocytes, and their cytokine secretions), all of which have significant but host-dependent atherogenic effects on their own. These components contribute to atherosclerosis in response to a wide range of stimulants and thus, their presence can mask the investigation of in vivo Cpn-specific biochemical pathways that lead to cell proliferation, vascular remodeling, and lesion formation. Understanding the biochemistry of Cpn “exclusive” pathways and effects may allow for the quantification and to determine the significance of Cpn in plaque formation in a host environment. Ultimately, it is only with knowledge such as this that we can develop and design new vaccines or more potent treatments against the persistent infection.

Acknowledgments: This work was supported by a grant from the Canadian Institutes for Health Research (CIHR). P. Cheung was a CIHR/Heart and Stroke Foundation of Canada IMPACT postdoctoral fellow. G. Pierce was a CIHR senior scientist.

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