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Am J Physiol Heart Circ Physiol 290: H1510-H1519, 2006. First published November 4, 2005; doi:10.1152/ajpheart.01055.2005
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Chlamydia pneumoniae in foci of "early" calcification of the tunica media in arteriosclerotic arteries: an incidental presence?

Yuri V. Bobryshev,1,3 Reginald S. A. Lord,1 and Dinh Tran2

1Surgical Professorial Unit and 2Division of Anatomical Pathology, St. Vincent's Hospital, and 3School of Medical Sciences, University of New South Wales, Sydney, New South Wales, Australia

Submitted 7 October 2005 ; accepted in final form 30 October 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Only a few previous works investigated the involvement of Chlamydia pneumoniae (Chlamydophila pneumoniae) in arterial calcification. The present study investigated a possible association between C. pneumoniae and medial calcification. Carotid artery segments obtained by endarterectomy from 60 patients were examined by PCR and immunohistochemistry to identify the presence of C. pneumoniae. Arterial specimens showing double-positive (n = 17), double-negative (n = 22), and single-positive results (n = 21) were further analyzed by a combination of histology, immunohistochemistry, and electron microscopy. Medial calcification occurred in 10 of 17 (58.8%) C. pneumoniae double-positive arterial specimens, but no medial calcification was observed in any of 22 C. pneumoniae double-negative arterial specimens. Electron microscopy indicated C. pneumoniae in smooth muscle cells (SMCs) in foci of medial calcification. Medial SMCs showing damage to the cytoplasm and basement membrane contained the structures with the appearance of elementary, reticulate, and aberrant bodies of C. pneumoniae. The presence of C. pneumoniae in SMCs was confirmed by electron-microscopic immunocytochemistry. In the extracellular matrix, calcification was observed in C. pneumoniae aberrant bodies that exited the SMCs. The findings offer a new hypothesis of arterial calcification: they suggest that C. pneumoniae infection of medial SMCs may be associated with the pathophysiological events of arteriosclerotic calcification of the tunica media.

arteriosclerosis; smooth muscle cells

STUDIES OVER THE LAST DECADE indicate that Chlamydia pneumoniae (Chlamydophila pneumoniae) may play a role in atherosclerosis and other vascular diseases (12, 15, 24). A large number of in vitro studies have elucidated the molecular mechanisms involved in functional alterations of vascular cells infected by C. pneumoniae (12, 15, 24), although a possible etiologic involvement of C. pneumoniae in vascular diseases remains controversial (12, 15, 24). Some studies have failed to detect C. pneumoniae in atherosclerotic lesions (6, 30, 49), while others have provided evidence of an association of C. pneumoniae with atherosclerosis by means of seroepidemiology analysis, detection of the microorganism in atheromas by culture, PCR, electron microscopy, immunohistochemistry, and in situ hybridization (12, 15, 24).

In a previous work, we used a combination of immunohistochemistry and PCR to detect C. pneumoniae in atherosclerotic plaques obtained by endarterectomy (8). C. pneumoniae was identified by immunohistochemistry (IH) and PCR (PCR+/IH+ specimens) in 17 of 60 (28%) arterial specimens (8). In two (3%) specimens, C. pneumoniae was identified by immunohistochemistry only (PCR–/IH+ specimens) and in 19 (32%) specimens, C. pneumoniae was detected by PCR only (PCR+/IH– specimens). In 22 (37%) specimens, no C. pneumoniae was detected by either technique (PCR–/IH– specimens). The presence of C. pneumoniae in the arterial specimens did not relate to the age or gender of the patient, the length of clinical history, or any clinical data available (8). During that study, we noted C. pneumoniae immunopositivity in some C. pneumoniae-positive specimens in areas of calcification in the tunica media (unpublished observation).

The present study investigated a possible association between C. pneumoniae and medial calcification.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Archival tissue specimens. Carotid artery segments were obtained by endarterectomy from 60 patients (33 men and 27 women, 46–75 yr old) (8). The present study conforms with the principles outlined in the Declaration of Helsinki and was approved by the institutional review board (8).

The presence or absence of C. pneumoniae in the specimens was established by a combination of immunohistochemistry and PCR, carried out according to Campbell et al. (14) as described previously (8). In the present study, specimens showing double-positive, double-negative, and single-positive results (8) were examined.

Histology and immunohistochemical staining. Histological sections stained with Mayer's hematoxylin and alizarin red were examined to determine areas of arterial calcification.

Anti-C. pneumoniae monoclonal antibody (clone RR402, DakoCytomation) was used to detect C. pneumoniae in the sections as described previously (8). The avidin-biotin complex (ABC) method was utilized for single immunostaining (8), and procedures for negative controls were carried out according to the recommendations of Dowell et al. (16) as previously described (8). Cell type-specific antibodies for smooth muscle cells (SMCs; {alpha}-smooth muscle actin-positive cells), macrophages (CD68+), T cells (CD3+), mast cells (mast cell tryptase-positive), and dendritic cells (fascin-positive and CD1a+) were used as previously described (10). Double immunostaining was used to analyze a possible association of C. pneumoniae with SMCs and was carried out as previously described (8, 10). A combination of alizarin red staining with immunohistochemical reactions precluded the identification of antigens; therefore, in immunohistochemical procedures, counterstaining with Mayer's hematoxylin was used for the visualization of calcified deposits.

Electron-microscopic analysis. For routine electron microscopy, tissue samples fixed in 2.5% and 1% OsO4 were dehydrated and embedded in Araldite resin as previously described (9).

For electron-microscopic immunohistochemistry, tissue pieces were fixed in 0.2% glutaraldehyde and 3% paraformaldehyde in PBS (pH 7.4) and embedded in LR White resin according to the protocol described by Keita et al. (25). Postembedding immunohistochemistry on ultrathin sections was carried out using anti-C. pneumoniae antibody according to technique of Keita et al. with gold-labeled secondary antibody prepared according to Simmons et al. (41). Colloidal gold particles were prepared by the reduction of a solution of HAuCl4 by a mixture of sodium citrate and tannic acid (41).

Nonstained ultrathin sections of LR White resin-embedded tissue were subjected to elemental microanalysis using a LINK QX 200J energy-dispersive X-ray microanalysis system attached to an electron microscope (model H7000, Hitachi).

Statistical analysis. Statistical tests of significance were carried out using the {chi}2 test in SPSS statistical software.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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All arterial specimens contained advanced atherosclerotic plaques with well-developed necrotic cores. All atherosclerotic plaques were complicated by neovascularization and calcification [atherosclerotic lesions of types V and VI according to Stary et al. (44)]. The distribution patterns and the peculiarities of localization of C. pneumoniae in atherosclerotic plaques (the tunica intima) were previously reported (8).

The examination of the occurrence of medial calcification in double-positive (PCR+/IH+) and double-negative (PCR–/IH–) arterial specimens revealed no calcification in any of 22 PCR–/IH– arterial specimens but calcification in 10 of 17 (58.8%) PCR+/IH+ specimens (Table 1). All specimens with medial calcification were characterized by multiple-foci calcification (Fig. 1A). In 7 of 10 specimens with medial calcification, C. pneumoniae immunopositivity was detected in areas of medial calcification (Table 1). There was no C. pneumoniae immunopositivity in areas without calcification and no immunopositivity in negative controls. In specimens with medial calcification, C. pneumoniae immunopositivity was present only in areas of the media exhibiting low levels of calcification ("early" foci of calcification; Fig. 1, B and C). These early calcified foci measured up to 0.02 mm2 and consisted of numerous calcified deposits with diameters <2 µm (Fig. 1). In zones of early calcification, only a few larger (<25-µm-diameter) calcified deposits were observed (Fig. 2). No C. pneumoniae immunopositivity was detected in calcified areas that consisted of calcified deposits with diameters >25 µm. Statistical analysis showed completely significant association between early calcification and the incidence of C. pneumoniae immunopositivity. There was also significant association between total medial calcification and the incidence of C. pneumoniae immunopositivity (P < 0.0005).


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  		Table 1. Calcification and presence of Chlamydia pneumoniae in calcification zones in tunica media of carotid artery specimens

 

Figure 1
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  		Fig. 1. Foci of calcification and Chlamydia pneumoniae immunopositivity in tunica media of carotid artery specimens. A: calcified areas (arrows) formed between and within layers of medial smooth muscle cells (SMCs). SMCs (rose) were identified using alkaline phosphatase-antialkaline phosphatase (APAAP) technique, and calcified deposits (blue) were revealed by counterstaining with Mayer's hematoxylin. B: C. pneumoniae immunopositivity (brown) among calcified deposits shown by avidin-biotin complex (ABC) immunoperoxidase technique with Mayer's hematoxylin counterstaining. C: double immunostaining showing C. pneumoniae-positive SMCs in zones of early medial calcification. SMCs were identified using APAAP technique, and C. pneumoniae was visualized by ABC method with Mayer's hematoxylin counterstaining. Scale bars, 100 (A) and 25 (B and C) µm.

 

Figure 2
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  		Fig. 2. Colocalization of C. pneumoniae-immunopositive cells with calcified deposits in tunica media of carotid artery specimens. A: double immunostaining showing C. pneumoniae-positive cells in zones of medial calcification. Large arrows, C. pneumoniae-infected SMCs; small arrows, calcified deposits. B: low-magnification image showing expansive areas of calcification (arrows) surrounded by {alpha}-smooth muscle actin-positive cells (rose). C: C. pneumoniae-immunopositive cells (large arrows) with ill-defined contours, suggesting cell destruction and calcified deposits (small arrows). SMCs were identified using APAAP technique (rose), and C. pneumoniae was visualized by ABC method (brown) with Mayer's hematoxylin counterstaining. Scale bars, 10 (A and C) and 40 (B) µm.

 
Immunostaining utilizing cell type-specific antibodies revealed that no immunoinflammatory cells, including macrophages, T cells, mast cells, and dendritic cells, were present within or around areas of medial calcification. Immunostaining with anti-{alpha}-smooth muscle actin antibody suggested that SMCs were the only cell type in areas affected by calcification (Figs. 1A and 2B). Double immunostaining demonstrated C. pneumoniae in SMCs in zones of early calcification (Fig. 1C and Fig. 2, A and C). Some C. pneumoniae-positive SMCs displayed ill-defined contours, suggesting cell destruction (Fig. 2C).

Areas of expansive medial calcification consisting of massive calcified deposits were not accessible by electron-microscopic analysis, but areas of early medial calcification were analyzed. In nonstained ultrathin sections, calcified deposits displayed a high electron density, and X-ray microanalysis showed that they contained high levels of calcium and phosphorus, with a mean calcium-to-phosphorus ratio of 1.63 ± 0.18. Contrasting the sections with uranyl acetate and lead citrate revealed calcifying deposits in areas where arterial cells underwent destructive alterations (Figs. 3 and 4). Calcified deposits markedly varied in size and appearance (Fig. 4). Some were round, and some had needlelike or fine threadlike formations of high electron density extending from the surface of their electron-dense core (Fig. 4, A and B). Some calcifying deposits were round 0.1- to 0.3-µm-diameter structures with electron-dense cores (Fig. 4D). Others did not contain an electron-dense core and were filled with amorphous material of intermediate or low electron density (Fig. 4E). Transitional forms between small and large calcified deposits were also observed (Fig. 4A).


Figure 3
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  		Fig. 3. A: electron micrograph showing expansive areas of cell destruction in tunica media with numerous membrane-bound structures of different shapes and sizes. B and C: enlargements of areas in A enclosed in rectangles. Arrows, extracellular structures displaying signs of "early" calcification. Scale bar, 5 µm.

 

Figure 4
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  		Fig. 4. A: heterogeneity of calcified deposits in tunica media. B: enlargement of area in A enclosed in rectangle. Note needlelike formations along the border of a calcified deposit. C: aggregates of calcifying vesicles. D: calcifying 0.3-µm-diameter structure (arrow) with an electron-dense border and core. E: dotlike foci of calcification of membrane-covered structures (arrows). Scale bars, 1 (A and B) and 0.5 (C and D) µm.

 
Electron-microscopic analysis showed that areas of medial calcification consisted of cells with a well-developed basement (basal) membrane and myofilaments. These cells were identified as SMCs of contractile phenotype. [Ultrastructural criteria for identification of arterial cell types have been described elsewhere (5, 9).] No other cell types, including myofibroblasts, were observed in foci of medial calcification.

In areas of calcification, some SMCs showed signs of destructive alterations, and these were found to contain the structures consistent with the appearance of inclusions, elementary bodies (EBs) and reticulate bodies (RBs) of C. pneumoniae (Figs. 5 and 6A). [Ultrastructural criteria for identification of C. pneumoniae are well defined (13, 26, 50).] In some C. pneumoniae-infected SMCs with disorganized myofilaments and edematous cytoplasm, the membranes surrounding C. pneumoniae inclusions were interrupted, and RBs and EBs, as well as intermediate forms, of C. pneumoniae were seen as having been released into the cytoplasm. Furthermore, in some C. pneumoniae-infected SMCs, the basal membrane was completely destroyed, and large numbers of EBs and aberrant bodies (ABs) of C. pneumoniae were observed in the surrounding extracellular matrix (Fig. 6, B–D). In degenerating SMCs, in which the basal membrane was completely destroyed, calcification of RBs, EBs, and ABs was evident (Fig. 7). Electron-microscopic immunocytochemistry confirmed that the microorganisms identified ultrastructurally as C. pneumoniae were immunopositive for anti-C. pneumoniae antibody (clone RR402, DakoCytomation; Fig. 8, A–D) and demonstrated the presence of C. pneumoniae immunopositivity in growing calcified deposits (Fig. 8, E and F).


Figure 5
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  		Fig. 5. A: electron micrograph demonstrating C. pneumoniae at different stages of development within inclusions in cytoplasm of medial SMCs. B and C: zones of adhesion (arrows) between inclusions. Scale bars, 0.5 µm.

 

Figure 6
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  		Fig. 6. Electron micrographs showing elementary bodies (EBs), reticulate bodies (RBs), and intermediate forms of C. pneumoniae in and around degenerating SMCs in tunica media. A: zone of destruction of inclusion membrane in the cytoplasm of a medial SMC (small arrows) and myofilaments (large arrow). B: zones of destruction of basal membrane of an SMC infected by C. pneumoniae (large arrows) and zone of intact external cell membrane (small arrows). {star}, Areas of cytoplasm containing myofilaments. N, nucleus. In B–D, note large number of C. pneumoniae RBs, intermediate forms, and aberrant bodies in surrounding extracellular matrix. In C, RBs are shown by small arrows and EB by large arrow. Scale bars, 0.5 (A, C, and D) and 1 (B) µm.

 

Figure 7
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  		Fig. 7. A: electron micrograph showing destructive alterations of an infected SMC. Cytoplasm contains aggregates of C. pneumoniae RBs and EBs, some of which show signs of calcification. B–D: enlargements of areas in A enclosed in rectangles. C: miniature bodies within cytoplasm of EB (arrows). Scale bar, 1 µm.

 

Figure 8
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  		Fig. 8. Electron-microscopic immunohistochemical identification of C. pneumoniae in SMC cytoplasm (A–C) and extracellular matrix (D). E: calcified deposit containing immunogold-labeled C. pneumoniae antigen (arrows). F: X-ray spectrum showing distinct peaks for calcium and phosphorus in a calcified deposit. Scale bars, 0.5 (A–D) and 1 (E) µm.

 
Degenerating C. pneumoniae-infected medial SMCs displayed ultrastructural signs of necrotic death, including chromatin condensation, edema, and vacuolization of the cytoplasm, as well as destruction of the basal membrane (Fig. 6B), but no fragmentation of chromatin or formation of apoptotic bodies was observed. [Ultrastructural criteria for distinguishing between necrosis and apoptosis are well defined (28, 48).]


   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
A large number of studies have tried to elucidate the role of C. pneumoniae in atherosclerosis (6, 8, 13, 15, 24, 30, 49), but whether C. pneumoniae is a causative or concomitant phenomenon has not been clarified (6, 8, 13, 15, 24, 30, 49).

Only a few previous works investigated a possible association between C. pneumoniae and arterial calcification (2, 4, 19, 22, 23, 27, 39). Lehto et al. (27) reported that C. pneumoniae antibodies were strongly associated with intimal calcification of the femoral arteries. Other studies examined a possible role of C. pneumoniae in calcification in aortic valve stenosis and reported variable results (2, 4, 19, 22, 23, 39).

In the present study, medial calcification was observed in 10 of 17 (58.8%) C. pneumoniae double-positive arterial specimens, whereas no medial calcification was found in any of 22 C. pneumoniae double-negative arterial specimens, strongly suggesting that the presence of C. pneumoniae in the arterial wall contributes to medial calcification. In the present study, C. pneumoniae immunopositivity was detected only in foci of early medial calcification, whereas "mature" calcified depositions were free of C. pneumoniae immunopositivity. Calcification of C. pneumoniae EBs, RBs, and ABs, 0.1–0.4 µm diameter, was observed in the tunica media. In the same locations, there were transitional forms between small (0.1- to 0.4-µm-diameter) calcified deposits and larger calcified deposits, suggesting that the smaller calcified deposits were precursors of the larger ones.

Vascular calcification is known to occur in two distinct forms: intimal calcification, which always occurs in the context of atherosclerosis, and medial calcification, which can occur in its absence (31). These two processes differ not only in morphology, but also in the pathological mechanisms involved (29, 31). Inflammatory cells such as macrophages and mast cells infiltrating plaque lipid-rich regions play an important role in atherosclerotic calcification (21, 31). In contrast to intimal calcification, medial calcification is known to occur in the absence of inflammatory cell infiltration and lipid deposition (21, 31). In this respect, the present study provides confirmatory observations demonstrating that SMCs are the only cell type present in foci of medial calcification. Apart from arteriosclerotic calcification of the media in large elastic-type arteries of nondiabetic individuals, the calcification process, known as Monckeberg's sclerosis, commonly affects the media of peripheral medium-sized arteries in aged and diabetic individuals (29, 31, 40). Calcification in Monckeberg's sclerosis has been suggested to be an active process orchestrated by phenotypically modified vascular SMCs (40).

Over the last decade, the involvement of SMCs in arterial calcification has been studied intensely (29, 30, 33–37), and calcification of human vascular SMCs in vitro has been found to correlate with high levels of matrix Gla protein and low levels of osteopontin expression, both of which are bone-associated proteins (32). Calcium deposits in the arterial wall exist mainly in the form of hydroxyapatite, which is a type of calcium phosphate normally found in bone (3, 31), and it has been postulated that the molecular mechanisms of arterial calcification may be similar to those operating during bone formation (1, 31, 32, 40, 43, 45, 47). Aortic SMCs, microvascular smooth muscle-like cells, and pericytes can differentiate in culture to form osteoblast-like cells, producing a calcified matrix (1, 29, 45, 47), and the concept of multipotent calcifying vascular cells capable of forming calcifying nodules has been developed (31, 40, 45–47). Transdifferentiation of some arterial SMCs into chondrocytes occurs in human arteries, and this process is accompanied by the formation of numerous calcifying vesicles (7). Recent findings indicate that a variety of different mechanisms might contribute to vascular calcification (31, 40, 45–47). Apoptosis of vascular SMCs has also been implicated in atherosclerotic calcification (33, 34), but, in the present study, no typical features of apoptotic death were detected in medial SMCs infected by C. pneumoniae. The present study suggests that the destruction of C. pneumoniae-infected medial SMCs occurred through necrosis. It has been recently appreciated that dying cells may display features of necrosis and apoptosis, and the distinction between apoptosis and necrosis can sometimes appear blurred (17).

In vitro studies have shown that C. pneumoniae affects proliferation of SMCs (20, 38), increases production of matrix metalloproteinases 1 and 3 by SMCs (37), induces production of cytokines by SMCs (18, 35, 36), and, finally, induces SMC death (17). These in vitro findings support the view that C. pneumoniae may be not just an incidental presence in the arterial wall (12, 15, 24).

The present study shows that C. pneumoniae participates in medial calcification by providing the structural basis for the formation of early calcified deposits along with other structural components of the extracellular matrix that can be "impregnated" by calcium salts. In some cases, alterations of C. pneumoniae in the cytoplasm of medial SMCs may be an initial step that triggers arteriosclerotic calcification. In the present study, C. pneumoniae immunopositivity was detected only in foci of early medial calcification, which may suggest that C. pneumoniae plays a role in the initial stage of medial calcification, but as the calcification process proceeds, C. pneumoniae antigens become undetectable or are destroyed. Statistical analysis has shown completely significant association between early calcification and the incidence of C. pneumoniae immunopositivity. There was also significant association between the total medial calcification and the incidence of C. pneumoniae immunopositivity (P < 0.0005). Although this study has shown that calcification of aberrant bodies of C. pneumoniae originating from infected medial smooth muscle cells is a casual event, further studies are needed to provide evidence of the physiological relations between C. pneumoniae and medial calcification.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
The research was supported by the Vascular Fund, Surgical Professorial Unit, and by St. Vincent's Clinic Foundation (Sydney, Australia).


   ACKNOWLEDGMENTS
 
We thank I. Nivison-Smith for statistical analysis.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Y. V. Bobryshev, Surgical Professorial Unit, St. Vincent's Hospital Sydney, 234 Victoria St., Darlinghurst NSW 2010, Australia (e-mail: y.bobryshev{at}unsw.edu.au)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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