Nanobacteria-Associated Calcific Aortic Valve Stenosis Tomislav M. Jelic1, Ho-Huang Chang1, Rod Roque1, Amer M. Malas2, Stafford G. Warren3, Andrei P. Sommer4 Departments of 1Pathology and Laboratory Medicine, Charleston Area Medical Center, 2Internal Medicine, Robert C. Byrd Health Science Center, West Virginia University, Charleston Division, 3Cardiovascular Medicine, Charleston Area Medical Center, Charleston, WV, USA, 4Materials Division, University of Ulm, Ulm, Germany
Calcific aortic valve stenosis is the most common valvular disease in developed countries, and the major reason for operative valve replacement. In the US, the current annual cost of this surgery is approximately $1 billion. Despite increasing morbidity and mortality, little is known of the cellular basis of the calcifications, which occur in high-perfusion zones of the heart. The case is presented of a patient with calcific aortic valve stenosis and colonies of progres-
sively mineralized nanobacteria in the fibrocalcific nodules of the aortic cusps, as revealed by transmission electron microscopy. Consistent with their outstanding bioadhesivity, nanobacteria might serve as causative agents in the development of calcific aortic valve stenosis.
Calcific aortic valve stenosis occurs predominantly in older persons, and has been linked to the same risk factors as atherosclerosis (1). The calcific deposits are composed of apatite-based mineral products in association with an organic phase (2). Their occurrence on aortic cusps is a pathological enigma because the cusps are in a high-perfusion milieu of the systolic pressure blood jet, which should actually preclude mineral deposition. Consequently, the existence of a causative agent - or inducer - of calcific aortic valve stenosis, serving as a liaison agent between tissues and blood calcium, seems plausible. Nanobacteria are powerful inducers of calcification, both on and within tissues, and have been identified repeatedly in the human heart. Thus, they might serve as primary inducers of spontaneous calcification processes (3). Originally, this calcification function was deduced from models, but subsequently validated in vitro. Notably, an extreme bioadhesive capacity of nanoscale apatite in general and of nanobacteria in particular - was demonstrated in laboratory experiments. Herein, observational evidence is provided for the presence of a massive colony of progressively mineralized nanobacteria in the aortic cusp.
Case report
Address for correspondence: Dr. Tomislav M. Jelic, Memorial Hospital, Charleston Area Medical Center, 3200 MacCorkle Ave., Charleston, WV, 25304, USA e-mail:
[email protected]
The Journal of Heart Valve Disease 2007;16:101-105
An 87-year-old woman suffering from increased shortness of breath, chest pressure and nausea was admitted to the authors’ institution. Clinical and laboratory evaluations showed the patient to have aortic valve stenosis, unstable angina pectoris, congestive heart failure, and pulmonary edema. Despite intensive treatment, she developed an acute myocardial infarction and died with a clinical picture of progressive left heart failure. The patient’s history included arterial hypertension, aortic stenosis with a mean pressure gradient of 21 mmHg across the stenotic valve, atrial fibrillation, and chronic renal failure (serum creatinine 3 mg/dl). At autopsy, pronounced calcific aortic valve stenosis was demonstrated with associated concentric left ventricular hypertrophy (2.5 cm) and cardiomegaly (720 g). The aortic cusps were heavily deformed with multiple fibrocalcific nodules of up to 0.4 cm diameter. Bilateral nephroangiosclerosis was also identified. On histological examination, hematoxylin and eosinstained sections of the aortic valve tissue revealed the presence of calcified nodules (Fig. 1). Calcium phosphate, contained in multifocal calcifications, was located in superficial portions of the nodules, or directly beneath the endothelium of the aortic cusps, and confirmed by von Kossa staining. Transmission electron microscopy (TEM) of the calcified area (Fig. 2) revealed numerous nanoscale-sized objects with sharp contours and a spherical architecture typical of nanobacteria.
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Figure 1: Light microscopy images of fibrocalcific nodule of aortic cusp. Hematoxylin and eosin staining. Original magnification: Left, ×20; right, ×100.
Some of the bacteria exhibited a vesicular morphology with electron-dense shells (Fig. 3). Based on measurements (see Fig. 2), the mineralized nanovesicles ranged in size from 60 to 300 nm, a signature considered distinctive of nanobacteria.
Discussion The etiology of calcific aortic valve stenosis is largely unknown. Although linked to the same risk factors as atherosclerosis, the early lesion of aortic valve stenosis has been found to be an active inflammatory process presenting some similarities (lipid deposition, macrophage and T-cell infiltration) and some dissimilarities (prominent mineralization and small numbers of smooth muscle cells) to atherosclerosis (4). Importantly, nanobacteria - which are protected by a 60- to 300-nm shell consisting of apatite - were isolated from human atherosclerotic tissue and could be cultured (5,6). Presently, nanobacteria have been detected in humans on four continents, in the heart (5,7), in human immunodeficiency virus (HIV) -infected blood (8), urine (9), arthritic synovial fluid (10), and kidney stones (11,12). They have also been detected in nasopharyngeal carcinoma tissue (13), and are probably one of the causative agents of severe peripheral neuropathy (14,15). A list of their prevalence indicates their potential to contribute to disease in both highperfusion organs (heart and kidney) and low-perfusion zones of the body such as the peripheral nerves and arthritic synovial fluid. Clinical evidence has also been obtained for transplacental or perinatal transmission of nanobacteria from HIV-infected mothers to their babies (8), as well as for a presence of nanobacteria in the blood of healthy people (16). Although neither the exact port of entry of nanobacteria in adults, nor the precise modality of infection is known, recently acquired data have suggested that humans might serve as the principal reservoir of nanobacteria, and that uptake of viable nanobacteria from the environment is possible (17). Presumably, these bacteria thrive in the human body for extended periods, but are too small to be identified using light
Figure 2: Transmission electron microscopy images of a colony of electron-dense nanoparticles on aortic valve tissue, revealing the distinct nature of the calcifications. The shape, size, size distribution and chain formation coincide precisely with structures observed in nanobacteria, both in vivo and in vitro. Original magnification: Left, ×7,000; right, ×20,000. microscopy. Tsurumoto et al. (10) provided impressive evidence for precisely this situation by culturing synovial fluid from arthritic patients. The fluid was first filtered (pore size 0.22 µm), whereupon nanoparticles became visible by using conventional light microscopy after two months (10), thus signaling growth. It is known that a fraction of nanobacteria is excreted from the body via the urine (9), which indicates the existence of a possible equilibrium between growth and excretion. Thus, nanobacteria may prevail in patients for life, without causing manifest damage. Their implication in calcific aortic valve stenosis is compelling, and can be interpreted on the basis of the presently assessed catalogue of nanobacterial functions (17). It is
Figure 3: Transmission electron microscopy image revealing the characteristic vesicular structure of nanobacteria. Original magnification, ×20,000.
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physically unlikely that spontaneous calcifications, starting with the deposition of solitary molecules, occur on the aortic valve, as the high-pressure bloodstream would instantly wash away any depositions of calcium phosphate before a stable mineral phase could be established. Thus, the presence of a nucleator (a factor facilitating calcification) is proposed. The present data suggest that, at least in some cases, nanobacteria might serve this role as they are sufficiently small to pass through the pores between endothelial cells on the aortic valve. Laboratory experiments and predictive models have helped to elucidate the mechanism of processes of spontaneous crystallization and biomineralization, as could be induced cooperatively by a number of nanobacteria on tissues, even on ultrasmooth hydrophobic surfaces in a non-stationary milieu (18), thereby indicating their probable participation in the poorly understood calcification of elastins (19). Moreover, exposure to stress (equivalent to physiological and/or biomechanical variations in the blood) enhanced the bioadhesive capability of the apatite nanovesicles by stimulating them to secrete slime (20). Slime is assumed to be the key determinant in the role of nanobacteria as possible nucleators of elastin calcification. Because of their nanoscale size, nanobacteria could colonize subendothelial layers of the aortic valve, thus facilitating the formation of calcific aortic valve stenosis. Shiekh et al. found that cultured nanobacteria injected intravenously into rats became trapped in the kidneys (21), adhering specifically to the renal epithelial surface. The same authors also provided data which showed clearly that the presence of nanobacteria accelerated secondary biomineralization processes in vitro. It is tempting, therefore, to establish a connection between this experimental model and the present observations, and to arrive at the premise that surface-covering calcification in aortic tissue could be initiated by nanobacteria. Based on published evidence (PubMed), it is concluded that viable nanobacteria may exist in the human body for extended periods, without causing manifest disease. However, with increasing slime synthesis provoked by stress-equivalent variations in their milieu (i.e., in the blood) (20), the rapid formation of networks of nanobacteria on tissues, established by their simultaneous immobilization, becomes increasingly probable (3). In the present case, the absence of any inflammatory reaction suggested that infection of the aortic cusps was remote and that any inflammatory response to nanobacteria had already faded. This scenario was in accord with the inflammatory character of early lesion in calcific aortic valve stenosis (4). In an initial phase of the disease, when the nanobacteria are smaller, less mineralized, and less anchored to the tissue, prevention of their attachment to the tissue
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might in turn prevent calcific aortic valve stenosis. Prerequisite to the design of methods that would control such nanobacterial attachment is an analysis of the response patterns of nanobacteria to physical and chemical stimuli. For example, slime synthesis in vitro was blocked by the irradiation of nanobacteria with moderate light intensities (ca. 1000 W/m2) (22), but stimulated by the administration of aminoglycosides (e.g., gentamycin) (23). The finding that slime synthesis was a dose dependent-phenomenon might have profound implications, and adds concern to an uncritical use of aminoglycosides, and possibly of other antibiotics not yet tested in this context. This would be especially important in elderly patients infected with nanobacteria, setting limits to perspectives of combating nanobacterial infections with some antibiotics (24). The response of nanobacteria to light stimulation has already been exploited, both in theory and from a practical, therapeutic standpoint (3,15). Their early detection in the heart is a precondition for therapeutic strategies to prevent secondary calcification processes, which could eventually result in aortic valve stenosis. In order to arrive at a clearer perspective, it would be necessary to consider the totality of the elements participating in the pathogenesis of calcific aortic valve stenosis, and to integrate them preferably into one ‘big picture’, thereby elucidating their interplay. Novel elements could be of particular interest in this context, including the important relationship between calcific aortic valve stenosis and leukocyte telomere shortening (25). At present, it is not clear how a shorter telomere length might contribute to calcific aortic valve stenosis, although the present studies provide evidence that nanobacteria may be linked to this condition. Indeed, nanobacteria were recently tentatively linked to telomere shortening in leukocytes of the heart (3). While additional studies in this area are required and are indeed under way - any advances could be hampered by insecurities regarding the biological nature of nanobacteria. The provocative name ‘nanobacteria’ - which was conceived during the 1990s and is suggestive of a bacterial nature - was apparently justified by reports on the identification of DNA content (11). Indications that nanobacteria contained RNA rather than DNA inspired the name ‘living nanovesicles’ (26), but this was short-lived when attempts were made to replace the original name with ‘calcifying nanoparticles’ (27). The new term, which is descriptive of non-biological objects, leaves room for concern regarding the DNA content of nanobacteria. As noted previously (3), this play on names might cause the scientific community to withdraw from research into nanobacteria. While the first name irritated many, the new name might create further confusion
104 Nanobacteria-associated calcific aortic valve stenosis T. M. Jelic et al. and cast additional doubts on the credibility of the DNA content of nanobacteria (28). In biology, the adequacy of names is crucial, and a conservative attitude might restrict progress in the field (29). The name nanobacteria has been used by all texts of biology and biomedicine, and has provoked much controversy (30). However, rather than being carried away with creating new words, it might be prudent to adhere strictly to the facts (which can be measured using highresolution imaging techniques) and remain patient with regard to names until the biological content has been definitively clarified. TEM, whilst not presently used routinely for imaging of the heart valves, is an exceptionally versatile high-resolution imaging method for studying nanobacterial ultrastructure. By using this technique the present authors first identified nanobacteria embedded in the tissue of aortic valves. Miller et al. (5) used TEM to image nanobacteria in isolated pathological samples, while Piper et al. (31) reported that TEM of aortic valves tissues from patients undergoing valve replacement for severe degenerative aortic valve stenosis provided no evidence of nanobacterial infection. Thus, it is hoped that the present findings will stimulate further studies, which are clearly necessary. References 1. Rajamannan NM, Gersh B, Bonow RO. Calcific aortic stenosis: From bench to the bedside - emerging clinical and cellular concepts. Heart 2003;89:801-805 2. Tomazic BB, Edwards WD, Schoen FJ. Physicochemical characterization of natural and bioprosthetic heart valve calcific deposits: Implications for prevention. Ann Thorac Surg 1995;60(Suppl.2):S322-S327 3. Sommer AP, Milankovits M, Mester AR. Nanobacteria, HIV and magic bullets - update of perspectives 2005. Chemotherapy 2006;52:95-97 4. Otto CM, Kuusisto J, Reichenbach DD, Gown AM, O’Brien KD. Characterization of the early lesion of ‘degenerative’ valvular aortic stenosis. Histological and immunohistochemical studies. Circulation 1994;90:844-853 5. Miller VM, Rodgers G, Charlesworth JA, et al. Evidence of nanobacterial-like structures in calcified human arteries and cardiac valves. Am J Physiol Heart Circ Physiol 2004;287:H1115-H1124 6. Puskas LG, Tiszlavicz L, Razga Z, Torday LL, Krenacs T, Papp JG. Detection of nanobacteria-like particles in human atherosclerotic plaques. Acta Biol Hung 2005;56:233-245 7. Jelic TM, Malas AM, Groves SS, et al. Nanobacteriacaused mitral valve calciphylaxis in a man with diabetic renal failure. South Med J 2004;97:194-198 8. Pretorius AM, Sommer AP, Aho KM, Kajander EO.
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HIV and nanobacteria. HIV Medicine 2004;5:391393 9. Shoskes DA, Thomas KD, Gomez E. Anti-nanobacterial therapy for men with chronic prostatitis/chronic pelvic pain syndrome and prostatic stones: Preliminary experience. J Urol 2005;173:474-477 10. Tsurumoto T, Matsumoto T, Yonekura A, Shindo H. Nanobacteria-like particles in human arthritic synovial fluids. J Proteome Res 2006;5:1276-1278 11. Kajander EO, Ciftcioglu N. Nanobacteria: An alternative mechanism for pathogenic intra- and extracellular calcification and stone formation. Proc Natl Acad Sci USA 1998;9:8274-8279 12. Khullar M, Sharma SK, Singh SK, et al. Morphological and immunological characteristics of nanobacteria from human renal stones of a north Indian population. Urol Res 2004;32:190-195 13. Zhou HD, Li GY, Yang YX, et al. Intracellular colocalization of SPLUNC1 protein with nanobacteria in nasopharyngeal carcinoma epithelia HNE1 cells depended on the bactericidal permeability increasing protein domain. Mol Immunol 2000;43,18641871 14. King RHM. The role of glycation in the pathogenesis of diabetic polyneuropathy. J Clin Pathol: Mol Pathol 2001;54:400-408 15. Sommer AP. Peripheral neuropathy and light - preliminary report indicating prevalence of nanobacteria in HIV. J Proteome Res 2003;2:665-666 16. Wang XJ, Liu W, Yang ZL, et al. The detection of nanobacteria infection in serum of healthy Chinese people. Zhonghua Liu Xing Bing Xue Za Zhi 2004;25:492-494 17. Sommer AP, Pavláth AE. Nanobioaerosols - reconsidering agricultural irrigation in a warming world. J Environ Monit 2006;8:341-346 18. Sommer AP, Rozlosnik N. Formation of crystalline ring patterns on extremely hydrophobic supersmooth substrates: Extension of ring formation paradigms. Cryst Growth Des 2005;5:551-557 19. Sommer AP, Gheorghiu E. Proteobionics: Biomimetics in proteomics. J Proteome Res 2006;5:611-618 20. Sommer AP, Pretorius AM, Kajander EO, Oron U. Biomineralization induced by stressed nanobacteria. Cryst Growth Des 2004;4:45-46 21. Shiekh FA, Khullar M, Singh SK. Lithogenesis: Induction of renal calcifications by nanobacteria. Urol Res 2006;34:53-57 22. Sommer AP, Oron U, Pretorius AM, et al. A preliminary investigation into light-modulated replication of nanobacteria and heart disease. J Clin Laser Med Surg 2003;21:231-235 23. Björklund M, Ciftcioglu N, Kajander EO.
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Extraordinary survival of nanobacteria under extreme conditions. Proc. SPIE, Instruments, Methods, and Missions for Astrobiology. July 20-22, San Diego, California 1998;3441:123-129 24. Ciftcioglu N, Miller-Hjelle MA, Hjelle JT, Kajander EO. Inhibition of nanobacteria by antimicrobial drugs as measured by a modified microdilution method. Antimicrob Agents Chemother 2002;46:2077-2086 25. Kurz DJ, Kloeckener-Gruissem B, Akhmedov A, et al. Degenerative aortic valve stenosis, but not coronary disease, is associated with shorter telomere length in the elderly. Arterioscler Thromb Vasc Biol 2006;26:e114-e117
Nanobacteria-associated calcific aortic valve stenosis T. M. Jelic et al.
105
26. Sommer AP, McKay DS, Ciftcioglu N, Oron U, Mester AR, Kajander EO. Living nanovesicles: Chemical and physical survival strategies of primordial biosystems. J Proteome Res 2003;2:441-443 27. Kajander EO. Nanobacteria - propagating calcifying nanoparticles. Lett Appl Microbiol 2006;42:549552 28. Abbott A. Battle lines drawn between ‘nanobacteria’ researchers. Nature 1999;401:105 29. Pace NR. Time for a change. Nature 2006;441:289 30. Hogan J. Nanobacteria revelations provoke new controversy. New Scientist 2004;May 19 31. Piper C, Dreier J, Kleesiek K, Korfer R, Horstkotte D. Nanobacteria to promote degenerative aortic valve stenosis? J Heart Valve Dis 2006;15:499-501
