Evidence of nanobacterial-like structures in human calcified arteries and cardiac valves

Virginia M. Miller1*, George Rodgers2, Jon A. Charlesworth3, Brenda Kirkland3, Sandra R. Severson4, Todd E. Rasmussen4, Marineh Yagubyan4, Jeri C. Rodgers5, Franklin R. Cockerill6, Robert L. Folk5, Vivek Kumar7, Gerard Farell-Baril7, and John C. Lieske7

1 Department of Surgery, Mayo Clinic, Rochester, MN, USA; Department of Physiology and Biophysics, Mayo Clinic, Rochester, MN, USA
2 Heart Hospital of Austin, Austin, TX, USA
3 Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA
4 Department of Surgery, Mayo Clinic, Rochester, MN, USA
5 Department of Geological Sciences and Internal Medicine, The University of Texas at Austin, Austin, TX, USA
6 Department of Clinical Microbiology, Mayo Clinic, Rochester, MN, USA
7 Department of Internal Medicine, Division of Nephrology, Mayo Clinic, Rochester, MN, USA

* To whom correspondence should be addressed. E-mail: miller.virginia@mayo.edu .

Mechanisms mediating vascular calcification remain incompletely understood. Nanometer scale objects hypothesized to be a type of bacteria (nanobacteria) are associated with calcified geological specimens, human kidney stones and psammona bodies in ovarian cancer. Experiments were designed to evaluate human vascular tissue for the presence of similar nanometer-scale objects. Calcified human aneurysms (n=8), carotid plaques (n=2), femoral arterial plaques (n=2) and cardiac valves (n=2) and non-calcified aneurysms from patients with bicuspid aortic valve disease (n=2) were collected as surgical waste from the Heart Hospital of Austin, Austin, Texas, and Mayo Clinic, Rochester, Minnesota. Whole mounts or adjacent sections from each specimen were examined by electron microscopy, stained for calcium phosphate, or stained with a commercially available antibody (8D10). Filtered (0.2 micron) homogenates of aneurysms were cultured and co-stained with 8D10 antibody followed by PicoGreen to detect DNA or incubated with [3H]-radiolabeled uridine. Staining for calcium phosphate was heterogeneously distributed within all calcified tissues. Immunological staining with 8D10 was also heterogeneously distributed in areas with and without calcium phosphate. Analysis of areas with positive immunostaining identified spheres ranging in size from 30-100 nm with a spectral pattern of calcium and phosphorus (high-energy dispersive spectroscopy). Nano-sized particles cultured from calcified but not from non-calcified aneurysms were recognized by a DNA-specific dye, incorporated radiolabeled uridine, and after decalcification, appeared via electron microscopy to contain cell walls. Therefore, nanometer-scale particles similar to those described as nanobacteria isolated from geological specimens and human kidney stones can be visualized in and cultured from human calcified cardiovascular tissue.