Sheldon Weinbaum

Vascular calcification predicts atherosclerotic plaque rupture and cardiovascular events. Retrospective studies of women taking bisphosphonates (BiPs), a proposed therapy for vascular calcification, showed that BiPs paradoxically increased morbidity in patients with prior acute cardiovascular events but decreased mortality in event-free patients. Calcifying extracellular vesicles (EVs), released by cells within atherosclerotic plaques, aggregate and nucleate calcification. We hypothesized that BiPs block EV aggregation and modify existing mineral growth, potentially altering microcalcification morphology and the risk of plaque rupture. Three-dimensional (3D) collagen hydrogels incubated with calcifying EVs were used to mimic fibrous cap calcification in vitro, while an ApoE^−/− mouse was used as a model of atherosclerosis in vivo. EV aggregation and formation of stress-inducing microcalcifications was imaged via scanning electron microscopy (SEM) and atomic force microscopy (AFM). In both models, BiP (ibandronate) treatment resulted in time-dependent changes in microcalcification size and mineral morphology, dependent on whether BiP treatment was initiated before or after the expected onset of microcalcification formation. Following BiP treatment at any time, microcalcifications formed in vitro were predicted to have an associated threefold decrease in fibrous cap tensile stress compared to untreated controls, estimated using finite element analysis (FEA). These findings support our hypothesis that BiPs alter EV-driven calcification. The study also confirmed that our 3D hydrogel is a viable platform to study EV-mediated mineral nucleation and evaluate potential therapies for cardiovascular calcification. Significance The most common cause of heart attacks or strokes is the rupture of thin fibrous caps that cover vulnerable plaques within blood vessels. Small mineral deposits, called microcalcifications, increase local tissue stress and thereby increase the risk of cap rupture. We report here the use of a three-dimensional collagen hydrogel model of fibrous cap calcification and a complementary mouse model of plaque formation to determine whether bisphosphonate (BiP) therapy, commonly used to treat bone loss, alters microcalcification formation. The results showed that BiP treatment resulted in time-dependent changes in microcalcification size and mineral morphology, dependent on whether BiP treatment was initiated before or after the expected onset of microcalcification formation.

We have been asked by friends, many who are not scientists, how far droplets from a sneeze,

Osteocytes are considered to be the major mechanosensory cells of bone, but how osteocytes in vivo process, perceive, and respond to mechanical loading remains poorly understood. Intracellular calcium (Ca²⁺) signaling resulting from mechanical stimulation has been widely studied in osteocytes in vitro and in bone explants, but has yet to be examined in vivo. This is achieved herein by using a three-point bending device which is capable of delivering well-defined mechanical loads to metatarsal bones of living mice while simultaneously monitoring the intracellular Ca²⁺ responses of individual osteocytes by using a genetically encoded fluorescent Ca²⁺ indicator. Osteocyte responses are imaged by using multiphoton fluorescence microscopy. We investigated the in vivo responses of osteocytes to strains ranging from 250 to 3,000 ????ϵ𝝁ϵ and frequencies from 0.5 to 2 Hz, which are characteristic of physiological conditions reported for bone. At all loading frequencies examined, the number of responding osteocytes increased strongly with applied strain magnitude. However, Ca²⁺ intensity within responding osteocytes did not change significantly with physiological loading magnitudes. Our studies offer a glimpse into how these critical bone cells respond to mechanical load in vivo, as well as provide a technique to determine how the cells encode magnitude and frequency of loading.

Significance: Although polarized mechanotransduction in osteocytes has been established, participation of α _V β ₃ integrin still remains to be conclusively demonstrated. We addressed this issue using the novel Stokesian fluid stimulus probe to discretely stimulate the osteocyte processes and cell body with physiologically relevant hydrodynamic forces in the piconewton range while imaging intracellular Ca ²⁺ signals. We demonstrate that osteocyte cell processes but not the cell bodies are mechanosensitive through discrete attachment sites provided by α _V β ₃ integrin, thereby revealing that integrin attachment sites provide the substrate for polarized osteocyte mechanosignaling and mechanotransduction.

Osteocytes are bone cells that form cellular networks that sense mechanical loads distributed throughout the bone tissue. Interstitial fluid flow in the lacunar canalicular system produces focal strains at localized attachment sites around the osteocyte cell process. These regions of periodic attachment between the osteocyte cell membrane and its canalicular wall are sites where pN-level fluid-flow induced forces are generated in vivo. In this study, we show that focally applied forces of this magnitude using a newly developed Stokesian fluid stimulus probe initiate rapid and transient intercellular electrical signals in vitro. Our experiments demonstrate both direct gap junction coupling and extracellular purinergic P2 receptor signaling between MLO-Y4 cells in a connected bone cell network. Intercellular signaling was initiated by pN-level forces applied at integrin attachment sites along both appositional and distal unapposed cell processes, but not initiated at their cell bodies with equivalent forces. Electrical coupling was evident in 58% of all cell pairs tested with appositional connections; coupling strength increased with the increasing number of junctional connections. Apyrase, a nucleotide-degrading enzyme, suppressed and abolished force-induced effector responses, indicating a contribution from ATP released by the stimulated cell. This work extends the understanding of how osteocytes modulate their microenvironment in response to mechanical signals and highlights mechanisms of intercellular relay of mechanoresponsive signals in the bone network.

This review examines changing perspectives on the biomechanics of vulnerable plaque rupture over the past 25 years from the first finite element analyses (FEA) showing that the presence of a lipid pool significantly increases the local tissue stress in the atheroma cap to the latest imaging and 3D FEA studies revealing numerous microcalcifications in the cap proper and a new paradigm for cap rupture. The first part of the review summarizes studies describing the role of the fibrous cap thickness, tissue properties, and lesion geometry as main determinants of the risk of rupture. Advantages and limitations of current imaging technologies for assessment of vulnerable plaques are also discussed. However, the basic paradoxes as to why ruptures frequently did not coincide with location of PCS and why caps >65 μm thickness could rupture at tissue stresses significantly below the 300 kPa critical threshold still remained unresolved. The second part of the review describes recent studies in the role of microcalcifications, their origin, shape, and clustering in explaining these unresolved issues including the actual mechanism of rupture due to the explosive growth of tiny voids (cavitation) in local regions of high stress concentration between closely spaced microinclusions oriented along their tensile axis.

Using 2.1-µm high-resolution microcomputed tomography, we have examined the spatial distribution, clustering, and shape of nearly 35,000 microcalcifications (µCalcs) ≥ 5 µm in the fibrous caps of 22 nonruptured human atherosclerotic plaques. The vast majority of these µCalcs were h )/particle diameter ( D ) < 0.4 if particles are oriented along the tensile axis of the cap. Of the thousands of µCalcs observed, there were 193 particle pairs with h / D ≤ 2 (tissue stress factor > 2), but only 3 of these pairs had h / D ≤ 0.4, where the local tissue stress could increase a factor > 5. Using nondecalcified histology, we also show that nearly all caps have µCalcs between 0.5 and 5 µm and that the µCalcs ≥ 5 µm observed in high-resolution microcomputed tomography are agglomerations of smaller calcified matrix vesicles. µCalcs < 5 µm are predicted to be not harmful, because the tiny voids associated with these very small particles will not explosively grow under tensile forces because of their large surface energy. These observations strongly support the hypothesis that nearly all fibrous caps have µCalcs, but only a small subset has the potential for rupture.