Sergio Bertazzo

Vascular Smooth Muscle Cell Calcification Is Mediated by Regulated Exosome Secretion

RATIONALE Matrix vesicles (MVs), secreted by vascular smooth muscle cells (VSMCs), form the first nidus for mineralization and fetuin-A, a potent circulating inhibitor of calcification, is specifically loaded into MVs. However, the processes of fetuin-A intracellular trafficking and MV biogenesis are poorly understood. OBJECTIVE The objective of this study is to investigate the regulation, and role, of MV biogenesis in VSMC calcification. METHODS AND RESULTS Alexa488-labeled fetuin-A was internalized by human VSMCs, trafficked via the endosomal system, and exocytosed from multivesicular bodies via exosome release. VSMC-derived exosomes were enriched with the tetraspanins CD9, CD63, and CD81, and their release was regulated by sphingomyelin phosphodiesterase 3. Comparative proteomics showed that VSMC-derived exosomes were compositionally similar to exosomes from other cell sources but also shared components with osteoblast-derived MVs including calcium-binding and extracellular matrix proteins. Elevated extracellular calcium was found to induce sphingomyelin phosphodiesterase 3 expression and the secretion of calcifying exosomes from VSMCs in vitro, and chemical inhibition of sphingomyelin phosphodiesterase 3 prevented VSMC calcification. In vivo, multivesicular bodies containing exosomes were observed in vessels from chronic kidney disease patients on dialysis, and CD63 was found to colocalize with calcification. Importantly, factors such as tumor necrosis factor-α and platelet derived growth factor-BB were also found to increase exosome production, leading to increased calcification of VSMCs in response to calcifying conditions. CONCLUSIONS This study identifies MVs as exosomes and shows that factors that can increase exosome release can promote vascular calcification in response to environmental calcium stress. Modulation of the exosome release pathway may be as a novel therapeutic target for prevention.

Nano-analytical electron microscopy reveals fundamental insights into human cardiovascular tissue calcification

The accumulation of calcified material in cardiovascular tissue is thought to involve cytochemical, extracellular matrix and systemic signals; however, its precise composition and nanoscale architecture remain largely unexplored. Using nano-analytical electron microscopy techniques, we examined valves, aortae and coronary arteries from patients with and without calcific cardiovascular disease and detected spherical calcium phosphate particles, regardless of the presence of calcific lesions. We also examined lesions after sectioning with a focused ion beam and found that the spherical particles are composed of highly crystalline hydroxyapatite that crystallographically and structurally differs from bone mineral. Taken together, these data suggest that mineralized spherical particles may play a fundamental role in calcific lesion formation. Their ubiquitous presence in varied cardiovascular tissues and from patients with a spectrum of diseases further suggests that lesion formation may follow a common process. Indeed, applying materials science techniques to ectopic and orthotopic calcification has great potential to lend critical insights into pathophysiological processes underlying calcific cardiovascular disease.

Hydroxyapatite surface solubility and effect on cell adhesion.

In living organisms the biological hydroxyapatite is in constant contact with body fluids, such as blood serum and saliva. Thus, dissolution, solubility and precipitation take place as part of the interaction of this material with biological fluids in tissues. In this work we have obtained the solubility constant for the system formed from aqueous solutions in equilibrium with hydroxyapatite and thus indirectly obtained the composition of the modified hydroxyapatite surface. In order to check the effects of this equilibrium and of the modification that the surface of hydroxyapatite suffers in aqueous solutions, we cultured pre-osteoblasts onto hydroxyapatite discs before and after equilibrium. The results revealed key steps of the mechanism for the bioactivity of hydroxyapatite, which are the solubilization of hydroxyapatite and the equilibrium that is formed on the surface. These processes modify the hydroxyapatite surface, whose composition is changed to a new calcium phosphate compound with the chemical formula of CaHPO4. A clear description of the transformations that occur on the surface of hydroxyapatite and of the interplay between these transformations and cell activity are two fundamental aspects of processes in which hydroxyapatite takes part, such as bone substitution, bone remodeling, osteoporosis and caries.

Genesis and growth of extracellular-vesicle-derived microcalcification in atherosclerotic plaques

Clinical evidence links arterial calcification and cardiovascular risk. Finite-element modelling of the stress distribution within atherosclerotic plaques has suggested that subcellular microcalcifications in the fibrous cap may promote material failure of the plaque, but that large calcifications can stabilize it. Yet the physicochemical mechanisms underlying such mineral formation and growth in atheromata remain unknown. Here, by using three-dimensional collagen hydrogels that mimic structural features of the atherosclerotic fibrous cap, and high-resolution microscopic and spectroscopic analyses of both the hydrogels and of calcified human plaques, we demonstrate that calcific mineral formation and maturation results from a series of events involving the aggregation of calcifying extracellular vesicles, and the formation of microcalcifications and ultimately large calcification zones. We also show that calcification morphology and the plaque’s collagen content – two determinants of atherosclerotic plaque stability - are interlinked.

Gold–silica quantum rattles for multimodal imaging and therapy

Significance Therapeutic and diagnostic nanoparticles combine multiple functionalities to improve efficacy of treatment but often require assembling complex systems at the expense of overall performance. Here we present a simple strategy to synthesize a hybrid, rattle-like, gold–silica nanoparticle that very efficiently combines therapy and imaging in an animal model. The nanoparticle design is uniquely centered on enabling the use of gold quantum dots (<2 nm) in biological systems. The resulting nanoparticles are highly biocompatible and display emergent photonic and magnetic properties matching and in some instances outperforming state-of-the-art nanotechnology-based medical agents for each of the functionalities investigated, promising a tighter integration between imaging and therapy. Gold quantum dots exhibit distinctive optical and magnetic behaviors compared with larger gold nanoparticles. However, their unfavorable interaction with living systems and lack of stability in aqueous solvents has so far prevented their adoption in biology and medicine. Here, a simple synthetic pathway integrates gold quantum dots within a mesoporous silica shell, alongside larger gold nanoparticles within the shell’s central cavity. This “quantum rattle” structure is stable in aqueous solutions, does not elicit cell toxicity, preserves the attractive near-infrared photonics and paramagnetism of gold quantum dots, and enhances the drug-carrier performance of the silica shell. In vivo, the quantum rattles reduced tumor burden in a single course of photothermal therapy while coupling three complementary imaging modalities: near-infrared fluorescence, photoacoustic, and magnetic resonance imaging. The incorporation of gold within the quantum rattles significantly enhanced the drug-carrier performance of the silica shell. This innovative material design based on the mutually beneficial interaction of gold and silica introduces the use of gold quantum dots for imaging and therapeutic applications.

Bioactivation of alumina by surface modification: a possibility for improving the applicability of alumina in bone and oral repair

OBJECTIVES In regenerative medicine, surface engineering of bioinert synthetic materials is often required in order to introduce bioactive species that can promote cell adhesion, proliferation, viability and enhanced ECM-secretion functions. The aim of this work is to study cell interaction with alumina-modified surfaces. MATERIAL AND METHODS In this work, chemical properties of alumina surface were changed by a reaction at the surface of alumina with low molecular weight dicarboxylic acid, which produced carboxyl groups. RESULTS These carboxyl groups were able to complex with Ca2+ on the surface, forming sites of precipitation for calcium phosphates that make alumina biocompatible, as indicated by cell culture of pre-osteoblasts (MC3T3-E1 cell line). CONCLUSIONS The procedure presented in this work shows that the insertion of specific functional groups on the surface of alumina increases cell interaction with the surface of alumina. This knowledge can be important in oral science and orthopedics, for the construction of prosthesis.

Biologically-active laminin-111 fragment that modulates the epithelial-to-mesenchymal transition in embryonic stem cells.

Significance Laminin-111 is one of the first extracellular matrix proteins expressed during embryogenesis and has been studied for decades, mainly because of its major role in assembling the basement membrane, but also because it has now become one of the most popular cell culture substrates for embryonic stem cells. However, considering the importance of this protein, the role laminin-111 plays during matrix remodeling—which is not only of great interest when seeking to understand cell–matrix interactions, but also when using laminin as a substrate for tissue engineering—is still unclear. Our findings propose a previously unidentified role of laminin-111 that goes beyond basement membrane assembly and an important involvement in the regulation of the epithelial-to-mesenchymal transition. The dynamic interplay between the extracellular matrix and embryonic stem cells (ESCs) constitutes one of the key steps in understanding stem cell differentiation in vitro. Here we report a biologically-active laminin-111 fragment generated by matrix metalloproteinase 2 (MMP2) processing, which is highly up-regulated during differentiation. We show that the β1-chain–derived fragment interacts via α3β1-integrins, thereby triggering the down-regulation of MMP2 in mouse and human ESCs. Additionally, the expression of MMP9 and E-cadherin is up-regulated in mouse ESCs—key players in the epithelial-to-mesenchymal transition. We also demonstrate that the fragment acts through the α3β1-integrin/extracellular matrix metalloproteinase inducer complex. This study reveals a previously unidentified role of laminin-111 in early stem cell differentiation that goes far beyond basement membrane assembly and a mechanism by which an MMP2-cleaved laminin fragment regulates the expression of E-cadherin, MMP2, and MMP9.

The living aortic valve: From molecules to function

The aortic valve lies in a unique hemodynamic environment, one characterized by a range of stresses (shear stress, bending forces, loading forces and strain) that vary in intensity and direction throughout the cardiac cycle. Yet, despite its changing environment, the aortic valve opens and closes over 100,000 times a day and, in the majority of human beings, will function normally over a lifespan of 70–90 years. Until relatively recently heart valves were considered passive structures that play no active role in the functioning of a valve, or in the maintenance of its integrity and durability. However, through clinical experience and basic research the aortic valve can now be characterized as a living, dynamic organ with the capacity to adapt to its complex mechanical and biomechanical environment through active and passive communication between its constituent parts. The clinical relevance of a living valve substitute in patients requiring aortic valve replacement has been confirmed. This highlights the importance of using tissue engineering to develop heart valve substitutes containing living cells which have the ability to assume the complex functioning of the native valve.

Fibres and cellular structures preserved in 75-million–year-old dinosaur specimens

Exceptionally preserved organic remains are known throughout the vertebrate fossil record, and recently, evidence has emerged that such soft tissue might contain original components. We examined samples from eight Cretaceous dinosaur bones using nano-analytical techniques; the bones are not exceptionally preserved and show no external indication of soft tissue. In one sample, we observe structures consistent with endogenous collagen fibre remains displaying ∼67 nm banding, indicating the possible preservation of the original quaternary structure. Using ToF-SIMS, we identify amino-acid fragments typical of collagen fibrils. Furthermore, we observe structures consistent with putative erythrocyte remains that exhibit mass spectra similar to emu whole blood. Using advanced material characterization approaches, we find that these putative biological structures can be well preserved over geological timescales, and their preservation is more common than previously thought. The preservation of protein over geological timescales offers the opportunity to investigate relationships, physiology and behaviour of long extinct animals.

Peptide-directed spatial organization of biomolecules in dynamic gradient scaffolds.

Specific binding peptides are used to spatially organize biomolecule gradients within an electrospun fiber scaffold. Different biomolecule-binding peptide-polymer conjugates are sequentially co-electrospun with a fiber-forming host polymer to generate opposing gradients of peptide functionalization. The binding peptides specifically and non-covalently guide the spatial arrangement of biomolecules into dynamic gradients within the scaffold, mimicking biological gradients found in native tissues.