INTERACTIONS BETWEEN COLLAGEN MATRIX AND PROTEOLIPOSOMES AS EXTRACELLULAR VESICLES BIOMIMETICS

Ectopic vascular calcifications represent a growing clinical problem associated with cardiovascular disease in the aging population. Vascular smooth muscle cells (VSMC) play an integral role in mediating vessel calcification by undergoing transdifferentiation to osteoblast-like phenotype. Osteoblasts control the deposit in the extracellular matrix and release a specific class of extracellular vesicles called matrix vesicles (MVs), which serve as the initial sites of hydroxyapatite mineral formation, also in the vessel wall. Annexin V (AnxA5) is acidic phospholipid-dependent Ca2+-binding protein with Ca2+-channel activity in the MV membrane. Tissue-nonspecific alkaline phosphatase (TNAP) is attached to the outer MVs membrane and plays a role of a pyrophosphatase hydrolyzing inorganic pyrophosphate. We have described the production of DPPC and DPPC:DPPS 10%-proteoliposomes harboring AnxA5, TNAP and AnxA5+TNAP, and their use as mimetic systems of MVs. Enzymatic activity, calcium uptake and phase contrast microscopy of giant proteoliposomes demonstrated the functional incorporation of both proteins. Proteoliposomes were found to bind to type II collagen fibers in both lipid compositions. Proteoliposomes-AnxA5 bound with the highest affinity and the DPPS presence in the lipid composition enhanced the degree of binding significantly to 74%. Proteoliposomes-TNAP in both lipid compositions poorly bound to the collagen matrix (< 20%). Proteoliposomes harboring both proteins showed 30% of binding at most. Of different collagens tested (I, II and III types), the best affinity it was found for proteoliposomes binding to type II collagen fibers. Besides, the AnxA5 binding to type II collagen occurs in a calcium-independent manner. These findings indicate that AnxA5 acts both: as a calcium-selective channel creating a Ca2+-rich microenvironment inside MVs, and via collagen binding of vesicles anchoring MVs to the matrix of calcifying sites. Understanding the molecular mechanisms that regulate the mineralization is essential for the development of novel therapeutic strategies to prevent or inhibit ectopic mineralization, especially of VSMC. The use of proteoliposomes represents greatly helpful models in these studies.
Acknowledgment: FAPESP 2019/08568-2, CAPES (Finance Code 001, #88887.320304/2019- 00), CNPq 305426/2021-4.