Avocado-derived extracellular vesicles loaded with ginkgetin and berberine prevent inflammation and macrophage foam cell formation.

Atherosclerosis, a chronic inflammatory disease of aorta, remains the major cause of morbidity and mortality among cardiovascular disease patients. Macrophage foam cell formation and inflammation are critically involved in early stages of atherosclerosis, hence chemopreventive targeting of foam cell formation by nutraceuticals may be a promising approach to curbing the progression of atherosclerosis. However, many nutraceuticals including berberine and ginkgetin have low stability, tissue/cell penetration and bioavailability resulting in inadequate chemotherapeutic effects of these nutraceuticals. We have used avocado-derived extracellular vesicles (EV) isolated from avocado (EVAvo ) as a novel carrier of nutraceuticals, in a strategy to alleviate the build-up of macrophage foam cells and expression of inflammatory genes. Our key findings are: (i) Avocado is a natural source of plant-derived EVs as shown by the results from transmission electron microscopy, dynamic light scattering and NanoBrook Omni analysis and atomic force microscopy; (ii) EVAvo are taken up by macrophages, a critical cell type in atherosclerosis; (iii) EVAvo can be loaded with high amounts of ginkgetin and berberine; (iv) ginkgetin plus berberine-loaded EVAvo (EVAvo(B+G) ) suppress activation of NFκB and NLRP3, and inhibit expression of pro-inflammatory and atherogenic genes, specifically Cd36, Tnfα, Il1ß and Il6; (v) EVAvo(B+G) attenuate oxidized low-density lipoprotein (oxLDL)-induced macrophage foam cell formation and (vi) EVAvo(B+G) inhibit oxLDL uptake but not its cell surface binding during foam cell formation. Overall, our results suggest that using EVAvo as a natural carrier of nutraceuticals may improve strategies to curb the progression of atherosclerosis by limiting inflammation and pro-atherogenic responses.

Mechanosensing and Mechanosignal Transduction in Atherosclerosis.

PURPOSE OF REVIEW: This review aims to summarize the latest findings on mechanosensing in atherosclerosis, elucidating the molecular mechanisms, cellular players, and potential therapeutic targets. RECENT FINDINGS: Atherosclerosis, a chronic inflammatory disease characterized by the buildup of lipid-laden plaque within arterial walls, is a major contributor to cardiovascular disease-related mortality and morbidity. Interestingly, atherosclerosis predominantly occurs in arterial areas with curves and branches. In these regions, endothelial cells encounter irregular blood flow with distinctive low-intensity fluctuating shear stress. On the other hand, straight sections of arteries, subjected to a consistent flow and related high-intensity, one-way shear stress, are relatively safeguarded against atherosclerosis due to shear-dependent, disease-preventing endothelial cell reactions. In recent years, researchers have been investigating the role of mechanosensing in the development and progression of atherosclerosis. At the core of mechanosensing is the ability of various cells to sense and respond to biomechanical forces in their environment. In the context of atherosclerosis, endothelial cells, smooth muscle cells, and immune cells are subjected to various mechanical or physical stimuli, including shear stress, cyclic strain, and matrix stiffness. These mechanical cues play a crucial role in regulating cellular behavior and contribute to the pathophysiology of atherosclerosis. Accumulating evidence suggests that various mechanical or physical cues play a critical role in the development and promotion of atherosclerosis.

Role of mechanosensitive channels/receptors in atherosclerosis.

Mechanical forces are critical physical cues that can affect numerous cellular processes regulating the development, tissue maintenance, and functionality of cells. The contribution of mechanical forces is especially crucial in the vascular system where it is required for embryogenesis and for maintenance of physiological function in vascular cells including aortic endothelial cells, resident macrophages, and smooth muscle cells. Emerging evidence has also identified a role of these mechanical cues in pathological conditions of the vascular system such as atherosclerosis and associated diseases like hypertension. Of the different mechanotransducers, mechanosensitive ion channels/receptors are gaining prominence due to their involvement in numerous physiological and pathological conditions. However, only a handful of potential mechanosensory ion channels/receptors have been shown to be involved in atherosclerosis, and their precise role in disease development and progression remains poorly understood. Here, we provide a comprehensive account of recent studies investigating the role of mechanosensitive ion channels/receptors in atherosclerosis. We discuss the different groups of mechanosensitive proteins and their specific roles in inflammation, endothelial dysfunction, macrophage foam cell formation, and lesion development, which are crucial for the development and progression of atherosclerosis. Results of the studies discussed here will help in developing an understanding of the current state of mechanobiology in vascular diseases, specifically in atherosclerosis, which may be important for the development of innovative and targeted therapeutics for this disease.

Role of macrophage TRPV4 in inflammation.

Transient receptor ion channels have emerged as immensely important channels/receptors in diverse physiological and pathological responses. Of particular interest is the transient receptor potential channel subfamily V member 4 (TRPV4), which is a polymodal, nonselective, calcium-permeant cation channel, and is activated by both endogenous and exogenous stimuli. Both neuronal and nonneuronal cells express functional TRPV4, which is responsive to a variety of biochemical and biomechanical stimuli. Emerging discoveries have advanced our understanding of the role of macrophage TRPV4 in numerous inflammatory diseases. In lung injury, TRPV4 mediates macrophage phagocytosis, secretion of pro-resolution cytokines, and generation of reactive oxygen species. TRPV4 regulates lipid-laden macrophage foam cell formation, the hallmark of atheroinflammatory conditions, in response to matrix stiffness and lipopolysaccharide stimulation. Accumulating data also point to a role of macrophage TRPV4 in the pathogenesis of the foreign body response, a chronic inflammatory condition, through the formation of foreign body giant cells. Deletion of TRPV4 in macrophages suppresses the allergic and nonallergic itch in a mouse model, suggesting a role of TRPV4 in skin disease. Here, we discuss the current understanding of the role of macrophage TRPV4 in various inflammatory conditions.

Mechanisms underlying TRPV4-mediated regulation of miR-146a expression.

Persistent inflammation is a major contributor in the development of various inflammatory diseases like atherosclerosis. Our study investigates how transient receptor potential vanilloid 4 (TRPV4), a mechanosensitive ion channel, interacts with microRNA-146a (miR-146a), within the context of inflammation and atherosclerosis. Micro-RNAs play a critical role in controlling gene expression, and miR-146a is notable for its anti-inflammatory actions. TRPV4 is activated by diverse soluble and mechanical stimuli, and often associated with inflammatory responses in various diseases. Here, we find that TRPV4 negatively regulates miR-146a expression in macrophages, especially following stimulation by lipopolysaccharides or alterations in matrix stiffness. We show that in atherosclerosis, a condition characterized by matrix stiffening, TRPV4 decreases miR-146a expression in aortic tissue macrophages. We find that TRPV4's impact on miR-146a is independent of activation of NFκB, Stat1, P38, and AKT, but is rather mediated through a mechanism involving histone deacetylation instead of DNA methylation at the miR-146a promoter site. Furthermore, we show that N-terminal residues 1 to 130 in TRPV4 is essential in suppression of miR-146a expression in LPS-stimulated macrophages. Altogether, this study identifies a regulatory mechanism of miR-146a expression by TRPV4 which may open new potential therapeutic strategies for managing inflammatory diseases.

Macrophage microRNA-146a is a central regulator of the foreign body response to biomaterial implants.

Host recognition and immune-mediated foreign body response (FBR) to biomaterials can adversely affect the functionality of implanted materials. To identify key targets underlying the generation of FBR, here we perform analysis of microRNAs (miR) and mRNAs responses to implanted biomaterials. We found that (a) miR-146a levels inversely affect macrophage accumulation, foreign body giant cell (FBGC) formation, and fibrosis in a murine implant model; (b) macrophage-derived miR-146a is a crucial regulator of the FBR and FBGC formation, as confirmed by global and cell-specific knockout of miR-146a; (c) miR-146a modulates genes related to inflammation, fibrosis, and mechanosensing; (d) miR-146a modulates tissue stiffness near the implant during FBR; and (e) miR-146a is linked to F-actin production and cellular traction force induction, which are vital for FBGC formation. These novel findings suggest that targeting macrophage miR-146a could be a selective strategy to inhibit FBR, potentially improving the biocompatibility of biomaterials.

Identification and functional analysis of a biflavone as a novel inhibitor of transient receptor potential vanilloid 4-dependent atherogenic processes.

Atherosclerosis, a chronic inflammatory disease of large arteries, is the major contributor to the growing burden of cardiovascular disease-related mortality and morbidity. During early atherogenesis, as a result of inflammation and endothelial dysfunction, monocytes transmigrate into the aortic intimal areas, and differentiate into lipid-laden foam cells, a critical process in atherosclerosis. Numerous natural compounds such as flavonoids and polyphenols are known to have anti-inflammatory and anti-atherogenic properties. Herein, using a fluorometric imaging plate reader-supported Ca2+ influx assay, we report semi high-throughput screening-based identification of ginkgetin, a biflavone, as a novel inhibitor of transient receptor potential vanilloid 4 (TRPV4)-dependent proatherogenic and inflammatory processes in macrophages. We found that ginkgetin (1) blocks TRPV4-elicited Ca2+ influx into macrophages, (2) inhibits oxidized low-density lipoprotein (oxLDL)-induced foam cell formation by suppressing the uptake but not the binding of oxLDL in macrophages, and (3) attenuates oxLDL-induced phosphorylation of JNK2, expression of TRPV4 proteins, and induction of inflammatory mRNAs. Considered all together, the results of this study show that ginkgetin inhibits proatherogenic/inflammatory macrophage function in a TRPV4-dependent manner, thus strengthening the rationale for the use of natural compounds for developing therapeutic and/or chemopreventive molecules.

Mechanosensing by TRPV4 mediates stiffness-induced foreign body response and giant cell formation.

Implantation of biomaterials or devices into soft tissue often leads to the development of the foreign body response (FBR), an inflammatory condition that can cause implant failure, tissue injury, and death of the patient. Macrophages accumulate and fuse to generate destructive foreign body giant cells (FBGCs) at the tissue-implant interface, leading to the development of fibrous scar tissue around the implant that is generated by myofibroblasts. We previously showed that the FBR in vivo and FBGC formation in vitro require transient receptor potential vanilloid 4 (TRPV4), a mechanosensitive ion channel. Here, we report that TRPV4 was required specifically for the FBR induced by implant stiffness independently of biochemical cues and for intracellular stiffening that promotes FBGC formation in vitro. TRPV4 deficiency reduced collagen deposition and the accumulation of macrophages, FBGCs, and myofibroblasts at stiff, but not soft, implants in vivo and inhibited macrophage-induced differentiation of wild-type fibroblasts into myofibroblasts in vitro. Atomic force microscopy demonstrated that TRPV4 was required for implant-adjacent tissue stiffening in vivo and for cytoskeletal remodeling and intracellular stiffening induced by fusogenic cytokines in vitro. Together, these data suggest a mechanism whereby a reciprocal functional interaction between TRPV4 and substrate stiffness leads to cytoskeletal remodeling and cellular force generation to promote FBGC formation during the FBR.

TRPV4 Plays a Role in Matrix Stiffness-Induced Macrophage Polarization.

Phenotypic polarization of macrophages is deemed essential in innate immunity and various pathophysiological conditions. We have now determined key aspects of the molecular mechanism by which mechanical cues regulate macrophage polarization. We show that Transient Receptor Potential Vanilloid 4 (TRPV4), a mechanosensitive ion channel, mediates substrate stiffness-induced macrophage polarization. Using atomic force microscopy, we showed that genetic ablation of TRPV4 function abrogated fibrosis-induced matrix stiffness generation in skin tissues. We have determined that stiffer skin tissue promotes the M1 macrophage subtype in a TRPV4-dependent manner; soft tissue does not. These findings were further validated by our in vitro results which showed that stiff matrix (50 kPa) alone increased expression of macrophage M1 markers in a TRPV4-dependent manner, and this response was further augmented by the addition of soluble factors; neither of which occurred with soft matrix (1 kPa). A direct requirement for TRPV4 in M1 macrophage polarization spectrum in response to increased stiffness was evident from results of gain-of-function assays, where reintroduction of TRPV4 significantly upregulated the expression of M1 markers in TRPV4 KO macrophages. Together, these data provide new insights regarding the role of TRPV4 in matrix stiffness-induced macrophage polarization spectrum that may be explored in tissue engineering and regenerative medicine and targeted therapeutics.