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.

Streptococcus gordonii Supragingival Bacterium Oral Infection-Induced Periodontitis and Robust miRNA Expression Kinetics.

Streptococcus gordonii (S. gordonii, Sg) is one of the early colonizing, supragingival commensal bacterium normally associated with oral health in human dental plaque. MicroRNAs (miRNAs) play an important role in the inflammation-mediated pathways and are involved in periodontal disease (PD) pathogenesis. PD is a polymicrobial dysbiotic immune-inflammatory disease initiated by microbes in the gingival sulcus/pockets. The objective of this study is to determine the global miRNA expression kinetics in S. gordonii DL1-infected C57BL/6J mice. All mice were randomly divided into four groups (n = 10 mice/group; 5 males and 5 females). Bacterial infection was performed in mice at 8 weeks and 16 weeks, mice were euthanized, and tissues harvested for analysis. We analyzed differentially expressed (DE) miRNAs in the mandibles of S. gordonii-infected mice. Gingival colonization/infection by S. gordonii and alveolar bone resorption (ABR) was confirmed. All the S. gordonii-infected mice at two specific time points showed bacterial colonization (100%) in the gingival surface, and a significant increase in mandible and maxilla ABR (p < 0.0001). miRNA profiling revealed 191 upregulated miRNAs (miR-375, miR-34b-5p) and 22 downregulated miRNAs (miR-133, miR-1224) in the mandibles of S. gordonii-infected mice at the 8-week mark. Conversely, at 16 weeks post-infection, 10 miRNAs (miR-1902, miR-203) were upregulated and 32 miRNAs (miR-1937c, miR-720) were downregulated. Two miRNAs, miR-210 and miR-423-5p, were commonly upregulated, and miR-2135 and miR-145 were commonly downregulated in both 8- and 16-week-infected mice mandibles. Furthermore, we employed five machine learning (ML) algorithms to assess how the number of miRNA copies correlates with S. gordonii infections in mice. In the ML analyses, miR-22 and miR-30c (8-week), miR-720 and miR-339-5p (16-week), and miR-720, miR-22, and miR-339-5p (combined 8- and 16-week) emerged as the most influential miRNAs.

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.

Specific microRNA Signature Kinetics in Porphyromonas gingivalis-Induced Periodontitis.

Porphyromonas gingivalis is one of the major bacteria constituting the subgingival pathogenic polymicrobial milieu during periodontitis. Our objective is to determine the global microRNA (miRNA, miR) expression kinetics in 8- and 16-weeks duration of P. gingivalis infection in C57BL/6J mice and to identify the miRNA signatures at specific time-points in mice. We evaluated differential expression (DE) miRNAs in mandibles (n = 10) using high-throughput NanoString nCounter® miRNA expression panels. The bacterial colonization, alveolar bone resorption (ABR), serum immunoglobulin G (IgG) antibodies, and bacterial dissemination were confirmed. In addition, all the infected mice showed bacterial colonization on the gingival surface, significant increases in ABR (p < 0.0001), and specific IgG antibody responses (p < 0.05-0.001). The miRNA profiling showed 26 upregulated miRNAs (e.g., miR-804, miR-690) and 14 downregulated miRNAs (e.g., miR-1902, miR-1937a) during an 8-weeks infection, whereas 7 upregulated miRNAs (e.g., miR-145, miR-195) and one downregulated miR-302b were identified during a 16-weeks infection. Both miR-103 and miR-30d were commonly upregulated at both time-points, and all the DE miRNAs were unique to the specific time-points. However, miR-31, miR-125b, miR-15a, and miR-195 observed in P. gingivalis-infected mouse mandibles were also identified in the gingival tissues of periodontitis patients. None of the previously identified miRNAs reported in in vitro studies using cell lines (periodontal ligament cells, gingival epithelial cells, human leukemia monocytic cell line (THP-1), and B cells) exposed to P. gingivalis lipopolysaccharide were observed in the in vivo study. Most of the pathways (endocytosis, bacterial invasion, and FcR-mediated phagocytosis) targeted by the DE miRNAs were linked with bacterial pathogen recognition and clearance. Further, eighteen miRNAs were closely associated with the bacterial invasion of epithelial cells. This study highlights the altered expression of miRNA in gingiva, and their expression depends on the time-points of infection. This is the first in vivo study that identified specific signature miRNAs (miR-103 and miR-30d) in P. gingivalis invasion of epithelial cells, establishes a link between miRNA and development of periodontitis and helping to better understand the pathobiology of periodontitis.

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.

Mechanotransduction via a TRPV4-Rac1 signaling axis plays a role in multinucleated giant cell formation.

Multinucleated giant cells are formed by the fusion of macrophages and are a characteristic feature in numerous pathophysiological conditions including the foreign body response (FBR). Foreign body giant cells (FBGCs) are inflammatory and destructive multinucleated macrophages and may cause damage and/or rejection of implants. However, while these features of FBGCs are well established, the molecular mechanisms underlying their formation remain elusive. Improved understanding of the molecular mechanisms underlying the formation of FBGCs may permit the development of novel implants that eliminate or reduce the FBR. Our previous study showed that transient receptor potential vanilloid 4 (TRPV4), a mechanosensitive ion channel/receptor, is required for FBGC formation and FBR to biomaterials. Here, we have determined that (a) TRPV4 is directly involved in fusogenic cytokine (interleukin-4 plus granulocyte macrophage-colony stimulating factor)-induced activation of Rac1, in bone marrow-derived macrophages; (b) TRPV4 directly interacts with Rac1, and their interaction is further augmented in the presence of fusogenic cytokines; (c) TRPV4-dependent activation of Rac1 is essential for the augmentation of intracellular stiffness and regulation of cytoskeletal remodeling; and (d) TRPV4-Rac1 signaling axis is critical in fusogenic cytokine-induced FBGC formation. Together, these data suggest a novel mechanism whereby a functional interaction between TRPV4 and Rac1 leads to cytoskeletal remodeling and intracellular stiffness generation to modulate FBGC formation.

TRPV4 is dispensable for the development of airway allergic asthma.

Allergic asthma is one of the most common immune-mediated disorders affecting the lungs. It is characterized clinically by airway hyperresponsiveness, eosinophilia, enhanced IL-4 and IL-13, peribronchial inflammation with mononuclear cell infiltration, and goblet cell hyperplasia associated with increased mucus production. However, chronic asthma with repeated exposures to inhaled allergens can result in subepithelial pulmonary fibrosis. The transient receptor potential cation channel subfamily V member 4 (TRPV4) protein can promote the generation of myofibroblasts and pulmonary fibrosis. Here, we investigated the possibility that TPRV4 facilitates the development of allergic asthma and subsequent pulmonary fibrosis in the lung. To test this, wild-type (WT) and TPRV4 gene knockout (KO) mice were repeatedly sensitized with chicken ovalbumin (OVA) and repeatedly subjected to aerosol challenge with 1% OVA. We found that there were no significant differences in the development of allergic asthma between the WT and TPRV4 KO mice. Both groups of mice exhibited similar levels of airway hyperresponsiveness, IL-13, IL-5, OVA-specific IgE, eosinophilia, mucus-secreting goblet cell hyperplasia, and deposition of collagen fiber, which is a hallmark of the pulmonary fibrosis. Thus, these data suggest that TPRV4 protein is dispensable in the initiation and development of airway asthma and subsequent fibrosis.

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.

Transient Receptor Potential Vanilloid 4 Is Required for Foreign Body Response and Giant Cell Formation.

The presence of biomaterials and devices implanted into soft tissue is associated with development of a foreign body response (FBR), a chronic inflammatory condition that can ultimately lead to implant failure, which may cause harm to or death of the patient. Development of FBR includes activation of macrophages at the tissue-implant interface, generation of destructive foreign body giant cells (FBGCs), and generation of fibrous tissue that encapsulates the implant. However, the mechanisms underlying the FBR remain poorly understood, as neither the materials composing the implants nor their chemical properties can explain triggering of the FBR. Herein, we report that genetic ablation of transient receptor potential vanilloid 4 (TRPV4), a Ca2+-permeable mechanosensitive cation channel in the transient receptor potential vanilloid family, protects TRPV4 knockout mice from FBR-related events. The mice showed diminished collagen deposition along with reduced macrophage accumulation and FBGC formation compared with wild-type mice in a s.c. implantation model. Analysis of macrophage markers in spleen tissues and peritoneal cavity showed that the TRPV4 deficiency did not impair basal macrophage maturation. Furthermore, genetic deficiency or pharmacologic antagonism of TRPV4 blocked cytokine-induced FBGC formation, which was restored by lentivirus-mediated TRPV4 reintroduction. Taken together, these results suggest an important, previously unknown, role for TRPV4 in FBR.

Role of TRPV4 in matrix stiffness-induced expression of EMT-specific LncRNA.

Long non-coding RNAs (LncRNAs) are long (> 200 bases), non-coding, single-stranded RNAs that have emerged as major regulators of gene expression, cell differentiation, development, and oncogenesis. In view of the fact that matrix stiffness plays a role in cellular functions associated with these processes, it is important to ask what role matrix stiffness plays in regulating expression of LncRNAs. In this report, we show that (i) matrix stiffness causes differential expression of epithelial-mesenchymal transition (EMT)-related LncRNAs and mRNAs in primary mouse normal epidermal keratinocytes, (ii) differential expression of EMT-related LncRNAs and mRNAs occurs in response to combined stimulation of transforming growth factor ß1 and matrix stiffness, and (iii) transient receptor potential (TRP) channel of the vanilloid subfamily, TRPV4, a matrix stiffness-sensitive ion channel, plays a role in differential expression of EMT-related LncRNAs and mRNAs in response to combined stimulation by TGFß1 and matrix stiffness. These data identify TRPV4 as a candidate plasma membrane mechanosensor that transmits matrix-sensing signals essential to LncRNA expression. Our results also show that we have established and validated an assay system capable of discovering novel LncRNAs and mRNAs sensitive to matrix stiffening.

Effectiveness of probiotics, prebiotics, and prebiotic-like components in common functional foods.

The bioactive ingredients in commonly consumed foods include, but are not limited to, prebiotics, prebiotic-like components, probiotics, and postbiotics. The bioactive ingredients in functional foods have also been associated with beneficial effects on human health. For example, they aid in shaping of gut microflora and promotion of immunity. These functional components also contribute in preventing serious diseases such as cardiovascular malfunction and tumorigenesis. However, the specific mechanisms of these positive influences on human health are still under investigation. In this review, we aim to emphasize the major contents of probiotics, prebiotics, and prebiotic-like components commonly found in consumable functional foods, and we present an overview of direct and indirect benefits they provide on human health. The major contributors are certain families of metabolites, specifically short-chain fatty acids and polyunsaturated fatty acids produced by probiotics, and prebiotics, or prebiotic-like components such as flavonoids, polyphenols, and vitamins that are found in functional foods. These functional ingredients in foods influence the gut microbiota by stimulating the growth of beneficial microbes and the production of beneficial metabolites that, in turn, have direct benefits to the host, while also providing protection from pathogens and maintaining a balanced gut ecosystem. The complex interactions that arise among functional food ingredients, human physiology, the gut microbiota, and their respective metabolic pathways have been found to minimize several factors that contribute to the incidence of chronic disease, such as inflammation oxidative stress.

TRPV4 regulates matrix stiffness and TGFß1-induced epithelial-mesenchymal transition.

Substrate stiffness (or rigidity) of the extracellular matrix has important functions in numerous pathophysiological processes including fibrosis. Emerging data support a role for both a mechanical signal, for example, matrix stiffness, and a biochemical signal, for example, transforming growth factor ß1 (TGFß1), in epithelial-mesenchymal transition (EMT), a process critically involved in fibrosis. Here, we report evidence showing that transient receptor potential vanilloid 4 (TRPV4), a mechanosensitive channel, is the likely mediator of EMT in response to both TGFß1 and matrix stiffness. Specifically, we found that: (a) genetic ablation or pharmacological inhibition of TRPV4 blocked matrix stiffness and TGFß1-induced EMT in normal mouse primary epidermal keratinocytes (NMEKs) as determined by changes in morphology, adhesion, migration and alterations of expression of EMT markers including E-cadherin, N-cadherin (NCAD) and α-smooth muscle actin (α-SMA), and (b) TRPV4 deficiency prevented matrix stiffness-induced EMT in NMEKs over a pathophysiological range. Intriguingly, TRPV4 deletion in mice suppressed expression of mesenchymal markers, NCAD and α-SMA, in a bleomycin-induced murine skin fibrosis model. Mechanistically, we found that: (a) TRPV4 was essential for the nuclear translocation of YAP/TAZ (yes-associated protein/transcriptional coactivator with PDZ-binding motif) in response to matrix stiffness and TGFß1, (b) TRPV4 deletion inhibited both matrix stiffness- and TGFß1-induced expression of YAP/TAZ proteins and (c) TRPV4 deletion abrogated both matrix stiffness- and TGFß1-induced activation of AKT, but not Smad2/3, suggesting a mechanism by which TRPV4 activity regulates EMT in NMEKs. Altogether, these data identify a novel role for TRPV4 in regulating EMT.

Increased carotid intima-media thickness in African American pediatric kidney transplant recipients.

Early signs of subclinical CV dysfunction can be detected by ultrasound for CIMT. Although A-A are at high risk for CV disease, CIMT of A-A kidney transplant recipients has not been previously investigated. The aim of this prospective, controlled, longitudinal study was to investigate determinants of CIMT in a multiracial pediatric kidney transplant population, with a focus on A-A. Transplant recipients (n = 42) had BMI, waist-to-height ratio, fasting glucose, lipid panel, HbA1c%, and CIMT measured at 1, 18, and 30 months post-transplant. Twenty-four healthy children (14 A-A) served as controls. CIMT of A-A transplant (0.49, 0.49, and 0.48 mm) was higher than non-AA transplant (0.43, 0.44, and 0.44 mm) at 1, 18, and 30 months and higher than A-A controls (0.47 mm). Hyperparathyroidism prior to transplant predicted high CIMT-for-race. A-A race was associated with 10% higher CIMT vs non-A-A transplant. Metabolic syndrome was associated with 0.03 ± 0.01 mm increase in CIMT among A-A transplant recipients only. In conclusion, A-A kidney transplant recipients have increased CIMT. Metabolic syndrome disproportionately affects CIMT of A-A children post-transplant. Identification of subclinical CV damage, detected by CIMT, may provide an opportunity for early detection of CV risk in this vulnerable population.

Correction to: Effects of obesity and metabolic syndrome on cardiovascular outcomes in pediatric kidney transplant recipients: a longitudinal study.

As originally published, this article contained errors owing to oversights in typesetting. The article has now been amended accordingly.

Effects of obesity and metabolic syndrome on cardiovascular outcomes in pediatric kidney transplant recipients: a longitudinal study.

BACKGROUND: Obesity and metabolic syndrome (MS) are common after kidney transplantation, but their contribution to adverse cardiovascular (CV) outcomes in children are not well known. A prospective, controlled, longitudinal cohort study was conducted to investigate the effects of obesity and MS on left ventricular hypertrophy (LVH) and myocardial strain in pediatric kidney transplant recipients. METHODS: Transplant recipients (n = 42) had anthropometrics [body mass index (BMI), waist circumference, waist-to-height ratio], biochemical parameters (fasting glucose, lipid panel, HbA1c%), and echocardiogram with speckle tracking analysis for strain measured at 1, 18, and 30 months post-transplant. Additionally, 35 pre-transplant echocardiograms were analyzed retrospectively. Healthy children (n = 24) served as controls. RESULTS: Waist-to-height ratio detected abdominal obesity in 46% of transplant patients, whereas only 8.1% were identified as obese by waist circumference. Ejection fraction and fractional shortening of the transplant group were normal. Prevalence of LVH was 35.2%, 17.1%, and 35.5% at 1, 18, and 30 months respectively. The longitudinal strain of transplant group was worse than controls at all time points (p < 0.001). Hemodialysis was independently associated with 21% worse longitudinal strain during the pre-transplant period (p = 0.04). After transplantation, obesity, MS, and systolic hypertension predicted increased odds of LVH (p < 0.04). Worse longitudinal strain was independently associated with obesity, MS, hypertension, and the combination of MS with elevated low density lipoprotein (LDL) cholesterol (p < 0.04), whereas higher estimated glomerular filtration rate (eGFR) conferred a protective effect (p < 0.001). CONCLUSION: Obesity and MS adversely affect CV outcomes after transplantation. Further studies are needed to investigate speckle tracking echocardiography as a tool for early detection of subclinical myocardial dysfunction in this population.

TRPV4 ion channel is a novel regulator of dermal myofibroblast differentiation.

Scleroderma is a multisystem fibroproliferative disease with no effective medical treatment. Myofibroblasts are critical to the fibrogenic tissue repair process in the skin and many internal organs. Emerging data support a role for both matrix stiffness, and transforming growth factor ß1 (TGFß1), in myofibroblast differentiation. Transient receptor potential vanilloid 4 (TRPV4) is a mechanosensitive ion channel activated by both mechanical and biochemical stimuli. The objective of this study was to determine the role of TRPV4 in TGFß1- and matrix stiffness-induced differentiation of dermal fibroblasts. We found that TRPV4 channels are expressed and functional in both human (HDF) and mouse (MDF) dermal fibroblasts. TRPV4 activity (agonist-induced Ca2+ influx) was induced by both matrix stiffness and TGFß1 in dermal fibroblasts. TGFß1 induced expression of TRPV4 proteins in a dose-dependent manner. Genetic ablation or pharmacological antagonism of TRPV4 channel abrogated Ca2+ influx and both TGFß1-induced and matrix stiffness-induced myofibroblast differentiation as assessed by 1) α-smooth muscle actin expression/incorporation into stress fibers, 2) generation of polymerized actin, and 3) expression of collagen-1. We found that TRPV4 inhibition abrogated TGFß1-induced activation of AKT but not of Smad2/3, suggesting that the mechanism by which profibrotic TGFß1 signaling in dermal fibroblasts is modified by TRPV4 may be through non-Smad pathways. Altogether, these data identify a novel reciprocal functional link between TRPV4 activation and TGFß1 signals regulating dermal myofibroblast differentiation. These findings suggest that therapeutic inhibition of TRPV4 activity may provide a targeted approach to the treatment of scleroderma.

Matrix-driven Myosin II Mediates the Pro-fibrotic Fibroblast Phenotype.

Pro-fibrotic mesenchymal cells are known to be the key effector cells of fibroproliferative disease, but the specific matrix signals and the induced cellular responses that drive the fibrogenic phenotype remain to be elucidated. The key mediators of the fibroblast fibrogenic phenotype were characterized using a novel assay system that measures fibroblast behavior in response to actual normal and fibrotic lung tissue. Using this system, we demonstrate that normal lung promotes fibroblast motility and polarization, while fibrotic lung immobilizes the fibroblast and promotes myofibroblast differentiation. These context-specific phenotypes are surprisingly both mediated by myosin II. The role of myosin II is supported by the observation of an increase in myosin phosphorylation and a change in intracellular distribution in fibroblasts on fibrotic lung, as compared with normal lung. Moreover, loss of myosin II activity has opposing effects on protrusive activity in fibroblasts on normal and fibrotic lung. Loss of myosin II also selectively inhibits myofibroblast differentiation in fibroblasts on fibrotic lung. Importantly, these findings are recapitulated by varying the matrix stiffness of polyacrylamide gels in the range of normal and fibrotic lung tissue. Comparison of the effects of myosin inhibition on lung tissue with that of polyacrylamide gels suggests that matrix fiber organization drives the fibroblast phenotype under conditions of normal/soft lung, while matrix stiffness drives the phenotype under conditions of fibrotic/stiff lung. This work defines novel roles for myosin II as a key regulatory effector molecule of the pro-fibrotic phenotype, in response to biophysical properties of the matrix.

Urokinase-type plasminogen activator receptor (uPAR) ligation induces a raft-localized integrin signaling switch that mediates the hypermotile phenotype of fibrotic fibroblasts.

The urokinase-type plasminogen activator receptor (uPAR) is a glycosylphosphatidylinositol-linked membrane protein with no cytosolic domain that localizes to lipid raft microdomains. Our laboratory and others have documented that lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) exhibit a hypermotile phenotype. This study was undertaken to elucidate the molecular mechanism whereby uPAR ligation with its cognate ligand, urokinase, induces a motile phenotype in human lung fibroblasts. We found that uPAR ligation with the urokinase receptor binding domain (amino-terminal fragment) leads to enhanced migration of fibroblasts on fibronectin in a protease-independent, lipid raft-dependent manner. Ligation of uPAR with the amino-terminal fragment recruited α5ß1 integrin and the acylated form of the Src family kinase, Fyn, to lipid rafts. The biological consequences of this translocation were an increase in fibroblast motility and a switch of the integrin-initiated signal pathway for migration away from the lipid raft-independent focal adhesion kinase pathway and toward a lipid raft-dependent caveolin-Fyn-Shc pathway. Furthermore, an integrin homologous peptide as well as an antibody that competes with ß1 for uPAR binding have the ability to block this effect. In addition, its relative insensitivity to cholesterol depletion suggests that the interactions of α5ß1 integrin and uPAR drive the translocation of α5ß1 integrin-acylated Fyn signaling complexes into lipid rafts upon uPAR ligation through protein-protein interactions. This signal switch is a novel pathway leading to the hypermotile phenotype of IPF patient-derived fibroblasts, seen with uPAR ligation. This uPAR dependent, fibrotic matrix-selective, and profibrotic fibroblast phenotype may be amenable to targeted therapeutics designed to ameliorate IPF.

Vav Guanine nucleotide exchange factors regulate atherosclerotic lesion development in mice.

OBJECTIVE: Atherosclerosis requires migration of monocytes to the arterial intima, with subsequent differentiation into foam cells. We showed previously that the scavenger receptor CD36 contributes to the activation of Vav family guanine nucleotide exchange factors (Vavs) in aortae from hyperlipidemic apoE-null mice and that oxidatively modified low-density lipoprotein induced CD36-dependent activation of macrophage Vavs in vitro. We also discovered that CD36-dependent uptake of oxidized low-density lipoprotein and foam cell formation were reduced in Vav-deficient macrophages. We now tested the hypothesis that Vavs play a role in atherosclerotic lesion development. APPROACH AND RESULTS: We showed that apoE/vav1 double-null mice fed a Western diet had significant reduction in total aortic lesion area (by en face analysis) compared with apoE-null mice, with no significant differences in body weight or plasma lipid profiles. Histological analysis of aortic sinus lesions showed fewer macrophages and foam cells in double-null mice compared with apoE-null mice, indicating impaired foam cell generation and homing of macrophages to atherosclerotic lesions. An intravital video microscopy-based adhesion assay with fluorescent (Qtracker655)-labeled monocytes showed reduced adhesion of vav1-null monocytes to hyperlipidemic carotid arteries compared with wild-type monocytes. Furthermore, fewer fluorescently labeled vav1-null monocytes accumulated in aortic sinus lesions in hyperlipidemic apoE-null mice. We also found that activation of RhoGTPase Rac and mitogen-activated protein kinase c-Jun N-terminal kinase-2 by CD36-specific oxidized phospholipids was dependent on Vavs. CONCLUSIONS: These results for the first time link Vavs to atherosclerotic lesion development and suggest that Vavs act as critical molecular links coupling hyperlipidemia with proatherogenic monocyte/macrophage responses.

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.