No abstract
The ability to measure real-time mechanosensitive events at the subcellular level in response to discrete mechanical stimulation is a critical component in understanding mechanically-induced cellular remodeling. Vascular smooth muscle cells (VSMC) were transfected with RhoA constructs (wild type, dominant negative or constitutively active) or treated with ML-7 to induce specific cytoskeletal tension characteristics prior to mechanical stimulation. Tensile stress was applied to live VSMC using an atomic force microscope probe functionalized with extracellular matrix (ECM) proteins. The ECM induces selective integrin activation and focal adhesion formation, enabling direct manipulation of cortical actin through an active ECM-integrin-actin linkage. Therefore, locally induced mechanosensitive events triggered downstream activation of intracellular signaling pathways responsible for actin and focal adhesion remodeling throughout the cell. Integration of mechanical stimulation with simultaneous fluorescence imaging by spinning-disk confocal and total internal reflection fluorescence microscopy enabled visualization and quantification of molecular dynamic events at the sub-cellular level in real-time. Results provide evidence that the pre-existing cytoskeletal tension affects the actomyosin apparatus which in turn coordinates the ability of the cell to adapt to the externally applied stress. RhoA activation induced high cytoskeletal tension that correlated with increased stress fiber formation, cell stiffness, integrin activation and myosin phosphorylation. In contrast, blocking Rho-kinase or myosin function was characterized by low cytoskeletal tension with a decreased level of stress fiber formation, lower cell stiffness and integrin activation. Our findings show that VSMC sense and adapt to physical microenvironmental changes by a coordinated response of the actomyosin apparatus necessary to establish a new homeostatic state.
Both monomeric and dimeric tetraacetylglucose-containing {Fe(NO)} dinitrosyl iron complexes (DNICs) were prepared and examined for NO release in the presence of both chemical NO-trapping agents and endothelial cells.
Mechanical force is an important stimulus and determinant of many vascular smooth muscle cell functions including contraction, proliferation, migration, and cell attachment. Transmission of force from outside the cell through focal adhesions controls the dynamics of these adhesion sites and initiates intracellular signaling cascades that alter cellular behavior. To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a critical first step is to develop a new technology to investigate cellular behavior at subcellular level that integrates an atomic force microscope (AFM) with total internal reflection fluorescence (TIRF) and fast-spinning disk (FSD) confocal microscopy, providing high spatial and temporal resolution. AFM uses a nanosensor to measure the cell surface topography and can apply and measure mechanical force with high precision. TIRF microscopy is an optical imaging technique that provides high-contrast images with high z-resolution of fluorescently labeled molecules in the immediate vicinity of the cell-coverslip interface. FSD confocal microscopy allows rapid 3-D imaging throughout the cell in real time. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real time.
Understanding cellular remodeling in response to mechanical stimuli is a critical step in elucidating mechanical activation of biochemical signaling pathways. Experimental evidence indicates that external stress-induced subcellular adaptation is accomplished through dynamic cytoskeletal reorganization. To study the interactions between subcellular structures involved in transducing mechanical signals, we combined experimental data and computational simulations to evaluate real-time mechanical adaptation of the actin cytoskeletal network. Actin cytoskeleton was imaged at the same time as an external tensile force was applied to live vascular smooth muscle cells using a fibronectin-functionalized atomic force microscope probe. Moreover, we performed computational simulations of active cytoskeletal networks under an external tensile force. The experimental data and simulation results suggest that mechanical structural adaptation occurs before chemical adaptation during filament bundle formation: actin filaments first align in the direction of the external force by initializing anisotropic filament orientations, then the chemical evolution of the network follows the anisotropic structures to further develop the bundle-like geometry. Our findings present an alternative two-step explanation for the formation of actin bundles due to mechanical stimulation and provide new insights into the mechanism of mechanotransduction.
In this study, dinitrosyl iron complexes (DNICs) are shown to deliver nitric oxide (NO) into the cytosol of vascular smooth muscle cells (SMCs), which play a major role in vascular relaxation and contraction. Malfunction of SMCs can lead to hypertension, asthma, and erectile dysfunction, among other disorders. For comparison of the five DNIC derivatives, the following protocols were examined: (a) the Griess assay to detect nitrite (derived from NO conversion) in the absence and presence of SMCs; (b) the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium (MTS) assay for cell viability; (c) an immunotoxicity assay to establish if DNICs stimulate immune response; and (d) a fluorometric assay to detect intracellular NO from treatment with DNICs. Dimeric Roussin's red ester (RRE)-type {Fe(NO) 2 } 9 complexes containing phenylthiolate bridges, [(μ-SPh)Fe(NO) 2 ] 2 or SPhRRE, were found to deliver NO with the lowest effect on cell toxicity (i.e., highest IC 50 ). In contrast, the RRE-DNIC with the biocompatible thioglucose moiety, [(μ-SGlu)Fe(NO) 2 ] 2 (SGlu = 1-thio-β-D-glucose tetraacetate) or SGluRRE, delivered a higher concentration of NO to the cytosol of SMCs with a 10-fold decrease in IC 50 . Additionally, monomeric DNICs stabilized by a bulky N-heterocyclic carbene (NHC), namely, 1,3-bis(2,4,6-trimethylphenyl)imidazolidene (IMes), were synthesized and yielded the DNIC complexes SGluNHC, [IMes(SGlu)Fe(NO) 2 ], and SPhNHC, [IMes(SPh)Fe(NO) 2 ]. These oxidized {Fe(NO) 2 } 9 NHC DNICs have an IC 50 of ∼7 μM; however, the NHC-based complexes did not transfer NO into the SMC. Per contra, the reduced, mononuclear {Fe(NO) 2 } 10 neocuproine-based DNIC, neoDNIC, depressed the viability of the SMCs, as well as generated an increase of intracellular NO. Regardless of the coordination environment or oxidation state, all DNICs showed a dinitrosyl iron unit (DNIU)-dependent increase in viability. This study demonstrates a structure−function relationship between the DNIU coordination environment and the efficacy of the DNIC treatments.
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