Biomedical subjects

Andreea Trache

Publications and source records attributed to Andreea Trache.

6 recordsLinked to original sources

PubMed2006

Quantification and confocal imaging of protein specific molecularly imprinted polymers.

We have employed FITC--albumin as the protein template molecule in an aqueous phase molecular imprinted polymer (HydroMIP) strategy. For the first time, the use of a fluorescently labeled template is reported, with subsequent characterization of the smart material to show that the HydroMIP possesses a significant molecular memory in comparison to that of the nonimprinted control polymer (HydroNIP). The imaging of the FITC--albumin imprinted HydroMIP using confocal microscopy is described, with the in situ removal of the imprinted protein displayed in terms of observed changes in the fluorescence of the imprinted polymer, both before and after template elution (using a 10% SDS/10% AcOH (w/v) solution). We also report the imaging of a bovine hemoglobin (BHb) imprinted HydroMIP using two-photon confocal microscopy and describe the effects of template elution upon protein autofluorescence. The findings further contribute to the understanding of aqueous phase molecular imprinting protocols and document the use of fluorescence as a useful tool in template labeling/detection and novel imaging strategies.

Albumins↗

PubMed2005

Mechanical properties of the interaction between fibronectin and alpha5beta1-integrin on vascular smooth muscle cells studied using atomic force microscopy.

The mechanical properties of integrin-extracellular matrix (ECM) interactions are important for the mechanotransduction of vascular smooth muscle cells (VSMC), a process that is associated with focal adhesions, and can be of particular significance in cardiovascular disease. In this study, we characterized the unbinding force and binding activity of the initial fibronectin (FN)-alpha5beta1 interaction on the surface of VSMC using atomic force microscopy (AFM). It is postulated that these initial binding events are important to the subsequent focal adhesion assembly. FN-VSMC adhesions were selectively blocked by antibodies against alpha5- and beta1-integrins as well as RGD-containing peptides but not by antibodies against alpha4- and beta3-integrins, indicating that FN primarily bound to alpha5beta1. A characteristic unbinding force of 39 +/- 8 pN was observed and interpreted to represent the FN-alpha5beta1 single-bond strength. The ability of FN to adhere to VSMC (binding probability) was significantly reduced by integrin antagonists, serum starvation, and platelet-derived growth factor (PDGF)-BB, whereas lysophosphatidic acid (LPA) increased FN binding. However, no significant change in the resolved unbinding force was observed. After engagement, the force required to dislodge the FN-coated bead from VSMC increased with increasing of contact time, suggesting a time-dependent increase in number of adhesions and/or altered binding affinity. LPA enhanced this process, whereas PDGF reduced it, suggesting that these factors also affect the multimolecular process of focal contact assembly. Thus AFM is a powerful tool for the characterization of the mechanical properties of integrin-ECM interactions and their regulation. Our results indicate that the functional activity of alpha5beta1 and focal contact assembly can be rapidly regulated.

Animals↗

PubMed2005

Histamine effects on endothelial cell fibronectin interaction studied by atomic force microscopy.

Atomic force microscopy was used to investigate the cellular response to histamine, one of the major inflammatory mediators that cause endothelial hyperpermeability and vascular leakage. AFM probes were labeled with fibronectin and used to measure binding strength between alpha5beta1 integrin and fibronectin by quantifying the force required to break single fibronectin-integrin bonds. The cytoskeletal changes, binding probability, and adhesion force before and after histamine treatment on endothelial cells were monitored. Cell topography measurements indicated that histamine induces cell shrinkage. Local cell stiffness and binding probability increased twofold after histamine treatment. The force necessary to rupture single alpha5beta1-fibronectin bond increased from 34.0 +/- 0.5 pN in control cells to 39 +/- 1 pN after histamine treatment. Experiments were also conducted to confirm the specificity of the alpha5beta1-fibronectin interaction. In the presence of soluble GRGDdSP the probability of adhesion events decreased >50% whereas the adhesion force between alpha5beta1 and fibronectin remained unchanged. These data indicate that extracellular matrix-integrin interactions play an important role in the endothelial cell response to changes of external chemical mediators. These changes can be recorded as direct measurements on live endothelial cells by using atomic force microscopy.

Animals↗

PubMedDate not supplied

Integrins and regulation of the microcirculation: from arterioles to molecular studies using atomic force microscopy.

Integrins are an important class of receptors for extracellular matrix proteins that can mediate both force transmission, by virtue of their connections with the cell matrix and cytoskeleton; and signal transduction, resulting from the assemblages of signaling proteins that associate with focal contacts. Consequently, integrins have been proposed to be the mechanosensor in vascular smooth muscle and endothelial cells and to play a central role in mechanotransduction. In this regard, mechanical force is an important stimulus for many vascular functions, including contractile and relaxation processes,proliferation, migration, attachment, and cell phenotype determination. Collectively, these functions define physiological properties of the vasculature such as control of blood flow, capillary pressure,permeability, and peripheral vascular resistance, and play a role in pathophysiological processes like hypertension, diabetes, and arteriosclerosis. Our knowledge concerning how integrins sense and transduce physical forces into cellular signals and which integrins are involved is incomplete. Compared to other cell surface receptors, integrins have a relatively low affinity for their binding sites on the extracellular matrix and their affinity can be regulated. These characteristics of integrin-ligand interaction may facilitate dynamic processes such as cell migration, cell remodeling, and contractile activation in response to external forces. Important questions remain concerning the nature and origin of integrin-mediated signaling in the vascular wall.

Animals↗

PubMedDate not supplied

Atomic force-multi-optical imaging integrated microscope for monitoring molecular dynamics in live cells.

A novel hybrid imaging system is constructed integrating atomic force microscopy (AFM) with a combination of optical imaging techniques that offer high spatial resolution. The main application of this instrument (the NanoFluor microscope) is the study of mechanotransduction with an emphasis on extracellular matrix-integrin-cytoskeletal interactions and their role in the cellular responses to changes in external chemical and mechanical factors. The AFM allows the quantitative assessment of cytoskeletal changes, binding probability, adhesion forces, and micromechanical properties of the cells, while the optical imaging applications allow thin sectioning of the cell body at the coverslip-cell interface, permitting the study of focal adhesions using total internal reflection fluorescence (TIRF) and internal reflection microscopy (IRM). Combined AFM-optical imaging experiments show that mechanical stimulation at the apical surface of cells induces a force-generating cytoskeletal response, resulting in focal contact reorganization on the basal surface that can be monitored in real time. The NanoFluor system is also equipped with a novel mechanically aligned dual camera acquisition system for synthesized Forster resonance energy transfer (FRET). The integrated NanoFluor microscope system is described, including its characteristics, applications, and limitations.

Animals↗

PubMedDate not supplied

Use of surface-enhanced Raman spectroscopy for the detection of human integrins.

Current research has revealed the importance of a class of cell surface proteins called integrins in various vital physiological functions such as blood clotting, regulation of blood pressure, tissue blood flow, and vascular remodeling. The key to integrin functionality is its ability to mediate force transmission by interacting with the extracellular matrix and cytoskeleton. In addition, they play a role in signal transduction via their connection with the proteins in focal adhesion (FA) points. To understand the complex mechanism of cell-cell and cell-extracellular matrix (ECM) adhesion that is responsible for these diverse biochemical interactions, it is necessary to identify the integrins on cells and monitor their interaction with various ligands. To this end, for the first time, we employ surface-enhanced Raman spectroscopy (SERS) to detect integrins. The results show the capability using SERS to detect the integrins to the nanomolar concentration regime and to distinguish between two different kinds of integrins, alphaVbeta3 and alpha5beta1, that are present in vascular smooth muscle cells (VSMCs). It is anticipated that the SERS approach will potentially help elucidate the mechanism of integrin-ligand interactions in a variety of phenomena of physiological importance.

Colloids↗