Irina Akopova

Reactive Oxygen Species-mediated TRPC6 Protein Activation in Vascular Myocytes, a Mechanism for Vasoconstrictor-regulated Vascular Tone

Both TRPC6 and reactive oxygen species (ROS) play an important role in regulating vascular function. However, their interplay has not been explored. The present study examined whether activation of TRPC6 in vascular smooth muscle cells (VSMCs) by ROS was a physiological mechanism for regulating vascular tone by vasoconstrictors. In A7r5 cells, arginine vasopressin (AVP) evoked a striking Ca2+ entry response that was significantly attenuated by either knocking down TRPC6 using siRNA or inhibition of NADPH oxidases with apocynin or diphenyleneiodonium. Inhibition of TRPC6 or ROS production also decreased AVP-stimulated membrane currents. In primary cultured aortic VSMCs, catalase and diphenyleneiodonium significantly suppressed AVP- and angiotensin II-induced whole cell currents and Ca2+ entry, respectively. In freshly isolated and endothelium-denuded thoracic aortas, hyperforin (an activator of TRPC6), but not its vehicle, induced dose- and time-dependent constriction in TRPC6 wide type (WT) mice. This response was not observed in TRPC6 knock-out (KO) mice. Consistent with the ex vivo study, hyperforin stimulated a robust Ca2+ entry in the aortic VSMCs from WT mice but not from KO mice. Phenylephrine induced a dose-dependent contraction of WT aortic segments, and this response was inhibited by catalase. Moreover, H2O2 itself evoked Ca2+ influx and inward currents in A7r5 cells, and these responses were significantly attenuated by either inhibition of TRPC6 or blocking vesicle trafficking. H2O2 also induced inward currents in primary VSMCs from WT but not from TRPC6 KO mice. Additionally, H2O2 stimulated a dose-dependent constriction of the aortas from WT mice but not from the vessels of KO mice. Furthermore, TIRFM showed that H2O2 triggered membrane trafficking of TRPC6 in A7r5 cells. These results suggest a new signaling pathway of ROS-TRPC6 in controlling vessel contraction by vasoconstrictors.

Changes in Orientation of Actin during Contraction of Muscle

It is well documented that muscle contraction results from cyclic rotations of actin-bound myosin cross-bridges. The role of actin is hypothesized to be limited to accelerating phosphate release from myosin and to serving as a rigid substrate for cross-bridge rotations. To test this hypothesis, we have measured actin rotations during contraction of a skeletal muscle. Actin filaments of rabbit psoas fiber were labeled with rhodamine-phalloidin. Muscle contraction was induced by a pulse of ATP photogenerated from caged precursor. ATP induced a single turnover of cross-bridges. The rotations were measured by anisotropy of fluorescence originating from a small volume defined by a narrow aperture of a confocal microscope. The anisotropy of phalloidin-actin changed rapidly at first and was followed by a slow relaxation to a steady-state value. The kinetics of orientation changes of actin and myosin were the same. Extracting myosin abolished anisotropy changes. To test whether the rotation of actin was imposed by cross-bridges or whether it reflected hydrolytic activity of actin itself, we labeled actin with fluorescent ADP. The time-course of anisotropy change of fluorescent nucleotide was similar to that of phalloidin-actin. These results suggest that orientation changes of actin are caused by dissociation and rebinding of myosin cross-bridges, and that during contraction, nucleotide does not dissociate from actin.

Sigma-1 receptor stimulation attenuates calcium influx through activated L-type Voltage Gated Calcium Channels in purified retinal ganglion cells

Sigma-1 receptors (σ-1rs) exert neuroprotective effects on retinal ganglion cells (RGCs) both in vivo and in vitro. This receptor has unique properties through its actions on several voltage-gated and ligand-gated channels. The purpose of this study was to investigate the role that σ-1rs play in regulating cell calcium dynamics through activated L-type Voltage Gated Calcium Channels (L-type VGCCs) in purified RGCs. RGCs were isolated from P3-P7 Sprague-Dawley rats and purified by sequential immunopanning using a Thy1.1 antibody. Calcium imaging was used to measure changes in intracellular calcium after depolarizing the cells with potassium chloride (KCl) in the presence or absence of two σ-1r agonists [(+)-SKF10047 and (+)-Pentazocine], one σ-1r antagonist (BD1047), and one L-type VGCC antagonist (Verapamil). Finally, co-localization studies were completed to assess the proximity of σ-1r with L-type VGCCs in purified RGCs. VGCCs were activated using KCl (20 mM). Pre-treatment with a known L-type VGCC blocker demonstrated a 57% decrease of calcium ion influx through activated VGCCs. Calcium imaging results also demonstrated that σ-1r agonists, (+)-N-allylnormetazocine hydrochloride [(+)-SKF10047] and (+)-Pentazocine, inhibited calcium ion influx through activated VGCCs. Antagonist treatment using BD1047 demonstrated a potentiation of calcium ion influx through activated VGCCs and abolished all inhibitory effects of the σ-1r agonists on VGCCs, implying that these ligands were acting through the σ-1r. An L-type VGCC blocker (Verapamil) also inhibited KCl activated VGCCs and when combined with the σ-1r agonists there was not a further decline in calcium entry suggesting similar mechanisms. Lastly, co-localization studies demonstrated that σ-1rs and L-type VGCCs are co-localized in purified RGCs. Taken together, these results indicated that σ-1r agonists can inhibit KCl induced calcium ion influx through activated L-type VGCCs in purified RGCs. This is the first report of attenuation of L-type VGCC signaling through the activation of σ-1rs in purified RGCs. The ability of σ-1rs to co-localize with L-type VGCCs in purified RGCs implied that these two proteins are in close proximity to each other and that such interactions regulate L-type VGCCs.

Nanostructured Silver-Based Surfaces: New Emergent Methodologies for an Easy Detection of Analytes

In this work, we describe how to realize a new sensing platform for an easy and fast detection of analytes. In particular, we utilized enhanced fluorescence emission on silver island films (SIFs) coupled to the total internal reflection fluorescence mode (TIRF) to develop a new assay format for the detection of target analytes. Here, as an example, we report on the detection of the toxic peptides present in gliadin (Gli). Our assay was performed as follows: (1) gliadin was first captured on surfaces coated with anti-Gli antibodies; (2) the surfaces were then incubated with fluorophore-labeled anti-Gli antibodies; (3) the signal from the fluorophore-labeled anti-Gli antibody bound to the antigen was detected by TIRF. The system was examined on glass surfaces and on SIFs. We observed a relevant enhancement of the signal from SIFs compared to the signal from the glass substrate not modified with a SIF. In addition, the estimated detection limit (EDL) of our methodology was 60 ng/mL (or lower). This limit is therefore lower than the clinical cut-off for Gli presence in food for celiac patients. The advantage of our method is a reduced number of testing steps, which allows for easy detection of residual toxic peptides in food labeled as gluten free. The proposed technology can be easily expanded to the determination of different target analytes.

Rotation of actin monomers during isometric contraction of skeletal muscle

Cyclic interactions of myosin and actin are responsible for contraction of muscle. It is not self-evident, however, that the mechanical cycle occurs during steady-state isometric contraction where no work is produced. Studying cross-bridge dynamics during isometric steady-state contraction requires an equilibrium time-resolved method (not involving application of a transient). This work introduces such a method, which analyzes fluctuations of anisotropy of a few actin molecules in muscle. Fluorescence anisotropy, indicating orientation of an actin protomer, is collected from a volume of a few attoliters (10(-18) L) by confocal total internal reflection (CTIR) microscopy. In this method, the detection volume is made shallow by TIR illumination, and narrow by confocal aperture inserted in the conjugate image plane. The signal is contributed by approximately 12 labeled actin molecules. Shortening of a myofibril during contraction is prevented by light cross-linking with 1-ethyl-3-[3-dimethylamino)-propyl]-carbodiimide. The root mean-squared anisotropy fluctuations are greater in isometrically contracting than in rigor myofibrils. The results support the view that during isometric contraction, cross-bridges undergo a mechanical cycle.

Exosomal Annexin II Promotes Angiogenesis and Breast Cancer Metastasis

Tumor-derived exosomes are emerging mediators of tumorigenesis and tissue-specific metastasis. Proteomic profiling has identified Annexin II as one of the most highly expressed proteins in exosomes; however, studies focused on the biological role of exosomal Annexin II (exo-Anx II) are still lacking. In this study, mechanistic insight was sought regarding exo-Anx II and its function in angiogenesis and breast cancer metastasis. Multiple in vitro and in vivo techniques were used to study the role of exo-Anx II in angiogenesis. Using atomic force microscopy and Western blotting, exo-Anx II expression was characterized in normal and breast cancer cells. In addition, organ-specific metastatic breast cancer cells and animal models were used to define the role exo-Anx II in breast cancer metastasis. Results revealed that exo-Anx II expression is significantly higher in malignant cells than normal and premetastatic breast cancer cells. In vitro and in vivo studies demonstrated that exo-Anx II promotes tPA-dependent angiogenesis. Furthermore, in vivo analysis indicated that metastatic exosomes create a favorable microenvironment for metastasis, and exo-Anx II plays an important role in this process, as priming with Anx II-depleted exosomes reduces brain (∼4-fold) and lung (∼2-fold) metastasis. Upon delineating the mechanism, it was discovered that exo-Anx II causes macrophage-mediated activation of the p38MAPK, NF-κB, and STAT3 pathways and increased secretion of IL6 and TNFα. These data demonstrate an important role for exo-Anx II in breast cancer pathogenesis. Implications: Exosome-associated Annexin II plays an important role in angiogenesis and breast cancer metastasis, which can be exploited as a potential biomarker as well as a therapeutic target for diagnosis and treatment of metastatic breast cancer. Mol Cancer Res; 15(1); 93–105. ©2016 AACR.

Pre-Clinical Evaluation of rHDL Encapsulated Retinoids for the Treatment of Neuroblastoma.

Despite major advances in pediatric cancer research, there has been only modest progress in the survival of children with high risk neuroblastoma (NB) (HRNB). The long term survival rates of HRNB in the United States are still only 30–50%. Due to resistance that often develops during therapy, development of new effective strategies is essential to improve the survival and overcome the tendency of HRNB patients to relapse subsequent to initial treatment. Current chemotherapy regimens also have a serious limitation due to off target toxicity. In the present work, we evaluated the potential application of reconstituted high density lipoprotein (rHDL) containing fenretinide (FR) nanoparticles as a novel approach to current NB therapeutics. The characterization and stability studies of rHDL-FR nanoparticles showed small size (<40 nm) and high encapsulation efficiency. The cytotoxicity studies of free FR vs. rHDL/FR toward the NB cell lines SK-N-SH and SMS-KCNR showed 2.8- and 2-fold lower IC50 values for the rHDL encapsulated FR vs. free FR. More importantly, the IC50 value for retinal pigment epithelial cells (ARPE-19), a recipient of off target toxicity during FR therapy, was over 40 times higher for the rHDL/FR as compared to that of free FR. The overall improvement in in vitro selective therapeutic efficiency was thus about 100-fold upon encapsulation of the drug into the rHDL nanoparticles. These studies support the potential value of this novel drug delivery platform for treating pediatric cancers in general, and NB in particular.

Monolayers of Silver Nanoparticles Decrease Photobleaching: Application to Muscle Myofibrils

Studying single molecules in a cell has the essential advantage that kinetic information is not averaged out. However, since fluorescence is faint, such studies require that the sample be illuminated with the intense light beam. This causes photodamage of labeled proteins and rapid photobleaching of the fluorophores. Here, we show that a substantial reduction of these types of photodamage can be achieved by imaging samples on coverslips coated with monolayers of silver nanoparticles. The mechanism responsible for this effect is the interaction of localized surface plasmon polaritons excited in the metallic nanoparticles with the transition dipoles of fluorophores of a sample. This leads to a significant enhancement of fluorescence and a decrease of fluorescence lifetime of a fluorophore. Enhancement of fluorescence leads to the reduction of photodamage, because the sample can be illuminated with a dim light, and decrease of fluorescence lifetime leads to reduction of photobleaching because the fluorophore spends less time in the excited state, where it is susceptible to oxygen attack. Fluorescence enhancement and reduction of photobleaching on rough metallic surfaces are usually accompanied by a loss of optical resolution due to refraction of light by particles. In the case of monolayers of silver nanoparticles, however, the surface is smooth and glossy. The fluorescence enhancement and the reduction of photobleaching are achieved without sacrificing the optical resolution of a microscope. Skeletal muscle myofibrils were used as an example, because they contain submicron structures conveniently used to define optical resolution. Small nanoparticles (diameter approximately 60 nm) did not cause loss of optical resolution, and they enhanced fluorescence approximately 500-fold and caused the appearance of a major picosecond component of lifetime decay. As a result, the sample photobleached approximately 20-fold more slowly than the sample on glass coverslips.

Decreasing photobleaching by silver nanoparticles on metal surfaces: application to muscle myofibrils

Recently it has become possible to study single protein molecules in a cell. However, such experiments are plagued by rapid photobleaching. We recently showed that the interaction of fluorophores with localized surface plasmon polaritons (LSPs) induced in the metallic nanoparticles led to a substantial reduction of photobleaching. We now investigate whether the photobleaching could be further reduced when the excited fluorophore interacts with the LSP excited in the metallic nanoparticles resident on mirrored surface. As an example we use myofibrils, subcellular structures within skeletal muscle. We compare nanoparticle-enhanced fluorescence of myofibrils in the presence and in the absence of a mirrored surface. The proximity of the mirrored surface led to enhancement of fluorescence and to a decrease in fluorescent lifetime, much greater than that observed in the presence of nanoparticles alone. We think that the effect is caused by the near-field interactions between fluorophores and LSP, and between fluorophores and propagating surface plasmons (PSPs) produced in the metallic surface by the nanoparticles. Photobleaching is decreased because fluorescence enhancement enables illumination with a weaker laser beam and because the decrease in fluorescence lifetime minimizes the probability of oxygen attack during the time a molecule is in the exited state.

Simultaneous measurement of rotations of myosin, actin and ADP in a contracting skeletal muscle fiber

The rotation of myosin heads and actin were measured simultaneously with an indicator of the enzymatic activity of myosin. To minimize complications due to averaging of signals from many molecules, the signal was measured in a small population residing in a femtoliter volume of a muscle fiber. The onset of rotation was synchronized by a sudden release of caged ATP. The orientation of cross-bridges was measured by anisotropy of recombinant fluorescent regulatory light chains exchanged with native regulatory light chains. The orientation of actin was measured by anisotropy of phalloidin added to actin filaments. The enzymatic activity of myosin was measured by dissociation of fluorescent ADP from the active site. The onset of all three events occurred at the same time. This suggests that in contracting muscle, actin does not move independently of myosin and that ATP hydrolysis is strongly coupled to the rotation of cross-bridges.