The role of the actomyosin based cytoskeleton in the regulation of physiological cell responses.

The role of the actomyosin based cytoskeleton in the regulation of physiological cell responses

Shape-to-graph mapping method for efficient characterization and classification of complex geometries in biological images.

With the ever-increasing quality and quantity of imaging data in biomedical research comes the demand for computational methodologies that enable efficient and reliable automated extraction of the quantitative information contained within these images. One of the challenges in providing such methodology is the need for tailoring algorithms to the specifics of the data, limiting their areas of application. Here we present a broadly applicable approach to quantification and classification of complex shapes and patterns in biological or other multi-component formations. This approach integrates the mapping of all shape boundaries within an image onto a global information-rich graph and machine learning on the multidimensional measures of the graph. We demonstrated the power of this method by (1) extracting subtle structural differences from visually indistinguishable images in our phenotype rescue experiments using the endothelial tube formations assay, (2) training the algorithm to identify biophysical parameters underlying the formation of different multicellular networks in our simulation model of collective cell behavior, and (3) analyzing the response of U2OS cell cultures to a broad array of small molecule perturbations

Nitroglycerin Tolerance in Caveolin-1 Deficient Mice

<div><p>Nitrate tolerance developed after persistent nitroglycerin (GTN) exposure limits its clinical utility. Previously, we have shown that the vasodilatory action of GTN is dependent on endothelial nitric oxide synthase (eNOS/NOS3) activity. Caveolin-1 (Cav-1) is known to interact with NOS3 on the cytoplasmic side of cholesterol-enriched plasma membrane microdomains (caveolae) and to inhibit NOS3 activity. Loss of Cav-1 expression results in NOS3 hyperactivation and uncoupling, converting NOS3 into a source of superoxide radicals, peroxynitrite, and oxidative stress. Therefore, we hypothesized that nitrate tolerance induced by persistent GTN treatment results from NOS3 dysfunction and vascular toxicity. Exposure to GTN for 48–72 h resulted in nitrosation and depletion (>50%) of Cav-1, NOS3 uncoupling as measured by an increase in peroxynitrite production (>100%), and endothelial toxicity in cultured cells. In the Cav-1 deficient mice, NOS3 dysfunction was accompanied by GTN tolerance (>50% dilation inhibition at low GTN concentrations). In conclusion, GTN tolerance results from Cav-1 modification and depletion by GTN that causes persistent NOS3 activation and uncoupling, preventing it from participating in GTN-medicated vasodilation.</p></div

PETN has less adverse effect on Cav-1 and eNOS.

<p>(A) Representative western blot of Cav-1 oligomer/monomer distribution in HLMVECs exposed to GTN (20 µM) and PETN (10 µM) for indicated time (24, 48 and 72 h). (B) Nitric oxide (NO) and peroxynitrite (ONOO-) measurement in HLMVECs exposed to GTN and PETN for indicated time. ** <i>p</i><0.01, n = 4–6 (C) Quantified eNOS monomer/dimer ratio in HLMVECs exposed to GTN and PETN for 72 h. * <i>p</i><0.05, n = 5.</p

NOS3 dysfunction and peroxynitrite production in GTN exposed endothelial cells.

<p>(A) Exposure of human lung microvascular endothelial cells (HLMVEC) to the GTN alters NOS3 dimer/monomer distribution and provokes NOS3 Ser1177 phosphorylation. Cell lysates were subjected to low-temperature SDS-PAGE (LT-PAGE) to assess NOS3 dimer/monomer ration. Representative Western blots show endothelial nitric oxide synthase dimer/monomer in unprocessed (top) and denatured samples boiled in presence of 2-mercaptoethanol (second top). (B) Representative chemiluminescence measurement of basal NO and peroxynitrite production in GTN tolerant HLMVECs. Cells were treated with 20 µM GTN for 72 h. n = 6, *** <i>p</i><0.001 (C) Representative peroxynitrite measurement by coumarin-7-boronic acid (CBA), a specific fluorescent peroxynitrite probe. Primary mouse endothelial cells were pretreated with GTN and then incubated with 10 µM CBA for 1 hour. Measurement was performed using HPLC, insert shows an example of typical fluorescence signals, n = 4, ** <i>p</i><0.01 (D) L-NIO inhibition of peroxynitrite in tolerant cells. HLMVEC were treated with GTN for 72 h and pre-incubated with NOS3 inhibitor L-NIO (50 µM) for 1 h in serum free media before measurement. n = 3, ** <i>p<</i>0.01 (E) NO and peroxynitrite production in WT and NOS3 KO MECs are measured basally and under stimulation of either GTN (100 nM) or NOS3 activator A23187 (10 µM). Results showing NOS3 KO MECs were incapable of responding to GTN and do not produce peroxynitrite after chronic exposure to GTN, indicating NOS3 is the effector of GTN action and toxicity. n = 6, * <i>p</i><0.05 (F) Stimulation of mouse endothelial cells (MECs) with activators of NOS3 (vasodilatory agonists) in cells chronically exposed to GTN (20 µM, 72 h) induced peroxynitrite production, supporting the concept that GTN poisoning converts a vasodilating response into a vasocontricting behavior, elicited by NOS3 dysregulation via ONOO⁻. n = 4–6, * <i>p</i><0.05, ** <i>p</i><0.01</p

Continuous GTN-exposure leads to Cav-1 loss, NOS3 hyperactivation and dysfunction.

<p>Representative western blot image of at least three independent experiments showing GTN-induced cav-1 depletion in (A) human umbilical vein endothelial cell (HUVEC) and (B) primary mouse lung microvascular endothelial cells (MEC). Cells were treated with 20 µM GTN or vehicle control of indicated time. Cav-1 levels were examined by western blot. (C) Representative western blot of GTN-induction of cav-1 depletion paralleled by NOS3 activation in mouse aorta. Mice were exposed to GTN in the form of ointment at 2% concentration continuously for 72 h. Controls were exposed to vehicle-white petrolatum base paste. Bar graph shows the relative amounts of cav-1 compared with control group (mean ± SD), n = 3, * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001</p

Caveolin-1 knockout mice are tolerant to GTN.

<p>Vasoreactivity experiments were performed in WT and Cav-1 -/- mouse mesenteric arteries. Results revealed that Cav-1 KO mice are resistant to both Acetylcholine (Ach, left panel) and low concentrations of GTN (middle panel). This indicates resistance is not due to defective signaling downstream of NO as shown by the normal responses of Cav-1 KO to sodium nitroprusside (SNP, right panel), a direct NO donor.</p