Hazel Y. Stevens

New fluorescent probes for the measurement of cell membrane viscosity

BACKGROUND Molecular rotors are fluorescent molecules that exhibit viscosity-dependent fluorescence quantum yield, potentially allowing direct measurements of cell membrane viscosity in cultured cells. Commercially available rotors, however, stain not only the cell membrane, but also bind to tubulin and migrate into the cytoplasm. We synthesized molecules related to 9-(dicyanovinyl)-julolidine (DCVJ), which featured hydrocarbon chains of different length to increase membrane compatibility. RESULTS Longer hydrocarbon chains attached to the fluorescent rotor reduce the migration of the dye into the cytoplasm and internal compartments of the cell. The amplitude of the fluorescence response to fluid shear stress, known to decrease membrane viscosity, is significantly higher than the response obtained from DCVJ. Notably a farnesyl chain showed a more than 20-fold amplitude over DCVJ and allowed detection of membrane viscosity changes at markedly lower shear stresses. CONCLUSIONS The modification of molecular rotors towards increased cell membrane association provides a new research tool for membrane viscosity measurements. The use of these rotors complements established methods such as fluorescence recovery after photobleaching with its limited spatial and temporal resolution and fluorescence anisotropy, which has low sensitivity and may be subject to other effects such as deformation.

PECAM-1 Interacts With Nitric Oxide Synthase in Human Endothelial Cells Implication for Flow-Induced Nitric Oxide Synthase Activation

Objective—We have previously shown that fluid shear stress (FSS) triggers endothelial nitric oxide synthase (eNOS) activity in endothelial cells and that the mechanotransduction mechanisms responsible for activation discriminate between rapid changes in FSS and FSS per se. We hypothesized that the particular sublocalization of eNOS at the cell–cell junction would render it responsive to activation by FSS temporal gradients. Methods and Results—In human umbilical vein endothelial cells (HUVECs), immunofluorescence revealed strong eNOS membrane staining at the cell–cell junction colocalizing with platelet/endothelial cell adhesion molecule-1 (PECAM-1). In PECAM-1 −/− mouse aorta, eNOS junctional localization seen in the wild type was absent. Similarly, junctional staining was lost in wild-type aorta near intercostal artery branches. eNOS/PECAM-1 association in HUVECs was confirmed by coimmunoprecipitation. When HUVECs were subjected to a 0.5s impulse of 12 dynes/cm2, a transient disruption of the eNOS/PECAM-1 complex was observed, accompanied by an increase in eNOS activity (cGMP production). Ramped flow did not trigger complex dissociation or an increase in cGMP production. In a cell-free system, a direct inhibition of eNOS activity by PECAM-1 is shown. Conclusions—These results suggest that eNOS is complexed with PECAM-1 at the cell–cell junction and is likely involved in the modulation of eNOS activity by FSS temporal gradients but not by FSS itself.

PECAM-1 is a critical mediator of atherosclerosis

SUMMARY Atherosclerosis is a chronic inflammatory disease of large arteries in which lesion development preferentially occurs at vessel sites exposed to rapid changes in flow. We have previously shown that platelet endothelial cell adhesion molecule (PECAM-1), a surface receptor of the immunoglobulin superfamily, is involved in mechanosensing of rapid changes in flow. We wondered whether apolipoprotein E deficient (ApoE−/−) mice, predisposed to development of atheromas, would be protected from atherosclerosis by deficiency in PECAM-1. Using double knockout (DKO) mice for both PECAM-1 and ApoE (ApoE−/−/PECAM-1−/−) we found a significant reduction of sudanophilic lesions in their aortae compared to single knockout (SKO) (ApoE−/−/PECAM-1+/+) mice maintained on a high-fat Western diet. Immunostaining of aortic sinus cross sections demonstrated significantly lower ICAM-1 expression in DKO lesions compared with SKO lesions, and en face preparations of vessel regions subjected to disturbed and laminar flow showed less disruption of junctional connexin 43 in DKO than in SKO mice. Thus, PECAM-1 deficiency reduced the extent of lesions at the aortic arch and the aortic sinus, and lowered atherogenic indices. These results suggest that PECAM-1 is an important factor in the atherogenic changes seen in the ApoE-deficient mouse model and thus should be considered as a potential target for protection against atherosclerosis.

Phospholipid-bound molecular rotors: synthesis and characterization.

Molecular rotors are fluorescent molecules with a viscosity-sensitive quantum yield that are often used to measure viscosity changes in cell membranes and liposomes. However, commercially available molecular rotors, such as DCVJ (1) do not localize in cell membranes but rapidly migrate into the cytoplasm leading to unreliable measurements of cell membrane viscosity. To overcome this problem, we synthesized molecular rotors covalently attached to a phospholipid scaffold. Attaching the rotor group to the hydrophobic end of phosphatidylcholine (PC) did not affect the rotors viscosity sensitivity and allowed adequate integration into artificial bilayers as well as complete localization in the plasma membrane of an endothelial cell line. Moreover, these new rotors enabled the monitoring of phospholipid transition temperature. However, attachment of the rotor groups to the hydrophilic head of the phospholipid led to a partial loss of viscosity sensitivity. The improved sensitivity and exclusive localization in the cell plasma membrane exhibited by the phospholipid-bound molecular rotors suggest that these probes can be used for the study of membrane microviscosity.

Rapid changes in shear stress induce dissociation of a Gαq/11–platelet endothelial cell adhesion molecule‐1 complex

It has been recently shown that endothelial platelet endothelial cell adhesion molecule‐1 (PECAM‐1) expression is pro‐atherogenic. PECAM‐1 is involved in sensing rapid changes in fluid shear stress but the mechanisms for activating signalling complexes at the endothelial cell junction have yet to be elucidated. Additional studies suggest the activation of membrane‐bound G proteins Gαq/11 also mediate flow‐induced responses. Here, we investigated whether PECAM‐1 and Gαq/11 could act in unison to rapidly respond to fluid shear stress. With immunohistochemistry, we observed a co‐localization of Gαq/11 and PECAM‐1 at the cell–cell junction in the atheroprotected section of mouse aortae. In contrast, Gαq/11 was absent from junctions in atheroprone areas as well as in all arterial sections of PECAM‐1 knockout mice. In primary human endothelial cells, temporal gradients in shear stress led to a rapid dissociation of the Gαq/11–PECAM‐1 complex within 30 s and a partial relocalization of the Gαq/11 staining to perinuclear areas within 150 min, whereas transitioning fluid flow devoid of temporal gradients did not disrupt the complex. Inhibition of G protein activation eliminated temporal gradient flow‐induced Gαq/11–PECAM‐1 dissociation. These results allow us to conclude that Gαq/11–PECAM‐1 forms a mechanosensitive complex and its localization suggests the Gαq/11–PECAM‐1 complex is a critical mediator of vascular diseases.

Extracellular signal‐regulated kinase activation and endothelin‐1 production in human endothelial cells exposed to vibration

Hand–arm vibration syndrome is a vascular disease of occupational origin and a form of secondary Raynauds phenomenon. Chronic exposure to hand‐held vibrating tools may cause endothelial injury. This study investigates the biomechanical forces involved in the transduction of fluid vibration in the endothelium. Human endothelial cells were exposed to direct vibration and rapid low‐volume fluid oscillation. Rapid low‐volume fluid oscillation was used to simulate the effects of vibration by generating defined temporal gradients in fluid shear stress across an endothelial monolayer. Extracellular signal‐regulated kinase (ERK1/2) phosphorylation and endothelin‐1 (ET‐1) release were monitored as specific biochemical markers for temporal gradients and endothelial response, respectively. Both vibrational methods were found to phosphorylate ERK1/2 in a similar pattern. At a fixed frequency of fluid oscillation where the duration of each pulse cycle remained constant, ERK1/2 phosphorylation increased with the increasing magnitude of the applied temporal gradient. However, when the frequency of flow oscillation was increased (thus decreasing the duration of each pulse cycle), ERK1/2 phosphorylation was attenuated across all temporal gradient flow profiles. Fluid oscillation significantly stimulated ET‐1 release compared to steady flow, and endothelin‐1 was also attenuated with the increase in oscillation frequency. Taken together, these results show that both the absolute magnitude of the temporal gradient and the frequency/duration of each pulse cycle play a role in the biomechanical transduction of fluid vibrational forces in endothelial cells. Furthermore, this study reports for the first time a link between the ERK1/2 signal transduction pathway and transmission of vibrational forces in the endothelium.

Computerized methods for X-ray-based small bone densitometry

Animal models have been widely used to correlate in vivo changes in bone mineral density (BMD) with changes in disease state of bone. In small animal models, e.g. the hindlimb suspension model of bone loss, a non-invasive assessment of BMD is required. X-ray radiography has been surpassed in some cases by quantitative computed tomography (QCT) and dual X-ray absorptiometry (DEXA) quantitation. However, there are drawbacks in using the computerized methods, especially for small animals. In this paper, we present image-processing algorithms to quantitatively determine bone area and mineral density in digitized radiographs. Image calibration is based on a calibration step wedge, and the algorithm automatically detects the steps and computes the calibration data. In addition, we demonstrate how the algorithm can accurately determine the cortical outline of the bone and provide reliable data and statistics for small animal studies. A downloadable implementation example for the popular NIH Image package is provided.

Cell Membrane Fluidity Changes and Membrane Undulations Observed Using a Laser Scattering Technique

Local transversal micromotions of cell membranes have been reported. These may provide the physical basis for changes in membrane fluidity. In this study, tissue scattering properties are used to measure the magnitude of transversal micromotions under varying stimuli. Laser light is directed at the cell surface at a low incident angle of 12° so that reflected or refracted light does not enter the microscope objective. Scattered light showed strong low-frequency intensity fluctuations with random-walk behavior. Fluctuations were quantified by the median coefficient of variation over the entire observed cell area. Incubation of the cells with protein crosslinkers (paraformaldehyde) and metabolic suppressors (sodium azide) suppressed these fluctuations by 62 and 44%, respectively. The application of hypoosmotic media caused an increase of fluctuation magnitude by 23% (p<0.005). Agents that increase membrane fluidity (2% ethanol and xenon) increased fluctuation magnitude by 15 and 31%, respectively (p<0.05). Cessation of the ethanol and xenon exposure led to partial recovery of the fluctuation magnitude, which was nonsignificant for ethanol. This study shows a strong link between membrane fluidity and transversal membrane undulations and provides an important step in the understanding of the mechanosensing function of the cell membrane.

Rescue of a primary myelofibrosis model by retinoid-antagonist therapy

Significance Our work identifies a therapeutically tractable yet previously unrecognized repressive epigenetic feature of primary myelofibrosis that controls a key cytokine circuit in the bone marrow microenvironment. Though lacking previous clinical utility, we show that an orally active retinoic acid receptor antagonist can normalize thrombopoietin production, restore bone integrity, and dramatically reduce fibrosis. This identification of a pathway-specific drug for primary myelofibrosis opens up an avenue of treatment options and provides insight into the complex mechanisms underlying myelofibrosis. Molecular targeting of the two receptor interaction domains of the epigenetic repressor silencing mediator of retinoid and thyroid hormone receptors (SMRTmRID) produced a transplantable skeletal syndrome that reduced radial bone growth, increased numbers of bone-resorbing periosteal osteoclasts, and increased bone fracture risk. Furthermore, SMRTmRID mice develop spontaneous primary myelofibrosis, a chronic, usually idiopathic disorder characterized by progressive bone marrow fibrosis. Frequently linked to polycythemia vera and chronic myeloid leukemia, myelofibrosis displays high patient morbidity and mortality, and current treatment is mostly palliative. To decipher the etiology of this disease, we identified the thrombopoietin (Tpo) gene as a target of the SMRT–retinoic acid receptor signaling pathway in bone marrow stromal cells. Chronic induction of Tpo in SMRTmRID mice results in up-regulation of TGF-β and PDGF in megakaryocytes, uncontrolled proliferation of bone marrow reticular cells, and fibrosis of the marrow compartment. Of therapeutic relevance, we show that this syndrome can be rescued by retinoid antagonists, demonstrating that the physical interface between SMRT and retinoic acid receptor can be a potential therapeutic target to block primary myelofibrosis disease progression.

Fluid shear-induced membrane fluctuations measured by laser dark field microscopy

The mechanism of mechanochemical signal transduction remains to be elucidated. There is mounting evidence that mechanochemical transduction in response to fluid shear stress occurs at the cell membrane. We previously demonstrated that fluid shear stress increases membrane free volume. Here we present evidence that fluid shear amplifies thermal fluctuations in the cell membrane. These results present further support of the membrane free volume theory of mechanochemical transduction.