Targeting SRSF2 mutations in leukemia with RKI-1447: A strategy to impair cellular division and nuclear structure.

Spliceosome machinery mutations are common early mutations in myeloid malignancies; however, effective targeted therapies against them are still lacking. In the current study, we used an in vitro high-throughput drug screen among four different isogenic cell lines and identified RKI-1447, a Rho-associated protein kinase inhibitor, as selective cytotoxic effector of SRSF2 mutant cells. RKI-1447 targeted SRSF2 mutated primary human samples in xenografts models. RKI-1447 induced mitotic catastrophe and induced major reorganization of the microtubule system and severe nuclear deformation. Transmission electron microscopy and 3D light microscopy revealed that SRSF2 mutations induce deep nuclear indentation and segmentation that are apparently driven by microtubule-rich cytoplasmic intrusions, which are exacerbated by RKI-1447. The severe nuclear deformation in RKI-1447-treated SRSF2 mutant cells prevents cells from completing mitosis. These findings shed new light on the interplay between microtubules and the nucleus and offers new ways for targeting pre-leukemic SRSF2 mutant cells.

Multisite assessment of reproducibility in high-content cell migration imaging data.

High-content image-based cell phenotyping provides fundamental insights into a broad variety of life science disciplines. Striving for accurate conclusions and meaningful impact demands high reproducibility standards, with particular relevance for high-quality open-access data sharing and meta-analysis. However, the sources and degree of biological and technical variability, and thus the reproducibility and usefulness of meta-analysis of results from live-cell microscopy, have not been systematically investigated. Here, using high-content data describing features of cell migration and morphology, we determine the sources of variability across different scales, including between laboratories, persons, experiments, technical repeats, cells, and time points. Significant technical variability occurred between laboratories and, to lesser extent, between persons, providing low value to direct meta-analysis on the data from different laboratories. However, batch effect removal markedly improved the possibility to combine image-based datasets of perturbation experiments. Thus, reproducible quantitative high-content cell image analysis of perturbation effects and meta-analysis depend on standardized procedures combined with batch correction.

Different hotspot p53 mutants exert distinct phenotypes and predict outcome of colorectal cancer patients.

The TP53 gene is mutated in approximately 60% of all colorectal cancer (CRC) cases. Over 20% of all TP53-mutated CRC tumors carry missense mutations at position R175 or R273. Here we report that CRC tumors harboring R273 mutations are more prone to progress to metastatic disease, with decreased survival, than those with R175 mutations. We identify a distinct transcriptional signature orchestrated by p53R273H, implicating activation of oncogenic signaling pathways and predicting worse outcome. These features are shared also with the hotspot mutants p53R248Q and p53R248W. p53R273H selectively promotes rapid CRC cell spreading, migration, invasion and metastasis. The transcriptional output of p53R273H is associated with preferential binding to regulatory elements of R273 signature genes. Thus, different TP53 missense mutations contribute differently to cancer progression. Elucidation of the differential impact of distinct TP53 mutations on disease features may make TP53 mutational information more actionable, holding potential for better precision-based medicine.

Biomechanical regulation of focal adhesion and invadopodia formation.

Integrin adhesions are a structurally and functionally diverse family of transmembrane, multi-protein complexes that link the intracellular cytoskeleton to the extracellular matrix (ECM). The different members of this family, including focal adhesions (FAs), focal complexes, fibrillar adhesions, podosomes and invadopodia, contain many shared scaffolding and signaling 'adhesome' components, as well as distinct molecules that perform specific functions, unique to each adhesion form. In this Hypothesis, we address the pivotal roles of mechanical forces, generated by local actin polymerization or actomyosin-based contractility, in the formation, maturation and functionality of two members of the integrin adhesions family, namely FAs and invadopodia, which display distinct structures and functional properties. FAs are robust and stable ECM contacts, associated with contractile stress fibers, while invadopodia are invasive adhesions that degrade the underlying matrix and penetrate into it. We discuss here the mechanisms, whereby these two types of adhesion utilize a similar molecular machinery to drive very different - often opposing cellular activities, and hypothesize that early stages of FAs and invadopodia assembly use similar biomechanical principles, whereas maturation of the two structures, and their 'adhesive' and 'invasive' functionalities require distinct sources of biomechanical reinforcement.

High-Throughput Screen Identifies Host and Microbiota Regulators of Intestinal Barrier Function.

BACKGROUND & AIMS: The intestinal barrier protects intestinal cells from microbes and antigens in the lumen-breaches can alter the composition of the intestinal microbiota, the enteric immune system, and metabolism. We performed a screen to identify molecules that disrupt and support the intestinal epithelial barrier and tested their effects in mice. METHODS: We performed an imaging-based, quantitative, high-throughput screen (using CaCo-2 and T84 cells incubated with lipopolysaccharide; tumor necrosis factor; histamine; receptor antagonists; and libraries of secreted proteins, microbial metabolites, and drugs) to identify molecules that altered epithelial tight junction (TJ) and focal adhesion morphology. We then tested the effects of TJ stabilizers on these changes. Molecules we found to disrupt or stabilize TJs were administered mice with dextran sodium sulfate-induced colitis or Citrobacter rodentium-induced intestinal inflammation. Colon tissues were collected and analyzed by histology, fluorescence microscopy, and RNA sequencing. RESULTS: The screen identified numerous compounds that disrupted or stabilized (after disruption) TJs and monolayers of epithelial cells. We associated distinct morphologic alterations with changes in barrier function, and identified a variety of cytokines, metabolites, and drugs (including inhibitors of actomyosin contractility) that prevent disruption of TJs and restore TJ integrity. One of these disruptors (putrescine) disrupted TJ integrity in ex vivo mouse colon tissues; administration to mice exacerbated colon inflammation, increased gut permeability, reduced colon transepithelial electrical resistance, increased pattern recognition receptor ligands in mesenteric lymph nodes, and decreased colon length and survival times. Putrescine also increased intestine levels and fecal shedding of viable C rodentium, increased bacterial attachment to the colonic epithelium, and increased levels of inflammatory cytokines in colon tissues. Colonic epithelial cells from mice given putrescine increased expression of genes that regulate metal binding, oxidative stress, and cytoskeletal organization and contractility. Co-administration of taurine with putrescine blocked disruption of TJs and the exacerbated inflammation. CONCLUSIONS: We identified molecules that disrupt and stabilize intestinal epithelial TJs and barrier function and affect development of colon inflammation in mice. These agents might be developed for treatment of barrier intestinal impairment-associated and inflammatory disorders in patients, or avoided to prevent inflammation.

TLR4 (Toll-Like Receptor 4)-Dependent Signaling Drives Extracellular Catabolism of LDL (Low-Density Lipoprotein) Aggregates.

OBJECTIVE: Aggregation and modification of LDLs (low-density lipoproteins) promote their retention and accumulation in the arteries. This is a critical initiating factor during atherosclerosis. Macrophage catabolism of agLDL (aggregated LDL) occurs using a specialized extracellular, hydrolytic compartment, the lysosomal synapse. Compartment formation by local actin polymerization and delivery of lysosomal contents by exocytosis promotes acidification of the compartment and degradation of agLDL. Internalization of metabolites, such as cholesterol, promotes foam cell formation, a process that drives atherogenesis. Furthermore, there is accumulating evidence for the involvement of TLR4 (Toll-like receptor 4) and its adaptor protein MyD88 (myeloid differentiation primary response 88) in atherosclerosis. Here, we investigated the role of TLR4 in catabolism of agLDL using the lysosomal synapse and foam cell formation. Approach and Results: Using bone marrow-derived macrophages from knockout mice, we find that TLR4 and MyD88 regulate compartment formation, lysosome exocytosis, acidification of the compartment, and foam cell formation. Using siRNA (small interfering RNA), pharmacological inhibition and knockout bone marrow-derived macrophages, we implicate SYK (spleen tyrosine kinase), PI3K (phosphoinositide 3-kinase), and Akt in agLDL catabolism using the lysosomal synapse. Using bone marrow transplantation of LDL receptor knockout mice with TLR4 knockout bone marrow, we show that deficiency of TLR4 protects macrophages from lipid accumulation during atherosclerosis. Finally, we demonstrate that macrophages in vivo form an extracellular compartment and exocytose lysosome contents similar to that observed in vitro for degradation of agLDL. CONCLUSIONS: We present a mechanism in which interaction of macrophages with agLDL initiates a TLR4 signaling pathway, resulting in formation of the lysosomal synapse, catabolism of agLDL, and lipid accumulation in vitro and in vivo.

Hyperglycemia drives intestinal barrier dysfunction and risk for enteric infection.

Obesity, diabetes, and related manifestations are associated with an enhanced, but poorly understood, risk for mucosal infection and systemic inflammation. Here, we show in mouse models of obesity and diabetes that hyperglycemia drives intestinal barrier permeability, through GLUT2-dependent transcriptional reprogramming of intestinal epithelial cells and alteration of tight and adherence junction integrity. Consequently, hyperglycemia-mediated barrier disruption leads to systemic influx of microbial products and enhanced dissemination of enteric infection. Treatment of hyperglycemia, intestinal epithelial-specific GLUT2 deletion, or inhibition of glucose metabolism restores barrier function and bacterial containment. In humans, systemic influx of intestinal microbiome products correlates with individualized glycemic control, indicated by glycated hemoglobin levels. Together, our results mechanistically link hyperglycemia and intestinal barrier function with systemic infectious and inflammatory consequences of obesity and diabetes.

Ceramide activation of RhoA/Rho kinase impairs actin polymerization during aggregated LDL catabolism.

Macrophages use an extracellular, hydrolytic compartment formed by local actin polymerization to digest aggregated LDL (agLDL). Catabolism of agLDL promotes foam cell formation and creates an environment rich in LDL catabolites, including cholesterol and ceramide. Increased ceramide levels are present in lesional LDL, but the effect of ceramide on macrophage proatherogenic processes remains unknown. Here, we show that macrophages accumulate ceramide in atherosclerotic lesions. Using macrophages from sphingosine kinase 2 KO (SK2KO) mice to mimic ceramide-rich conditions of atherosclerotic lesions, we show that SK2KO macrophages display impaired actin polymerization and foam cell formation in response to contact with agLDL. C16-ceramide treatment impaired wild-type but not SK2KO macrophage actin polymerization, confirming that this effect is due to increased ceramide levels. We demonstrate that knockdown of RhoA or inhibition of Rho kinase restores agLDL-induced actin polymerization in SK2KO macrophages. Activation of RhoA in macrophages was sufficient to impair actin polymerization and foam cell formation in response to agLDL. Finally, we establish that during catabolism, macrophages take up ceramide from agLDL, and inhibition of ceramide generation modulates actin polymerization. These findings highlight a critical regulatory pathway by which ceramide impairs actin polymerization through increased RhoA/Rho kinase signaling and regulates foam cell formation.

α-Spectrin and integrins act together to regulate actomyosin and columnarization, and to maintain a monolayered follicular epithelium.

The spectrin cytoskeleton crosslinks actin to the membrane, and although it has been greatly studied in erythrocytes, much is unknown about its function in epithelia. We have studied the role of spectrins during epithelia morphogenesis using the Drosophila follicular epithelium (FE). As previously described, we show that α-Spectrin and ß-Spectrin are essential to maintain a monolayered FE, but, contrary to previous work, spectrins are not required to control proliferation. Furthermore, spectrin mutant cells show differentiation and polarity defects only in the ectopic layers of stratified epithelia, similar to integrin mutants. Our results identify α-Spectrin and integrins as novel regulators of apical constriction-independent cell elongation, as α-Spectrin and integrin mutant cells fail to columnarize. Finally, we show that increasing and reducing the activity of the Rho1-Myosin II pathway enhances and decreases multilayering of α-Spectrin cells, respectively. Similarly, higher Myosin II activity enhances the integrin multilayering phenotype. This work identifies a primary role for α-Spectrin in controlling cell shape, perhaps by modulating actomyosin. In summary, we suggest that a functional spectrin-integrin complex is essential to balance adequate forces, in order to maintain a monolayered epithelium.

Myosin light-chain phosphatase regulates basal actomyosin oscillations during morphogenesis.

Contractile actomyosin networks generate forces that drive tissue morphogenesis. Actomyosin contractility is controlled primarily by reversible phosphorylation of the myosin-II regulatory light chain through the action of myosin kinases and phosphatases. While the role of myosin light-chain kinase in regulating contractility during morphogenesis has been largely characterized, there is surprisingly little information on myosin light-chain phosphatase (MLCP) function in this context. Here, we use live imaging of Drosophila follicle cells combined with mathematical modelling to demonstrate that the MLCP subunit flapwing (flw) is a key regulator of basal myosin oscillations and cell contractions underlying egg chamber elongation. Flw expression decreases specifically on the basal side of follicle cells at the onset of contraction and flw controls the initiation and periodicity of basal actomyosin oscillations. Contrary to previous reports, basal F-actin pulsates similarly to myosin. Finally, we propose a quantitative model in which periodic basal actomyosin oscillations arise in a cell-autonomous fashion from intrinsic properties of motor assemblies.

Degradation of aggregated LDL occurs in complex extracellular sub-compartments of the lysosomal synapse.

Monocyte-derived cells use an extracellular, acidic, lytic compartment (a lysosomal synapse) for initial degradation of large objects or species bound to the extracellular matrix. Akin to osteoclast degradation of bone, extracellular catabolism is used by macrophages to degrade aggregates of low density lipoprotein (LDL) similar to those encountered during atherogenesis. However, unlike osteoclast catabolism, the lysosomal synapse is a highly dynamic and intricate structure. In this study, we use high resolution three dimensional imaging to visualize compartments formed by macrophages to catabolize aggregated LDL. We show that these compartments are topologically complex, have a convoluted structure and contain sub-regions that are acidified. These sub-regions are characterized by a close apposition of the macrophage plasma membrane and aggregates of LDL that are still connected to the extracellular space. Compartment formation is dependent on local actin polymerization. However, once formed, compartments are able to maintain a pH gradient when actin is depolymerized. These observations explain how compartments are able to maintain a proton gradient while remaining outside the boundaries of the plasma membrane.

Monocyte-Derived Dendritic Cells Upregulate Extracellular Catabolism of Aggregated Low-Density Lipoprotein on Maturation, Leading to Foam Cell Formation.

OBJECTIVE: Although dendritic cells are known to play a role in atherosclerosis, few studies have examined the contribution of the wide variety of dendritic cell subsets. Accordingly, their roles in atherogenesis remain largely unknown. We investigated the ability of different dendritic cell subsets to become foam cells after contact with aggregated low-density lipoprotein (LDL; the predominant form of LDL found in atherosclerotic plaques). APPROACH AND RESULTS: We demonstrate that both murine and human monocyte-derived dendritic cells use exophagy to degrade aggregated LDL, leading to foam cell formation, whereas monocyte-independent dendritic cells are unable to clear LDL aggregates by this mechanism. Exophagy is a catabolic process in which objects that cannot be internalized by phagocytosis (because of their size or association with extracellular structures) are initially digested in an extracellular acidic lytic compartment. Surprisingly, we found that monocyte-derived dendritic cells upregulate exophagy on maturation. This contrasts various forms of endocytic internalization in dendritic cells, which decrease on maturation. Finally, we show that our in vitro results are consistent with dendritic cell lipid accumulation in plaques of an ApoE(-/-) mouse model of atherosclerosis. CONCLUSIONS: Our results show that monocyte-derived dendritic cells use exophagy to degrade aggregated LDL and become foam cells, whereas monocyte-independent dendritic cells are unable to clear LDL deposits. Furthermore, we find that exophagy is upregulated on dendritic cell maturation. Thus, exophagy-mediated foam cell formation in monocyte-derived dendritic cells could play a significant role in atherogenesis.

Plasmin promotes foam cell formation by increasing macrophage catabolism of aggregated low-density lipoprotein.

OBJECTIVE: The plasmin/plasminogen system is involved in atherosclerosis. However, the mechanisms by which it stimulates disease are not fully defined. A key event in atherogenesis is the deposition of low-density lipoprotein (LDL) on arterial walls where it is modified, aggregated, and retained. Macrophages are recruited to clear the lipoproteins, and they become foam cells. The goal of this study was to assess the role of plasmin in macrophage uptake of aggregated LDL and foam cell formation. APPROACH AND RESULTS: Plasminogen treatment of macrophages catabolizing aggregated LDL significantly accelerated foam cell formation. Macrophage interaction with aggregated LDL increased the surface expression of urokinase-type plasminogen activator receptor and plasminogen activator activity, resulting in increased ability to generate plasmin at the cell surface. The high local level of plasmin cleaves cell-associated aggregated LDL, allowing a portion of the aggregate to become sequestered in a nearly sealed, yet extracellular, acidic compartment. The low pH in the plasmin-induced compartment allows lysosomal enzymes, delivered via lysosome exocytosis, greater activity, resulting in more efficient cholesteryl ester hydrolysis and delivery of a large cholesterol load to the macrophage, thereby promoting foam cell formation. CONCLUSIONS: These findings highlight a critical role for plasmin in the catabolism of aggregated LDL by macrophages and provide a new context for considering the atherogenic role of plasmin.

Macrophages create an acidic extracellular hydrolytic compartment to digest aggregated lipoproteins.

A critical event in atherogenesis is the interaction of macrophages with subendothelial lipoproteins. Although most studies model this interaction by incubating macrophages with monomeric lipoproteins, macrophages in vivo encounter lipoproteins that are aggregated. The physical features of the lipoproteins require distinctive mechanisms for their uptake. We show that macrophages create an extracellular, acidic, hydrolytic compartment to carry out digestion of aggregated low-density lipoproteins. We demonstrate delivery of lysosomal contents to these specialized compartments and their acidification by vacuolar ATPase, enabling aggregate catabolism by lysosomal acid hydrolases. We observe transient sealing of portions of the compartments, allowing formation of an "extracellular" proton gradient. An increase in free cholesterol is observed in aggregates contained in these compartments. Thus, cholesteryl ester hydrolysis can occur extracellularly in a specialized compartment, a lysosomal synapse, during the interaction of macrophages with aggregated low-density lipoprotein. A detailed understanding of these processes is essential for developing strategies to prevent atherosclerosis.

Aggregated LDL in contact with macrophages induces local increases in free cholesterol levels that regulate local actin polymerization.

OBJECTIVE: Interaction of macrophages with aggregated matrix-anchored lipoprotein deposits is an important initial step in atherogenesis. Aggregated lipoproteins require different cellular uptake processes than those used for endocytosis of monomeric lipoproteins. In this study, we tested the hypothesis that engagement of aggregated LDL (agLDL) by macrophages could lead to local increases in free cholesterol levels and that these increases in free cholesterol regulate signals that control cellular actin. METHODS AND RESULTS: AgLDL resides for prolonged periods in surface-connected compartments. Although agLDL is still extracellular, we demonstrate that an increase in free cholesterol occurs at sites of contact between agLDL and cells because of hydrolysis of agLDL-derived cholesteryl ester. This increase in free cholesterol causes enhanced actin polymerization around the agLDL. Inhibition of cholesteryl ester hydrolysis results in decreased actin polymerization. CONCLUSIONS: We describe a novel process that occurs during agLDL-macrophage interactions in which local release of free cholesterol causes local actin polymerization, promoting a pathological positive feedback loop for increased catabolism of agLDL and eventual foam cell formation.

p120 catenin regulates lamellipodial dynamics and cell adhesion in cooperation with cortactin.

The armadillo-family protein, p120 catenin (p120), binds to the juxtamembrane domain of classical cadherins and increases cell-cell junction stability. Overexpression of p120 modulates the activity of Rho family GTPases and augments cell migratory ability. Here we show that down-regulation of p120 in epithelial MCF-7 cells by siRNA leads to a striking decrease in lamellipodial persistence and focal adhesion formation. Similar alterations in lamellipodial activity were observed in MCF-7 cells treated with siRNA to cortactin, an activator of Arp2/3-dependent actin polymerization. We found that, in many cell types, p120 is colocalized with cortactin-containing actin structures not only at cell-cell junctions, but also at extrajunctional sites including membrane ruffles and actin-rich halos around endocytotic vesicles. p120 depletion led to dramatic loss of cortactin and its partner, Arp3, from the cell leading edges. Cortactin and p120 are shown to directly interact with each other via the cortactin N-terminal region. We propose that the mechanism underlying p120 functions at the leading edge involves its cooperation with cortactin.

Elevated cholesterol levels in the plasma membranes of macrophages inhibit migration by disrupting RhoA regulation.

OBJECTIVE: Atherogenesis begins as small subendothelial accumulations of foam cells that develop through unregulated uptake of modified and aggregated low-density lipoprotein (LDL). The reason why foam cells remain in the atherosclerotic plaque rather than migrating out of the area is unclear. We tested the hypothesis that elevated membrane cholesterol levels, which may result from interactions with aggregated LDL, affect macrophage migration. METHODS AND RESULTS: Cholesterol loading by incubation with cholesterol-chelated methyl-beta-cyclodextrin decreased migration of J774A.1 macrophages toward complement 5a (C5a) in transwell migration assays, even though cholesterol-loaded macrophages responded to a bath application of C5a. In a micropipette polarization assay, cholesterol-loaded cells polarized toward a C5a gradient. In a transwell migration assay, cholesterol-loaded cells extended lamellae through the filter pores but were unable to translocate their cell bodies. Cholesterol loading decreased both the cellular levels of GTP-bound active RhoA and the phosphorylation of myosin light chain. Expression of constitutively active RhoA largely prevented the inhibition of cell migration by cholesterol loading. CONCLUSIONS: These results suggest that increases in plasma membrane cholesterol content alter RhoA activation, resulting in inhibition of cell migration. These findings provide one possible explanation for the retention of foam cells in atherosclerotic lesions.

Caldesmon effects on the actin cytoskeleton and cell adhesion in cultured HTM cells.

Caldesmon is a multifunctional ubiquitous regulator of the actin cytoskeleton, which can affect both actomyosin contractility and actin polymerization. Previous studies showed that caldesmon over-expression in cultured fibroblasts produces effects that resemble those of chemical inhibitors of cellular contractility. Since these inhibitors (H-7, Y-27632, etc.) have been shown to lower intraocular pressure and increase outflow facility from the anterior chamber of the eye, we proposed that caldesmon might be used for gene therapy of glaucoma. In the present study we examined the effects of expression of adenovirus-delivered rat non-muscle caldesmon fused with green fluorescent protein (AdCaldGFP) on the actin cytoskeleton and matrix adhesions in cultured human trabecular meshwork (HTM) cells. In addition, we assessed the effect of caldesmon on the stability of cell-cell junctions in kidney epithelial MDCK cells. Cultured HTM cells demonstrate a well-developed actin cytoskeleton, comprising mainly arrays of parallel actomyosin bundles (stress fibers). Lamellipodial protrusions containing dense actin networks are also observed. Cell-matrix adhesions are dominated by focal adhesions (FAs) associated with the ends of the stress fibers, focal complexes in lamellipodia, and fibrillar adhesions in the central part of the spread cells. Treatment of HTM cells with AdCaldGFP resulted in dose-dependent morphological changes within 24-48 hr post-infection. Cells expressing moderate levels of caldesmon exhibited straight bundles containing actin and myosin II, which were considerably shorter than those in control cells. Short filament bundles in caldesmon over-expressing cells formed arrays consisting of triangular actin structures with small vinculin-positive FAs at their vertices. In addition, the fraction of cells displaying large lamellipodia increased. About 40-50% of the population of caldesmon-expressing cells demonstrated high levels of GFP-caldesmon expression and severe changes in the actin cytoskeleton, manifested by the disappearance of stress fibers and the formation of curved actin- and myosin-containing bundles. These bundles formed together a dynamic network consisting of pulsating loops filling the entire cytoplasm. Addition of thapsigargin, which increases intracellular Ca++ concentration, resulted in a straightening of the curved bundles. Another type of novel actin structures induced by caldesmon over-expression were highly dynamic circular waves that propagated over the affected cells with a velocity about 10 microm min. In cells with disrupted stress fibers, vinculin-containing FAs and tensin-rich fibrillar adhesions had also essentially vanished. However, phosphotyrosine-positive focal complexes were still prominent throughout the lamellipodia of these cells. Over-expression of caldesmon in MDCK cells reduced, in a dose dependent manner, the beta-catenin content at cell-cell adherens junctions and in some cases led to physical disruption of adherens junctions. Thus, caldesmon over-expression induces unique reorganization of the actin cytoskeleton in affected cells, accompanied by disruption of focal and fibrillar cell-matrix adhesions, and destabilization of cell-cell adherens junctions. Inducing such changes in the contractility and actin cytoskeleton of HTM cells in glaucomatous eyes in vivo could produce a therapeutically useful increase in outflow facility.

Caldesmon transgene expression disrupts focal adhesions in HTM cells and increases outflow facility in organ-cultured human and monkey anterior segments.

Cytoskeleton modulating compounds have been shown to lower intraocular pressure (IOP) and increase outflow facility. Caldesmon is one protein that is involved in the regulation of actin stress fiber formation. The effects of rat non-muscle caldesmon (Cald) gene over-expression on focal adhesions in human trabecular meshwork (HTM) cells and on outflow facility in organ-cultured human and monkey anterior segments were determined. Treatment of HTM cells with adenovirus-delivered caldesmon (AdCaldGFP) resulted in characteristic changes in the actin cytoskeleton and matrix adhesions within 24-48 hr post-transduction. Stress fibers gradually disappeared and novel actin structures were formed (see manuscript by Grosheva et al., this issue). In cells with disrupted stress fibers, vinculin-containing focal adhesions were also disrupted. In organ-cultured anterior segments, baseline outflow facility (microl min-1 mmHg-1) for all anterior segments averaged (mean+/-sem): human, 0.19+/-0.03 (n=12); monkey, 0.36+/-0.02 (n=19). In human anterior segments, transduction with 10(7) plaque forming units of AdGFPCald increased outflow facility by 43+/-21% (p

Elevated plasma membrane cholesterol content alters macrophage signaling and function.

OBJECTIVE: During atherogenesis, macrophages migrate into the subendothelial space where they ingest deposited lipoproteins, accumulate lipids, and transform into foam cells. It is unclear why these macrophages do not remove their lipid loads from the region. This study was aimed at testing the hypothesis that macrophage behavior is altered when membrane cholesterol levels are elevated, as might be the case for cells in contact with lipoproteins within atherosclerotic lesions. METHODS AND RESULTS: We examined the effects of elevating membrane cholesterol on macrophage behavior. J774 macrophages were treated with either acetylated low-density lipoprotein (ac-LDL) and ACAT inhibitor or cholesterol-chelated methyl-beta-cyclodextrin (chol-MbetaCD) to increase membrane cholesterol levels. Our results show that elevating the membrane cholesterol of J774 macrophages induced dramatic ruffling, stimulated cell spreading, and affected F-actin organization. Cellular adhesion was required for these effects, and Rac-mediated signaling pathways were involved. Additionally, 3-dimensional transwell chemotaxis assays showed that migration of J774 macrophages was significantly inhibited when membrane cholesterol levels were raised. CONCLUSIONS: These findings indicate that increased membrane cholesterol causes dramatic effects on macrophage cellular functions related to the actin cytoskeleton. They should provide new insights into the early steps of atherogenesis.