Emerging mechanisms to modulate VWF release from endothelial cells.

Agonist-mediated exocytosis of Weibel-Palade bodies underpins the endothelium's ability to respond to injury or infection. Much of this important response is mediated by the major constituent of Weibel-Palade bodies: the ultra-large glycoprotein von Willebrand factor. Upon regulated WPB exocytosis, von Willebrand factor multimers unfurl into long, platelet-catching 'strings' which instigate the pro-haemostatic response. Accordingly, excessive levels of VWF are associated with thrombotic pathologies, including myocardial infarction and ischaemic stroke. Failure to appropriately cleave von Willebrand Factor strings results in thrombotic thrombocytopenic purpura, a life-threatening pathology characterised by tissue ischaemia and multiple microvascular occlusions. Historically, treatment of thrombotic thrombocytopenic purpura has relied heavily on plasma exchange therapy. However, the demonstrated efficacy of Rituximab and Caplacizumab in the treatment of acquired thrombotic thrombocytopenic purpura highlights how insights into pathophysiology can improve treatment options for von Willebrand factor-related disease. Directly limiting von Willebrand factor release from Weibel-Palade bodies has the potential as a therapeutic for cardiovascular disease. Cell biologists aim to map the WPB biogenesis and secretory pathways in order to find novel ways to control von Willebrand factor release. Emerging paradigms include the modulation of Weibel-Palade body size, trafficking and mechanism of fusion. This review focuses on the promise, progress and challenges of targeting Weibel-Palade bodies as a means to inhibit von Willebrand factor release from endothelial cells.

Modulation of endothelial organelle size as an antithrombotic strategy.

BACKGROUND: It is long established that von Willebrand factor (VWF) is central to hemostasis and thrombosis. Endothelial VWF is stored in cell-specific secretory granules, Weibel-Palade bodies (WPBs), organelles generated in a wide range of lengths (0.5-5.0 µm). WPB size responds to physiological cues and pharmacological treatment, and VWF secretion from shortened WPBs dramatically reduces platelet and plasma VWF adhesion to an endothelial surface. OBJECTIVE: We hypothesized that WPB-shortening represented a novel target for antithrombotic therapy. Our objective was to determine whether compounds exhibiting this activity do exist. METHODS: Using a microscopy approach coupled to automated image analysis, we measured the size of WPB bodies in primary human endothelial cells treated with licensed compounds for 24 hours. RESULTS AND CONCLUSIONS: A novel approach to identification of antithrombotic compounds generated a significant number of candidates with the ability to shorten WPBs. In vitro assays of two selected compounds confirm that they inhibit the pro-hemostatic activity of secreted VWF. This set of compounds acting at a very early stage of the hemostatic process could well prove to be a useful adjunct to current antithrombotic therapeutics. Further, in the current SARS-CoV-2 pandemic, with a considerable fraction of critically ill COVID-19 patients affected by hypercoagulability, these WPB size-reducing drugs might also provide welcome therapeutic leads for frontline clinicians and researchers.

Editor's Highlight: Multiparametric Image Analysis of Rat Dorsal Root Ganglion Cultures to Evaluate Peripheral Neuropathy-Inducing Chemotherapeutics.

Chemotherapy-induced peripheral neuropathy (CIPN) is a major, dose-limiting adverse effect experienced by cancer patients. Advancements in mechanism-based risk mitigation and effective treatments for CIPN can be aided by suitable in vitro assays. To this end, we developed a multiparametric morphology-centered rat dorsal root ganglion (DRG) assay. Morphologic alterations in subcellular structures of neurons and non-neurons were analyzed with an automated microscopy system. Stains for NeuN (a neuron-specific nuclear protein) and Tuj-1 (ß-III tubulin) were used to identify neuronal cell nuclei and neuronal cell bodies/neurites, respectively. Vimentin staining (a component of Schwann cell intermediate filaments) was used to label non-neuronal supporting cells. Nuclei that stained with DAPI, but lacked NeuN represented non-neuronal cells. Images were analyzed following 24 h of continuous exposure to CIPN-inducing agents and 72 h after drug removal to provide a dynamic measure of recovery from initial drug effects. Treatment with bortezomib, cisplatin, eribulin, paclitaxel or vincristine induced a dose-dependent loss of neurite/process areas, mimicking the 'dying back' degeneration of axons, a histopathological hallmark of clinical CIPN in vivo. The IC50 for neurite loss was within 3-fold of the maximal clinical exposure (Cmax) for all five CIPN-inducing drugs, but was >4- or ≥ 28-fold of the Cmax for 2 non-CIPN-inducing agents. Compound-specific effects, eg, neurite fragmentation by cisplatin or bortezomib and enlarged neuronal cell bodies by paclitaxel, were also observed. Collectively, these results support the use of a quantitative, morphologic evaluation and a DRG cell culture model to inform risk and examine mechanisms of CIPN.

Novel insights into renal D-amino acid oxidase accumulation: propiverine changes DAAO localization and peroxisomal size in vivo.

Chronic exposure to propiverine, a frequently prescribed pharmaceutical for treatment of overactive bladder and incontinence, provokes massive protein accumulation in the cytosol and nucleus of renal proximal tubule epithelial cells in rats. Previously, the accumulating protein was identified as D-amino acid oxidase (DAAO), a peroxisomal flavoenzyme expressed in kidney, liver and brain. The cellular mechanism of propiverine-induced DAAO accumulation, however, remains unexplained and poorly characterized. Therefore, to further increase the understanding of DAAO accumulation in rat kidney, this study aimed to characterize DAAO accumulations using differential immunofluorescent staining of rat kidney sections as well as in vitro binding analyses and proteasomal activity studies. We demonstrated that propiverine is neither a ligand of DAAO nor an inhibitor of the proteasome in vitro. However, propiverine treatment resulted in a significant decrease of peroxisomal size in rat proximal tubule epithelial cells. Moreover, peroxisomal catalase also accumulated in the cytosol and nuclei of propiverine-treated rats concurrently with DAAO. Taken together, our study indicates that propiverine treatment affects the trafficking and/or degradation of peroxisomal proteins such as DAAO and catalase by a so far unique and unknown mechanism.

The Parkinson-associated human P5B-ATPase ATP13A2 protects against the iron-induced cytotoxicity.

P-type ion pumps are membrane transporters that have been classified into five subfamilies termed P1-P5. The ion transported by the P5-ATPases is not known. Five genes named ATP13A1-ATP13A5 that belong to the P5-ATPase group are present in humans. Loss-of-function mutations in the ATP13A2 gene (PARK9, OMIM 610513) underlay a form of Parkinson's disease (PD) known as the Kufor-Rakeb syndrome (KRS), which belongs to the group of syndromes of neurodegeneration with brain iron accumulation (NBIA). Here we report that the cytotoxicity induced by iron exposure was two-fold reduced in CHO cells stably expressing the ATP13A2 recombinant protein (ATP13A2). Moreover, the iron content in ATP13A2 cells was lower than control cells stably expressing an inactive mutant of ATP13A2. ATP13A2 expression caused an enlargement of lysosomes and late endosomes. ATP13A2 cells exhibited a reduced iron-induced lysosome membrane permeabilization (LMP). These results suggest that ATP13A2 overexpression improves the lysosome membrane integrity and protects against the iron-induced cell damage.

Regulation of lipid droplet size in mammary epithelial cells by remodeling of membrane lipid composition-a potential mechanism.

Milk fat globule size is determined by the size of its precursors-intracellular lipid droplets-and is tightly associated with its composition. We examined the relationship between phospholipid composition of mammary epithelial cells and the size of both intracellular and secreted milk fat globules. Primary culture of mammary epithelial cells was cultured in medium without free fatty acids (control) or with 0.1 mM free capric, palmitic or oleic acid for 24 h. The amount and composition of the cellular lipids and the size of the lipid droplets were determined in the cells and medium. Mitochondrial quantity and expression levels of genes associated with mitochondrial biogenesis and polar lipid composition were determined. Cells cultured with oleic and palmitic acids contained similar quantities of triglycerides, 3.1- and 3.8-fold higher than in controls, respectively (P < 0.0001). When cultured with oleic acid, 22% of the cells contained large lipid droplets (>3 µm) and phosphatidylethanolamine concentration was higher by 23 and 63% compared with that in the control and palmitic acid treatments, respectively (P < 0.0001). In the presence of palmitic acid, only 4% of the cells contained large lipid droplets and the membrane phosphatidylcholine concentration was 22% and 16% higher than that in the control and oleic acid treatments, respectively (P < 0.0001). In the oleic acid treatment, approximately 40% of the lipid droplets were larger than 5 µm whereas in that of the palmitic acid treatment, only 16% of the droplets were in this size range. Triglyceride secretion in the oleic acid treatment was 2- and 12-fold higher compared with that in the palmitic acid and control treatments, respectively. Results imply that membrane composition of bovine mammary epithelial cells plays a role in controlling intracellular and secreted lipid droplets size, and that this process is not associated with cellular triglyceride content.

Organelle size scaling of the budding yeast vacuole is tuned by membrane trafficking rates.

Organelles serve as biochemical reactors in the cell, and often display characteristic scaling trends with cell size, suggesting mechanisms that coordinate their sizes. In this study, we measure the vacuole-cell size scaling trends in budding yeast using optical microscopy and a novel, to our knowledge, image analysis algorithm. Vacuole volume and surface area both show characteristic scaling trends with respect to cell size that are consistent among different strains. Rapamycin treatment was found to increase vacuole-cell size scaling trends for both volume and surface area. Unexpectedly, these increases did not depend on macroautophagy, as similar increases in vacuole size were observed in the autophagy deficient mutants atg1Δ and atg5Δ. Rather, rapamycin appears to act on vacuole size by inhibiting retrograde membrane trafficking, as the atg18Δ mutant, which is defective in retrograde trafficking, shows similar vacuole size scaling to rapamycin-treated cells and is itself insensitive to rapamycin treatment. Disruption of anterograde membrane trafficking in the apl5Δ mutant leads to complementary changes in vacuole size scaling. These quantitative results lead to a simple model for vacuole size scaling based on proportionality between cell growth rates and vacuole growth rates.

Chloroplast downsizing under nitrate nutrition restrained mesophyll conductance and photosynthesis in rice (Oryza sativa L.) under drought conditions.

The phenomenon whereby ammonium enhances the tolerance of rice seedlings (Oryza sativa L., cv. 'Shanyou 63' hybrid indica China) to water stress has been reported in previous studies. To study the intrinsic mechanism of biomass synthesis related to photosynthesis, hydroponic experiments supplying different nitrogen (N) forms were conducted; water stress was simulated by the addition of polyethylene glycol. Water stress decreased leaf water potential (Ψ(leaf)) under nitrate nutrition, while it had no negative effect under ammonium nutrition. The decreased Ψ(leaf) under nitrate nutrition resulted in chloroplast downsizing and subsequently decreased mesophyll conductance to CO(2) (g(m)). The decreased g(m) and stomatal conductance (g(s)) under nitrate nutrition with water stress restrained the CO(2) supply to the chloroplast and Rubisco. The relatively higher distribution of leaf N to Rubisco under ammonium nutrition might also be of benefit for photosynthesis under water stress. In conclusion, chloroplast downsizing induced a decline in g(m), a relatively higher decrease in g(s) under nitrate nutrition with water stress, restrained the CO(2) supply to Rubisco and finally decreased the photosynthetic rate.

PICK1 inhibition of the Arp2/3 complex controls dendritic spine size and synaptic plasticity.

Activity-dependent remodelling of dendritic spines is essential for neural circuit development and synaptic plasticity, but the precise molecular mechanisms that regulate this process are unclear. Activators of Arp2/3-mediated actin polymerisation are required for spine enlargement; however, during long-term depression (LTD), spines shrink via actin depolymerisation and Arp2/3 inhibitors in this process have not yet been identified. Here, we show that PICK1 regulates spine size in hippocampal neurons via inhibition of the Arp2/3 complex. PICK1 knockdown increases spine size, whereas PICK1 overexpression reduces spine size. NMDA receptor activation results in spine shrinkage, which is blocked by PICK1 knockdown or overexpression of a PICK1 mutant that cannot bind Arp2/3. Furthermore, we show that PICK1-Arp2/3 interactions are required for functional hippocampal LTD. This work demonstrates that PICK1 is a novel regulator of spine dynamics. Via Arp2/3 inhibition, PICK1 has complementary yet distinct roles during LTD to regulate AMPA receptor trafficking and spine size, and therefore functions as a crucial factor in both structural and functional plasticity.

Ghrelin attenuates heat-induced degenerative effects in the rat testis.

This study was conducted to examine the efficacy of ghrelin in prevention of deleterious effects of heat stress in rat testicular tissue. Forty five adult male rats were scheduled for this study and were divided equally into three groups: heat-saline, heat-ghrelin and control-saline. The scrota of heated-designed rats were immersed once in water bath at 43 °C for 15 min. Immediately upon heating, 2 nmol of ghrelin were given subcutaneously to heat-ghrelin animals every other day up to day 60 and physiological saline to the other two groups using the same method. The animals were sacrificed at 10, 30 and 60 days after heat treatment and their testes were taken for later photomicrograph and immunohistochemical analysis. Testicular histopathology revealed a significant reduction in the means of seminiferous tubules and Sertoli cell nucleus diameters as well as germinal epithelium height on day 10 in both heated groups. Furthermore, other testicular components including miotic index, spermatogenesis rate, presence of spermatocytes and volume densities were dramatically decreased following heat exposure. Notably, ghrelin caused a partial recovery in all of the above-mentioned parameters and accelerated testicular regeneration process by day 30 compared to the heat-saline group (P<0.05). Because of testicular progressive recovery, these indices were similar among groups on day 60 (P>0.05). However, immunohistochemistry evaluation for in situ detection of Bcl-2 protein did not exhibit any germ cells-positive of this factor among groups at different experimental days. In conclusion, the results of the present study indicate for the first time the novel evidences of ghrelin ability in attenuation of heat-induced testicular damage and also that ghrelin therapy may be useful as a suppressor of degenerative effects following testicular hyperthermia.

Mitochondrial decay and impairment of antioxidant defenses in aging RPE cells.

In the eye, the retinal pigment epithelium (RPE) is exposed to a highly oxidative environment, partly due to elevated oxygen partial pressure from the choriocapillaris and to digestion of polyunsaturated fatty acid laden photoreceptor outer segments. Here we examined the vulnerability of RPE cells to stress and changes in their mitochondria with increased chronological aging and showed that there is greater sensitivity of the cells to oxidative stress, alterations in their mitochondrial number, size, shape, matrix density, cristae architecture, and membrane integrity as a function of age. These features correlate with reduced cellular levels of ATP, ROS, and [Ca(2+)](c), lower Deltapsim, increased [Ca(2+)](m) sequestration and decreased expression of mtHsp70, UCP2, and SOD3. Mitochondrial decay, bioenergetic deficiencies, and weakened antioxidant defenses in RPE cells occur as early as age 62. With increased severity, these conditions may significantly reduce RPE function in the retina and contribute to age related retinal anomalies.

Effect of selenium on characteristics of rape chloroplasts modified by cadmium.

Selenium appears to be an important protective agent that decreases cadmium-induced toxic effects in animals and plants. The aim of these studies was to investigate the changes of properties of chloroplast membranes obtained from Cd-treated rape seedlings caused by Se additions. Chloroplasts were isolated from leaves of 3-week-old rape plants cultured on Murashige-Skoog media supplied with 2 microM Na(2)SeO(4) and/or 400 microM CdCl(2) under in vitro conditions. The following physicochemical characteristics of chloroplasts were chosen as indicators of Se-effects: average size, zeta potential, ultrastructure, lipid and fatty acid composition and fluidity of envelope membrane. The results suggest that Se can partly counterbalance the destructive effects of Cd. This protective action led to an increase of chloroplast size reduced by Cd treatment and rebuilt, to some extent, the chloroplast ultrastructure. Lipid and fatty acid composition of chloroplast envelopes modified by Cd showed a decrease in digalactosyl-diacylglycerol content and an increase of content of monogalactosyl-diacylglycerol and phospholipid fractions, as well as an increase of fatty acid saturation of all lipids studied. The change in fatty acid saturation correlated well with a decrease of membrane fluidity and with a diminishing of absolute values of zeta potential. The presence of selenium in cultured media caused a partial reversal of the detected changes, which was especially visible in properties related to the hydrophobic part of an envelope, i.e. fatty acid saturation and fluidity.

Phosphatidylethanolamine synthesized by three different pathways is supplied to peroxisomes of the yeast Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae three pathways lead to the formation of phosphatidylethanolamine (PE), namely decarboxylation of phosphatidylserine (PS) (i) by Psd1p in mitochondria, and (ii) by Psd2p in a Golgi/vacuolar compartment; and (iii) synthesis via CDP-ethanolamine pathway in the endoplasmic reticulum. To determine the contribution of these pathways to the supply of PE to peroxisomes, we subjected mutants bearing defects in the respective metabolic routes to biochemical and cell biological analysis. Despite these defects in PE formation mutants were able to grow on oleic acid indicating induction of peroxisome proliferation. Biochemical analysis revealed that PE formed through all three pathways was supplied to peroxisomes. These analyses also demonstrated that selective as well as equilibrium interorganelle flux of PE appear to be equally important for cellular homeostasis of this phospholipid. Electron microscopic inspection confirmed that defects in PE synthesis still allowed formation of peroxisomes, although these organelles from strains lacking PSD1 were significantly smaller than wild type. The fact that peroxisomes were always found in close vicinity to mitochondria, ER and lipid particles supported the view that membrane contact may play a role in lipid traffic between these organelles.

Effect of transient and permanent permeability transition pore opening on NAD(P)H localization in intact cells.

To study the effect of mitochondrial permeability transition pore (PTP) opening on NAD(P)H localization, intact cells were exposed to the Ca(2+) ionophore A23187. PTP opening, mitochondrial membrane potential, mitochondrial volume, and NAD(P)H localization were assessed by time-lapse laser confocal microscopy using the calcein-cobalt technique, tetramethylrhodamine methyl ester, MitoTracker, and NAD(P)H autofluorescence, respectively. Concomitant with PTP opening, NAD(P)H fluorescence increased outside mitochondria. These events occurred in all cells and were prevented by cyclosporin A. Mitochondrial membrane potential was not systematically collapsed, whereas mitochondrial volume did not change, confirming that A23187 induced transient PTP opening in a subpopulation of cells and suggesting that mitochondrial swelling did not immediately occur after PTP opening in intact cells. NAD(P)H autofluorescence remained elevated after PTP opening, particularly after membrane potential had been collapsed by an uncoupler. Extraction of nucleotide for NAD(P)H quantification confirmed that PTP opening led to an increase in NAD(P)H content. Because the oxygen consumption rate decreased, whereas the lactate/pyruvate ratio increased after PTP opening in intact cells, we conclude that PTP opening inhibits respiration and dramatically affects the cytosolic redox potential in intact cells.

Classification of cell subpopulations using multiple cellular parameters from high-content imaging studies.

Cell samples from in vitro studies are almost never uniform; individual cells of a population may differ with respect to their developmental stage, phase in the cell cycle, state of transfection, or by natural variability. Therefore, a sample of cells is usually composed of several subcategories that depend on the experiment setting and research interest. A critical interest of much molecular research is to deconvolute the mixture population and quantitatively assess the subcategories by their proportions in the cell population. In this research, subpopulation classification analysis using the simultaneous analysis of multiple cellular parameters is proposed. In particular, an iterative K-means clustering algorithm is used to establish subpopulation fingerprints, which are then used to classify unknown cell samples by support vector machines. The analyses demonstrate that the multivariate subpopulation analysis is statistically powerful and biologically relevant.

Dysferlin domain-containing proteins, Pex30p and Pex31p, localized to two compartments, control the number and size of oleate-induced peroxisomes in Pichia pastoris.

Yarrowia lipolytica Pex23p and Saccharomyces cerevisiae Pex30p, Pex31p, and Pex32p comprise a family of dysferlin domain-containing peroxins. We show that the deletion of their Pichia pastoris homologues, PEX30 and PEX31, does not affect the function or division of methanol-induced peroxisomes but results in fewer and enlarged, functional, oleate-induced peroxisomes. Synthesis of Pex30p is constitutive, whereas that of Pex31p is oleate-induced but at a much lower level relative to Pex30p. Pex30p interacts with Pex31p and is required for its stability. At steady state, both Pex30p and Pex31p exhibit a dual localization to the endoplasmic reticulum (ER) and peroxisomes. However, Pex30p is localized mostly to the ER, whereas Pex31p is predominantly on peroxisomes. Consistent with ER-to-peroxisome trafficking of these proteins, Pex30p accumulates on peroxisomes upon overexpression of Pex31p. Additionally, Pex31p colocalizes with Pex30p at the ER in pex19Delta cells and can be chased from the ER to peroxisomes in a Pex19p-dependent manner. The dysferlin domains of Pex30p and Pex31p, which are dispensable for their interaction, stability, and subcellular localization, are essential for normal peroxisome number and size. The growth environment-specific role of these peroxins, their dual localization, and the function of their dysferlin domains provide novel insights into peroxisome morphogenesis.

Inhibition of proteasome activity impairs centrosome-dependent microtubule nucleation and organization.

Centrosomes are dynamic organelles that consist of a pair of cylindrical centrioles, surrounded by pericentriolar material. The pericentriolar material contains factors that are involved in microtubule nucleation and organization, and its recruitment varies during the cell cycle. We report here that proteasome inhibition in HeLa cells induces the accumulation of several proteins at the pericentriolar material, including gamma-tubulin, GCP4, NEDD1, ninein, pericentrin, dynactin, and PCM-1. The effect of proteasome inhibition on centrosome proteins does not require intact microtubules and is reversed after removal of proteasome inhibitors. This accrual of centrosome proteins is paralleled by accumulation of ubiquitin in the same area and increased polyubiquitylation of nonsoluble gamma-tubulin. Cells that have accumulated centrosome proteins in response to proteasome inhibition are impaired in microtubule aster formation. Our data point toward a role of the proteasome in the turnover of centrosome proteins, to maintain proper centrosome function.

Inhibition of adenylate cyclase activity in the goldfish melanophore is mediated by α2-adrenoceptors and a pertussis toxin-sensitive GTP-binding protein.

The signal-transduction system that mediates the melanosome-aggregating response in melanophores of the black-moor goldfish, Carassius auratus, was investigated by examining the inhibition of adenylate cyclase activity mediated by α-adrenoceptors in cultured cells. When the melanophores were incubated with 1 mmol·1⁻¹ 3-isobutyl-1-methylxanthine for 5 min, the intracellular level of cyclic adenosine-3',5'-monophosphate increased two- to three-fold. Norepinephrine at 100 nmol· 1⁻¹ and naphazoline at 1 µmol· 1⁻¹ inhibited the 3-isobutyl-1-methylxanthine-induced accumulation of cyclic adenosine-3',5'-monophosphate in the cells in both the presence and the absence of isoproterenol, a beta-adrenergic agonist. Methoxamine and phenylephrine also reduced the extent of accumulation of cyclic adenosine-3',5'-monophosphate, but only when they were present at relatively high concentrations (above 100 µmol·1⁻¹). The range of concentrations at which norepinephrine inhibited the accumulation of cyclic adenosine-3',5'-monophosphate was consistent with the range at which it induced the aggregation of melanosomes. Pretreatment of the cells with pertussis toxin (1 µg·m1⁻¹) for 15 h or treatment with 100 nmol·1⁻¹ yohimbine (an α2-adrenergic antagonist) inhibited the effects of the α-adrenergic agonists on both the aggregation of melanosomes and the 3-isobutyl-1-methylxanthine-induced accumulation of cyclic adenosine-3',5'-monophosphate, but prazosin (an α1-adrenergic antagonist) at 100 nmol·1⁻¹ was not inhibitory. These results indicate that the melanosome-aggregating response of the goldfish melanophore is induced mainly via inhibition of the activity of adenylate cyclase,which occurs as a result of stimulation of a pathway that involves α2-adrenoceptors and a inhibitory GTP-binding protein.