Tuning the endothelial response: differential release of exocytic cargos from Weibel-Palade bodies.

Essentials Endothelial activation initiates multiple processes, including hemostasis and inflammation. The molecules that contribute to these processes are co-stored in secretory granules. How can the cells control release of granule content to allow differentiated responses? Selected agonists recruit an exocytosis-linked actin ring to boost release of a subset of cargo. SUMMARY: Background Endothelial cells harbor specialized storage organelles, Weibel-Palade bodies (WPBs). Exocytosis of WPB content into the vascular lumen initiates primary hemostasis, mediated by von Willebrand factor (VWF), and inflammation, mediated by several proteins including P-selectin. During full fusion, secretion of this large hemostatic protein and smaller pro-inflammatory proteins are thought to be inextricably linked. Objective To determine if secretagogue-dependent differential release of WPB cargo occurs, and whether this is mediated by the formation of an actomyosin ring during exocytosis. Methods We used VWF string analysis, leukocyte rolling assays, ELISA, spinning disk confocal microscopy, high-throughput confocal microscopy and inhibitor and siRNA treatments to demonstrate the existence of cellular machinery that allows differential release of WPB cargo proteins. Results Inhibition of the actomyosin ring differentially effects two processes regulated by WPB exocytosis; it perturbs VWF string formation but has no effect on leukocyte rolling. The efficiency of ring recruitment correlates with VWF release; the ratio of release of VWF to small cargoes decreases when ring recruitment is inhibited. The recruitment of the actin ring is time dependent (fusion events occurring directly after stimulation are less likely to initiate hemostasis than later events) and is activated by protein kinase C (PKC) isoforms. Conclusions Secretagogues differentially recruit the actomyosin ring, thus demonstrating one mechanism by which the prothrombotic effect of endothelial activation can be modulated. This potentially limits thrombosis whilst permitting a normal inflammatory response. These results have implications for the assessment of WPB fusion, cargo-content release and the treatment of patients with von Willebrand disease.

Predicting physical activity energy expenditure in wheelchair users with a multisensor device.

AIM: To assess the error in predicting physical activity energy expenditure (PAEE), using a multisensor device in wheelchair users, and to examine the efficacy of using an individual heart rate calibration (IC) method. METHODS: 15 manual wheelchair users (36±10 years, 72±11 kg) completed 10 activities: resting, folding clothes, wheelchair propulsion on a 1% gradient (3456 and 7 km/h) and propulsion at 4 km/h (with an additional 8% of body mass, 2% and 3% gradient) on a motorised wheelchair treadmill. Criterion PAEE was measured using a computerised indirect calorimetry system. Participants wore a combined accelerometer and heart rate monitor (Actiheart). They also performed an incremental arm crank ergometry test to exhaustion which permitted retrospective individual calibration of the Actiheart for the activity protocol. Linear regression analysis was conducted between criterion (indirect calorimetry) and estimated PAEE from the Actiheart using the manufacturer's proprietary algorithms (group calibration, GC) or IC. Bland-Altman plots were used and mean absolute error was calculated to assess the agreement between criterion values and estimated PAEE. RESULTS: Predicted PAEE was significantly (p<0.01) correlated with criterion PAEE (GC, r=0.76 and IC, r=0.95). The absolute bias ±95% limits of agreement were 0.51±3.75 and -0.22±0.96 kcal/min for GC and IC, respectively. Mean absolute errors across the activity protocol were 51.4±38.9% using GC and 16.8±15.8% using IC. SUMMARY: PAEE can be accurately and precisely estimated using a combined accelerometer and heart rate monitor device, with integration of an IC. Interindividual variance in cardiovascular function and response to exercise is high in this population. Therefore, in manual wheelchair users, we advocate the use of an IC when using the Actiheart to predict PAEE.

The secretion of von Willebrand factor from endothelial cells; an increasingly complicated story.

von Willebrand factor (VWF) plays key roles in both primary and secondary hemostasis by capturing platelets and chaperoning clotting factor VIII, respectively. It is stored within the Weibel-Palade bodies (WPBs) of endothelial cells as a highly prothrombotic protein, and its release is thus necessarily under tight control. Regulating the secretion of VWF involves multiple layers of cellular machinery that act together at different stages, leading to the exocytic fusion of WPBs with the plasma membrane and the consequent release of VWF. This review aims to provide a snapshot of the current understanding of those components, in particular the members of the Rab family, acting in the increasingly complex story of VWF secretion.

A role for Rab10 in von Willebrand factor release discovered by an AP-1 interactor screen in C. elegans.

BACKGROUND: Endothelial von Willebrand factor (VWF) mediates platelet adhesion and acts as a protective chaperone to clotting factor VIII. Rapid release of highly multimerized VWF is particularly effective in promoting hemostasis. To produce this protein, an elaborate biogenesis is required, culminating at the trans-Golgi network (TGN) in storage within secretory granules called Weibel-Palade bodies (WPB). Failure to correctly form these organelles can lead to uncontrolled secretion of low-molecular-weight multimers of VWF. The TGN-associated adaptor AP-1 and its interactors clathrin, aftiphilin and γ-synergin are essential to initial WPB formation at the Golgi apparatus, and thus to VWF storage and secretion. OBJECTIVES: To identify new proteins implicated in VWF storage and/or secretion. METHODS: A genomewide RNA interference (RNAi) screen was performed in the Nematode C. elegans to identify new AP-1 genetic interactors. RESULTS: The small GTPase Rab10 was found to genetically interact with a partial loss of function of AP-1 in C. elegans. We investigated Rab10 in human primary umbilical vein endothelial cells (HUVECs). We report that Rab10 is enriched at the Golgi apparatus, where WPB are formed, and that in cells where Rab10 expression has been suppressed by siRNA, VWF secretion is altered: the amount of rapidly released VWF was significantly reduced. We also found that Rab8A has a similar function. CONCLUSION: Rab10 and Rab8A are new cytoplasmic factors implicated in WPB biogenesis that play a role in generating granules that can rapidly respond to secretagogue.

Modeling vibrational energy transmission at bolted junctions between a plate and a stiffening rib.

An analytical model is presented for structure-borne sound transmission at a bolted junction in a rib-stiffened plate structure. The model is based on the wave approach for junctions of semi-infinite plates and calculates coupling loss factors required by statistical energy analysis. The stiffening rib is modeled as a plate strip and the junction is represented by an elastic interlayer with a spatially dependent stiffness. Experimental verification is carried out on a series of Plexiglas plate structures with varying rib depth and bolt spacing. A well-defined connection length at the junction was created by inserting thin spacers between the plate and the rib at each bolt. Comparison between numerical and experimental data for this case showed good agreement. Measured results for the bolted junction without spacers suggested that structure-borne sound transmission could be modeled as a series of connections characterized by a finite connection length. This concept is explored further by determining an equivalent connection length which gives the best agreement between numerical and experimental data.

A model of unloaded human intervertebral disk based on NMR relaxation.

NMR relaxation rates were related to the composition of the nucleus pulposus from 11 and anulus fibrosus from six human intervertebral disks. Tissue water was proportional to glycosaminoglycan (GAG) and residue, the noncollagen, non-GAG portion of the dry weight (R2 = 0.74). The solid signal fraction depended on collagen and residue protons (R2 = 0.89). 1/T1 was proportional to collagen and residue (R2 = 0.97). T2 showed 2-4 components labeled A, B, C, and D, with means +/- standard deviations of 3.1 +/- 1.6, 17.5 +/- 9.5, 64 +/- 22, and 347 +/- 162 msec. Signal fractions of A and B depended on the collagen-associated water protons (R2 = 0.94 and 0.85), C on residue-associated water protons (R2 = 0.82), and D on GAG-associated water protons (R2 = 0.74). The data led to a model of disk architecture in which the collagen and residue were largely solid, forming distinct water compartments; the remaining water was present in a proteoglycan gel.

Effect of atmospheric radiance errors in radiometric sea-surface skin temperature measurements.

Errors in measurements of sea-surface skin temperature (SSST) caused by inappropriate measurements of sky radiance are discussed; both model simulations and in situ data obtained in the Atlantic Ocean are used. These errors are typically caused by incorrect radiometer view geometry (pointing), temporal mismatches between the sea surface and atmospheric views, and the effect of wind on the sea surface. For clear-sky, overcast, or high-humidity atmospheric conditions, SSST is relatively insensitive (<0.1 K) to sky-pointing errors of ?10 degrees and to temporal mismatches between the sea and sky views. In mixed-cloud conditions, SSST errors greater than ?0.25 K are possible as a result either of poor radiometer pointing or of a temporal mismatch between the sea and sky views. Sea-surface emissivity also changes with sea view pointing angle. Sea view pointing errors should remain below 5 degrees for SSST errors of <0.1 K. We conclude that the clear-sky requirement of satellite infrared SSST observations means that sky-pointing errors are small when one is obtaining in situ SSST validation data at zenith angles of <40 degrees . At zenith angles greater than this, large errors are possible in high-wind-speed conditions. We recommend that high-resolution inclinometer measurements always be used, together with regular alternating sea and sky views, and that the temporal mismatch between sea and sky views be as small as possible. These results have important implications for the development of operational autonomous instruments for determining SSST for the long-term validation of satellite SSST.

Social stress and atherosclerosis in roosters.

1. Socially stressed roosters fed either plain mash or an atherogenic diet had a greater incidence and severity of aortic atherogenesis than similarly fed non-stressed birds. 2. Results demonstrated a significant atherogenic effect of stress in chickens even in the absence of hyperlipidaemia. 3. Lack of appreciable differences in plasma lipids between stressed and non-stressed birds suggested that the atherogenic effects of stress may be attributable to neuroendocrine responses. 4. Levels of HDLc of plain mash groups were significantly higher than in the atherogenic fed groups. 5. Haemodynamic data showed no treatment-related differences.

Effect of valium (diazepam) on experimental atherosclerosis in roosters.

The development of aortic and coronary atherosclerotic plaques were investigated in roosters fed an atherogenic diet with and without the addition of Valium (0.2 mg/kg twice daily) over a period of 5 months, as a step toward understanding the role of emotional factors in atherogenesis. Plasma levels of cholesterol and triglycerides, hemodynamic parameters, and body weight were measured. There was a progressive, and quantitatively similar, body weight gain in all birds. In addition, there were no significant differences in values for blood pressure, cardiac output, and heart rate between the various experimental groups. Conversely, birds receiving the atherogenic diet, as well as those receiving the atherogenic diet along with Valium, exhibited a marked hypercholesterolemia which reached a peak of 600 mg/dl in 4-6 weeks, before decreasing to between 200 and 300 mg/dl by the 10th week. Plasma triglyceride levels followed a qualitatively similar pattern as plasma cholesterol. Those birds fed an atherogenic diet alone developed atherosclerotic lesions on the aortic surface which was more pronounced on the abdominal than the thoracic aorta. Aortas from birds given Valium along with the atherogenic diet were completely free of lesions despite the fact that plasma cholesterol and triglyceride levels were not significantly different in the two groups. Histological sections of coronary arteries showed severe lesions in 4 out of 7 birds which were fed the atherogenic diet alone, whereas the birds given Valium along with the atherogenic diet had only an occasional slight lipid deposit. It was concluded that Valium provides some protection against the development of atherosclerosis in roosters fed an atherogenic diet.

Comparison of brachial venous and mixed blood gas tensions and pH values in the chicken.

Differences in blood gas tensions and pH between brachial venous blood (BVB) and mixed venous blood obtained from the pulmonary artery (PAB) were compared in anesthetized male White Leghorn chickens to determine if BVB as obtained in routine venipuncture cold be used to estimate mixed venous values of pO2, pCO2, and pH. When paired samples were compared over the range of 25 to 68 mm Hg, brachial pO2 was 5.7 mm Hg higher (P less than .001) than PAB pO2. Brachial pCO2 was 4.25 mm Hg higher (P less than .001) than PAB pCO2 over the range of 17 to 56 mm Hg. Brachial pH was .066 units lower (p less than .001) than comparable values for PAB over the range of 7.2-7.6 pH units. Regression equations are given for estimating mixed venous blood gas tensions and pH values from blood samples taken from the brachial vein.

Pancreatic elastase activation as a possible indicator of the relative hazard of different cigarettes.

Levels of elastase activity in homogenates of pancreas from beagle dogs exposed to cigarette smoke on a daily basis for 600 d were previously reported. Elastase activity in pancreatic homogenates from animals exposed to smoke from high-nicotine cigarettes was significantly greater than in sham-exposed controls or in animals smoking low-nicotine cigarettes. In the study described here, dogs were exposed on an acute basis to smoke from high-nicotine and from nicotine-free cigarettes. Anesthetized animals were prepared by laparotomy, duodenotomy, and pancreatic duct cannulation. Pancreatic fluid was collected and preexposure elastase levels were determined. Samples were then taken during exposure to smoke from cigarettes of both types. There was no measurable elastase activity in the baseline samples. Similarly, no activity was found in fluids collected before and during exposure to smoke from nicotine-free cigarettes. Enzyme activity was detected, however, in samples collected during exposure to smoke from high-nicotine cigarettes once blood levels of nicotine reached 50-70 ng/ml. The results suggest that cigarette smoke may provoke activation of elastase and indicate the need for further study of any association of cigarette smoking with pancreatic disease.

Long-term effects of diazepam on plasma lipids and atheroma in roosters fed in atherogenic diet.

Long-term effects of diazepam on plasma lipids and atheroma in roosters fed an atherogenic diet have been studied. Our data indicate that long-term administration (8 months) of diazepam (Valium) 0.2 mg/kg orally lowered the plasma cholesterol level when compared to the atherogenic group, but this was not statistically significant (P greater than 0.05). However, at the dose of 0.4 mg/kg of Valium the plasma cholesterol was significantly lowered when compared to the controls on an atherogenic diet (P less than 0.05). Both doses of Valium significantly decreased the incidence and severity of lesions on the endothelial surface of the aorta and coronary artery in roosters fed an atherogenic diet. No changes in plasma triglyceride values were noted. Administration of 0.4 mg/kg of Valium to birds receiving the atherogenic diet produced a small but significant increase of mean, systolic and diastolic blood pressures as compared to the birds receiving an atherogenic diet but no Valium. A similar increase in blood pressure was also noted in the control group on plain mash diet receiving 0.2 mg/kg of Valium. Microscopic examination of the coronary arteries showed that birds on an atherogenic regimen receiving Valium had less atherosclerosis than those not receiving Valium.

Comparison of Cardiopulmonary Parameters in awake and anesthetized chickens.

Steady state cardiovascular and respiratory parameters in adult male chickens while they were awake and after anesthetization with a mixture of chloral hydrate, magnesium sulfate, and pentobarbital were compared. Blood pressure (BP), heart rate (HR), cardiac output (CO), stroke volume (SV), peripheral resistance (TPR), tidal volume (VT), respiratory rate (RR), minute ventilation (V), end-experatory carbon dioxide partial pressure (PACO2), and arterial blood gases and pH were measured simultaneously on birds spontaneously breathing air. Anesthetization resulted in increased HR and RR and decreased BP, CO, TPR, VT, PACO2, and blood gas tension. The data indicate a depression of cardiovascular function but no change in total ventilation although the relative contributions of VT and RR were changed. Anesthetization increased variability in SV although the other parameters were maintained in a steady-state condition over a 2-h period.

Acute isovolemic anemia in anesthetized chickens.

Acute isovolemic anemia was produced in anesthetized chickens by serial exchanges of 6% dextran 70 equal to 1% of body weight to quantitate cardiovascular and metabolic parameters. When hematocrit (Hct) and hemoglobin (Hb) levels were reduced by 50% (from 33.3 to 16.3 vol %, and from 10.3 to 5.4 g/100 g, respectively, P less than 0.001), tissue oxygen delivery was maintained by increases in cardiac output (CO), stroke volume (SV), oxygen extraction, and reduced total peripheral resistance (TPR). Heart rate, right atrial pressure, and oxygen consumption (Vo2) were unchanged. Further reductions in Hct and Hb (to 10.8 vol % and 3.7 g/100 g, respectively), were accompanied by cardiovascular failure, as evidenced by falling CO, SV, tissue oxygen delivery, and Vo2. Relative apparent viscosity determinations on the exchanged blood-dextran mixtures indicated that large viscosity changes occurred with the first exchange whereas subsequent exchanges had small incremental viscosity changes. These data indicate that in acutely anemic chickens, oxygen transport capacity was maintained by increased cardiac output and decreased peripheral resistance, unless the severity of the anemia resulted in cardiovascular failure.

Functional significance of thoracic vagal branches in the chicken.

One minute electrical stimulation was used to excite the right and left cervical vagi as well as specific points on, or branches of, the left thoracic vagus. Respiratory, heart rate and blood pressure responses were observed with the nerves intact and cut. Stimulation of either intact cervical vagus produced apnea, bradycardia and blood pressure depression. Stimulation of the cut ends after nerve section demonstrated that the heart rate and blood pressure effects were efferent and the respiratory change was afferent. No responses were observed due to stimulation of the vagus caudal to the lungs. Stimulation of cardiac branches reduced heart rate and blood pressure but did not produce significant respiratory effects. Middle and anterior pulmonary branches were found to contribute only to respiratory changes through afferent nerves. Sudden, sustained reduction of CO2 in the airways produced immediate, sustained apnea. The data suggest that CO2 sensitive thoracic receptors important in regulation of respiration are confined primarily to the lungs and that these receptors play no direct role in cardiovascular function.