Role of Chronic Shear Stress in Endothelial Form and Function

Endothelial cells in vivo exist in a dynamic environment, subject to the physical forces of blood flow as it is regulated through the cardiac cycle. Arguably, the most important force endothelial cells are subject to is shear stress, the frictional force of blood flow across the cell surface. Areas of the vasculature that experience laminar shear stress appear resistant to the development of atherosclerotic plaques, whereas those that experience low shear stress, due to complex patterns of blood flow, appear susceptible. In vitro study of the effects of chronic shear stress on the endothelium has been somewhat limited, due to the methods of modelling shear stress available, which are for the most part only suitable for culture for up to 24 hours. I have validated an orbital shaker method of modelling two flow environments seen in the vasculature, unidirectional flow and non-directional flow, with associated shear stress profiles, for chronic time periods of up to 7 days. I have shown clear differences between the two environments in terms of endothelial cell morphology and protein expression and identified many ways in which sheared cells differ from their static counterparts, in terms of morphology, protein expression, vascular mediator release and transcriptional profile. Shear stress appears to be a protective force, inhibiting expression of inflammatory mediators and significantly altering response to inflammatory stimulus. The orbital shaker may prove a useful model for in vitro study of the endothelium in a situation similar to that of physiological conditions

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The thermophysiological and ergogenic response to heat stress intervention strategies

A thesis submitted to the University of Bedfordshire, in partial fulfilment of the requirements for the degree of Master of Science by ResearchEndurance exercise in hot environments puts a great strain on both the physiological and cellular mechanisms of the body to maintain efficient heat dissipation and thermal homeostasis. Once the body is unable to dissipate more heat than is gained thermal stress increases core (TCore) and skin temperature (TSkin) impairing performance. Athletes and military personnel train and compete/work in many extreme environments; the utilisation of intervention strategies prior to exercise will delay the onset of fatigue and reduce thermal strain at a physiological and cellular level. The purpose of the first experiment was to investigate the combined effect of hyperhydration and pre-cooling methods on endurance cycling performance in the heat. Five healthy males completed a mile (16.1 km) self-paced time trial (TT) in a hot and humid environment (30°C & 50% RH) on 4 occasions: Glycerol hyperhydration (HH), pre-cooling (PC), glycerol hyperhydration and pre-cooling (HH+PC) and control (C). The cellular stress response was assessed via Heat Shock 70 kDa Protein 2 (HSP72) mRNA expression within leukocytes. There was a significant difference in completion time between the conditions (p = 0.025). On average, completion time during the PC trial was 6% faster than C (p = 0.03, 95% CI = -15 to - 210 s) and 4% faster than HH (p = 0.02, 95% CI = - 21 to -132 s). There was no significant difference in HSP72 mRNA expression between conditions (p = 0.26). PC via CWI alone or in combination with HH, enhanced endurance performance in hot and humid environments with no further ergogenic effect seen when HH was used in combination with PC. In light of the findings from the first experimental chapter, experiment 2 looked at the kinetics and mechanisms of G-HH compared to hyperhydration with water (W-HH) at rest. 16 resting males’ on 2 occasions: ingested one of two solutions evenly over a 90 min period. Glycerol solution (G-HH) or a water solution (W-HH). It was revealed that peak change in 2 plasma volume (%ΔPV) was significantly higher after G-HH (19.1 ± 6.3%) than W-HH (10.2 ± 4.5%) (F1, 9.3 = 14.37, p = 0.004). G-HH effectively expanded PV more than water hyperhydration for the full 120 min observation period (p = 0.02). It is recommended that exercise and extreme environment occupational pursuits (such as military and bush firefighters), commences immediately post the 90 min ingestion period when PV expansion is highest, to delay the onset of dehydration. The third experimental chapter investigated the potential pre-cooling action of an acute dose of acetaminophen and its comparison to established pre-cooling methods: cold water immersion and ice slurry ingestion on exercise in extreme heat. Evaluated from physiological and cellular perspective. 8 recreationally active males completed a 40 min sub-maximal run in extreme heat (40°C & 30% RH) on 4 occasions: cold water immersion (CWI), ice slurry ingestion (ICE), acetaminophen ingestion (ACT) and control (CON). There was significant reduction in TRectal (-0.48°C) and Tskin (4°C lower than all other conditions) after CWI compared to ICE, CON and ACT. A significant down regulation of HSP72 expression post exercise after ACT compared to CWI. ACT did not elicit a thermoregulatory reduction but did however reduce strain on a cellular level during exercise in extreme heat. CWI proved to be the most effective form of pre-cooling through the reduction of TRectal and Tskin prior to exercise. These findings confirm previous research that cold water immersion alters the robust PV expansion produced by glycerol hyperhydration (Gordon, Fogarty, Greenleaf et al., 2003). Cold water immersion is the most effective pre-cooling method to reduce thermal strain and improve performance it does however lack practical application. Acetaminophen did not prove to effectively reduce thermoregulatory strain but did however reduce strain on a cellular level. These results suggest that individuals participating in prolonged exercise in hot conditions due to its practicality for use in the field further research needs to be conducted in 3 to acetaminophen’s mechanisms of action and potential to reduce thermal strain at a cellular and possibly in the correct settings physiological level

Shape and Compliance of Endothelial Cells after Shear Stress In Vitro or from Different Aortic Regions: Scanning Ion Conductance Microscopy Study

Objective: To measure the elongation and compliance of endothelial cells subjected to different patterns of shear stress in vitro, and to compare these parameters with the elongation and compliance of endothelial cells from different regions of the intact aorta. Materials and Methods: Porcine aortic endothelial cells were cultured for 6 days under static conditions or on an orbital shaker. The shaker generated a wave of medium, inducing pulsatile shear stress with a preferred orientation at the edge of the well or steadier shear stress with changing orientation at its centre. The topography and compliance of these cells and cells from the inner and outer curvature of ex vivo porcine aortic arches were measured by scanning ion conductance microscopy (SICM). Results: Cells cultured under oriented shear stress were more elongated and less compliant than cells grown under static conditions or under shear stress with no preferred orientation. Cells from the outer curvature of the aorta were more elongated and less compliant than cells from the inner curvature. Conclusion: The elongation and compliance of cultured endothelial cells vary according to the pattern of applied shear stress, and are inversely correlated. A similar inverse correlation occurs in the aortic arch, with variation between region

A role for ATP in renal fibrosis as a downstream mediator of TGF-β1-evoked changes in hemichannel activity

Aims: Data from our group confirms that increased expression of Connexin-26 and Connexin-43 in biopsy material from patients with proven nephropathy is paralleled by TGF-1-evoked changes in hemichannel-mediated ATP release. With recent studies linking increased ATP to fibrosis in multiple tissue types, we have previously confirmed that incubation of proximal tubule cells with ATPS (1-100M) evokes increased expression of ECM markers. In the current study, we provide evidence of a direct role for ATP and associated downstream purinergic signalling as a mediator of TGF-1 induced altered hemichannel activity in ECM expression. Methods: Human kidney (HK2) proximal tubule cells were treated for 48hrs with either TGF-β1(10ng/mL) ± nucleotidase; apyrase (100µM) or ATPγS (10µM) ± purinergic receptor antagonist suramin (100µM). Expression of Collagen I, Collagen IV, Fibronectin and Laminin were determined by immunoblotting. Results: Immunoblotting confirmed that apyrase negated TGF-β1 upregulation of Collagen I, from 366.0±13.0% of control to 119.5% (n=3 P<0.001) and reversed loss of Collagen IV expression from 30.1±12.1% of control to 123.3±9.8% (n=3 P<0.01). The nucleotidase also negated an upregulation of Fibronectin, from 201.5±3.7% to 156.7±27.6% (n=3 P<0.01) and Laminin, from 339.5±43.1% to 173.4±42.8% (n=3 P<0.05). Suramin inhibited ATPγS-induced changes in expression of ECM, reducing expression of Collagen I from 452.9±20.6% of control to 192.1±16.0%; Collagen IV from 157.2±17.4% to 93.6±12.8%; Fibronectin from 222.6±9.5% to 103.7±5.2% and Laminin from 177.8±25.0% to 86.4±11.7% (n=3 P<0.05). Conclusions: The current study confirms that TGF-1 induced changes in hemichannel mediated ATP release may in part, contribute to tubular fibrosis in the diabetic kidney. Acknowledgement: This work is supported by Diabetes UK (BDA:16/0005427 and BDA:16/0005509)

A role for Collagen I in regulating Connexin-43 mediated hemichannel activity in the proximal region of the diabetic kidney.

Background and Aims: Tubulointerstitial fibrosis represents the key underlying pathology in diabetic kidney disease and is characterised by tubular injury and extracellular matrix remodelling. In diabetic nephropathy increased Collagen I deposition is linked to inflammation and fibrosis, yet, we know little as to how collagen facilitates these effects. Pivotal to early tubular injury is impaired epithelial (E)-cadherin mediated cell adhesion; loss of which inhibits docking of connexin-mediated hemichannels and prevents gap junction intercellular communication. Uncoupled hemi-channels respond via increased release of adenosine triphosphate (ATP), an effect we have previously confirmed is linked to increased expression of inflammatory and fibrotic markers. The current study investigates if Collagen I mediates its effects through altered connexin-43 (Cx43) mediated hemichannel activity in human proximal tubule cells. Materials and Methods: Human kidney (HK-2) proximal tubule cells were cultured on Collagen I (50µg/mL) and treated for 48 hours ± TGF-β1 (2-10ng/mL). Uncoated plastic served as the control. Expression of candidate proteins was determined by immunoblotting. Cell-substrate interactions were measured using the Cytoselect cell adhesion assay. Carboxyfluorescein (200µM) dye uptake and ATP biosensing were used to measure hemi-channel activity and ATP release respectively in TGF-β1 treated HK-2 at 48 hours. Results: Immunoblot analysis confirmed that TGF-β1 increased Collagen I expression to 167±19%, 181±8% and 192±19% at 2, 4 and 10ng/mL respectively as compared to control, whilst adhesion assays confirmed that HK-2 cells exhibit increased affinity for Collagen I in the presence of TGF-β1 (10ng/mL) 0.36±0.06OD as compared to control (0.15±0.02D) (n=4, P<0.05). Interestingly, when cultured on Collagen I, cells exhibit increased expression of Cx43 to 107±16%, with co-incubation of TGF-1 exacerbating this effect to 136±7% as compared to control (n=5, P<0.001). This increase in expression was paralleled by increased hemichannel activity and ATP release. Carboxyfluorescein dye uptake increased in cells cultured on Collagen I ± TGF-1 to 149±6% (Collagen I) and 251±5% (Collagen I + TGF-1) respectively, as compared to control (n=5, P<0.001), whilst biosensing confirmed a significant increase in ATP, increased from 3.6±0.4µM (Collagen I) to 4.5±0.2µM (Collagen I + TGF-1) as compared to control (n=3, P<0.001). Conclusion: The current study confirms a role for Collagen I in regulating Connexin43 expression, changes which are exacerbated in the presence of pro-fibrotic TGF-1, the principal mediator of damage in the tubular region of the diabetic kidney. Increased expression of this protein, central to cell-cell communication, was paralleled by increased hemichannel mediated ATP release, most likely a consequence of diminished cell-cell adhesion and Gap Junction mediated intercellular communication as previously reported by the group. With studies linking increased Collagen I deposition to altered cell phenotype in tubulointerstitial fibrosis, and elevated ATP release to increased fibrosis and inflammation; this study highlights a potential role for Collagen I in exacerbating diabetic tubular injury by regulating connexin-mediated hemi-channel activity

'The live dynamic whole of feeling and behavior': capital punishment and the politics of emotion, 1945–1957

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Purinergic receptor (P2X7) activation reduces cell–cell adhesion between tubular epithelial cells of the proximal kidney

Loss of epithelial (E)-cadherin mediated cell-cell adhesion impairs gap junction formation and facilitates hemichannel-mediated ATP release in the diabetic kidney. Linked to inflammation and fibrosis, we hypothesized that local increases in inter-cellular ATP activate P2X7 receptors on neighbouring epithelial cells of the proximal tubule, to further impair cell-cell adhesion and ultimately exacerbate tubular injury. Immunoblotting confirmed changes in E-cadherin expression in human kidney cells treated with non-hydrolysable ATPγS ± the P2X7 antagonist, A438079. Atomic force microscopy based single-cell force spectroscopy quantified maximum unbinding force, tether rupture events, and work of detachment. Confocal microscopy assessed cytoskeletal reorganisation. Our studies confirmed that ATPγS downregulated E-cadherin expression in proximal kidney cells, loss of which was paralleled by a reduction in intercellular ligation forces, decreased tether rupture events and cytoskeletal remodelling. Co-incubation with A438079 restored loss of adhesion, suggesting that elevated extracellular ATP mediates tubular injury through P2X7 induced loss of E-cadherin mediated adhesion

Purinergic receptor (P2X7) activation contributes to disassembly of adherens & tight junctions in tubular epithelial cells of the diabetic kidney.

Background and aims: Preceded by a loss of cell-cell adhesion, glucose-evoked changes in connexin (Cx) expression/function are linked to increased hemichannel mediated release of adenosine triphosphate (ATP) in tubular epithelial cells of the diabetic kidney. Elevated ATP is associated with inflammation and fibrosis, and this study investigates a role for Cx43 hemichannel-mediated ATP release in regulating adherens- and tight-junction proteins, e!ects that initiate the morphological and phenotypic changes of tubular damage. Materials and methods: Human primary proximal tubule epithelial cells (HPTECs) and clonal tubular epithelial cells (HK2) were cultured in TGFβ1 (10ng/mL) ± apyrase (5U/ml), or non-hydrolysable ATPγS (100μM) at 48h. Immunoblotting assessed protein expression. Trans-epithelial electrical resistance assessed paracellular tight junction formation and atomic force microscopy force spectroscopy measured cell-cell adhesion. Carboxyfluorescein uptake and ATP biosensing measured hemichannel activity and nucleotide release. Co-incubation of cells with TGFβ1 ± Peptide5 (25μM) or A438079 (50μM) assessed e!ect of Cx43 hemichannel and purinergic receptor (P2X7) blockade respectively. Results: Immunoblotting confirmed that TGFβ1 downregulated E-cadherin (ECAD), claudin-2 and ZO-1 to 38.5±4.1%, 60.5±4.4% and 64.8±4.4% respectively, whilst N-cadherin (NCAD) expression increased to 213.3±28.0% compared to control (P<0.01; n=4). The e!ect was replicated by ATPγS, which decreased expression of ECAD, claudin-2 and ZO-1 to 43.4±6.1%, 42.0±2.6% and 45.9±1.4% respectively. NCAD increased to 181.3±6.3% (P<0.01; n=3). In a separate series of experiments, co-incubation with the ectonucleotidase apyrase partially restored ECAD expression to 51.2±3.2%, and NCAD to 133.3±9.1%, compared to control (P<0.001; n=3). Trans-epithelial resistance decreased in TGFβ1 and ATPγS treated cells from 67.7±5.5Ω.cm2to 27.6±2.0Ω.cm2 and 42.6±3.0Ω.cm2 respectively (P<0.05; n=3). Mean unbinding forces between ATPγS treated cells also decreased from 2.17±0.64nN in control cells to 1.60±0.48nN (P<0.001; n=3) confirming a loss of cell-cell adhesion. Increased carboxyfluorescein uptake (609.4±46.0%) and ATP release (6.10±0.36μM from 0.43±0.03μM) confirmed increased hemichannel mediated ATP release in TGFβ1 treated cells, an effect blocked by Cx43 mimetic, Peptide5 (163.0±10.2% and 0.60±0.20μM) (P<0.001; n=3). Co-incubation of HPTECs with TGFβ1 and Peptide5 restored expression of ECAD (108.9±17.1% from 31.5±9.2%), NCAD (154.7±10.6% from 280.5±16.7%), claudin-2 (100.9±10% from 65.3±5.4%) and ZO-1 (91.6±12.8% from 59.6±3.1%) compared to control (P<0.01; n=3). Blocking P2X7 with A438079 restored ECAD expression from 22.2±5.5% to 52.8±5.4% in TGFβ1 treated cells (P<0.001; n=3), with unbinding forces restored from 2.17±0.64nN to 2.45±0.89nN (P<0.001; n=3). Conclusion: Hemichannel mediated ATP release is downstream of TGFβ1-evoked changes to adherens- and tight-junction proteins, e!ects blocked by inhibiting P2X7 receptors or Cx43 hemichannel activity. Disassembly of these junctions is a pivotal event in progression of tubular injury in diabetic nephropathy and data suggests a potential role for Cx-mediated hemichannel activity as a future therapeutic target in diabetic kidney disease