Abstract not available

<p>Reduksjon av gassbobler i blodbanen: metoder for å forebygge ugunstige effekter av dekompresjon.</p><p>Når en dykker returnerer til overflaten etter dykking, kan det dannes gassbobler i kroppen som følge av overmetning av gasser. Slike gassbobler kan igjen føre til trykkfallsyke, men det gjenstår fremdeles å finne alle mekanismene bak denne sammenhengen. Gassbobler er derimot gode indikatorer på risiko for trykkfallsyke, og den gjennomgående arbeidshypotesen i denne avhandlingen har vært at gassbobler i blodbanen er den bakenforliggende årsaken til alvorlig trykkfallsyke. Det å redusere mengden gassbobler vil dermed øke sikkerheten for dykkeren.</p><p>Avhandlingen består av tre studier som på forskjellige måter forsøker å redusere boblemengden ved trykkreduksjon. Alle arbeidene er gjennomført med bruk av gris som forsøksdyr, og alle dykkene er simulert i trykk-kammer spesielt laget for slike studier. For å måle gassbobler har vi benyttet ultralydavbildning, samt at vi har tatt ut kar for å måle eventuelle funksjonelle endringer i disse i etterkant av dykkene.</p><p>Den første studien demonstrer en ny metode for å redusere gassbobledannelsen ved dekompresjon. Ved kortvarig å øke trykket under pågående trykkreduksjon kan boblemengden signifikant reduseres, resultatene viser at en modell som tar hensyn til bobledannelse beskriver resultatene bedre enn en tradisjonell modell som bare tar hensyn til overmetningen. I den andre studien har vi for første gang vist at gassbobler i blodbanen kan påvirkes medikamentelt også hos store dyr under dekompresjon fra metning. Ved å gi nitrater umiddelbart før dekompresjonen startet, ble mengden gassbobler signifikant redusert sammenlignet med kontrollene som ikke fikk tilført nitrater. Studien åpner veien for videre studier av biokjemiske prosesser involvert i både dannelsen av og effektene av gassbobler. I den siste studien undersøkte vi om en behandlingsprosedyre for trykkfallsyke til bruk når et trykk-kammer ikke er tilgjengelig ville være effektiv om behandlingstrykket ble redusert fra 190 kPa til 160 kPa med pusting av ren oksygen. Vi viste her at trykket var tilstrekkelig for å fjerne boblene etter dykket, men vi forhindret ikke skader på blodkarene.</p><p>Kandidat: Andreas Møllerløkken</p><p>Institutt: Institutt for sirkulasjon og bildediagnostikk</p><p>Veileder: Professor Alf O. Brubakk</p><p>Finansieringskilder: Statoil, Norsk Hydro, Phillips Petroleum Company Norway og Petroleumstilsynet gjennom programmet forskning og utvikling innen dykking, kontraktsnr. 4600002328 med Norsk Undervannsintervensjon (NUI).</p><p>Ovennevnte avhandling er funnet verdig til å forsvares offentlig</p><p>for graden Philosophia Doctor i medisinsk teknologi</p><p>Disputas finner sted i Auditoriet, Medisinsk teknisk forskningssenter</p><p>Tirsdag 15.01.08 , kl. 12.15</p>

Ph.D. / Biomedical Engineering / The cardiovascular disease atherosclerosis is directly linked to the functions of the endothelium, the monolayer of endothelial cells (ECs) that line the lumen of all blood vessels. EC functions are affected by fluid shear stress (FSS), the tangential force exerted by flowing blood. In vivo FSS is determined by vascular geometry with relatively straight vessels producing high, unidirectional FSS and vessel branch points and curvatures producing low, oscillatory FSS. While these distinct FSS conditions differentially regulate EC functions, they also dramatically affect EC shape and cytoskeletal structure. High and unidirectional FSS induces EC elongation and cytoskeletal alignment, while concurrently promoting EC functions that are atheroprotective. In contrast, low and oscillatory FSS induces cobblestone-shaped ECs with randomly oriented cytoskeletal features, while simultaneously promoting EC functions that create an athero-prone vascular environment. Whether these distinct EC shapes and cytoskeletal structures influence EC functions, independent of FSS, is largely unknown. The overall hypothesis of this study is that cell shape and cytoskeletal structure regulate EC functions through mechanisms that are independent of FSS. Due to advances in surface engineering in the field of micropatterning, EC shape can be controlled independent of external forces by creating spatially localized surface cues. In this research, lanes of protein were micropatterned on glass surfaces to induce EC elongated shape in the absence of FSS. In Aim 1, micropattern-elongated EC (MPEC) shape and cytoskeletal structure were fully characterized and determined to be comparable to FSS-elongated ECs. Thus, inducing EC elongation on micropatterned lanes provides a platform for studying the functional consequences of EC shape, independent of FSS. Using this model, the following important markers of EC functions related to atherosclerosis were evaluated to determine the influence of EC shape and cytoskeletal alignment: extracellular matrix deposition (Aim 2), inflammatory function(Aim 3), and thrombotic potential (Aim 4). The results indicate that EC-elongated shape and cytoskeletal alignment participate in promoting selected EC functions that are protective against atherosclerosis, independent of FSS. Since EC shape is governed by the cytoskeleton, this data suggests that the cytoskeleton plays an active role in the regulation of EC functions that promote cardiovascular health.

We hypothesized that the interaction between tumor necrosis factor alpha(TNF)/nuclear factor-kappaB (NFkB) via activation of IKK may amplify one anotherresulting in the evolution of vascular disease and insulin resistance associated withdiabetes. The interaction between TNFa and monocyte chemoattractant protein-1 (MCP-1) may contribute to the evolution of vascular inflammation and endothelial dysfunctionin coronary arterioles in type 2 diabetes. To test this hypothesis, endothelium-dependent(ACh) and –independent (SNP) vasodilation of isolated, pressurized coronary arterioles(40-100 μm) from mLeprdb (heterozygote, normal), Leprdb (homozygote, diabetic) andLeprdb mice null for TNF (dbTNF-/dbTNF-) were examined. Although dilation of vesselsto SNP was not different between Leprdb and mLeprdb mice, dilation to ACh was reducedin Leprdb mice. The NFkB antagonist, MG-132, IKK inhibitor, sodium salicylate(NaSal), or Anti-MCP-1 partially restored endothelium-dependent coronary arteriolardilation in Leprdb mice. Protein expression of IKK and IKK were higher in Leprdb thanin mLeprdb mice. The expression of IKK, but not the expression of IKK was increasedin dbTNF-/dbTNF- mice. Leprdb mice showed increased insulin resistance, but NaSal improved insulin sensitivity. Protein expression of TNFa, NFkB, phosphorylation ofIKK and JNK were greater in Leprdb mice, but NaSal attenuated protein expression ofthem in Leprdb mice. The ratio of phosphorylated IRS-1 at Ser307 (pIRS-1)/IRS-1protein expression was elevated in Leprdb mice; both NaSal and JNK inhibitor SP600125reduced pIRS-1/IRS-1 in Leprdb mice. MG-132 or neutralization of TNF reducedsuperoxide production in Leprdb mice. Anti-MCP-1 attenuated superoxide productionand protein expression of nitrotyrosine (N-Tyr), which is an indicator of peroxynitriteproduction, in isolated coronary arterioles of Leprdb mice. Immunostaining resultsshowed that expression of MCP-1 and vascular cellular adhesion molecule-1 (VCAM) isco-localized with endothelial cells and macrophages. Anti-TNFa or anti-MCP-1markedly reduced macrophage infiltration and the number of MCP-1 positive cells.Neutralization of TNFa or anti-MCP-1 reduced the expression of adhesion molecules. Inconclusion, our results indicate that the interaction between NFkB and TNFa signalinginduces activation of IKKb. In addition, TNFa and TNFa-related signaling, includingthe expression of MCP-1 and adhesion molecules, further exacerbates oxidative stressleading to endothelial dysfunction in type 2 diabetes.

Reduksjon av gassbobler i blodbanen: metoder for å forebygge ugunstige effekter av dekompresjon. Når en dykker returnerer til overflaten etter dykking, kan det dannes gassbobler i kroppen som følge av overmetning av gasser. Slike gassbobler kan igjen føre til trykkfallsyke, men det gjenstår fremdeles å finne alle mekanismene bak denne sammenhengen. Gassbobler er derimot gode indikatorer på risiko for trykkfallsyke, og den gjennomgående arbeidshypotesen i denne avhandlingen har vært at gassbobler i blodbanen er den bakenforliggende årsaken til alvorlig trykkfallsyke. Det å redusere mengden gassbobler vil dermed øke sikkerheten for dykkeren. Avhandlingen består av tre studier som på forskjellige måter forsøker å redusere boblemengden ved trykkreduksjon. Alle arbeidene er gjennomført med bruk av gris som forsøksdyr, og alle dykkene er simulert i trykk-kammer spesielt laget for slike studier. For å måle gassbobler har vi benyttet ultralydavbildning, samt at vi har tatt ut kar for å måle eventuelle funksjonelle endringer i disse i etterkant av dykkene. Den første studien demonstrer en ny metode for å redusere gassbobledannelsen ved dekompresjon. Ved kortvarig å øke trykket under pågående trykkreduksjon kan boblemengden signifikant reduseres, resultatene viser at en modell som tar hensyn til bobledannelse beskriver resultatene bedre enn en tradisjonell modell som bare tar hensyn til overmetningen. I den andre studien har vi for første gang vist at gassbobler i blodbanen kan påvirkes medikamentelt også hos store dyr under dekompresjon fra metning. Ved å gi nitrater umiddelbart før dekompresjonen startet, ble mengden gassbobler signifikant redusert sammenlignet med kontrollene som ikke fikk tilført nitrater. Studien åpner veien for videre studier av biokjemiske prosesser involvert i både dannelsen av og effektene av gassbobler. I den siste studien undersøkte vi om en behandlingsprosedyre for trykkfallsyke til bruk når et trykk-kammer ikke er tilgjengelig ville være effektiv om behandlingstrykket ble redusert fra 190 kPa til 160 kPa med pusting av ren oksygen. Vi viste her at trykket var tilstrekkelig for å fjerne boblene etter dykket, men vi forhindret ikke skader på blodkarene. Kandidat: Andreas Møllerløkken Institutt: Institutt for sirkulasjon og bildediagnostikk Veileder: Professor Alf O. Brubakk Finansieringskilder: Statoil, Norsk Hydro, Phillips Petroleum Company Norway og Petroleumstilsynet gjennom programmet forskning og utvikling innen dykking, kontraktsnr. 4600002328 med Norsk Undervannsintervensjon (NUI). Ovennevnte avhandling er funnet verdig til å forsvares offentlig for graden Philosophia Doctor i medisinsk teknologi Disputas finner sted i Auditoriet, Medisinsk teknisk forskningssenter Tirsdag 15.01.08 , kl. 12.15

Endothelial Progenitor Cells (EPCs) are thought to participate in the healing of injured vascular endothelium by incorporating into the defect sites to mediate endothelial recovery. Recently, osteopontin (OPN) was shown to be fundamental in accelerating estrogen-dependent healing of injured blood vessels. Here, we are investigating the effect OPN has on EPC behavior. Late outgrowth human EPCs (LEPCs) were derived from circulating monocytes isolated by leukophoresis, and grown in culture until passage six. L-EPCs were then assayed for adhesion, spreading, chemotaxis, and haptotaxis, as well as resistance to detachment by flow electric cellsubstrate impedance sensing (ECIS). The results of standard and ECIS methods showed both dose and time dependent responses in cell adhesion and spreading. In addition, OPN promoted haptotactic migration of EPCs in Boyden chamber assays. LEPCs seeded onto 10μM OPN substrates and exposed to laminar flow had grater survival and higher resistance to detachment than OPN/static and flow only conditions. CD44 and !1 integrins were only responsible for approximately 50% of LEPCs adhesion to OPN compared to the unblocked condition. Western blots showed that Rho GTPases were activated in L-EPCs seeded on OPN. However, this activation could not be completely blocked by either CD44 or !1 integrin antagonists. These data confirm the direct effects of OPN on EPCs adhesion, and suggest that OPN works by mediating cell adhesion during vascular injury.

Previous studies have shown that L-tryptophan treatment has been known to reduce blood pressure (BP) in hypertensive rats. L-tryptophan is converted to serotonin (5-HT), a potent vasoconstrictor agonist. The direct vascular effects of L-tryptophan, an essential amino acid, and the mechanism that contributes to the fall in BP have not been fully explored. The present study aims to examine the direct vascular responses to both D- and L- tryptophan using perfused mesenteric vascular bed, an ex-vivo preparation that represents the resistance function of circulation. Perfusion was maintained at a constant flow rate (5 mL/min) with Krebs buffer (pH 7.4, 37˚C) after isolation from 12 to 14 week old male Sprague-Dawley rats. The basal perfusion pressure (PP) (mean ± SEM) was 27 ± 3 mmHg. Inclusion of D- and L-isomers in the perfusion medium led to concentration-dependent increase in PP. While the maximal response (Emax) was similar, D-tryptophan (EC50: 0.25 ± 0.12* µmol; Emax: 128 ± 8 mmHg) was more potent (lower EC50 value; *p < 0.01) than L-tryptophan (EC50: 0.79 ± 0.30 µmol; Emax: 141 ± 7 mmHg). Inclusion of increasing concentrations (2, 5 and 10 nM) of the 5-HT2A selective antagonist, ketanserin, led to parallel right-ward shifts in the concentration-response curves to D- and L-tryptophan with restoration of their Emax. In contrast, the α1 selective agonist, methoxamine (30 µM), constricted preparations, both D- (IC50: 0.94 ± 0.30* µmol; Imax: 96 ± 2%) and L-tryptophan (IC50: 2.8 ± 1.0 µmol Imax: 88± 1%) evoked concentration-dependent vasodilatation, an effect that was resistant to blockade by either ketanserin or other 5-HT antagonists. Again, D-tryptophan was more potent than L-tryptophan in the presence of 5-HT antagonist (*p < 0.05). Neither the removal of endothelium nor incubation with selective inhibitors of dilatory mediators released from the endothelium, failed to alter the vasodilator responses to D- and L-tryptophan. In potassium chloride depolarized preparations, L-tryptophan evoked an additive vasoconstrictor response. The vasodilator responses to L-tryptophan persisted in the presence of glibenclamide, a KATP channel inhibitor, or tetraethyl ammonium, a BKCa channel inhibitor, or BaCl2, a Kir channel inhibitor, or ouabain, a Na+-K+-ATPase pump inhibitor. These data confirm that the essential amino acid, L-tryptophan, as well as its D-isomer, evoke a biphasic vasoconstrictor and vasodilator responses in the resistance type mesenteric vascular bed. While the vasoconstrictor responses are mediated by activation of vascular 5-HT receptors, the endothelium-independent vasodilator responses are not linked to activation of vascular 5-HT receptors, vascular potassium channels, Na+-K+-ATPase pump or via inhibition of voltage-operated Ca2+-channels. Plasma concentration of L-tryptophan is about 90 - 120 µM. The endothelium/5-HT independent direct vasodilator responses characterized here for the first time could account for the antihypertensive/ BP lowering effect of L-tryptophan reported earlier by other laboratories.

Endothelial Progenitor Cells (EPCs) are thought to participate in the healing of injured vascular endothelium by incorporating into the defect sites to mediate endothelial recovery. Recently, osteopontin (OPN) was shown to be fundamental in accelerating estrogen-dependent healing of injured blood vessels. Here, we are investigating the effect OPN has on EPC behavior. Late outgrowth human EPCs (LEPCs) were derived from circulating monocytes isolated by leukophoresis, and grown in culture until passage six. L-EPCs were then assayed for adhesion, spreading, chemotaxis, and haptotaxis, as well as resistance to detachment by flow electric cellsubstrate impedance sensing (ECIS). The results of standard and ECIS methods showed both dose and time dependent responses in cell adhesion and spreading. In addition, OPN promoted haptotactic migration of EPCs in Boyden chamber assays. LEPCs seeded onto 10μM OPN substrates and exposed to laminar flow had grater survival and higher resistance to detachment than OPN/static and flow only conditions. CD44 and !1 integrins were only responsible for approximately 50% of LEPCs adhesion to OPN compared to the unblocked condition. Western blots showed that Rho GTPases were activated in L-EPCs seeded on OPN. However, this activation could not be completely blocked by either CD44 or !1 integrin antagonists. These data confirm the direct effects of OPN on EPCs adhesion, and suggest that OPN works by mediating cell adhesion during vascular injury.

It is well established that hypertension decreases endothelium-dependent vasomotor function, partially by excessive generation and reduced scavenging of reactive oxygen species (ROS). Nevertheless, at appropriate levels, some ROS can act as signaling molecules in the vasculature and contribute to endothelium-dependent dilation. Recent evidence in healthy resistance arteries suggests that the ROS species hydrogen peroxide (H2O2) acts as an endogenous endothelium-dependent dilator through a non-nitric oxide, non-prostaglandin (3NP) pathway. The aim of this study was to investigate the role of endogenous H2O2 in 3NP-mediated endothelium-dependent dilation of rat mesenteric arteries, and the changes that occur in these vessels with essential hypertension. 18-20wk old male spontaneously hypertensive rats (SHR; n=24) had an elevated systolic blood pressure of 198±6mmHg compared to 93±4mmHg (p<0.001) in the age matched normotensive Wistar-Kyoto rat (WKY; n=22). Isolated mesenteric arteries were preconstricted with norepinephrine (NEPI), followed by exposure to increasing doses of the endothelium-dependent dilator acetylcholine (ACh), which revealed vasomotor dysfunction in the SHR (maximal dilation: WKY: 94.8±1.3% vs. SHR: 75.2±2.9%, p<0.001). Incubation of the vessels with the non-specific cyclooxygenase (COX) inhibitor indomethacin (INDO) restored the ACh response in the SHR to the level of the WKY control (area under the curve: WKY: 354.6±8.6 vs. SHR INDO: 350.2±12.2, p>0.05) indicating that the release of constrictory prostaglandins from COX contribute to endothelial vasomotor dysfunction. Co-incubation of vessels with INDO and the nitric oxide synthase inhibitor Nω-nitro-L-arginine (LN) inhibited dilation in SHR (46.2±4.8%, p<0.001) but not in WKY (98.3±1.5%, p>0.05), indicating an elevated 3NP component in WKY over SHR. Further co-incubation with the H2O2 scavenger catalase (CAT), LN, and INDO inhibited the 3NP component to a greater extent in SHR (29.7±3.1%, p=0.062) than in WKY (91.6±2.5%, p<0.05). The responses of SHR and WKY mesenteric arteries to the endothelium-independent dilator sodium nitroprusside, the receptor-mediated constrictor NEPI, and the electrochemical constrictor KCl were no different between LN INDO and CAT LN INDO conditions. These data suggest that endogenous H2O2 has a greater role in mediating endothelium-dependent dilation in the mesenteric resistance arteries of SHR. Interestingly, in SHR, co-incubation with LN INDO improved dilation over LN alone (46.2±4.8% vs 23.3±3.2±, p=0.001), and CAT LN INDO decreased dilation from LN INDO to a similar extent, suggesting that COX-inhibition could be a source of H2O2 for endogenous vasodilation. Western blotting revealed a 54% increase in COX-1 protein expression in the SHR mesenteric arteries (WKY: 1.00±0.18 (n=9) vs. SHR: 1.54±0.17 (n=13), p<0.05), but no difference in the expression of the pro-oxidant enzyme p47phox, and the anti-oxidant enzymes CAT, SOD-1, and SOD-2. Administration of exogenous H2O2 to NEPI preconstricted mesenteric arteries revealed a dose-dependent dilation that was no different between SHR and WKY, and incubation of isolated WKY and SHR mesenteric arteries with CAT reduced the accumulation of H2O2 to a similar extent, as assessed by the H2O2-specific fluorescent dye Amplex Red. In conclusion, endogenous H2O2 is a vasodilator in the mesenteric arteries of SHR and WKY rats in the absence of nitric oxide and prostaglandins. In the SHR, COX-1 inhibition may allow endogenous H2O2 to become bioavailable for vasodilation. This study is the first to show a role for endogenous H2O2 in maintaining endothelium-dependent dilation in hypertensive rat resistance arteries, and provides evidence to support a role for COX-1-inhibition in the increased availability of H2O2 for dilation.

It is well established that hypertension decreases endothelium-dependent vasomotor function, partially by excessive generation and reduced scavenging of reactive oxygen species (ROS). Nevertheless, at appropriate levels, some ROS can act as signaling molecules in the vasculature and contribute to endothelium-dependent dilation. Recent evidence in healthy resistance arteries suggests that the ROS species hydrogen peroxide (H2O2) acts as an endogenous endothelium-dependent dilator through a non-nitric oxide, non-prostaglandin (3NP) pathway. The aim of this study was to investigate the role of endogenous H2O2 in 3NP-mediated endothelium-dependent dilation of rat mesenteric arteries, and the changes that occur in these vessels with essential hypertension. 18-20wk old male spontaneously hypertensive rats (SHR; n=24) had an elevated systolic blood pressure of 198±6mmHg compared to 93±4mmHg (p<0.001) in the age matched normotensive Wistar-Kyoto rat (WKY; n=22). Isolated mesenteric arteries were preconstricted with norepinephrine (NEPI), followed by exposure to increasing doses of the endothelium-dependent dilator acetylcholine (ACh), which revealed vasomotor dysfunction in the SHR (maximal dilation: WKY: 94.8±1.3% vs. SHR: 75.2±2.9%, p<0.001). Incubation of the vessels with the non-specific cyclooxygenase (COX) inhibitor indomethacin (INDO) restored the ACh response in the SHR to the level of the WKY control (area under the curve: WKY: 354.6±8.6 vs. SHR INDO: 350.2±12.2, p>0.05) indicating that the release of constrictory prostaglandins from COX contribute to endothelial vasomotor dysfunction. Co-incubation of vessels with INDO and the nitric oxide synthase inhibitor Nω-nitro-L-arginine (LN) inhibited dilation in SHR (46.2±4.8%, p<0.001) but not in WKY (98.3±1.5%, p>0.05), indicating an elevated 3NP component in WKY over SHR. Further co-incubation with the H2O2 scavenger catalase (CAT), LN, and INDO inhibited the 3NP component to a greater extent in SHR (29.7±3.1%, p=0.062) than in WKY (91.6±2.5%, p<0.05). The responses of SHR and WKY mesenteric arteries to the endothelium-independent dilator sodium nitroprusside, the receptor-mediated constrictor NEPI, and the electrochemical constrictor KCl were no different between LN INDO and CAT LN INDO conditions. These data suggest that endogenous H2O2 has a greater role in mediating endothelium-dependent dilation in the mesenteric resistance arteries of SHR. Interestingly, in SHR, co-incubation with LN INDO improved dilation over LN alone (46.2±4.8% vs 23.3±3.2±, p=0.001), and CAT LN INDO decreased dilation from LN INDO to a similar extent, suggesting that COX-inhibition could be a source of H2O2 for endogenous vasodilation. Western blotting revealed a 54% increase in COX-1 protein expression in the SHR mesenteric arteries (WKY: 1.00±0.18 (n=9) vs. SHR: 1.54±0.17 (n=13), p<0.05), but no difference in the expression of the pro-oxidant enzyme p47phox, and the anti-oxidant enzymes CAT, SOD-1, and SOD-2. Administration of exogenous H2O2 to NEPI preconstricted mesenteric arteries revealed a dose-dependent dilation that was no different between SHR and WKY, and incubation of isolated WKY and SHR mesenteric arteries with CAT reduced the accumulation of H2O2 to a similar extent, as assessed by the H2O2-specific fluorescent dye Amplex Red. In conclusion, endogenous H2O2 is a vasodilator in the mesenteric arteries of SHR and WKY rats in the absence of nitric oxide and prostaglandins. In the SHR, COX-1 inhibition may allow endogenous H2O2 to become bioavailable for vasodilation. This study is the first to show a role for endogenous H2O2 in maintaining endothelium-dependent dilation in hypertensive rat resistance arteries, and provides evidence to support a role for COX-1-inhibition in the increased availability of H2O2 for dilation.