1. Kartonus Per Magnus Haram IB, 2012 Klinikk for Thoraxkirurgi Institutt for sirkulasjon og billeddiagnostikk

2. Ordbruk Relaksasjon = dilatasjon Blir mediert av en vasodilatator (dilator), en substans som relakserer Kontraksjon = konstriksjon Blir mediert av en konstriktor, substans som frembringer kontraksjon

3. Media (smooth muscle)Externa Intima (endothelium) Blood vessels and vasomotor tone Vasomotor tone Variations of the vascular diameter caused by changes in contraction and relaxation of smooth muscle cells vasodilation vasoconstriction Anatomy of blood vessel The main task of the arterial system is to secure an adequate supply of oxygen to organs The flow varies directly as the fourth power of radius of the vessel (Q=r 4 )

4. Control of vasomotor tone Why? To maintain blood pressure stabile Important during changes of posture and blood loss Maintains perfusion of vital organs during rest and activity All resistance and capacitance vessels under basal conditions exhibit some degree of smooth muscle contraction that determines the diameter and hence the flow through the vessel Basal vascular tone is different among organs Organs with a large vasodilatory capacity (e.g. myocardium, skeletal muscle, skin, splanchnic circulation) have high vascular tone Organs having low vasodilatory capacity (e.g. cerebral and renal circulation), have low vascular tone

5. Control of vasomotor tone How? ConstrictionDilation Extrinsic Intrinsic Neural Humoral Tissue metabolites Local hormones Myogenic Endothelial factors Vasoconstrictor (-) Vaodilators (+) - - - ± ± + The vasomotor tone at any given instant is determined by the balance of competing vasoconstrictor and vasodilator influences

6. Neural regulering Det autonome nervesystemet Det sympatiske systemet; ”fight or flight” Dilaterer til muskler og hjerte. Konstrigerer til hud, mage, tarm og nyre. Adrenalin og noradrenalin.

7. Medulla oblongata

8. Renin-Angiotensin-Aldosteron systemet.

9. Autoregulering Organenes evne til å opprettholde konstant blodstrøm selv om perfusjonstrykket synker. Antageligvis en kombinasjon av metabolske, myogene eller endotelderiverte faktorer. Nyre, hjerne og koronarkar har høy autoregulering. Skjelettmuskel har moderat autoregulering, mens huden har dårlig autoregulering.

10. Local Metabolites - Most Potent Regulation VASOCONSTRICTOR Oxygen normoxia constricts hypoxia dilates VASODILATOR Adenosine <= AMP<= ADP <= ATP specific receptors on smooth muscle + lactate, acetate, CO 2, H + Metabolic theory of blood flow regulation

11. Aktiv hyperemi Økt blodstrøm i et organ (hyperemi) ved økt metabolsk aktivitet i dette organet. Utløses av hypoxy, laktat, hypercarbia, osv Skjer ved trening, fordøyelse, infeksjon. Reaktiv hyperemi Forbigående økning i et organs blodstrøm som følge av en kort periode med ischemi. utløses av hypoxy, laktat, hypercarbia, osv.

12. Endothelial function Artery Phenylephrine Ach (10 -9 – 3x10 -5 M) Vessels were contracted with phenylephrine Acetylcholine was added in accumulating doses to stimulate endogenous NO-production Accumulating doses of acetycholine was also added in the presence of L-NAME. Nitroprusside was added to evaluate the response to exogenous NO. Super oxide dismutase was added to quench oxygen radicals EC50: The molar concentration of an agonist, which produces 50% of the maximal possible response for that agonist

13. What is EDRF? (Endothelium Derived Relaxing Factor) EDRF was first discovered by Furchgott and Zawadzki (Nature 288: 373-376 (1980)) Intact endothelium Endothelium rubbed away vasorelaxation no vasorelaxation Nitric Oxide (NO) Endothelial Derived Hyperpolarizing factor, EDHF Prostacyclin (PGI 2 )

14. Nobel Laureates for Nitric Oxide Research 1998

15. Endothelium derived relaxing factors NO eNOS l-arginine l-citrulline Shear stress from blood flow Acetylcholine Bradykinine Substance P Calcium ionophore [Ca i ] ↑ AMPK CaMKII Akt MAPk PI3K lumen Endothelial cell sGC GTP cGMP SMC M2 Arachidonate PGH 2 prostaglandin synthase prostacyclin synthase PGI 2 cAMP PL PLipase AC Ca + [Ca i ] ↓ vasorelaxation PKA K IR K+K+ BK ca K+K+

16. In vivo effects of prostacyclin Inhibits platelet aggregation Vasodilation Aspirin blocks the formation of prostacyclin, but has little effect upon blood-pressure Production decreases with age and decline in production is also associated with intima hyperplasia and growth of vascular smooth muscle

17. What is EDRF ? (Endothelium Derived Relaxing Factor) Nitric Oxide (NO) Endothelial Derived Hyperpolarizing factor, EDHF Prostacyclin (PGI 2 ) Not affected by blocking the NO-production Not affected by blocking the PGI 2 - production

18. [Ca 2+ ] (nM) 0 600 Membrane Potential (mV) -60 -40 Membrane Potential (mV) -60 -40 [Ca 2+ ] (nM) 0 600 Busse et al, 2002 Diameter Endothelium Vascular smooth muscle Endothelium derived hyperpolarising factor

19. Endothelium derived relaxing factors NO eNOS l-arginine l-citrulline Shear stress from blood flow Acetylcholine Bradykinine Substance P Calcium ionophore [Ca i ] ↑ AMPK CaMKII Akt MAPk PI3K lumen Endothelial cell sGC GTP cGMP SMC M Arachidonate PGH 2 prostaglandin synthase prostacyclin synthase PGI 2 cAMP M [Ca i ] ↑ PL PLipase CYP2C AC EET K ca K+K+ SK ca K+K+ IK ca K+K+ Hyperpolarisation BK ca K+K+ Gap junctions K IR K+K+ Ca + [Ca i ] ↓ Hyperpolarisation vasorelaxation ATP 2 K + 3 Na +

20. Clinical effects of EDHF Up till now, NO has been considered as the main contributor to endothelial function Disruption of the smooth muscle hyperpolarisation can influence the level of endothelial cell membrane potential and also reducing the level of NO bioavailability. Suggested candidates for EDHF Arachidonic acid product (Buss et al, 2002) Myoendothelial gap junctions (Chaytor et al, 1998) Increase in extracellular potassium (Edwards et al, 1998) Residual NO resistant to L-NOARG (Chauhan et al, 2003) Hydrogen peroxide (Matuba et al, 2000) Expression of EDHF might require the elimination of NO in order to be expressed (Cowan et al, 1991), (Adeagbo et al, 1993) EDHF is also shown to be attenuated by traditional risk factors such as diabetes and hyperlipidemia (Shimokawa et al, 1999)

21. NO may inhibit EDHF by inhibitiing CYP450 EDHF prevailed when NO production was blocked EDHF prevailed in eNOS(-/-) mice (Brandes et al, 2000) Which EDRF prevails? EDHF versus NO and prostacyclin There is also apparent crosstalk between EDHF and NO as hyperpolarisation of the endothelial cell can increase the driving force of calcium and also the production of NO (Qiu et al 2001, Luckhoff et al 1990) Peroxynitrite can inhibit K Ca – channels in vascular smooth muscle and inhibit the formation of EDHF (Liu et al 2002) Synergistic action between NO and EDHF which occurs in the endothelium and/or in the vascular smooth muscle (Freitas et al 2003) Endothelium dependent relaxations in eNOS(-/-) and COX-1(-/-) ♀ : preserved and normotensive ♂ : decreased and hypertensive (Scotland et al, 2005)

22. How is EDRF distributed along the vascular tree? EDHF is most prominent in smaller arteries and arterioles NO is most prominent in conduit arteries and large arteries This can be due to the fact that in arterioles the ratio between the endothelium and smooth muscle reaches one, and the influence of the membrane potential is therefore symmetric. The phenomenon of EDHF can therefore be explained as a spread of endothelial hyperpolarisation to the smooth muscle cells (Bény 1999) In human subcutaneus resistance arteries, 80% of the ach-mediated vasorelaxation is mediated by EDHF (Coats et al 2001) In large arteries, the membrane potential is strongly influenced by the smooth muscle, and spreading of the membrane potential is rapidly dissipated within the smooth muscle Action potential from the smooth muscle can be transmitted to the endothelium where it can affect the production of NO (Bény 1999) The importance of EDHF increases as the as the vessel size decreases as in the resistance arteries of the coronary, mesenteric, hepatic and renal circulation (McNeish et al 2003)

23. How to improve EDRF Exercise traing improves both NO and EDHF mediated vasorelaxation (Hambrecht et al 2000, Mombouli et al 1996) Estrogen replacement therapy improves both NO and EDHF mediated vasorelaxation (Tagawa et al 1997, Sakuma et al 2002) Antihypertensive treatment improves both EDHF and NO mediated vasorelaxation Eicosapentaenoic acid, a major component of fish oil, improves both NO and EDHF mediated vasorelaxation (Tagawa et al 1999, Tagawa et al 2002) Nifedipine, a calcium channel blocker, improves NO and EDHF mediated vasorelaxation (Yamagishi et al 2005, Fisslthaler et al 2000)

24. What is EDCF ? (Endothelium Derived Constricting Factor) Endothelin Thromboxane A2 Released by thrombin, angiotensin II, arginine-vasopressin, IL-1, TGFβ, catecholamines and anoxia, but inhibited by NO Stimulates vascular smooth cell proliferation Most potent known vasoconstrictor Vasoconstricor especially important during tissue injury and inflammation Potent platelet aggregator (inhibited by aspirin) Responsible for Prinzmetal’s angina (Smith et al 1981)

25. Endothelium derived constricting factors lumen Endothelial cell SMC Arachidonate PGH 2 prostaglandin synthase Thromboxane synthase TXA 2 PL PLipase Ca + [Ca i ] ↑ vasoconstriction Angiotensin II Thrombin Cytokines ROS Big ET-1 ECE NO Prostcyclin ANP ET-1 ET A PL-C RyR IP 3 ET B eNOS cGMP NO Inflammation Injury TR

26. Sensing of shear stress and initiation of NO-production NO eNOS l-arginine l-citrulline Shear stress from blood flow Blood vessel lumen Acetylcholine Bradykinine [Ca i ] ↑ AMPK CaMKII Akt MAPk PI3K Caveolae Hypercholesterolemia Hyperglycemia AGE Lack of BH 4 or HSP 90 Caveolin-1 TNF-α Hypertension HSP 90 HDL VEGF HMG-CoA reductase inhib. Endothelial cell sGC [Ca i ] ↓ GTP cGMP Smooth muscle cell vasorelaxation ”Caveolae signalling hypothesis” docking sites for several signalling molecules ↑ density by in vitro shear stress sensor of shear stress?

27. Impairment of arterial relaxation in experimental atherosclerosis

28. Vasomotor responses of atherosclerotic human coronary vessels

29. Effect of lovastatin and probucol on vasomotor response of atherosclerotic coronary vessels in patients

30. eNOS and metabolic disturbances eNOS +/- mice: (eNOS-expression ~50% of WT) Normal diet: -normal metabolic homeostasis High fat diet:-arterial hypertension - ↓ insulin sensitivity eNOS -/- mice: Normal diet: -hypertensive and insulin resistant -In humans, eNOS-gene polymorphims are associated with hypertension, coronary artery disease and myocardial infarction. -eNOS may represent the genetic link between cardiovascular and metabolic disease. Calorie restriction WT-mice:-increased mitochondrial biogenesis (PGC1α), gene- expression and function, eNOS-expression. eNOS -/- mice:-no effect upon mitochondrial markers

31. Trene endotel ?

32. The timecourse of improvement and decay of endothelial function In exercising athletes, cardiac output can be as much as 40 L min -1 This suggests that the transporting capacity of the vessels to the working musculature must be in excellent condition 6 weeks of high intensity exercise 7 groups x 6 rats One bout of high intensity exercise 7 groups x 6 rats 0612 24 48 96 192 0612 24 48 96 192 ( hours postexercise ) How is the improvement and decay of endothelial function after exercise?

33. The timecourse of improvement and decay of endothelial function * * Time post exercise ( h ) 0612244896192 -0,1 0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 Endothelial function following one bout of exercise EC 50 (exercise – control) * * One bout of exercise induced improvements in endothelial function 12-24 hours post-exercise *

34. The timecourse of improvement and decay of endothelial function * * * * * P < 0.05 * * * * * Time post-exercise ( h ) 0612244896192 -0,2 0,0 0,2 0,4 0,6 0,8 EC 50 (exercise – control) * * * * * Improvement in endothelial function achieved through 6 weeks of exercise, is lost within a week Endothelial function following 6 weeks of exercise

35. O2-O2- The decay of endothelial function at 0 hours post-exercise High intensity exercise generates oxygen radicals This might decrease the bioavailability of NO O2-O2- NO SOD H 2 O 2 + O 2 * P < 0.05 * * P < 0.01

36. The genetic makeup of Homo Sapiens has not changed since the late paleolithic era, about 100000 years ago, when the hunter-gatherer lifestyle was prevailing in order to survive Phenotypes not able to engage in physical activity nessecary to hunt prey or gather food, would increase the likelihood of random elimination Random elimination was less likely to occur in phenotypes able to perform physical activity at a level sufficient to support the hunter-gatherer lifestyle What happens when this ancient phenotype adapted to physical activity is subjected to a sedentary lifestyle? Genome with ”activity” genes selected for physical activity Sedentary lifestyle Genome unchanged last 100000 years Insulin resistance Vascular disease Diabetes mellitus 2 Hypertension Endothelial dysfunction Cancer Osteporosis Weak skeletal muscles Depression Obesity ” Exercise capacity is a more powerfull predictor of mortality than other established risk factors for cardiovascular disease” (Myers et al 2002) Historical perspective on aerobic capacity

37. Heterogeneous founder population Applied artificial selection for endurance capacity across 11 generations High Capacity Runners (HCR) Selection on low versus high intrinsic exercise performance Low Capacity Runners (LCR) Characteristics Running capacity 191 m 853 m VO 2ma x 43.1 mLmin -1 kg -0.75 67.8 mLmin -1 kg -0.75

38. Maximal oxygen uptake – VO 2max LCR HCR Post SE S E § † § Pre S Selection for endurance capacity is associated with diverging VO 2max Exercise training increased VO 2max by 45% in HCR and LCR († for p < 0.01) (§ for p < 0.01) VO 2max in LCR is equal to that in rats with post-infarction heart failure

39. High Capacity Runners (HCR) Selection on low versus high intrinsic exercise performance Low Capacity Runners (LCR) Characteristics Running capacity 191 m 853 m VO 2ma x 43.1 mLmin -1 kg -0.75 67.8 mLmin -1 kg -0.75 Body mass 212.5/309.5 g ♀ / ♂ 172.3/257.6 g ♀ / ♂ 101.5 mmHg89.7 mmHg Mean Bp 110.3 mg/dL 92.4 mg/dL Fasting blood glucose Adipose tissue 68.3 38.7 Plasma 67.1 mg/dL 25.1 mg/dL triglycerides Plasma free 0.64 mEq/L 0.33 mEq/L fatty acids Insulin 684 pM 296 pM Clustering of cardiovascular risk factors

40. IB: α-eNOS ReIB: α-Actin 120- 56- LCRHCR SEES eNOS Actin LCR SE HCR SE e-NOS Proteins involved in the production of NO eNOS Molecular link between cardiovascular and metabolic disease? hyperlipidemia TNFα hyperglycemia eNOS -/- mice: hypertensive and insulin resistant

41. IB: α-eNOS ReIB: α-Actin 120- 56- LCRHCR SEES eNOS Actin LCR SE HCR SE 56- IB: α-HSP 90 ReIB: α-Actin 98- Actin HSP 90 HSP-90 e-NOS Proteins involved in the production of NO IB: α-Caveolin-1 ReIB: α-Actin 19- 56- Actin Caveolin-1

42. HCR-S LCR-S HCR-E LCR-E Caveolin-1 and caveolae density * § ‡ LCR SE HCR SE Caveolae Density Caveolin-1 (-/-) are associated with increased vasorelaxation due to ach but also reduced flow mediated dilation (‡ p < 0.05) (§ p < 0.01)

43. The influence of NO on gene induction NO NF  B Oxidant stress (hydrogen peroxide) oxLDL? Gene induction Collagen biosynthesis Cell proliferation Vascular thickening Cell membrane Inhibition Activation + + –