Endothelial dysfunction in hypertension: from bench to bedside

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1 Endothelial dysfunction in hypertension: from bench to bedside Stefano Taddei Department of Clinical and Experimental Medicine University of Pisa, Italy

Press Release: The 1998 Nobel Prize in Physiology or

NOBEL ASSEMBLY AT KAROLINSKA INSTITUTET October 12, 1998

decided to award the Nobel Prize in Physiology or

Ignarro and Ferid Murad for their discoveries concerning

2 Press Release: The 1998 Nobel Prize in Physiology or Medicine NOBELFÖRSAMLINGEN KAROLINSKA INSTITUTET THE NOBEL ASSEMBLY AT KAROLINSKA INSTITUTET October 12, 1998 The Nobel Assembly at Karolinska Institutet has today decided to award the Nobel Prize in Physiology or Medicine for 1998 jointly to Robert F. Furchgott, Louis J. Ignarro and Ferid Murad for their discoveries concerning "nitric oxide as a signalling molecule in the cardiovascular system Figura 3

AGGREGATION INHIBITION Renin Shear TGF 1 Stress Thrombin

T AT1 A M Mitocondria NADPH-oxidase enos Xanthine-oxidase

Endothelium AT-II ET-1 PGH 2 O - 2 - TXA 2 NO PGI 2 EDHFs

3 AGGREGATION INHIBITION Renin Shear TGF 1 Stress Thrombin AT-II Ach ADP ET HT Platelets Bk Renin ATG ACE AT-I T AT1 A M Mitocondria NADPH-oxidase enos Xanthine-oxidase ET-1 Cyclooxygenase L-Arg NO P PGI 2 ET B S 1 EDHF BK Endothelium AT-II ET-1 PGH 2 O TXA 2 NO PGI 2 EDHFs AT1 ET A ET B TX CONTRACTION cgmp cgmp RELAXATION K + Smooth muscle cel

4 Endothelial dysfunction in human hypertension

5 Endothelium-dependent relaxation in WKY and SHR Lüscher TF & Vanhoutte PM, Hypertension 1986

6 Genetic Hypertension

7 1% 2% 0.5% 0.2% 0.1% 0.1% 0.02% Data acquisition and analysis Forearm blood flow (plethysmography) Cuff inflator O 1 MODE CUFF PRESET 05 0 Hokanson Intra-arterial infusion Plethysmograph AUT O Hokanson EC6 PO PLETHYSMOGRAPH VEI ART WE N ERY R POSITI ON RANGE CALIBRATIO N GALLER Y BALAN CE PHOTO SELECT BP and HR monitoring

8 Endothelium-dependent vasodilation in patients with essential hypertension or primary aldosteronism FBF Δ% p<0.05 Normotensive subjects Patients with essential hypertension Patients with primary aldosteronism Acetylcholine µg/100 ml/min Sodium Nitroprusside Taddei S et al. Hypertension 1993

COX is responsible for endotyhelial dysfunction in patients with essential hypertensive but not in patients with primary aldosteronism FBF Δ% Healthy Subjects Essential Hypertension Primary

9 COX is responsible for endotyhelial dysfunction in patients with essential hypertensive but not in patients with primary aldosteronism FBF Δ% Healthy Subjects Essential Hypertension Primary Aldosteronism saline indomethacin p<0.05 Acetylcholine Acetylcholine Acetylcholine µg/100 ml/min Taddei S et al. Hypertension 1993

10 FBF (D %) Cyclooxygenase inhibition restores NO activity in essential hypertension Healthy Subjects Hypertensive Patients Baseline Indomethacin saline L-NMMA p<0.05 Acetylcholine Acetylcholine µg/100 ml/min Acetylcholine Taddei S et al. Hypertension 1997

11 Cyclooxygenase is a major source of oxidative stress in essential hypertension Healthy Subjects Hypertensive Patients FBF D% salina indometacina vitamina C indo + vit C p< ACETYLCHOLINE µg/100 ml/min ACETYLCHOLINE Taddei S et al. Circulation 1998

12 NO COX

13 COX and endothelial dysfunction in essential hypertensive patients: unsolved questions Which COX isoenzyme is the predominant isoform contributing to ROS generation in essential hypertension? Is COX the only recognized ROS source, in small resistance arteries from essential hypertensive patients?

~350 µm) Isometric (wire myograph) Peripheral resistance

14 Micromiography Gluteal subcutaneous or intrasurgery biopsy Subcutaneous fat 3 rd branch Mesenteric artery(150 ~350 µm) Isometric (wire myograph) Peripheral resistance artery (150 ~350 µm) Isobaric (pressure myograph) (45-60 mmhg)

15 Relaxation response (%) Relaxation response (%) Identification of sources of oxidative stress in small arteries of essential hypertensive patients Essential hypertensive patients Essential hypertensive patients 0 20 saline L-NAME Asc. Acid Asc. Acid + L-NAME 0 20 saline L-NAME Dup-697 (COX-2 inhibitor) Dup L-NAME Acetylcholine (10 x mol/l) Acetylcholine (10 x mol/l) Virdis A et al, Hypertension 2013

16 Immunostaining of COX-1 and COX-2 in controls and hypertensive patients NORMOTENSIVE CONTROLS HYPERTENSIVE PATIENTS Virdis A et al, Hypertension 2013

17 Essential Hypertension Endothelial cells NO ROS - NAD(P)H oxidase COX-2 PGH 2 AA PGE 2 TXA 2 PGI 2 PGD 2 PGF 2 RELAXATION CONTRACTION Vascular Smooth Muscle Cells

18 AGGREGATION INHIBITION Renin Shear TGF 1 Stress Thrombin AT-II Ach ADP ET HT Platelets Bk Renin ATG ACE AT-I T AT1 A M Mitocondria NADPH-oxidase enos Xanthine-oxidase ET-1 Cyclooxygenase L-Arg NO P PGI 2 ET B S 1 EDHF BK Endothelium AT-II ET-1 PGH 2 O TXA 2 NO PGI 2 EDHFs AT1 ET A ET B TX CONTRACTION cgmp cgmp RELAXATION K + Smooth muscle cells

19 Effect of L-NMMA (to block NO-synthase) and ouabain (to block hyperpolarization) on response to bradykinin Normotensive Subjects Essential Hypertensive Patients FBT Δ% Saline L-NMMA Ouabain P<0.05 Bradykinin Control Bradykinin Vitamin C Bradykinin µg/100 ml/min Taddei S et al. Circulation 1999

20 Mechanisms responsible for endothelium-dependent vasodilation in human hypertension NO EDHF O NO EDHF O 2 - NO EDHF Relaxation Normotensive Subjects Relaxation Essential Hypertensive Patients Relaxation Essential Hypertensive Patients

21 Effects of L-NMMA and sulfaphenazole on vasodilation to bradykinin Normotensive subjects Hypertensive patients Saline L-NMMA Sulfaphenazole p< # Saline L-NMMA Sulfaphenazole p< # # Bradykinin (ng/100 ml/min) Bradykinin (ng/100 ml/min) Taddei S et al, JACC 2006

22 The isoenzyme 2C of the cytochrome P450 epoxygenase (named CYP 2C) is a major source of EDHF Ach, BK R K + Ach, BK R PL PLipase [Ca 2+ ]i AA EET s CYP 2C K Ca Hyperpolarization [Ca 2+ ]i SK Ca IK Ca Hyperpolarization, Relaxation BK Ca K + Na + - K + pump Ouabain K + K IR K + Hyperpolarization [Ca 2+ ]i Relaxation Hyperpolarization, Relaxation Busse R et al, Trends in Pharmacological Sciences, 2002

23 AGGREGATION INHIBITION Renin Shear TGF 1 Stress Thrombin AT-II Ach ADP ET HT Platelets Bk Renin ATG ACE AT-I T AT1 A M Mitocondria NADPH-oxidase enos Xanthine-oxidase ET-1 Cyclooxygenase L-Arg NO P PGI 2 ET B S 1 EDHF BK Endothelium AT-II ET-1 PGH 2 O TXA 2 NO PGI 2 EDHFs AT1 ET A ET B TX CONTRACTION cgmp cgmp RELAXATION K + Smooth muscle cells

24 Fibrinolitic properties of endothelial cells Pro-fibrinolitic platelets Anti-fibrinolitic + + coagulation t-pa + FIBRINOLYSIS - PAI-1 CV risk factors Hypertension Diabetes Smoking etc plasminogen fibrin plasmin FDP

Simultaneous bolood sampling for the determination of venous-arterial differences Venous value greater than arterial value RELEASE Venous value lower than arterial value

25 Simultaneous bolood sampling for the determination of venous-arterial differences Venous value greater than arterial value RELEASE Venous value lower than arterial value UPTAKE Arterial sample Net balance = (Cv-Ca) x [FBF x (1-Ht)] Deep venous sample vc= venous concentration ac= arterial concentration FBF= forearm blood flow Ht= hematocrit

26 t-pa balance (ng/100 ml/min) t-pa balance (ng/100 ml/min) Bradykinin, but not acetylcholine, can realese t-pa in hypertensive patients with impaired NO availability by a mechanism involving a sulfaphenazol (SULFA)-sensitive pathway (EDHF?) Acetylcholine induced t-pa release Bradykinin induced t-pa release 1.5 Normotensive subjects Hypertensive patients 1.5 Normotensive subjects Hypertensive patients p<0.01 vs baseline p<0.01 vs baseline ACh ACh + L- NMMA ACh ACh + L- NMMA 0 BDK BDK + L- BDK BDK + NMMA SULFA Giannarelli C et al. Hypertension 2007 Giannarelli C et al Circulation 2009

27 t-pa balance (ng/100 ml/min) t-pa balance (ng/100 ml/min) Bradykinin, but not acetylcholine, can realese t-pa in hypertensive patients with impaired NO availability by a mechanism involving a sulfaphenazol (SULFA)-sensitive pathway (EDHF?) Acetylcholine induced t-pa release Bradykinin induced t-pa release 1.5 Normotensive subjects Hypertensive patients 1.5 Normotensive subjects Hypertensive patients p<0.01 vs baseline p<0.01 vs baseline ACh ACh + L- NMMA ACh ACh + L- NMMA 0 BDK BDK + L- BDK BDK + NMMA SULFA Giannarelli C et al. Hypertension 2007 Giannarelli C et al Circulation 2009

28 Relaxation t-pa release Smooth muscle cells Healthy Conditions NO CYP 2C9-derived EDHF Endothelial cells Bradykinin

29 Relaxation t-pa release Smooth muscle cells Essential Hypertension Endothelial cells NO CYP 2C9-derived EDHF Bradykinin

30 Hypertension causes premature aging of endothelial function in humans normotensives hypertensives p<0.05 Normotensive individuals Hypertensive patients Age-group interaction = 0.25 Taddei S et al, Circulation 1996 Bruno RM et al, Hypertension mmHg

31 Clinical significance of endothelial dysfunction in hypertension: moving to non-invasive tests

32 Pathogenesis of atherosclerosis from endothelial dysfunction to clinical disease endothelial dysfunction plaque growth PGI EDHF NO stimuli-induced vasodilation (e.g, to shear stress) remodeling/ proliferation acute coronary syndrome ischemia / angina pectoris clinical manifestations

33 Brachial Artery Flow Mediated Dilation (FMD) Endothelium-dependent stumulus h shear stress = post-ischemic flow (Reactive Hyperemia) to 5 min. ischemia Endothelium-dependent response h Diameter following Reactive Hyperemia FMD Studio (

34 Prediction of future cardiovascular outcomes by brachial artery FMD: a meta-analysis +1% FMD -13% CV risk Inaba Y et al. Int J Cardiovasc Imaging 2010

35 In the MESA study FMD did not improve cardiovascular risk assessment in intermediate-risk individuals Jeboah J et al. JAMA. 2012;308(8):

36 FMD reproducibility in the MESA study: was it enough? Reproducibility in the MESA study: - CAC: intraobserver and interobserver agreement k=0.90 and IMT: coefficient of variation 7.07% - FMD: intraclass coefficient 0.54 Jeboah J et al. JAMA. 2012;308(8):

37 FMD standardization improves reproducibility 1 month CV: 12.9% Ghiadoni L et al. Curr Pharm Des 2008 Gemignani V et al. UM&B 2007 Ghiadoni et al. J Hypertens month CV: 18.3% Charakida M et al. Eur Heart J 2010 Charakida M et al. Eur Heart J 2013

FMD as a surrogate endpoint in clinical trials 466 patients with target LDL-C levels received dalcetrapib (a CETP-inhibitor) 600 mg/day or placebo for 36 weeks on

38 FMD as a surrogate endpoint in clinical trials 466 patients with target LDL-C levels received dalcetrapib (a CETP-inhibitor) 600 mg/day or placebo for 36 weeks on top of standard therapy (including statins). The primary outcome measures were the change from baseline of flow-mediated dilatation (%FMD) of the right brachial artery

39 Luscher TF et al, EHJ 2012 No change in FMD, despite increased HDL levels No benefit on CV events in the DALoutcome study

40 CV Risk Factors NO-Synthase Inflammation Oxidative Stress NO O Treatment Endothelial dysfunction Cardiovascular events

41 Aging, endothelyal dysfunction and aerobic physical exercise 900 FBF (D %) YOUNG ATHLETES YOUNG SEDENTARY SUBJECTS 900 FBF (D %) OLD ATHLETES OLD SEDENTARY SUBJECTS saline L-NMMA saline L-NMMA p < p < p < ACETYLCHOLINE µg/100 ml/tissue/min ACETYLCHOLINE ACETYLCHOLINE ACETYLCHOLINE µg/100 ml/tissue/min Taddei S et al. Circulation 2000

42 Different Effect of Antihypertensive Drugs on Conduit Artery Endothelial Function Normotensive subjects 7.1 Hypertensive patients (baseline) 5.2 p<0.001 after 6 months treatment Perindopril 6.4 Nifedipine Amlodipine p<0.01 vs other treatments Atenolol Nebivolol Telmisartan FMD (%) Ghiadoni L et al. Hypertension 2003

43 Ramipril dose-dependently increases nitric oxide availability in the radial artery of essential hypertension patients Ghiadoni L. et al J Hypertens 2007, 25:

44 Effect of pharmacological treatment on endothelial dysfunction Conduit arteries ACE-I AT 1 -Ant Ca-Ant coronary peripheral Subcutaneous microcirculation Muscle microcirculation acetylcholine, metacholine bradikynin no data + +

45 Beyond hypertension: endothelial dysfunction is a key player in the greatest epidemics of XXI century: Obesity Dementia

46 Relaxation response (%) Relaxation response (%) Nitric oxide availability in obese patients and control subjects Biodisponibilità di NO nei pazienti obesi e controlli Control Obese 0 Saline L-NAME 0 Saline L-NAME 20 P < P = Acetylcholine (10 x mol/l) Acetylcholine (10 x mol/l) Virdis A et al, JACC 2011

47 Relaxation response (%) Relaxation response (%) Role of ROS on endothelium-dependent relaxation in Produzione di ROS negli obesi e controlli obese patients and control subjects Control Obese 0 20 Saline Tempol 0 20 Saline Tempol Tempol + L-NAME P < P < Acetylcholine (10 x mol/l) Acetylcholine (10 x mol/l) Virdis A et al, JACC 2011

Relaxation response (%) Role of TNF-α on endothelium-dependent relaxation and NO availability in obese patients ell Infliximab sul rilasciamento endoteliodipendente nei pazienti obesi 0 20

48 Relaxation response (%) Role of TNF-α on endothelium-dependent relaxation and NO availability in obese patients ell Infliximab sul rilasciamento endoteliodipendente nei pazienti obesi 0 20 saline Infliximab Infliximab + L-NAME P < Endothelium R NOS L-arginine NO ROS 80 P < Acetylcholine (10 x mol/l) TNF-α Smooth Muscle cells Virdis A et al, JACC 2011

49 Blood brain barrier Endothelial nitric oxide: protector of an healthy mind Katusic ZS and Austin SA. Eur Heart J. 2013

The vascular basis of dementia Cardiovascular risk factors are involved in Amyloid-beta deposition and Alzheimer s dementia through two main

50 The vascular basis of dementia Cardiovascular risk factors are involved in Amyloid-beta deposition and Alzheimer s dementia through two main mechanisms: Blood-brain barrier dysfunction Brain hypoperfusion Endothelium-derived NO plays a role in both mechanisms Zlokovic BV. Nat Rev Neuroscience (2011)

Study The Train the Brain Consortium, Scientific

51 Cognitive-physical training in mild cognitive impairment: The Train the Brain Mind the Vessel Study The Train the Brain Consortium, Scientific reports 2017 Bruno RM et al, Hypertension 2017 in press

52 Cognitive-physical training improves cognitive function, cerebral blood flow and endothelial function MRI-cerebral blood flow Hippocampal and parahippocampal regions are crucial for memory and processing of non-verbal / spatial information The Train the Brain Consortium, Scientific reports 2017

FMD (%) Cognitive-physical training improves cognitive function, cerebral blood flow and endothelial function MRI-cerebral blood flow Brachial artery FMD non MCI MCI-training MCI-no training

53 FMD (%) Cognitive-physical training improves cognitive function, cerebral blood flow and endothelial function MRI-cerebral blood flow Brachial artery FMD non MCI MCI-training MCI-no training Hippocampal and parahippocampal regions are crucial for memory and processing of non-verbal / spatial information Bruno RM et al, Hypertension 2017 in press The Train the Brain Consortium, Scientific reports 2017

54 Parahippocampal CBF (ml/100g/min) Endothelial function is a determinant of parahippocampal blood flow in patients with mild cognitive impairment Clinical determinants: none Vascular determinants: - FMD (r=0,26, p=0,03) Multiple regression model: beta P value r 2 Age 0,15 0,47 0,01 Male sex -0,78 0,75 0,00 Mean BP 0,036 0,73 0,00 BMI -0,39 0,17 0,03 BAD 1,44 0,36 0,01 FMD 0,93 0,04 0,06 r=0.26, p=0,03 r=-0,13, p=0,48 MCI Non MCI Non MCI MCI FMD (%) Bruno RM et al, oral presentation at Artery 2017

55 I need to thank a lot of people!!!

56 My mentor Prof. Paul M. Vanhoutte

57 My group

59 My family

Classification of Endothelial Dysfunction. Stefano Taddei Department of Internal Medicine University of Pisa, Italy

Classification of Endothelial Dysfunction Stefano Taddei Department of Internal Medicine University of Pisa, Italy Pathogenesis of atherosclerosis from endothelial dysfunction to clinical disease endothelial