Nitric Oxide 3/14/ %-40% prevalence in sickle cell and thalassemia 1-3. Mild-moderate pulmonary hypertension 4

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1 Hemolysis-associated endothelial dysfunction and pulmonary hypertension: An emerging cause of death in the hemolytic anemias Mark T. Gladwin, MD Director, Hemostasis and Vascular Biology Research Institute Chief, Pulmonary, Allergy and Critical Care Medicine University of Pittsburgh, Institute for Transfusion Medicine, Hemophilia Center of Western Pennsylvania Hemolysis-Associated Pulmonary Hypertension (sickle cell disease, thalassemia, PNH,spherocytosis, stomatocytosis, microangiopathic hemolytic anemia) 2%-4% prevalence in sickle cell and thalassemia 1-3 Mild-moderate pulmonary hypertension 4 Up to 5% 2-year survival in sickle cell patients 2,3,5,6 Potential cause of sudden death in adults 1. Simmons et al. Arch Intern Med. 1988;148: Sutton et al. Am J Cardiol. 1994;74: Castro. Hematol Oncol Clin North Am. 1996;1: Norris et al. J Assoc Acad Minor Phys. 1992;3:2. 5. Powars et al. Medicine (Baltimore). 1988;67: Castro et al. Blood. 23;11:1257. Nitric Oxide What is wrong with hemolysis? N O 1

2 Regulates vasodilator tone High Reaction Rates of NO with hemoglobin NO Inhibits platelet aggregation and attachment Endothelial VCAM, ICAM, E- selectin α α β β O 2 + NO 1 7 M - 1 sec -1 K A α α NO β β Inhibits release of vasocontrictors and growth factors (ET-1) Inactivates superoxide: Diffusion-limited antioxidant α α β β + NO 1 7 M - 1 sec -1 α α I β β + NO Vasoconstriction X NO + Platelet aggregation and attachment + Release of procoagulant factors Plasma Hb + + Inflammatory cell attachment ( via VCAM-1) Release of growth factors Schechter and Gladwin; NEJM 23;348:1483 2

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4 /14/29 Plasma Contains Elevated Hemoglobin and Absent Haptoglobin AA SS 25 Heme (µm) SS AA NO Consumption Assay Plasma of Patients Consumes More NO than Plasma of Normal Volunteers 15 pa pa OxyHb (µm) Minute NO Trace Injection of NO donor Injection of NO consumer pa pa OxyHb (µm) Min NO consumption ( µm) Plasma heme (µm) 4

5 Plasma Hemoglobin Mediates NO Consumption SNP Free Hb 1A 1B 1C 2A 2B 2C A: Before IP B: After IP C: IP Eluate 3A 3B 3C NO consumption (µm) ENDOTHELIUM SMOOTH MUSCLE NO guanylyl cyclase (inactive) guanylyl cyclase (active) cgmp GTP Relaxation Venous Occlusion Strain-Gauge Plethysmography Blood Pressure Transducer Upper Arm Blood Pressure Cuff Venous Cannula Arterial Cannula Wrist Cuff Strain Gauge Connected to Plethysmograph Plasma Hemoglobin Impairs Forearm Blood Flow Responses to Nitroprusside Percent Change in Forearm Blood Flow During SNP Infusion Plasma Heme (µm) Reiter et al. Nature Med 22 5

6 Plasma Hemoglobin Impairs Cremasteric Blood Flow Responses to Nitroprusside R 2 =.94 P =.3 C57BL BERK-trait BERK+ γ BERK Plasma hemoglobin (µm heme) NO resistance state in SCD Blunted vasodilation to NONOate Nath et al. Am J. Physiol 2 Blunted vasodilation to sodium nitroprusside Kaul, et al. Am J Physiol 2; Kaul, et al. JCI 24 Blunted vasodilation to acetylcholine Aslan et al. PNAS 21 Blunted responses to nitroglycerin in humans Eberhardt et al. Am J Hematol 23 Kaul et al. JCI 24 Glutamate Small intestine Citrulline Kidney X Renal dysfunction ARGININE HEMOLYSIS Inflammation/cytokines Liver damage Arginine insufficiency & elevated arginase in sickle cell disease Inflammatory cytokines NOS Substrate Competition Arginase NO CONSUMPTION: HEMOLYSIS: Cell free Hb Superoxide RNOS NO Ornithine Polyamines Proline Vascular smooth muscle proliferation Airway remodeling Collagen production/deposition Lung fibrosis Airway remodeling SCD and PHT Morris et al. JAMA 25 6

7 Arg/Orn Ratio Low Arginine-to-Ornithine Ratio in patients with SCD and PAH 3.5 p< Control SCD Arg/Orn Ratio p=.3 Mild PHT Severe PHT SCD Patients Morris et al. JAMA 25 Association of plasma arginase activity with intravascular hemolysis Cell-Free Hb (µm) R =.56, p < Plasma Arginase (µ mol/ml/hr) Survival Proportions Low Global Arg Bioavailability - Risk of Death 1. Arg/Orn High Arg/Orn 1. Arg/[Orn+Cit] High Arg/(Orn+Cit).9 Medium Arg/Orn.9 Medium Arg/(Orn+Cit) Survival.8 Low Arg/Orn Survival.8 Low Arg/(Orn+Cit) Follow-Up Time (Months) Follow-Up Time (Months) RR: 2.2 [1.,4.9], p=.2 RR: 3.6 [1.5,8.3], p<.1 Morris et al. JAMA 25 7

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10 Plexogenic Vascular Lesions Concentric Medial and Intimal Hypertrophy Doppler Features of Right Heart Peak TR velocity measured RVSP calculated as 4v 2 + RAP Haque et al. Hum Pathol. 22;33:137. Peak TR velocity 4.1 m/s Peak gradient 67mm Hg Est. RVSP 72mmHg Distributions of Tricuspid Regurgitation Velocity in Patients With Sickle Cell Disease TR jet velocity (m/sec m/sec) Sickle cell patients 33% 67% African American controls Fraction survival Survival by TRV Group p <.1 Adults with SCD TRV 3 m/s TRV < 2.5 m/s Months TRV m/s Relative Risk 1 Gladwin et al. N Engl J Med. 24;35:886 Updated October 19, 24 1

11 Prospective mortality studies examining TRV > 2.5 PAH is the most common cause of high PA pressures in sickle cell disease 6 5 Ataga K, et al. Br J Haematol : relative risk, 9.24; 95% confidence interval: ; P =.1 Percent of Patients De Castro et al. American J. Hematology 28 83:19-25 hazard ratio: 15.9; p<.5 1 PAH DD Mixed RAP (mmhg) 8 ±.6 11 ± 1 18 ± 5 PAS (mmhg) 58 ±3 51 ± 3 6 ± 7 PAD (mmhg) 25 ± 1 23 ± 1 37 ±.9 mpap (mmhg) 36 ± 2 33 ± 2 42 ± 5 CO (L/min) 7.6 ± ± ± 1.3 PCWP (mmhg) 13 ±.7 2 ± 1 2 ± 2 PVR (dyn/sec cm -5 ) 259 ± ± ± 59 Exercise Capacity in Patients With Sickle Cell Disease Without and With Pulmonary Hypertension Without PHT With PHT P Value 6-min walk (m) 435 ± 4 32 ± 29.2 VO 2 max (% pred.) 52 ± 3 41 ± 2.24 Correlations with TRV in NIH cohort High TRV correlated with markers of hemolysis (high T. bili, LDH and low Hb) No correlation with WBC, platelet count, fetal Hb level No correlation with number of episodes of VOC and ACS AJRCCM 27 11

12 Logistic Regression Analysis for Tricuspid Regurgitation Velocity <2.5 m/sec and 2.5 m/sec Odds Ratio 95%Confidence Independent Variable P Value (OR) Interval for OR Systolic blood pressure < (1.6, 5.3) Transferrin (mg/l) <.1.17 (.7,.38) Brain Natriuretic Peptide Ventricular Cardiomyocyte Cardiomyocyte stretch (pressure or volume) Pre-Pro-BNP Pro-BNP aa signal sequence Alkaline phosphatase (units/l) (1., 2.8) Lactate dehydrogenase (units/l) (1.3, 5.9) Cardiovascular problems (1.2, 7.2) reported Renal problems reported (1.3, 22.9) Blood NT-Pro-BNP 1-76 BNP Vasodilation Natriuresis RAAS Brain Natriuretic Peptide is Elevated in Idiopathic Pulmonary Arterial Hypertension BNP (pg/ml) Survival rate (%) BNP < 15 pg/ml BNP 15 pg/ml PVR (dynes.sec - 1.cm - 5 ) Months Lauchte H et al. JACC 24;43: Nagaya N et al. Circulation 2;12:

13 High NT-proBNP in Sickle Cell Disease Pulmonary Hypertension NT-proBNP Correlates with the Severity of Pulmonary Hypertension NT-pro BNP (pg/ml) 5 **,# * AAC NO PH PH * P <.1 vs AAC ** P <.1 vs AAC # P <.1 vs < 2.5 Parameter R P TR jet velocity.5 <.1 Mean PA pressure.43.4 Pulmonary vascular resistance.51.1 Transpulmonary gradient.4.27 Cardiac output ROC Curve of NT-proBNP for the Diagnosis of Pulmonary Hypertension Survival Proportions 1 NT-pro BNP < 16 pg/ml Sensitivity % % - Specificity% Area under the curve =.81 ( ) P <.1 Cutoff (pg/ml) Sensitivity (%) Specificity (%) PPV (%) NPV (%) Percent Survival NT-pro BNP P < Months 16 pg/ml Machado et al. JAMA. 26; 296:31 13

14 Validation Cohort: MSH Follow Up Study Sample of 121 patients from the 1996 MSH patient s follow-up study Patients had received hydroxyurea or placebo in a randomized trial from 1992 to 1995, had severe disease, and had comprehensive follow-up starting in 1996 NT-proBNP measured in samples collected at enrollment visit in the 1996 MSH follow up study A priori Analytical Plan Prevalence NT-proBNP level of 16 pg/ml: 75 th percentile for the NIH cohort Predictive of death in NIH cohort Prospectively defined as indicative of pulmonary hypertension Used as a categorical value for data analysis 3% 7% NT-pro BNP level < 16 pg/ml ³ 16 pg/ml 14

15 Percent Survival Survival Proportions NT-pro BNP < 16 pg/ml NT-pro BNP P= Months 16 pg/ml Risk Factors High NT-proBNP levels correlated with: Older age (P<.1) Low hemoglobin level (P<.1) High plasma hemoglobin level (P=.14) High Creatinine level (P=.8) High BUN level (P=.4) High total bilirubin level (P=.2) High uric acid level (P=.14) NT-proBNP is Inversely Correlated to Hemoglobin Level CSSCD? Hemoglobin (g/dl) Off Hydroxyurea On Hydroxyurea R=.53 P<.9 R=.64 P< log NT-proBNP Cooperative Study of SCD: Did high NT-BNP predict death? 15

16 CSSCD Adult survival by NT-BNP CSSCD Adult survival by NT-BNP Fraction Surviving Fraction Surviving Conclusion In patients in the 1996 MSH and 1978 CSSCD cohorts pulmonary hypertension was common and the major risk factor for death A mechanism in the most common hemolytic disease in the world: Malaria? 16

17 Severe Malarial Anemia Severe Malarial Anemia Jacqueline J. Janka, Ousmane A. Koita, Broulaye Traoré, Maya T. Josépha, Fawaz Mzayek, Vandana Sachdev, Xunde Wang, Kassoum Sanogo, Lansana Sangaré, Laurel Mendelsohn, Gregory J. Kato, Henry Masur, Mark T. Gladwin, Donald J. Krogstad NHLBI, and University of Pittsburgh Roberto Machado, Greg Kato, Sruti Shiva, Jim Taylor, James Nichols, Wynona Coles, Lori Hunter, Mary K. Hall, Xunde Wang University of Pittsburgh Sidney Morris, Bruce Freeman NIDDK Alan Schechter, Griffin Rodgers NHLBI Vandana Sachdev, Richard Cannon III Wake Forrest University Daniel Kim-Shapiro Oakland Childrens Hospital Claudia Morris, Elliot Vichinsky 17