Lung Development, Injury and Repair

Transcription

1 Chronic Lung Disease of Prematurity: Where have all the Vessels gone? Role of Angiogenic Growth Factors, Nitric Oxide and Endothelial Progenitor Cells Faruqa Ladha, Arul Vadivel, Rajesh Anthuvan, Shumei Zhong, Bernard Thébaud Children s Hospital of Eastern Ontario/Division of Neonatology Ottawa Hospital Research Institute/Regenerative Medicine Program Sprott Centre for Stem Cell Research University of Ottawa Lung Development, Injury and Repair 1

2 Increased survival, but no decrease in morbidity for extreme premature infants Incidence of Bronchopulmonary Dysplasia by Gestational Age Percentage of babies Percentage of babies at 28 days at 36 weeks Extreme Prematurity Gestational age (weeks) Source: Incidence of Retinopathy of Prematurity 1 by Gestational Age Stage 1 &2 8 Stage 3& Extreme Prematurity Gestational age at birth (weeks) Incidence of Bronchopulmonary Dysplasia In compared to 6-7 Percentage of babies ,7 46, Study periods Shah, P et al. Canadian Neonatal Network. 1 BPD: An Arrest in Lung Development The new BPD Jobe A. Pediatr Res 1999 Northway et al, NEJM 1967 Survivors with BPD: 34 GA, 2234g Non-survivors: 31 GA, 166g IUGR Intrauterine Cytokine exposure Initial and Chronic Ventilation Postnatal Infection Nutrition Oxygen exposure Postnatal Glucocorticoids Decreased Alveolarization in BPD: Potential Long Term Consequences 2

3 Late Cardiopulmonary Disease in a Young Adult with BPD Slide courtesy Steven Abman Angiogenic growth factors and the making of a blood vessel HIF 3

4 Angiogenesis Promotes Normal Lung Development Normal alveolar sac (1) VEGF HIF NO Alveolar sac after VEGF inhibition (3) Maniscalco et al. Am J Respir Cell Mol Biol 1997 Jakkula et al. Am J Physiol 1999 Gerber et al. Development 1999 (2) Lassus et al. Am J Respir Crit Care Med 1 Galambos et al. Am J Respir Cell Mol Biol 2 Han et al. Circ Res 4 Delisser et al. J Biol Chem 6 Thébaud B and Abman SH. Am J Respir Crit Care Med 7 Alveolar sac in BPD Restored alveolarization after pro-angiogenic treatment Kunig et al. Am J Physiol 5/6 Thébaud et al Circulation 5 Bland et al, AJRCCM 5 McCurnin et al, Am J Physiol 5 Asikainen et al, FASEB J 6 Bland et al. Am J Physiol 7 Inhibition of VEGF reduces lung size, impairs alveolar development and decreases lung angiogenesis: Evidence that angiogenic growth factors promote lung development VEGF-trap Control-saline x 15µm hfc Thébaud et al. Circulation 5 In vivo intratracheal adenovirus-mediated VEGF gene transfer promotes lung vascular growth H&E stained sections Control + Ad5VEGF 145 +Ad5GFP A Vascular lung casts Scanning Electron Microscopy x 5µm 5µm + Ad5VEGF 145 B + Ad5-VEGF+Ang1 5µm 5µm 5µm Lung wet/dry weight Control +Ad5VEGF 145 +Ad5GFP +Ad5VEGF +Ang1 N=5, P<.5 vs Ctrl, P<.5 vs +VEGF+Ang1 Thébaud et al. Circulation 5 4

5 In vivo intratracheal adenovirus-mediated HIF gene transfer promotes lung vascular growth +Ad.GFP + Ad.HIF A B +Ad.GFP + Ad.HIF x x x x +Ad.GFP + Ad.HIF Vessels/HPF Vessel Density + Ad.GFP + Ad.HIF D Vadivel et al. AJRCMB 13 Angiogenic growth factors and the making of a blood vessel NO Ang1 Lin et al, Pediatr Res 5, Schreiber et al, NEJM 3 Ballard et al, NEJM 6, Kinsella et al, NEJM 6 Inhaled NO improves lung structure and function in chronically ventilated preterm lambs Bland et al. Am J Respir Crit Care Med. 5 5

6 Multiple Mechanisms of Action of ino Martin RJ, Walsh MC. New Engl J Med 5 Schreiber et al. New Engl J Med 3 Single centre RCT 7 newborns < 34 wks, <g, requiring MV ino 1ppm for 1d then 5ppm for 6 d or placebo gas 1 outcome death or BPD at 36 weeks 2 outcome IVH, PVL and PDA Main findings: ino survival without BPD (p=.3/rr.76 {95%CI.6-.97}) especially among infants with less severe lung disease (OI<7) (p=.2/rr.53 {95%CI }) 47% decrease in severe intracranial hemorrhage and PVL Neurodevelopmental Outcome at 2 years: Mestan et al. New Engl J Med children (82 % of survivors) abnormal neurodevelopmental outcomes defined as either: disability (CP, bilateral blindness, or bilateral hearing loss) or delay (no disability, but one score < 7 on the Bayley Scales of Infant Development II) in4 % abnormal vs 46 % in the placebo group (RR.53; 95 percent confidence interval,.33 to.87; P=.1) Effect persisted after adjustment for BW, gender, presence or absence of BPD and severe IVH or PVL 6

7 Medical and Functional Outcomes at Early School-Age: Patrianakos-Hoobler et al. American Academy of Pediatrics, May of 167 survivors (81%) at 6 years. Medical outcomes: somatic growth, hospitalizations and ongoing morbidities Functional outcomes: School readiness levels using standardized neurodevelopmental assessments of basic concepts (Bracken School Readiness Component), perceptual skills (Visual-Motor Integration), receptive vocabulary (Peabody Picture Vocabulary Test-3rd Ed.), daily living functional skills (WeeFIM), cerebral palsy, hearing impairment, vision impairment, and autism Compared to placebo (n=65), ino children (n=7) had no difference in: Weight, height, head circumference; hospitalizations. Compared to placebo, ino children were: more likely to have none/mild/single chronic ongoing morbidity less likely to have multiple chronic ongoing morbidities or technology-dependence Functional outcomes were similar between groups Kinsella et al. New Engl J Med 6 Multicentre RCT (16 centres) 793 newborns < 34 wks, <125g requiring MV <48H ino 5ppm or placebo gas for 21 d or until extubation 1 outcome composite of death or CLD at 36 weeks 2 outcome IVH, PVL or ventriculomegaly Early Inhaled NO Treatment of Premature Newborns with Respiratory Failure Kinsella et al, N Engl J Med 6 7

8 Effects of early treatment with low-dose ino on brain injury (grade 3-4 ICH, PVL, ventriculomegaly) in preterm infants Kinsella et al. New Engl J Med 6 Ballard et al. New Engl J Med 6 Multicentre RCT (21 centres) 582 newborns < 32 wks, <125gm requiring MV 7-21d ino at decreasing concentrations or placebo gas for 24 d 1 outcome death or survival without BPD at 36 weeks 2 outcome days of resp support,, length of stay Overall ino: survival without BPD (p=.4) LOS (p=.4), length on (p=.6) lung disease severity if enrolled from 7-14 d vs 15-21d Effects of ino on survival without BPD for infants enrolled between 7 and 21 days of age Ballard et al. New Engl J Med 6 Modfied by Kinsella et al. J Pediatr 7 8

9 EUNO. et al. Lancet 1 Multicentre RCT (36 EU centres) 8 newborns < 29 wks, >5gm requiring Resp support<24h ino at 5ppm or placebo gas for min 7 d - max 21d 1 Survival without BPD at 36 weeks 2 Alive without brain injury Overall ino: No effect on survival without BPD (66% vs 65%) Incidence of BPD: 27% vs 24% No effect on Alive without brain injury Kinsella et al, J Pediatr 14 To assess the efficacy and safety of early, noninvasive inhaled nitric oxide (ino) therapy in premature newborns who do not require mechanical ventilation Multicentre RCT, 124 newborns who required noninvasive supplemental oxygen within the first 72 hours after birth Stratified by birth weight (5-749, , g) prior to randomization to ino (1 ppm) or placebo gas (controls) until 3 weeks postmenstrual age No difference in incidence of death or BPD, BPD severity, need for mechanical ventilation, duration of mechanical ventilation, or safety outcomes including severe intracranial hemorrhage Summary of ino Trials Early rescue therapy for severe respiratory failure: No benefit Potential increased risk of severe ICH Selective indications? PPROM ± oligohydramnios Prevention of BPD in infants with mild respiratory distress: Safe Effective in a subset of preterm infants>1g Neuroprotective Economical considerations (high cost barrier to its use?) 9

10 Smith H et al. J Thorac Cardiovasc Surg 6 L-Citrulline preserves alveolar development in -induced BPD in newborn rats Normoxia Normoxia + L-Citrulline Hyperoxia Hyperoxia + L-Citrulline X A) Mean Linear Intercept Normoxia Normoxia+ L-Citrulline Hyperoxia Hyperoxia+ L-Citrulline B) Septal counts Vadivel et al. Pdiatr Res 1 Angiogenic growth factors and the making of a blood vessel HIF NO Ang1 1

11 Endothelial Progenitor Cells (EPCs): ECFCs fulfill the stringent criteria of a true EPC EPC hierarchy model based on proliferative and clonogenic potential of discrete populations of progenitor cells DN Prater, J Case, DA Ingram, MC Yoder. Leukemia 7 Modified from MC Yoder, DA Ingram. Blood 7 ECFCs exist in the Human Fetal Lung Cobblestone-looking ECFC Colony LDL-uptake and Ulex-lectin binding Ulex Di-Ac-LDLHoechst x 1x Cell Surface Markers CD31 CD146 CD15 1x Endothelial network formation CD144 (VE-Cad) CD14 CD45 Alphonse RS et al. Circulation 14 Human Lung ECFC Progeny Possess the Ability to Form de novo Blood Filled Vessels in vivo Human Lung ECFCs form de novo blood vessels that connect with the host vasculature vessels/mm 2 Slide courtesy Merv Yoder 11

12 Exposure to Oxygen Impairs Lung ECFC Function EPC hierarchy model based on proliferative and clonogenic potential of discrete populations of progenitor cells Network Formation Room Air % O2 Clonogenic Potential 25 Nb Intersections 15 (P<.5) 1 5 Room Air ECFC Function is impaired in -induced BPD EPC hierarchy model based on proliferative and clonogenic potential of discrete populations of progenitor cells Single Cell Clonogenic Potential >5 12

13 Therapeutic Potential of UCB-ECFC in Experimental -induced Neonatal Lung Injury Rag-/-mice RNU rats hucb-ecfc (jugular vein) Endpoints olung Compliance (Flexivent ) olung histology opulmonary artery acceleration time and right ventricular hypertrophy P- P-5 P-14 P-28 Hyperoxia(85% ) Human cord blood-derived ECFCs Restore Arrested Alveolar and Lung Vascular Growth in -induced Experimental BPD Alveolar Growth +ECFC Vascular Growth +ECFC +ECFC +ECFC Mean Linear Intercept Vessel Count A µm P <.1 vs all other groups; n = 4-6 animals/group +ECFC +ECFC B No. of vessels per HPF ECFC P <.5 vs all other groups; n = 4-6 animals/group +ECFC Human cord blood-derived ECFCs Attenuate Pulmonary Hypertension in -induced Experimental BPD +ECFC +ECFC Right Ventricular Hypertrophy P<.1 vs other groups n=4-9/group P<.1 vs other groups n=4-6/group +ECFC +ECFC RV/LV+S 13

14 Resident Lung ECFC Function is Restored by Exogenous Human cord blood-derived ECFCs +ECFC Colonies per 96-well plate ECFC < >5 Cells/Colony +ECFC P <.5; n = 3 trials per group (x 1 µm) X Cord Length P <.5 vs all 4 other groups; n = 6 per group ECFC Branch Points P <.5 vs all other groups; 15 n = 6 per group 1 5 A B +ECFC +ECFC +ECFC +ECFC No Adverse Effect of Human cord blood-derived ECFCs at 1 months +ECFC 5 Mean Linear intercept P<.5; n=3-6/group 3 µm +ECFC 1 B +ECFC +ECFC Human Lung Mouse Lung A Distance (m) 6 Exercise Capacity P <.5 vs P <.5 vs all groups n=3-6/group PAAT (ms) PAAT C + ECFC + ECFC D +ECFC +ECFC E Low Lung Engraftment of Cord Blood-derived ECFCs 5 Alu/18s (control as 1) 3 1 Control 2 hrs 3 days 14 days 21 days 14

15 Evidence that ECFCs act via a Paracrine Effect: Therapeutic Potential of cell-free Conditioned media In vitro 1- AEC wound healing 2- EC capillary-like network Rag-/-mice RNU rats In vivo hucb-ecfc CdM (i.p.) Endpoints olung Compliance (Flexivent ) olung histology opulmonary artery acceleration time and right ventricular hypertrophy P- P-5 P-14 P-21 Hyperoxia(85% ) Human cord blood-derived ECFC CdM preserves Alveolar and Vascular Growth in -induced BPD +DMEM +ECFC CdM Mean Linear intercept P<.1 vs other groups n=6/group +DMEM +ECFC CdM DMEM ECFC- CdM DMEM ECFC- CdM No. of vessels/1 HPF DMEM ECFC- CdM DMEM ECFC- CdM p<.1 vs O2-DMEM n= 5/group Angiogenesis promotes lung growth and repair and may have therapeutic benefit in BPD 1- Angiogenesis inhibition 2- Hyperoxia 3- Pro-angiogenic Therapies In experimental BPD VEGF ECFCs 15

16 Chronic Lung Disease of Prematurity: Where have all the Vessels gone? Role of Angiogenic Growth Factors, Nitric Oxide and Endothelial Progenitor Cells Faruqa Ladha, Arul Vadivel, Rajesh Anthuvan, Shumei Zhong, Bernard Thébaud Children s Hospital of Eastern Ontario/Division of Neonatology Ottawa Hospital Research Institute/Regenerative Medicine Program Sprott Centre for Stem Cell Research University of Ottawa Lung Development, Injury and Repair 1

17 Increased survival, but no decrease in morbidity for extreme premature infants Incidence of Bronchopulmonary Dysplasia by Gestational Age Percentage of babies Percentage of babies at 28 days at 36 weeks Extreme Prematurity Gestational age (weeks) Source: Incidence of Retinopathy of Prematurity 1 by Gestational Age Stage 1 &2 8 Stage 3& Extreme Prematurity Gestational age at birth (weeks) Incidence of Bronchopulmonary Dysplasia In compared to 6-7 Percentage of babies ,7 46, Study periods Shah, P et al. Canadian Neonatal Network. 1 BPD: An Arrest in Lung Development The new BPD Jobe A. Pediatr Res 1999 Northway et al, NEJM 1967 Survivors with BPD: 34 GA, 2234g Non-survivors: 31 GA, 166g IUGR Intrauterine Cytokine exposure Initial and Chronic Ventilation Postnatal Infection Nutrition Oxygen exposure Postnatal Glucocorticoids Decreased Alveolarization in BPD: Potential Long Term Consequences 2

18 Late Cardiopulmonary Disease in a Young Adult with BPD Slide courtesy Steven Abman Angiogenic growth factors and the making of a blood vessel HIF 3

19 Angiogenesis Promotes Normal Lung Development Normal alveolar sac (1) VEGF HIF NO Alveolar sac after VEGF inhibition (3) Maniscalco et al. Am J Respir Cell Mol Biol 1997 Jakkula et al. Am J Physiol 1999 Gerber et al. Development 1999 (2) Lassus et al. Am J Respir Crit Care Med 1 Galambos et al. Am J Respir Cell Mol Biol 2 Han et al. Circ Res 4 Delisser et al. J Biol Chem 6 Thébaud B and Abman SH. Am J Respir Crit Care Med 7 Alveolar sac in BPD Restored alveolarization after pro-angiogenic treatment Kunig et al. Am J Physiol 5/6 Thébaud et al Circulation 5 Bland et al, AJRCCM 5 McCurnin et al, Am J Physiol 5 Asikainen et al, FASEB J 6 Bland et al. Am J Physiol 7 Inhibition of VEGF reduces lung size, impairs alveolar development and decreases lung angiogenesis: Evidence that angiogenic growth factors promote lung development VEGF-trap Control-saline x 15µm hfc Thébaud et al. Circulation 5 In vivo intratracheal adenovirus-mediated VEGF gene transfer promotes lung vascular growth H&E stained sections Control + Ad5VEGF 145 +Ad5GFP A Vascular lung casts Scanning Electron Microscopy x 5µm 5µm + Ad5VEGF 145 B + Ad5-VEGF+Ang1 5µm 5µm 5µm Lung wet/dry weight Control +Ad5VEGF 145 +Ad5GFP +Ad5VEGF +Ang1 N=5, P<.5 vs Ctrl, P<.5 vs +VEGF+Ang1 Thébaud et al. Circulation 5 4

20 In vivo intratracheal adenovirus-mediated HIF gene transfer promotes lung vascular growth +Ad.GFP + Ad.HIF A B +Ad.GFP + Ad.HIF x x x x +Ad.GFP + Ad.HIF Vessels/HPF Vessel Density + Ad.GFP + Ad.HIF D Vadivel et al. AJRCMB 13 Angiogenic growth factors and the making of a blood vessel NO Ang1 Lin et al, Pediatr Res 5, Schreiber et al, NEJM 3 Ballard et al, NEJM 6, Kinsella et al, NEJM 6 Inhaled NO improves lung structure and function in chronically ventilated preterm lambs Bland et al. Am J Respir Crit Care Med. 5 5

21 Multiple Mechanisms of Action of ino Martin RJ, Walsh MC. New Engl J Med 5 Schreiber et al. New Engl J Med 3 Single centre RCT 7 newborns < 34 wks, <g, requiring MV ino 1ppm for 1d then 5ppm for 6 d or placebo gas 1 outcome death or BPD at 36 weeks 2 outcome IVH, PVL and PDA Main findings: ino survival without BPD (p=.3/rr.76 {95%CI.6-.97}) especially among infants with less severe lung disease (OI<7) (p=.2/rr.53 {95%CI }) 47% decrease in severe intracranial hemorrhage and PVL Neurodevelopmental Outcome at 2 years: Mestan et al. New Engl J Med children (82 % of survivors) abnormal neurodevelopmental outcomes defined as either: disability (CP, bilateral blindness, or bilateral hearing loss) or delay (no disability, but one score < 7 on the Bayley Scales of Infant Development II) in4 % abnormal vs 46 % in the placebo group (RR.53; 95 percent confidence interval,.33 to.87; P=.1) Effect persisted after adjustment for BW, gender, presence or absence of BPD and severe IVH or PVL 6

22 Medical and Functional Outcomes at Early School-Age: Patrianakos-Hoobler et al. American Academy of Pediatrics, May of 167 survivors (81%) at 6 years. Medical outcomes: somatic growth, hospitalizations and ongoing morbidities Functional outcomes: School readiness levels using standardized neurodevelopmental assessments of basic concepts (Bracken School Readiness Component), perceptual skills (Visual-Motor Integration), receptive vocabulary (Peabody Picture Vocabulary Test-3rd Ed.), daily living functional skills (WeeFIM), cerebral palsy, hearing impairment, vision impairment, and autism Compared to placebo (n=65), ino children (n=7) had no difference in: Weight, height, head circumference; hospitalizations. Compared to placebo, ino children were: more likely to have none/mild/single chronic ongoing morbidity less likely to have multiple chronic ongoing morbidities or technology-dependence Functional outcomes were similar between groups Kinsella et al. New Engl J Med 6 Multicentre RCT (16 centres) 793 newborns < 34 wks, <125g requiring MV <48H ino 5ppm or placebo gas for 21 d or until extubation 1 outcome composite of death or CLD at 36 weeks 2 outcome IVH, PVL or ventriculomegaly Early Inhaled NO Treatment of Premature Newborns with Respiratory Failure Kinsella et al, N Engl J Med 6 7

23 Effects of early treatment with low-dose ino on brain injury (grade 3-4 ICH, PVL, ventriculomegaly) in preterm infants Kinsella et al. New Engl J Med 6 Ballard et al. New Engl J Med 6 Multicentre RCT (21 centres) 582 newborns < 32 wks, <125gm requiring MV 7-21d ino at decreasing concentrations or placebo gas for 24 d 1 outcome death or survival without BPD at 36 weeks 2 outcome days of resp support,, length of stay Overall ino: survival without BPD (p=.4) LOS (p=.4), length on (p=.6) lung disease severity if enrolled from 7-14 d vs 15-21d Effects of ino on survival without BPD for infants enrolled between 7 and 21 days of age Ballard et al. New Engl J Med 6 Modfied by Kinsella et al. J Pediatr 7 8

24 EUNO. et al. Lancet 1 Multicentre RCT (36 EU centres) 8 newborns < 29 wks, >5gm requiring Resp support<24h ino at 5ppm or placebo gas for min 7 d - max 21d 1 Survival without BPD at 36 weeks 2 Alive without brain injury Overall ino: No effect on survival without BPD (66% vs 65%) Incidence of BPD: 27% vs 24% No effect on Alive without brain injury Kinsella et al, J Pediatr 14 To assess the efficacy and safety of early, noninvasive inhaled nitric oxide (ino) therapy in premature newborns who do not require mechanical ventilation Multicentre RCT, 124 newborns who required noninvasive supplemental oxygen within the first 72 hours after birth Stratified by birth weight (5-749, , g) prior to randomization to ino (1 ppm) or placebo gas (controls) until 3 weeks postmenstrual age No difference in incidence of death or BPD, BPD severity, need for mechanical ventilation, duration of mechanical ventilation, or safety outcomes including severe intracranial hemorrhage Summary of ino Trials Early rescue therapy for severe respiratory failure: No benefit Potential increased risk of severe ICH Selective indications? PPROM ± oligohydramnios Prevention of BPD in infants with mild respiratory distress: Safe Effective in a subset of preterm infants>1g Neuroprotective Economical considerations (high cost barrier to its use?) 9

25 Smith H et al. J Thorac Cardiovasc Surg 6 L-Citrulline preserves alveolar development in -induced BPD in newborn rats Normoxia Normoxia + L-Citrulline Hyperoxia Hyperoxia + L-Citrulline X A) Mean Linear Intercept Normoxia Normoxia+ L-Citrulline Hyperoxia Hyperoxia+ L-Citrulline B) Septal counts Vadivel et al. Pdiatr Res 1 Angiogenic growth factors and the making of a blood vessel HIF NO Ang1 1

26 Endothelial Progenitor Cells (EPCs): ECFCs fulfill the stringent criteria of a true EPC EPC hierarchy model based on proliferative and clonogenic potential of discrete populations of progenitor cells DN Prater, J Case, DA Ingram, MC Yoder. Leukemia 7 Modified from MC Yoder, DA Ingram. Blood 7 ECFCs exist in the Human Fetal Lung Cobblestone-looking ECFC Colony LDL-uptake and Ulex-lectin binding Ulex Di-Ac-LDLHoechst x 1x Cell Surface Markers CD31 CD146 CD15 1x Endothelial network formation CD144 (VE-Cad) CD14 CD45 Alphonse RS et al. Circulation 14 Human Lung ECFC Progeny Possess the Ability to Form de novo Blood Filled Vessels in vivo Human Lung ECFCs form de novo blood vessels that connect with the host vasculature vessels/mm 2 Slide courtesy Merv Yoder 11

27 Exposure to Oxygen Impairs Lung ECFC Function EPC hierarchy model based on proliferative and clonogenic potential of discrete populations of progenitor cells Network Formation Room Air % O2 Clonogenic Potential 25 Nb Intersections 15 (P<.5) 1 5 Room Air ECFC Function is impaired in -induced BPD EPC hierarchy model based on proliferative and clonogenic potential of discrete populations of progenitor cells Single Cell Clonogenic Potential >5 12

28 Therapeutic Potential of UCB-ECFC in Experimental -induced Neonatal Lung Injury Rag-/-mice RNU rats hucb-ecfc (jugular vein) Endpoints olung Compliance (Flexivent ) olung histology opulmonary artery acceleration time and right ventricular hypertrophy P- P-5 P-14 P-28 Hyperoxia(85% ) Human cord blood-derived ECFCs Restore Arrested Alveolar and Lung Vascular Growth in -induced Experimental BPD Alveolar Growth +ECFC Vascular Growth +ECFC +ECFC +ECFC Mean Linear Intercept Vessel Count A µm P <.1 vs all other groups; n = 4-6 animals/group +ECFC +ECFC B No. of vessels per HPF ECFC P <.5 vs all other groups; n = 4-6 animals/group +ECFC Human cord blood-derived ECFCs Attenuate Pulmonary Hypertension in -induced Experimental BPD +ECFC +ECFC Right Ventricular Hypertrophy P<.1 vs other groups n=4-9/group P<.1 vs other groups n=4-6/group +ECFC +ECFC RV/LV+S 13

29 Resident Lung ECFC Function is Restored by Exogenous Human cord blood-derived ECFCs +ECFC Colonies per 96-well plate ECFC < >5 Cells/Colony +ECFC P <.5; n = 3 trials per group (x 1 µm) X Cord Length P <.5 vs all 4 other groups; n = 6 per group ECFC Branch Points P <.5 vs all other groups; 15 n = 6 per group 1 5 A B +ECFC +ECFC +ECFC +ECFC No Adverse Effect of Human cord blood-derived ECFCs at 1 months +ECFC 5 Mean Linear intercept P<.5; n=3-6/group 3 µm +ECFC 1 B +ECFC +ECFC Human Lung Mouse Lung A Distance (m) 6 Exercise Capacity P <.5 vs P <.5 vs all groups n=3-6/group PAAT (ms) PAAT C + ECFC + ECFC D +ECFC +ECFC E Low Lung Engraftment of Cord Blood-derived ECFCs 5 Alu/18s (control as 1) 3 1 Control 2 hrs 3 days 14 days 21 days 14

30 Evidence that ECFCs act via a Paracrine Effect: Therapeutic Potential of cell-free Conditioned media In vitro 1- AEC wound healing 2- EC capillary-like network Rag-/-mice RNU rats In vivo hucb-ecfc CdM (i.p.) Endpoints olung Compliance (Flexivent ) olung histology opulmonary artery acceleration time and right ventricular hypertrophy P- P-5 P-14 P-21 Hyperoxia(85% ) Human cord blood-derived ECFC CdM preserves Alveolar and Vascular Growth in -induced BPD +DMEM +ECFC CdM Mean Linear intercept P<.1 vs other groups n=6/group +DMEM +ECFC CdM DMEM ECFC- CdM DMEM ECFC- CdM No. of vessels/1 HPF DMEM ECFC- CdM DMEM ECFC- CdM p<.1 vs O2-DMEM n= 5/group Angiogenesis promotes lung growth and repair and may have therapeutic benefit in BPD 1- Angiogenesis inhibition 2- Hyperoxia 3- Pro-angiogenic Therapies In experimental BPD VEGF ECFCs 15

31 Chronic Lung Disease of Prematurity: Where have all the Vessels gone? Role of Angiogenic Growth Factors, Nitric Oxide and Endothelial Progenitor Cells Faruqa Ladha, Arul Vadivel, Rajesh Anthuvan, Shumei Zhong, Bernard Thébaud Children s Hospital of Eastern Ontario/Division of Neonatology Ottawa Hospital Research Institute/Regenerative Medicine Program Sprott Centre for Stem Cell Research University of Ottawa Lung Development, Injury and Repair 1

32 Increased survival, but no decrease in morbidity for extreme premature infants Incidence of Bronchopulmonary Dysplasia by Gestational Age Percentage of babies Percentage of babies at 28 days at 36 weeks Extreme Prematurity Gestational age (weeks) Source: Incidence of Retinopathy of Prematurity 1 by Gestational Age Stage 1 &2 8 Stage 3& Extreme Prematurity Gestational age at birth (weeks) Incidence of Bronchopulmonary Dysplasia In compared to 6-7 Percentage of babies ,7 46, Study periods Shah, P et al. Canadian Neonatal Network. 1 BPD: An Arrest in Lung Development The new BPD Jobe A. Pediatr Res 1999 Northway et al, NEJM 1967 Survivors with BPD: 34 GA, 2234g Non-survivors: 31 GA, 166g IUGR Intrauterine Cytokine exposure Initial and Chronic Ventilation Postnatal Infection Nutrition Oxygen exposure Postnatal Glucocorticoids Decreased Alveolarization in BPD: Potential Long Term Consequences 2

33 Late Cardiopulmonary Disease in a Young Adult with BPD Slide courtesy Steven Abman Angiogenic growth factors and the making of a blood vessel HIF 3

34 Angiogenesis Promotes Normal Lung Development Normal alveolar sac (1) VEGF HIF NO Alveolar sac after VEGF inhibition (3) Maniscalco et al. Am J Respir Cell Mol Biol 1997 Jakkula et al. Am J Physiol 1999 Gerber et al. Development 1999 (2) Lassus et al. Am J Respir Crit Care Med 1 Galambos et al. Am J Respir Cell Mol Biol 2 Han et al. Circ Res 4 Delisser et al. J Biol Chem 6 Thébaud B and Abman SH. Am J Respir Crit Care Med 7 Alveolar sac in BPD Restored alveolarization after pro-angiogenic treatment Kunig et al. Am J Physiol 5/6 Thébaud et al Circulation 5 Bland et al, AJRCCM 5 McCurnin et al, Am J Physiol 5 Asikainen et al, FASEB J 6 Bland et al. Am J Physiol 7 Inhibition of VEGF reduces lung size, impairs alveolar development and decreases lung angiogenesis: Evidence that angiogenic growth factors promote lung development VEGF-trap Control-saline x 15µm hfc Thébaud et al. Circulation 5 In vivo intratracheal adenovirus-mediated VEGF gene transfer promotes lung vascular growth H&E stained sections Control + Ad5VEGF 145 +Ad5GFP A Vascular lung casts Scanning Electron Microscopy x 5µm 5µm + Ad5VEGF 145 B + Ad5-VEGF+Ang1 5µm 5µm 5µm Lung wet/dry weight Control +Ad5VEGF 145 +Ad5GFP +Ad5VEGF +Ang1 N=5, P<.5 vs Ctrl, P<.5 vs +VEGF+Ang1 Thébaud et al. Circulation 5 4

35 In vivo intratracheal adenovirus-mediated HIF gene transfer promotes lung vascular growth +Ad.GFP + Ad.HIF A B +Ad.GFP + Ad.HIF x x x x +Ad.GFP + Ad.HIF Vessels/HPF Vessel Density + Ad.GFP + Ad.HIF D Vadivel et al. AJRCMB 13 Angiogenic growth factors and the making of a blood vessel NO Ang1 Lin et al, Pediatr Res 5, Schreiber et al, NEJM 3 Ballard et al, NEJM 6, Kinsella et al, NEJM 6 Inhaled NO improves lung structure and function in chronically ventilated preterm lambs Bland et al. Am J Respir Crit Care Med. 5 5

36 Multiple Mechanisms of Action of ino Martin RJ, Walsh MC. New Engl J Med 5 Schreiber et al. New Engl J Med 3 Single centre RCT 7 newborns < 34 wks, <g, requiring MV ino 1ppm for 1d then 5ppm for 6 d or placebo gas 1 outcome death or BPD at 36 weeks 2 outcome IVH, PVL and PDA Main findings: ino survival without BPD (p=.3/rr.76 {95%CI.6-.97}) especially among infants with less severe lung disease (OI<7) (p=.2/rr.53 {95%CI }) 47% decrease in severe intracranial hemorrhage and PVL Neurodevelopmental Outcome at 2 years: Mestan et al. New Engl J Med children (82 % of survivors) abnormal neurodevelopmental outcomes defined as either: disability (CP, bilateral blindness, or bilateral hearing loss) or delay (no disability, but one score < 7 on the Bayley Scales of Infant Development II) in4 % abnormal vs 46 % in the placebo group (RR.53; 95 percent confidence interval,.33 to.87; P=.1) Effect persisted after adjustment for BW, gender, presence or absence of BPD and severe IVH or PVL 6

37 Medical and Functional Outcomes at Early School-Age: Patrianakos-Hoobler et al. American Academy of Pediatrics, May of 167 survivors (81%) at 6 years. Medical outcomes: somatic growth, hospitalizations and ongoing morbidities Functional outcomes: School readiness levels using standardized neurodevelopmental assessments of basic concepts (Bracken School Readiness Component), perceptual skills (Visual-Motor Integration), receptive vocabulary (Peabody Picture Vocabulary Test-3rd Ed.), daily living functional skills (WeeFIM), cerebral palsy, hearing impairment, vision impairment, and autism Compared to placebo (n=65), ino children (n=7) had no difference in: Weight, height, head circumference; hospitalizations. Compared to placebo, ino children were: more likely to have none/mild/single chronic ongoing morbidity less likely to have multiple chronic ongoing morbidities or technology-dependence Functional outcomes were similar between groups Kinsella et al. New Engl J Med 6 Multicentre RCT (16 centres) 793 newborns < 34 wks, <125g requiring MV <48H ino 5ppm or placebo gas for 21 d or until extubation 1 outcome composite of death or CLD at 36 weeks 2 outcome IVH, PVL or ventriculomegaly Early Inhaled NO Treatment of Premature Newborns with Respiratory Failure Kinsella et al, N Engl J Med 6 7

38 Effects of early treatment with low-dose ino on brain injury (grade 3-4 ICH, PVL, ventriculomegaly) in preterm infants Kinsella et al. New Engl J Med 6 Ballard et al. New Engl J Med 6 Multicentre RCT (21 centres) 582 newborns < 32 wks, <125gm requiring MV 7-21d ino at decreasing concentrations or placebo gas for 24 d 1 outcome death or survival without BPD at 36 weeks 2 outcome days of resp support,, length of stay Overall ino: survival without BPD (p=.4) LOS (p=.4), length on (p=.6) lung disease severity if enrolled from 7-14 d vs 15-21d Effects of ino on survival without BPD for infants enrolled between 7 and 21 days of age Ballard et al. New Engl J Med 6 Modfied by Kinsella et al. J Pediatr 7 8

39 EUNO. et al. Lancet 1 Multicentre RCT (36 EU centres) 8 newborns < 29 wks, >5gm requiring Resp support<24h ino at 5ppm or placebo gas for min 7 d - max 21d 1 Survival without BPD at 36 weeks 2 Alive without brain injury Overall ino: No effect on survival without BPD (66% vs 65%) Incidence of BPD: 27% vs 24% No effect on Alive without brain injury Kinsella et al, J Pediatr 14 To assess the efficacy and safety of early, noninvasive inhaled nitric oxide (ino) therapy in premature newborns who do not require mechanical ventilation Multicentre RCT, 124 newborns who required noninvasive supplemental oxygen within the first 72 hours after birth Stratified by birth weight (5-749, , g) prior to randomization to ino (1 ppm) or placebo gas (controls) until 3 weeks postmenstrual age No difference in incidence of death or BPD, BPD severity, need for mechanical ventilation, duration of mechanical ventilation, or safety outcomes including severe intracranial hemorrhage Summary of ino Trials Early rescue therapy for severe respiratory failure: No benefit Potential increased risk of severe ICH Selective indications? PPROM ± oligohydramnios Prevention of BPD in infants with mild respiratory distress: Safe Effective in a subset of preterm infants>1g Neuroprotective Economical considerations (high cost barrier to its use?) 9

40 Smith H et al. J Thorac Cardiovasc Surg 6 L-Citrulline preserves alveolar development in -induced BPD in newborn rats Normoxia Normoxia + L-Citrulline Hyperoxia Hyperoxia + L-Citrulline X A) Mean Linear Intercept Normoxia Normoxia+ L-Citrulline Hyperoxia Hyperoxia+ L-Citrulline B) Septal counts Vadivel et al. Pdiatr Res 1 Angiogenic growth factors and the making of a blood vessel HIF NO Ang1 1

41 Endothelial Progenitor Cells (EPCs): ECFCs fulfill the stringent criteria of a true EPC EPC hierarchy model based on proliferative and clonogenic potential of discrete populations of progenitor cells DN Prater, J Case, DA Ingram, MC Yoder. Leukemia 7 Modified from MC Yoder, DA Ingram. Blood 7 ECFCs exist in the Human Fetal Lung Cobblestone-looking ECFC Colony LDL-uptake and Ulex-lectin binding Ulex Di-Ac-LDLHoechst x 1x Cell Surface Markers CD31 CD146 CD15 1x Endothelial network formation CD144 (VE-Cad) CD14 CD45 Alphonse RS et al. Circulation 14 Human Lung ECFC Progeny Possess the Ability to Form de novo Blood Filled Vessels in vivo Human Lung ECFCs form de novo blood vessels that connect with the host vasculature vessels/mm 2 Slide courtesy Merv Yoder 11

42 Exposure to Oxygen Impairs Lung ECFC Function EPC hierarchy model based on proliferative and clonogenic potential of discrete populations of progenitor cells Network Formation Room Air % O2 Clonogenic Potential 25 Nb Intersections 15 (P<.5) 1 5 Room Air ECFC Function is impaired in -induced BPD EPC hierarchy model based on proliferative and clonogenic potential of discrete populations of progenitor cells Single Cell Clonogenic Potential >5 12

43 Therapeutic Potential of UCB-ECFC in Experimental -induced Neonatal Lung Injury Rag-/-mice RNU rats hucb-ecfc (jugular vein) Endpoints olung Compliance (Flexivent ) olung histology opulmonary artery acceleration time and right ventricular hypertrophy P- P-5 P-14 P-28 Hyperoxia(85% ) Human cord blood-derived ECFCs Restore Arrested Alveolar and Lung Vascular Growth in -induced Experimental BPD Alveolar Growth +ECFC Vascular Growth +ECFC +ECFC +ECFC Mean Linear Intercept Vessel Count A µm P <.1 vs all other groups; n = 4-6 animals/group +ECFC +ECFC B No. of vessels per HPF ECFC P <.5 vs all other groups; n = 4-6 animals/group +ECFC Human cord blood-derived ECFCs Attenuate Pulmonary Hypertension in -induced Experimental BPD +ECFC +ECFC Right Ventricular Hypertrophy P<.1 vs other groups n=4-9/group P<.1 vs other groups n=4-6/group +ECFC +ECFC RV/LV+S 13

44 Resident Lung ECFC Function is Restored by Exogenous Human cord blood-derived ECFCs +ECFC Colonies per 96-well plate ECFC < >5 Cells/Colony +ECFC P <.5; n = 3 trials per group (x 1 µm) X Cord Length P <.5 vs all 4 other groups; n = 6 per group ECFC Branch Points P <.5 vs all other groups; 15 n = 6 per group 1 5 A B +ECFC +ECFC +ECFC +ECFC No Adverse Effect of Human cord blood-derived ECFCs at 1 months +ECFC 5 Mean Linear intercept P<.5; n=3-6/group 3 µm +ECFC 1 B +ECFC +ECFC Human Lung Mouse Lung A Distance (m) 6 Exercise Capacity P <.5 vs P <.5 vs all groups n=3-6/group PAAT (ms) PAAT C + ECFC + ECFC D +ECFC +ECFC E Low Lung Engraftment of Cord Blood-derived ECFCs 5 Alu/18s (control as 1) 3 1 Control 2 hrs 3 days 14 days 21 days 14

45 Evidence that ECFCs act via a Paracrine Effect: Therapeutic Potential of cell-free Conditioned media In vitro 1- AEC wound healing 2- EC capillary-like network Rag-/-mice RNU rats In vivo hucb-ecfc CdM (i.p.) Endpoints olung Compliance (Flexivent ) olung histology opulmonary artery acceleration time and right ventricular hypertrophy P- P-5 P-14 P-21 Hyperoxia(85% ) Human cord blood-derived ECFC CdM preserves Alveolar and Vascular Growth in -induced BPD +DMEM +ECFC CdM Mean Linear intercept P<.1 vs other groups n=6/group +DMEM +ECFC CdM DMEM ECFC- CdM DMEM ECFC- CdM No. of vessels/1 HPF DMEM ECFC- CdM DMEM ECFC- CdM p<.1 vs O2-DMEM n= 5/group Angiogenesis promotes lung growth and repair and may have therapeutic benefit in BPD 1- Angiogenesis inhibition 2- Hyperoxia 3- Pro-angiogenic Therapies In experimental BPD VEGF ECFCs 15