Role of Inflammation in Pulmonary Hypertension

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1 Role of Inflammation in Pulmonary Hypertension K. R. Stenmark University of Colorado Denver, USA Prominent Fibroproliferative Changes are Observed in the Lung Vasculature of Infants With Pulmonary Arterial Hypertension (PAH) 1

Traditional Hypothesis Injury Resident Pulmonary Artery Cell Activation Modulation of Resident Cell Phenotype Proliferation, Migration, Matrix deposition

2 Traditional Hypothesis Injury Resident Pulmonary Artery Cell Activation Modulation of Resident Cell Phenotype Proliferation, Migration, Matrix deposition Structural Vascular Remodeling Inflammatory Cells, Including Monocytes / Macrophages (CD68+), Accumulate In and Around Vascular Lesions of Patients with PAH CD68 2

in the Vascular Lesions of Patients with PAH Plexiform Control CD31 Concentric Lesion

3 Accumulation of Inflammatory (CD68+) Cells is Associated with Fibroproliferative Vascular Changes in Infants with PAH Normal BPD Progenitor-like (CD133+, ckit+) Cells Accumulate in the Vascular Lesions of Patients with PAH Plexiform Control CD31 Concentric Lesion ckit Plexiform Lesion ckit/cd31 CD31 CD133 Toshner M., et al., Am J Respir Crit Care Med

Proliferation, Differentiation, Fibrosis

Perivascular Inflammation Mouse Rat Calf

4 Complimentary Hypothesis Traditional Hypothesis Injury Resident PA Cell Activation Circulating Inflammatory&Progenitor Cell Recruitment Modulation of Cell Phenotype Direct & Indirect Contribution to Cell Proliferation, Differentiation, Fibrosis Pulmonary Vascular Remodeling Experimentally-Induced Pulmonary Vascular Remodeling Varies Considerably Between Animal Species Perivascular Inflammation Mouse Rat Calf Vascular Remodeling (Medial / Adventitial Thickening) CD11b α-sm-actin 4

5 Sustained Hypoxia Induces Robust Accumulation of Monocytes in the PA Wall CD45 Normoxia Hypoxia Leukocytes Normoxia Hypoxia ED1 Monocytes Neutrophils OX62 Dendritic cells CD45RA B-lymphocytes CD3 T-lymphocytes Lymph node Lymph node Hypoxia-induced Accumulation of Monocytes in the PA Adventitia is Time-Dependent Controlol 1-wk Hypoxia 4-wk Hypoxia Time-course 5

Observation: Perivascular accumulation of monocytes is closely associated with fibrosis in PH Question: What is the cellular link between monocytic accumulation and fibrosis in PH?

6 Observation: Perivascular accumulation of monocytes is closely associated with fibrosis in PH Question: What is the cellular link between monocytic accumulation and fibrosis in PH? Fibrocytes A subset of circulating leukocytes that co-express leukocytic (CD45, etc) and mesenchymal (collagens) markers and: Rapidly recruited to the site of tissue injury; Transition into fibroblasts / myofibroblasts; Produce extracellular matrix proteins; Secrete inflammatory and pro-angiogenic factors (Bucala et al. 1994; 2005; Abe et al. 2001; Hartlapp et al. 2001; Yang et al. 2002; Schmidt et al. 2003; Metz 2003, etc.) 6

Fibrocytes (CD45+ / Collagen+) Comprise > 40% of the Recruited

Fibrocytes Transition into a Myofibrocyte Phenotype (CD68+ /

, 2006 Two Experimental Approaches Demonstrate that Fibrocytes

via DiI-liposomes; b) Identifying DiI-labeled monocytes in the

rats Approach 2: Using a natural bovine XY /XX chimera

7 Fibrocytes (CD45+ / Collagen+) Comprise > 40% of the Recruited Leukocytes in the Remodeled PA Wall Some of the Recruited Fibrocytes Transition into a Myofibrocyte Phenotype (CD68+ / α-sm-actin+) Confocal microscopy Frid et al. Am. J. Pathol., 2006 Two Experimental Approaches Demonstrate that Fibrocytes in the Hypoxic Vessel Wall Originate from Circulation / Bone Marrow. Approach 1: a) In vivo DiI-labeling of monocytes in circulation via DiI-liposomes; b) Identifying DiI-labeled monocytes in the adventitia of pulmonary (but not systemic) arteries of hypoxic rats Approach 2: Using a natural bovine XY /XX chimera (freemartin calf), identify Y-chromosome+ cells recruited to hypoxic PA wall. Normoxia M DiI+ cells Lung parenchyma Adv. Hypoxia M Lung parenchyma Adv 7

8 What is the Contribution of Circulating Monocytes / Fibrocytes to Pulmonary Vascular Remodeling and Fibrosis? Approach: In Vivo Depletion of Circulating Monocytes / Fibrocytes via i.v. Injections of Liposome-Encapsulated Clodronate When i.v.-injected, clodronate-liposomes are taken up only by circulating phagocytic cells (monocytes/ macrophages/ fibrocytes) - and intracellular excess clodronate induces apoptosis of these cells Not toxic to non-phagocytic cells, no reduction in the number or activation of T- cells Hypoxia-Induced Perivascular Remodeling & Fibrosis are Attenuated by Depletion of Circulating Monocytes/Fibrocytes via Clodronate-Liposomes (Clo-L) Normoxia Hypoxia Hyp + Clo-L CD11b pro-coll ED-A-FN TN-C Frid et al. Am J Pathol

9 What are the Local Mediators that: 1. Induce Recruitment and Retention of Monocytes / Fibrocytes in the PA Wall? PA Monocytes fibrocytes 2. Participate in the Differentiation of the Recruited Fibrocytes into Mesenchymal (collagen-producing) Cells? Hypoxia Induces PA-Specific mrna Upregulation (blue bars) of Chemokines & Receptors Known to be Involved in Monocyte Recruitment and Activation Moreover, regression of PH vascular remodeling is associated with downregulation of mrna for these chemokines / receptors (green bars) 9

level) of Chemokines, Adhesion Molecules, and Differentiation Factors Known to be Involved in Monocyte / Fibrocyte

10 Hypoxia Induces PA-Specific mrna Upregulation (blue bars) of Adhesion Molecules, Growth and Differentiation Factors Known to be Involved in Monocyte Retention and Fibrocyte Differentiation Hypoxia Induces Upregulation (at the protein level) of Chemokines, Adhesion Molecules, and Differentiation Factors Known to be Involved in Monocyte / Fibrocyte Recruitment, Retention and Differentiation SDF-1 VEGF OPN Fibronectin (ED-A isoform) VCAM-1 Hsp-47 (pro-collagen I) TGFβ1 10

11 Why is Inflammation Largely Perivascular in Nature? CD68 Human PH Hypoxic Rat PA Monocytes fibrocytes Hypothesis: Fibroblasts Orchestrate the Initiation and Perpetuation of Inflammation in the Vessel Wall Cytokines & Hormones (Ang II, TNFα, TGFβ-1, IL-1, ET-1) Adventitial Fibroblasts Mechanical Forces (stretch, injury) + NADPH Oxidase O 2 - H 2 O 2 TLR Integrins Hypoxia, Ischemia Chemokines / Cytokines / Adhesion Molecules Monocytes / Lymphocytes Vaso Vasovasorum + + Medial VSMC Activated SMC Haurani, et al. Cardiovascular Research,

Fibroblasts From the Hypoxic Hypertensive Calves Exhibit in vitro A Constituitively Activated Proinflammatory Phenotype Relative Gene Expression 2000 1500

Relative Gene Expression 250 CXCR4 Relative Gene Expression 500 VCAM-1 400 300 200 100 Relative Gene Expression 20000 15000 10000 5000 OPN 0 0 0 PA

12 Fibroblasts From the Hypoxic Hypertensive Calves Exhibit in vitro A Constituitively Activated Proinflammatory Phenotype Relative Gene Expression SDF-1 Relative Gene Expression S100A Relative Gene Expression IL Relative Gene Expression 250 CXCR4 Relative Gene Expression 500 VCAM Relative Gene Expression OPN PA Adventitial Fibroblasts From Hypoxic Hypertensive Calves Exhibit Constitutive Activation of pakt and p38mapk Under Basal Conditions CO-Fibs PH-Fibs CO-Fibs PH-Fibs 12

13 Fibroblasts From Hypoxic Hypertensive Calves Induce Greater Adhesion of Monocytes Than Control Fibroblasts CO-Fibs PH-Fibs CO-Fibs PH-Fibs Rapamycin (mtor inhibitor) Attenuates Monocyte Recruitment and Remodeling in Hypoxic Rats 13

14 Inhibition of the SDF1/CXCR4 Axis Prevents and Reverses PH in Neonatal Hypoxic Mice Young et al. Circ Res., 2009 Myocardial Inflammation and Fibrosis Contribute to Right Ventricular Dysfunction in Chronically Hypoxic Calves 14

15 Conclusions/ Speculations Hypoxia Fibrocyte Precursors CD45+ / CD11b+ Cytokines Chemokines adv. Bone Marrow PA inflammation Circulation matrix proteins Vascular Remodeling α SM-actin Circulating Fibrocytes Vascular remodeling in PAH is due, in part, to recruitment of circulating inflammatory/progenitor cells. The pathways involved in the recruitment and retention of these cells may serve as selective pharmacologic targets for PAH treatment. Acknowledgments Ped Critical Care CVP Research Renal Medicine Cardiology M. Frid M. Li A. Flockton D. Brown D. Burke R. Thukaram D. Strassheim E. Gerasimovskaya E. Grayck A. Anwar S. Riddle T. Carpenter M. Das N. Davie S. Majka J. Crossno S. Walchak J. Dempsey V. Karoor R. Nemenoff M. Weiser W. Tan M. Yeager K. Colvin 15

16 Control CD45 Hypoxia ckit Question: What are the mechanisms through which the recruited inflammatory / progenitor cells contribute to the remodeling process? 16

17 Cell Culture Model: Non-Resident (Recruited = R ) Cells Have Been Isolated from Distal PA of Hypoxic Calves SMC CD11b procoll-1 Fibrocyte-like (CD11b+/Coll+) R -cells In Culture, R -cells Transition (Differentiate) into Myofibroblasts (α-sm-actin+) CD11b CD11b αsma DAPI 17

18 R -Cells Exhibit Augmented Proliferative (including Autocrine Growth) and Migratory Capabilities Proliferation Serum-stimulated Migration Serum-deprived (autocrine growth) Autocrine Growth of R Cells is Due, in part, to PDGF and SDF-1 Production Expression pattern (mrna, protein) Inhibitory assays (μg/ml) (μg/ml) 18

19 R -Cells Produce Potent Pro-Mitogenic Activity for Resident PA Cells (SMC & Fibs) Pro-Mitogenic Effects of R - Conditioned Medium are Due, in part, to PDGF(s), SDF-1 and S100A4 (effect of neutralizing Abs) PDGF Abs SDF-1 Abs 3μg 6μg 12μg S100A4 Abs 19

20 In culture, R -Cells Express mrna for Progenitor- Associated Markers and Inflammatory Mediators What are the Routes of Delivery of Circulating Cells to the Pulmonary Vasculature? Appearance of CD11b+ Cells in Adventitia Correlates with enos+ Vasa Vessels 72 hr Hypoxia 20

the Bronchial Circulation b.c. Normoxic Normoxic b.

21 Extensive Neovascularization is Observed in PA Adventitia of Hypoxia Calves Marked expansion of the Bronchial Circulation b.c. Normoxic Normoxic b.c. Hypoxic Davie et al. Am. J. Physiol.,

Role of Inflammatory and Progenitor Cells in Pulmonary Vascular Remodeling: Potential Role for Targeted Therapies. Traditional Hypothesis Stress

3/1/212 Role of Inflammatory and Progenitor Cells in Pulmonary Vascular Remodeling: Potential Role for Targeted Therapies K.R. Stenmark University of Colorado Denver, CO 845 Prominent Fibroproliferative