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 Changes are Observed in the Lung Vasculature of Patients With Pulmonary Arterial Hypertension (PAH) M AW Traditional Hypothesis Stress Resident Pulmonary Artery Cell Activation However, rapidly accumulating experimental evidence suggests an important role of NON-RESIDENT cells in vascular remodeling (and specifically in perivascular / adventitial areas) 1. Robust accumulation of Inflammatory cells CD68 Modulation of Resident Cell Phenotype Proliferation, Migration, Matrix deposition 2. Accumulation of cells expressing Progenitor Markers Structural Vascular Remodeling CD133 CD34 ckit (CD117) 1
3/1/212 Complimentary Hypothesis Traditional Hypothesis Resident PA Cell Activation Stress Circulating / Resident Inflammatory & Progenitor Cell Recruitment / Activation Vascular Remodeling and Vascular Inflammation Are Common Characteristics of PH in Various Mammalian Species, Yet to a Different Degree Vascular Remodeling (α-sm-actin) Mouse (5wk Hypoxic) Rat (4wk Hypoxic) Calf (2wk Hypoxic) Human with PH Modulation of Cell Phenotype Direct & Indirect Contribution to Cell Proliferation, Differentiation, Fibrosis Pulmonary Vascular Remodeling Vascular Inflammation (CD45) Vascular Inflammation Vascular Remodeling Animal Models to Study Human PAH: Chronically Hypoxic Calves Perivascular Accumulation of Mononuclear Phagocytes is Closely Associated With Remodeling and Fibrosis in PH Human infant with PAH Calf with hypoxia-induced PAH Inflammation M M AW AW Fibrosis Control Hypoxic 2
3/1/212 Increased Numbers of Progenitor-like (ckit+) Cells are Observed in the PA Adventitia of Hypoxic Calves Normoxic Calf Hypoxic Calf Question: What is the cellular link between mononuclear phagocyte accumulation and fibrosis in PH? Davie et al. Am. J. Physiol., 24 Fibrocytes A subset of circulating leukocytes with a dual phenotype that co-express leukocytic and mesenchymal markers and: Are rapidly recruited to the site of tissue injury; Transition into fibroblasts / myofibroblasts; Produce extracellular matrix proteins; Secrete inflammatory, growth, pro-angiogenic factors Fibrocytes (CD45+ / Collagen+) Comprise Up to 4% of the Recruited Leukocytes in the Remodeled PA Wall Chonically hypoxic calf - PA (Bucala et al. 1994; 25; Abe et al. 21; Hartlapp et al. 21; Yang et al. 22; Schmidt et al. 23; Metz 23, etc.) 3
3/1/212 Accumulation of Alternatively Activated (M2) Macrophages (CD163+, CD26+) in the Adventitia and Media of Calves With Severe PH Expression of Markers on Monocytes, Macrophages, Fibrocytes, and Fibroblasts: M2 Macrophage and Fibrocyte Similarities CD26 CD163 Pilling et al. PLoS., 29 Potential Origins of the Mesenchymal and Endothelial Precursors in the Remodeled Vessels Bone Marrow / Circulation Resident Vascular Progenitors Two Distinct Experimental Approaches Demonstrate That Fibrocytes in the Hypoxic Vessel Wall Originate from Circulation / Bone Marrow. Approach 1: In vivo DiI-labeling of monocytes in circulation via DiI-liposomes DiI+ cells Approach 2: Using Freemartin calf (a natural bovine XY / XX chimera), identify Y-chromosome+ BM cells recruited to hypoxic PA wall. Normoxia M Lung parenchyma Adv. Hypoxia Frid et al. Am. J. Pathol., 26 M Lung parenchyma Adv 4
3/1/212 Fibrocyte-like Cells (termed R -cells Due to Their Rhomboidal morphology in culture) Can be Isolated From the PA Media of Hypoxic Calves SMC In Culture, Fibrocyte-like Cells Transition / Differentiate into Myofibroblasts (α-sm-actin+) CD11b CD11b αsma DAPI SMC R -cells Frid et al. Am. J. Physiol., 29 R -cells CD11b procoll-1 Similarity with in vivo observation by confocal microscopy: Fibrocyte-like (CD11b+/Coll+) R -cells Frid et al. Am. J. Pathol., 26 Fibrocyte-like R -Cells Express mrna for Progenitor- Associated Markers and Inflammatory Mediators CEACAM1 Fibrocyte-like R -Cells Produce Potent Pro-Mitogenic Activity for Resident Vascular Cells (SMCs & Fibs) CEACAM 1 or else? Frid et al. Am. J. Physiol., 29 Frid et al. Am. J. Physiol., 29 5
3/1/212 What are the Cellular Mechanisms Inducing Recruitment, Retention and Activation of Circulating Pro-Inflammatory Cells (including Fibrocytes and Mesenchymal Progenitor Cells) to the Pulmonary Artery Wall? Hypothesis: Fibroblasts Orchestrate the Initiation and Perpetuation of Inflammation in the Vessel Wall Cytokines & Hormones (Ang II, TNFα, TGFβ-1, IL-1, ET-1) Mechanical Forces (stretch, injury) TLR Integrins Activated Adventitial Fibroblast Hypoxia, Ischemia Leukocytes / Lymphocytes Vasa Vasorum Adhesion Molecules / Chemokines / Cytokines Medial SMC A Cytokines B Adventitial Fibroblasts from Hypertensive Calves Express a Pro-Inflammatory Phenotype Chemokines & Costimulatory Molecules C Adhesion Molecule(s) 2. 1.5 1..5. IL-1β CCL2/MCP-1 38 285 19 95 18 12 6 CCR7 GM-CSF 9 6 3 4 3 2 1 VCAM-1 IL-6 24 18 12 6 CO-Fibs CXCL12/SDF-1 13 975 65 325 12 8 4 CD4L 4 3 2 1 CCR2 n.s. 14. 1.5 7. 3.5. TNFα n.s. CCL5/RANTES 12 9 6 3 1 75 5 25 4 3 2 1 CO-Fibs CXCR4 CD4 Protein Expression Adventitial Fibroblasts from Hypertensive Calves () Induce Increased Migration, Adhesion and Pro-Inflammatory Activation of Monocytes 1. Monocyte Transwell Migration 2. Monocyte Adhesion to Fibroblasts Fold Change Relative Number of Migrated THP-1 2. 1.5 1..5. CO-Fib-CM PH-Fib-CM 11. 8.25 5.5 2.75 IL-1β 9. 6.75 4.5 2.25 % Bound THP-1 Cells 17 136 % Bound Cells (relative to control) 12 68 34 CO-Fibs 3. Monocyte Pro-Inflammatory Activation CCL2/MCP-1 Type I Collagen 5.... CO-Fib-CM -CM CO-Fib-CM PH-Fib-CM CO-Fib-CM PH-Fib-CM 3.75 2.5 1.25 6
3/1/212 Conditioned Media From (not CO-Fibs) Induce Bovine Bone-Marrow Derived Macrophages to Express CD163 and Mannose Receptor (markers of M2 activation) Relative Expression of CD163 (normalized to unstimulated) 3. 2.5 2. 1.5 1..5 Relative Expression of MR (normalized to unstimulated) 4. 35. 3. 25. 2. 15. 1. 5. Epigenetic Gene Silencing May Hold Key to Fatal Lung Vascular Disease Science Daily (June 7, 21) A rare but fatal disease of blood vessels in the lung may be caused in part by aberrant silencing of genes rather than genetic mutation, new research reports. S.A. Archer. Unx CO-CM PH-CM. blank CO-CM PH-CM Potential Mechanisms? Hypothesis: Pro-Inflammatory Fibroblast Phenotype is due to Abnormal Activity of Histone Modifying Enzymes (i.e. HDACs) Adventitial Fibroblast 1. Alterations in HDAC Activity 3. Monocyte recruitment Vasa Vasorum Fibroblasts from Hypertensive Calves () Exhibit Increased HDAC Activity and Protein Expression a. b. Class I HDAC Catalytic Activity (fluorescence X 1 3 ) 35 262 175 87.5 CO-Fibs + Apicidin 2. Chemokines / Cytokines c. HDAC1 HDAC2 HDAC3 1. 1. 6. 7.5 7.5 4.5 5. 5. 3. Histone Histone deacetylases (HDACs) are are a class a of enzymes that modify DNA-binding histones and and chromatin chromatin structure, structure, thus thus playing playing an important role role in in regulation of gene expression, and and in in epigenetics. 2.5. 2.5 1.5.. 7
3/1/212 Adventitial Fibroblasts as Cellular Targets: Inhibition of HDACs in results in attenuation of pro-inflammatory phenotype Inhibition of Class I HDACs Attenuates Pro-Inflammatory Phenotype of 23 IL-6 2 CCL2/MCP-1 2 CXCL12/SDF-1 Apicidin = Class I HDACi SAHA = pan HDACi; 1 um 184 138 92 46 Untreated +SAHA +Apicidin 15 1 5 Untreated + SAHA + Apicidin 15 1 5 Untreated +SAHA +Apicidin 25 GM-CSF 16 VCAM-1 115 CCL5/RANTES 2 15 1 12 8 92 69 46 5 4 23 Untreated +SAHA +Apicidin Untreated +SAHA +Apicidin Untreated +SAHA +Apicidin M. Li et al J. Immunol 211 Inhibition of Class I HDACs Attenuates Capability of to Induce Monocyte Migration and Pro-Inflammatory Activation Relative Number of Cells Migrated Monocyte Migration 1 75 5 25 PH-Fib-CM + Apicidin Monocyte Pro-Inflammatory Activation 19 1425 95 475 CCL2/MCP-1 PH-Fib-CM + Apicidin 35 IL-1β 2625 175 875 PH-Fib-CM + Apicidin Class 1 HDAC Activity is Increased in Tissue Derived Fibrocytes (R-cells) Copy/1 copies of HPRT 28 21 14 7 CO-SMC "R" Cells 8
3/1/212 HDAC Inhibitors Attenuate Fibrocyte Activation ckit 8 6 4 2 CO-SMC "R"Cells # +VPA (5mM) +SAHA (1µM) PDGFb 3. 22.5 15. # 7.5. CO-SMC "R"Cells +VPA (5mM) # +SAHA (1µM) 3. 2.25 1.5.75. S1A4 CO-SMC "R"Cells # +VPA (5mM) # +SAHA (1µM) "R"Cells "R"Cells "R"Cells 2.5 MCP-1 2. 1.5 1..5. CO-SMC "R"Cells +VPA +SAHA (5mM) (1µM) 1. SDF-1 7.5 5. 2.5. CO-SMC "R"Cells +VPA +SAHA (5mM) (1µM) 1. 7.5 5. 2.5. IL-6 CO-SMC "R"Cells +VPA +SAHA (5mM) (1µM) "R"Cells "R"Cells "R"Cells Class I HDAC Inhibition Suppresses Hypoxia- Induced Pulmonary Arterial Medial Thickening Class I HDAC Inhibition Suppresses Multiple Pathological Pathways in the RV 9
3/1/212 Conclusion: Better understanding of the mechanisms leading to inflammation / progenitor cell recruitment, retention and activation, including epigenetic alterations in control of protein expression, may lead to alternative therapeutic strategies in pulmonary hypertension Acknowledgments Developmental Lung Biology M. Frid M. Li A. Flockton D. Brown D. Burke M. Yeager K. Colvin E. Gerasimovskaya H. Nijmeh E. Grayck S. Riddle N. Davie CVP Research S. Majka S. Walchak Cardiology T. McKinsey Renal M. Weiser-Evans R. Nemenoff 1