Large Vessel Stiffening and Pulmonary Hypertension: Contribution of Extracellular Proteins and Inflammation

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1 6/21/213 Large Vessel Stiffening and Pulmonary Hypertension: Contribution of Extracellular Proteins and Inflammation RV Failure is the Proximate Cause of Death in Patients With Pulmonary Hypertension K. R. Stenmark University of Colorado Denver, CO 8262, USA Normotensive Hypertensive Reproduced from Ghio S et al. JACC, 21 Increased resistance to blood flow Historical understanding: resistance determines afterload. Drugs lower resistance, but have not adequately improved outcomes. Increased afterload PAH Right Heart Failure Increased stiffness of pulmonary artery It s now known that resistance and compliance determine afterload.. Pulmonary Circulation: Questions Is there evidence for central vascular stiffening in PH? What are the potential cellular mechanisms contributing to stiffening of the pulmonary artery? Death Note: stiffness is the inverse of compliance Should/can anything be done to directly reduce proximal vascular stiffening in PH? 1

2 6/21/213 Pulmonary Hypertensive Vessels Enlarge and Stiffen. Significant Increases in Impedance are Observed in Patients with PAH Normotensive PA Hypertensive PA D= 1.6 cm D= 2.5 cm Artery Wall At Systole Wall Deformation Over Cardiac Cycle Measured by MRI (Age, Gender and BSA Matched) Significant elevation in modulus of impedance for the first three harmonics in patients with PH. Z : resistance, Z 1, Z 2 : oscillatory function / capacitance Hunter et al., Am H.J., 28 PAH-Associated Vascular Remodeling in Human Physiologic Stiffness is Increased in Patients with PAH Normal PA Hypertensive PA Collagen Circumferential Stress HEALTHY PA Stiffening in Severe PH HYPERTENSIVE HEALTHY HYPERTENSIVE Elastin fragmentation Collagen accumulation 2-Photon Second Harmonic Generation Microscopy: Elastin ; Collagen 2

3 6/21/213 Chronic Hypoxia Results in Stiffening of the Extralobar Pulmonary Arteries in Mice and Rats Can Animal Models be Used to Study Proximal Vascular Stiffening in Pulmonary Hypertension? Mouse Ex Vivo biaxial stress strain testing of rat PAs Rat Marked Increases in Resistance and Stiffening in Chronically Hypoxic Neonatal Calves Interim Summary: PH-Associated Pulmonary Vascular Remodeling in Human Significantly elevated stiffness Neonatal (2 wk-old) Calves Control (n=12) Severe PH (n=8) Indices of Ventricular Resistance Peak Systolic Pressure (mm Hg) 31.±3 11.5±17.7 Mean PAP (mm Hg) 2.1±3 73.4±1 Total Pulmonary Resistance (mm Hg/L/min) 2.1±1. 8.±2. Indices of Pulmonary Vascular Stiffening Pulse Pressure (mm Hg) 2.1±2 51.2±16.9 MPA Stiffness (mm KPa) 112±35 Moderate PH (n=6) 59.4± ± ± ± ±51 287±11 Matrix Remodeling in PH STRUCTURAL: Elastin fragmentation Collagen accumulation FUNCTIONAL Early collagen engagement Significant anisotropy (different longitudinal vs. circumferential mechanics) Loss of elastin-dependent mechanics Near total loss of PA capacitive function 3

4 6/21/213 Pulmonary Circulation: Questions Is there evidence for central vascular stiffening in pulmonary hypertension? What are the potential cellular mechanisms contributing to stiffening of the pulmonary artery? Hypothesis: Large Vessel Stiffening is Due to Accumulation and Activation of Mononuclear Cells Should/can anything be done to directly reduce proximal vascular stiffening in PH? Elevated mpap, pulse pressure, and disturbed flow patterns High Afterload Right Heart Failure. Humans with PAH: Accumulation of Monocyte/Macrophage in Pulmonary Artery Media, Neointima, and Adventitia Calf Model Exhibits Greater Similarities to Human PAH than Rodent Models Calf Rat Medial resident immunomodulatory cells PA media normally contains resident immunomodulatory leukocytic cells; in PAH, numbers of these cells within vascular media markedly increase. Accumulation of inflammatory cells in PA Media, Adventitia, (and Neointima); Elastin fragmentation is observed Influx of inflammatory cells in PA Adventitia only. 4

5 6/21/213 Extracellular Matrix (ECM) Derived Fragments Can Drive a Vascular Inflammatory Response ECM peptides with immune / inflammatory effects: Elastin Collagen (I and IV) Fibronectin Laminin Thrombospondin Hyaluron Vascular Inflammation, Elastin Fragmentation, and Vascular Stiffening Chemotactic for leukocytes Activated Macrophages Elastin Fragments ( Elastokines ) Pro-Mitogenic for VSMCs & Fibs Degradation of Elastin Anti- Apoptotic Pro- Angiogenic Chronic Inflammation Vascular Remodeling Rabinovitch M. 1999; Antonicelli F. et al. 27 Duca L. et al 24 Expression of Pro-Inflammatory Mediators, Pro-Fibrotic Markers, and Proteolytic Enzymes is Augmented in Vascular Media of Hypoxic Hypertensive Calves (Hx) RT-PCR Expression relative to HPRT Il1b Il6 Ccl2 Ccl12 Ssp1 (MCP-1) (SDF-1) (Osteopontin) C O 6 H x CO Hx C O H x Col1a1 Col3a1? Thbs-1 Mmp1 (Collagen I) (Collagen III) (Thrombospondin-1) (MMP-1) CO Hx? CO Hx Bone Marrow-Derived Macrophages (BMDMs) but not SMCs Respond to Elastin Peptide Treatment by Upregulating Pro-Inflammmatory Mediators RT-PCR Relative Expression to HPRT Il1β Ccl2 (MCP-1) Νos2 (inos) C O H x CO Hx CO Hx CO Hx 5

6 6/21/213 Bone Marrow-Derived Macrophages (BMDMs) but not SMCs Responded to Elastin Peptide Treatment by Upregulating Pro-Fibrotic Markers Relative Expression to HPRT Relative Expression to HPRT Ccl12 (SDF-1) Ssp1 (Osteopontin) Tnc (Tenascin-C) Col1a1 (Collagen I) Col3a1 (Collagen III) Thbs1 (Thrombospondin- 1) Interim Summary: Normal conduit PA media (human and bovine, but not rodent) contains resident inflammatory/immunomodulatory leukocytic cells. In pulmonary hypertension, increased accumulation of numbers of these leukocytic cells is observed. Elastin peptides induce activation of naïve macrophages (BMDMs): upregulated mrna expression of pro-inflammatory and pro-fibrotic markers. Pulmonary Circulation: Questions Is there evidence for central vascular stiffening in pulmonary hypertension? What are the potential cellular mechanisms contributing to stiffening of the pulmonary artery? Should/can anything be done to directly reduce proximal vascular stiffening in PH? Compliance is Fundamental to PH Low compliance is the leading independent predictor of mortality Compliance correlates directly with exercise capacity and quality of life Low compliance accounts for up to 5% of RV afterload Low compliance stimulates progression of the disease Pulmonary vascular stiffness is increased 25- to 3-fold etc 1 Campo et al, Am J Respir Crit Care Med, 29, 2 Mahapatra et al, J Am Soc. Echocardiography, 26, 3 Mahapatra et al, J Am Col Card, 26, 4 Gan et al, Chest, 27, 5 Hunter et al, J Appl Physiol, 21, 6 Sanz et al, JACC Card. Imag. 29, 7 Li and Chesler, Pulm. Circ. 211, 8 Perrot et al, Chest, 211, 9 Bonderman et al, Chest, 211, 1 Saouti et al, Am J Respir Crit Care Med, 21, 11 Milnor et al, Circ Res, 1966, 12 Milnor et al, Circ Res, 1969, 13 Wang and Chesler, Pulm Circ, 211, 14 Stenmark et al, Circ Res, 26, 15 Birukov Andiox Redox Sig, 29, 16 Li et al, Ann Biomed Eng, 29, 17 Stevens et al, JACC, Cardiovascular Imaging, 212 6

7 6/21/213 Potential Mechanisms of HPF-Induced Perpetuation of Distal PA Remodeling In PH Therapies Aimed at Reducing Vascular Stiffening Renin Angiotensin Aldosterone System (RAAS) blockers AGE cross-link breakers (alagebrium) Statins Anti-Inflammatory agents Elafin (serine protease inhibitor) SM Ce SM Ce Fibs--Myofibs differentiation Fibroblst-myod SMC hypertrophy Aria CV s Device Restores Compliance to PA Bench: Hemodynamic Benefits Reservoir Pressure in Pulmonary Artery Conduit Gas filled balloon & anchor SQ access port Pa Pressure (mmhg) Systole 2 Diastole Time (s) Off On Implanted KV1 like a pacemaker Mimics function of elastic pulmonary artery Balloon collapses during systole & expands during diastole Acts like a passive IABP By restoring compliance, it allows more blood to flow for less heart work Device Compliance increased by.51 ml/mmhg (in this example) Hemodynamic Effects Lower systolic pressure reduces RV work & wall stress increases SV Higher diastolic pressure Increases blood flow Reduction of pulse pressure breaks the disease-stimulating feedback loop Expected Patient Effects Increase in exercise capacity & QOL Decrease in mortality likely 7

8 Slide 27 KV1 Do we want to say that it functions similar to an IABP? Stick in slide with benchtop data in it. Karl Vollmers, 6/17/213

9 6/21/213 Bovine Studies Device in vivo Device efficacy studies using high altitude cattle Brisket disease = RHF due to PH caused by chronic hypoxia Histology of the pulmonary vessels resembles vessels of PAH patients Device increased Pulmonary Vascular Compliance by 1.3 ml/mmhg (~ 47% increase) Implications for PH Patients: Mayo Clinic Study Compliance is Fundamental to PH Normal person Q 4 Q 2 Q 3 Q 1 drug & device An increase of compliance by 1.3 ml/mmhg (as in Bovine Study 3) would move patients up two quartiles. Good chance of Survival Benefit drug only Relationship of Capacitance and Mortality in Patients with ipah (JACC 26, McGoon, et al) 1 Bonderman et al, Chest, 211, 2 Lankhaar et al, Eur Heart J, 28, 8

10 6/21/213 Conclusion: Pulmonary Hypertension is a Disease of Disordered Coupling of the RV to the Pulmonary Circulation, Where Large Vessel Stiffening is Critical Cardiovascular Pulmonary Laboratories Dunbar Ivy Maria Frid Suzette Riddle Min Li Mike Yeager Dale Brown Joe Albietz Lori Walker Peter Buttrick UC Denver Department of Bioengineering Robin Shandas Steve Lammers Kendall Hunter Jerry Qi Wei Tan Luciano Mazzaro University of Pittsburgh Hunter Champion et al University of Minnesota John Scandura (Aria) Ken Weir Elevated mpap, pulse pressure, and disturbed flow patterns High Afterload Right Heart Failure. Large Vessel Stiffening, via Effects on Pulse Wave Intensity, Modulates Distal Vessel Structure and Function High Pulsatility Flow (HPF) Stimulates Bovine Distal PA-EC Inflammation Mimetic Circulatory System Pressure (mmhg) 9

11 6/21/213 Ventricular-Arterial Coupling in the Pulmonary Circulation Potential Mechanisms Contributing to Vascular Stiffening Endothelium: Endothelial dysfunction Increased permeability Media: Collagen, VSMCs, AGE s, MMPs, MΦ? Elastin, (elastases ) Intima: Collagen, VSMCs, Leukocytes ( MΦ ), MMPs, AGE s, TGF-β, etc etc. Elastin from: Zieman SJ et al., ATVB 25 Adventitia: Collagen, Fibroblasts MΦ, T-cells Extrinsic Influences: NaCl, Lipids, AngII, Sympathetic Neurohormones, Shear Stress, Increased Luminal Diameter In Vivo Depletion of Macrophages via Clodronate- Liposomes Results in Attenuation of Hypoxia-Induced Fibrosis and Vascular Remodeling in Rats Hypothesis In pulmonary hypertension, vascular remodeling and stiffening are the result of augmented Accumulation and Activation of Inflammatory / Immunomodulatory cells within the media of large PAs. 1

12 6/21/213 Potential Mechanisms Contributing to Vascular Stiffening Endothelium: Endothelial dysfunction Increased permeability Media: Collagen, VSMCs, AGE s, MMPs, MΦ? Elastin, (elastases ) Hypothesis Elastin peptides, generated in the setting of PH in the media of large elastic PAs, perpetuate pro-inflammatory and pro-fibrogenic gene programs in monocytes/macrophages. Intima: Collagen, VSMCs, Leukocytes ( MΦ ), MMPs, AGE s, TGF-β, etc etc. Elastin Adventitia: Collagen, Fibroblasts MΦ T-cells Extrinsic Influences: NaCl, Lipids, AngII, Sympathetic Neurohormones, Shear Stress, Increased Luminal Diameter MMP s Elafin Treatment Reverses Established Severe PH in Rats Non-transgenic Elafin-transgenic Non-transgenic Elafin-transgenic Injured Injured Sham Sham P <.5, One-way ANOVA with Dunnett's Multiple Comparison Movat s pentachrome stain Mac-3 antigen stain N. Nickel and M. Rabinovitch unpublished 11