Molecular Mechanisms of Vascular Calcification Catherine Shanahan, PhD Cardiovascular Division, King s College London, UK ESC, Munich, August 2012
CONFLICTS OF INTEREST: NONE TO DECLARE
Vascular smooth muscle cell phenotype Differential cdna screening CONTRACTILE media a-sm actin g-sm actin SM22a calponin phospholamban ubiquitin vinculin elastin osteoglycin aquaporin-1 Nesprin-1 (1RA1) Development Vascular Injury Atherosclerosis SYNTHETIC Intima matrix Gla protein osteopontin Shanahan CM et al Circ Res 1993
Human VSMCs exhibit phenotypic plasticity Runx2 BMP2 TGFb ismad osteogenic chondrocytic adipocytic osterix msx2 cbfa1 sox9 SREBP1 calcification matrix production lipid accumulation CKD diabetes Atherosclerosis ageing Stability? Matrix degradation
Vascular calcification is a detrimental process MEDIAL INTIMAL Diabetes Renal Disease Ageing Atherosclerosis Stiffness Plaque Rupture EBCT Major cause of cardiovascular mortality in CKD Increased risk of myocardial infarction In Health Inhibitors Prevent Calcification of Soft Tissues
Vascular Calcification is a Regulated Process similar to bone calcification
Vascular calcification is a regulated process 1. Mineral is hydroxyapatite SMC 2. Matrix vesicles identified in the vessel wall Media 3. Bone formation occurs in the vessel wall Intima
Vascular calcification is a regulated process 4. Mouse gene knockouts develop vascular calcification MGP (matrix Gla protein) Fetuin** Klotho/ FGF23** Osteoprotegerin carbonic anhydrase Smad 6 matrix KO s 5. Human single gene defects Keutel Syndrome (MGP null) Idiopathic calcification in newborn (Enpp1) Genetic Component Luo et al Nature 1997
Expression of osteogenic markers by VSMCs in calcified human media RT-PCR in situ hybridization calcified normal a-sm actin ALK MGP ALK VK BSP BSP BGP control negative control BGP Tyson et al, ATVB, 2003
Human VSMCs in vitro default to an osteo/chondrocytic phenotype N Ca N Ca SM22 OP MGP Runx2 COL II Sox9 Days in nodules 3 7 14 21 ALK ALK BSP BGP BSP BGP SPARC control Shanahan et al, Circulation, 1999. Tyson et al, ATVB, 2003.
Ca (mg/ml) / protein (mg/ml) Ca (mg) / protein (mg) Apoptosis promotes calcification in VSMCs 5 * p=0.002 4 * Death signal Fas TNFa Ca/P 3 2 1 ZVAD.fmk 0 Control anti-fas Caspase cleavage 0.07 0.06 * p=0.0011 Confocal hoechst/glowover Showing apoptosis in Day 7 nodule Apoptosis 0.05 0.04 * 0.03 0.02 0.01 SEM of calcified apoptotic bodies 0.00 Control +ZVAD Proudfoot et al 2001
BONE matrix vesicles osteoblast matrix Requirements for mineralization INITIATION NUCLEATION CRYSTAL GROWTH VASCULATURE apoptosis and matrix vesicles loss of inhibitors MGP Fetuin A osteoblastic conversion of VSMCs elaboration of calcifying matrix source of Ca and P osteoclasts osteoclast-like macrophages? REABSORPTION
Mechanisms of VSMC calcification 1. What initiates calcification? APOPTOSIS and VESICLE RELEASE 2. What is the role of mineralization-regulating proteins? INHIBITION 3. What regulates the osteoblastic phenotype of VSMCs? CBFA1/Runx2 + TGFb-superfamily
GFR Renal osteodystrophy Vascular insults Disease Calcium Phosphate Ca x PO4 Oxidized lipids Inflammation Hypertension Advanced glycation endproducts PTH VSMC Damage Treatment Vitamin D Calcium-based phosphate binders Warfarin Time on dialysis Pre-existing vascular calcification (genetic predisposition)
One of the earliest events in vascular calcification is vesicle release In vivo In vitro Smooth muscle cell Collagen fibrils Induced by VSMC death (apoptosis) and Calcium overload/stress Hypercalcemia (>2.8mM), Hyperphosphatemia (>2.0mM), Atherosclerosis (Ca> 30mM) End-Stage Renal Disease (Ca x P), Hypertension (intracellular Ca-overload)
Calcification of VSMCs is mediated by vesicles Calcification induced by extracellular Ca and P in vitro Matrix vesicles 0.5hrs 22 hrs i ii iii 50-150nm Apoptotic bodies Reynolds et al, JASN, 2004 Reynolds et al, JASN, 2005 200-800nm Two Vesicle Populations
Mean fold increase in 45 Ca Incorporation MV from Ca + P Treated VSMCs are mineralization competent Vesicle Isolation and Calcification Assay flask tapped 90 80 70 60 50 Matrix Vesicles p<0.0001 ** Apoptotic Bodies AB pelleted at 10,000 rpm supernatant collected 40 30 20 10 0 p<0.004 p<0.04 ** ** p<0.0001 p<0.02 ** ** ABs incubated in calcifying media + 45 Ca 37 C, 24 hr reaction mixtures pelleted at 10,000 rpm % 45 Ca incorporation
Mechanisms of VSMC vesicle calcification Do vesicles normally contain proteins that inhibit their ability to calcify? LOCAL CIRCULATING Matrix Gla Protein Fetuin-A Vitamin K dependent Inhibited by warfarin Schäfer et al. JCI 2003 3 months Undercarboxylated in CKD 11 months Luo et al Nature 1997 Levels reduced in CKD and Dialysis Serum free + Serum VSMC take up Fetuin from serum
MV SF MV +5%HS recmgp MV Fold Increase Above Control In Health Vesicles contain Inhibitors that Reduce their Calcification Potential Local Inhibitor 4 3.5 ** p<0.03 MGP 30kDa - 15kDa - MGP 3 2.5 2 1.5 1 0.5 0 Control Warfarin Circulating Inhibitor m g protein 1 2 5000 4000 ** Fetuin-A 55kDa - Fetuin-A 45 Ca incorporation/ 3000 2000 1000 0 MV ** Figure 5.7 Mineral Deposition in Hypercalcemic Mode with Apoptotic bodies and Vesicles. Pellets of hypercalcemic VSMCs were frozen, as described in materials and methods. U showed the presence of mineral depositi associated with the internal and external surf vesicles (2). AB 500nM EM immunogold for fetuin-a
Death Signal Damage Uremic milieu OSTEOGENIC DIFFERENTIATION Ca increase +? Apoptotic Bodies Matrix Vesicles Protection from Ca-overload? Phagocytosis Ca 2+ Calcification Pi PS ALK P i P i P i Ca i Ca i Ca i Ca i P i Fetuin MGP Annexins Pit-1
Is there evidence for similar mechanisms in vivo? Studied vessels from children on dialysis who develop rapid medial vascular calcification pristine vessels -no atherosclerosis Intact - vascular matrix structure maintained Measured: Correlated with : CALCIUM LOAD VESSEL HISTOLOGY VASCULAR MEASURES BIOCHEMICAL DATA
Ca load in the vessel wall (m gm/ m L) Children on dialysis develop rapid medial calcification p = 0.0005 50 p = 0.02 Pre-dialysis M 40 30 Ad 20 Dialysis M 10 0 Normal Pre-dialysis Dialysis n = 6 n = 10 n = 24 Ad Only dialysis vessels had overt calcification on von kossa, CT scan and PWV.
Dialysis vessels show increased osteogenic differentiation Alkaline Phosphatase (IU/mg) 25 20 15 10 5 p = 0.88 p = 0.02 % Runx2 positive areas /unit area 20 15 10 5 2.0 0.8 p < 0.0001 13.4 1.9 4.4 1.2 0 Normal Pre-dialysis Dialysis n = 6 n = 10 n = 18 0 Normal Pre-dialysis Dialysis n = 4 n = 4 n = 6 Normal Pre-dialysis Dialysis M M M Runx2 Ad Ad Ad M M M Osterix Ad Ad Ad (Shroff et al 2008,Circulation)
number of VSMCs / unit area Calcification correlates with VSMC loss via apoptosis 150 p < 0.001 Normal Normal 125 100 Dialysis Dialysis 75 50 Normal Pre-dialysis Dialysis n = 4 n = 8 n = 10 a-sm actin TUNEL
Non-functional inhibitors (Glu-MGP) predominate in dialysis vessels Normal Pre-dialysis Dialysis M M Gla-MGP M Ad Ad Ad M M M Glu-MGP Ad Ad Ad
% annexin positive areas /unit area Calcification is a vesicle mediated process 70 60 50 40 30 20 10 0 p = 0.009 42.9 15.6 7.8 4.6 1.7 0.7 Normal Pre-dialysis Dialysis n = 4 n = 4 n = 6 Annexin VI staining Normal Dialysis
Ca load is associated with increased vesicle deposition by VSMCs a b c e d (Shroff et al 2008, Circulation)
Contractile VSMC elevated P other? MGP Fetuin-A Runx2 Secretory VSMC adaptation/repair elevated P elevated Ca other? loss of inhibitors Apoptosis Calcifying VSMC Nanocrystals deposited Increased osteogenesis Endocytosis of nano-crystals
DISEASE PROMOTERS HEALTH INHIBITORS Ageing Ca x P Ca P Inflammation Warfarin Lipid AGEs Oxidative Stress Vitamin D? APOPTOSIS Vesicle release osteo/chondrocytic VSMCs nucleation of calcium crystals + proliferation on appropriate matrix vascular calcification ARTERIAL STIFFENING PLAQUE RUPTURE Fetuin MGP Pyrophosphate Klotho FGF23 Vitamin K PTH/PTHrP? Vitamin D?
Acknowledgements King s College London Alex Kapustin Diane Proudfoot Joanne Reynolds Valentine Iyemere Michael Schoppet Afshin Far John Davies Kerry Tyson Rosamund McNair Cambridge University Department of Chemistry Melinda Duer Dave Reid Multi-Imaging Centre, Anatomy Jeremy Skepper Maastricht University Leon Schurgers Aachen University Willi Jahnen-Dechent Great Ormond Street Hospital Lesley Rees John Deanfield Rukshana Shroff King s College London Manuel Mayr