THE PATHOPHYSIOLOGY OF VASCULARCALCIFICATION

Catherine M. ShanahanProfessor of Cellular Signalling

King’s College London

KDIGO

DISCLOSURES

• None

KDIGO

PRESENTATION OVERVIEW:• Vascular smooth muscle cell-mediated calcification

– VSMC phenotypes– Setting in which it occurs

• Processes that promote or drive vascular calcification– Hyperphosphatemia, hypercalcemia– Loss of inhibitory mechanisms– Oxidative stress

• How CKD specifically leads to vascular calcification• Are patterns of coronary and/or peripheral calcifications – in a clinical and/or

pathophysiological way - different in CKD-patients?

KDIGO

Vascular calcification occurs at two sites

MEDIAL INTIMAL

Stiffness Plaque Rupture

• Major cause of cardiovascular mortality in CKD • Increased risk of myocardial infarction and all cause mortality• Surgical complications and amputations• Valve calcification

Diabetes Renal Disease

AgeingAtherosclerosis

EBCT

KDIGO

Vascular Calcification is a

Cell-Mediated Process similar to

bone calcificationKDIGO

Shanahan, C. M. (2013)

Mechanisms of Vascular Smooth Muscle Cell Calcification

CKDDiabetesAgeing

ROS?

DAMAGE/STRESS

LOSS OF INHIBITORS

OSTEOGENIC DIFFERENTIATION

CELL DEATH/VESICLE RELEASE

MINERALIZATION

KDIGO

Ageing

HypertensionVascular Calcification Osteoporosis CKD

Ageing is the Strongest Risk Factor for Defects inKidney-Bone-Vascular Axis Tissues

• Elevated Phosphate/FGF23/Klotho• Low Vitamin D• DNA damage• Oxidative Stress• Systemic Inflammation

KDIGO

Inhibitors Prevent Vascular Calcification

Mouse gene knockouts develop vascular calcificationand bone defects (eg. osteoporosis).

• MGP (matrix Gla protein)• Fetuin**• Osteoprotegerin• Klotho/ FGF23** - Phosphate and Vit D

metabolism – premature ageing• Pyrophosphate metabolism (ENPP1)• carbonic anhydrase• Smad 6

Luo et al Nature 1997

KDIGO

*

**

* *****

****

**

****** *** **** *** *******0

200

400

20 40 60 80

Ca

(mm

ol/k

g)

AGE (years)

Aorta (r=0.84)

Internal Iliac (r=0.75)

Ibels et al. Am J Med 1979

Incidence of Vascular Calcification with Age

KDIGO

normaldialysis

0.001

0.01

0.1

1

10

100

25-34 35-44 45-54 55-64 65-74 75-84 >85

Ann

ual m

orta

lity(%)

age range (years)Adapted from Foley et al. Am J Kid Dis 1998

Adolescents and young adult with CKD:– structural and functional abnormalities in the large vessels – present even in the second decade of life– linked to disorders in mineral metabolism

Goodman, NEJM, 2000; Litwin, JASN, 2005; Mitsnefes, JASN 2005; Goldsmith, NDT, 2006

Cardiovascular mortality in CKD patients

KDIGO

¯ GFR Renal osteodystrophy

Disease

­Calcium ­Phosphate ­Ca x PO4 Oxidized lipids InflammationHypertensionPTHAdvanced glycation end-products

Vascular insults

Treatment

Vitamin DCalcium-based phosphate bindersWarfarin

VSMC Damage

• Time on dialysis• Pre-existing vascular calcification (once present rapidly progresses)

KDIGO

Vessel Rings from Children in vivo and ex vivo

Studied vessels from children on dialysis whodevelop rapid medial vascular calcification• pristine vessels -no atherosclerosis

• Intact - vascular matrix structure maintainedMeasured: CALCIUM LOAD

VESSEL HISTOLOGY

Correlated with : VASCULAR MEASURESBIOCHEMICAL DATA

Alk P

1mM P +1.8mM Ca

2mM P + 1.8mM Ca

2mM P + 2.7mM Ca

ImmunoCa

KDIGO

Normal Pre-dialysis Dialysis0

10

20

30

40

50p = 0.02

p = 0.0005

n = 6 n = 10 n = 24

Ca

load

in th

e ve

ssel

wal

l (µg

m/µ

L)

Ad

M

M

Ad

Dialysis

Pre-dialysis

Children on Dialysis develop rapid medial calcification

High Circulating Phosphate Levels, Transient Hypercalcemia?

KDIGO

Calcification correlates with VSMC loss via apoptosis

Normal Pre-dialysis Dialysis50

75

100

125

150 p < 0.001

n = 4 n = 8 n = 10

num

ber o

f VSM

Cs

/ uni

tarea

Dialysis

Normal Normal

Dialysis

a-SM actin TUNEL

KDIGO

MAd

M

Ad

Normal Pre-dialysis Dialysis

Gla-MGP

Glu-MGP

Ad

M

Ad

Ad

M

Ad

M

M

Loss of Calcification InhibitorsNon-functional Glu-MGP predominates in

Dialysis vessels

KDIGO

M

Ad

Runx2

Normal Pre-dialysis DialysisM

Ad

M

Ad

Osterix

M

Ad Ad

MM

Ad

Normal Pre-dialysis Dialysis0

5

10

15

20

25

n = 6 n = 10 n = 18

p = 0.02

p = 0.88

Alk

alin

e P

hosp

hata

se (I

U/µ

g)0

5

10

15

20

n = 4 n = 4 n = 6

2.0 ± 0.8

13.4 ± 1.9

4.4 ± 1.2

p < 0.0001

Normal Pre-dialysis Dialysis

% R

unx2

pos

itive

are

as /u

nit a

rea

Dialysis vessels show increased osteogenic differentiation

(Shroff et al 2008,Circulation)

KDIGO

Ca load is associated with increased vesicle deposition by VSMCs

(Shroff et al 2008, Circulation)

b

c

a

d

eKDIGO

Is there Evidence that the VSMCs from Children on Dialysis

are Prematurely Aged?

ENVIRONMENTAL TOXINS

DNA DAMAGE

SENESCENCEand

CELL DEATH

KDIGO

Vessels from Children on dialysis have increased senescence markers and oxidative DNA damage

Oxidative DNA damage increasespredialysis

p16 is increased in Dialysis

KDIGO

Calcium and Phosphate Drive Oxidative DNA damage in Dialysis Vessels

KDIGO

Cell growth

020406080100120140

0 10 20 30 40 50

Days

Nº

cells

(x 1

0^6)

NormalDialysis

Normal P6

Dialysis P7

VSMCs from children on dialysis senesce early in culture

Phase SAbG

SAbG = senescence associated b-galactosidase

KDIGO

Calcium and Phosphate Induce DNA damage

DOXO

p ATM / ATR

p H2AX

b - actin

Base

line

H 2O2

Ca +

P

No treatment

Ca + P

No treatment

Ca + P

DAPI

DAPI

DAPI

DAPI

pATM

gH2AX

gH2AX

pATM

Normal-A P6

0

20

40

60

80

100

Baseline DOXO H O Ca + P%

Pos

itive

cel

ls

p ATM / ATRp H2AX

a aaa

aa

2 2

KDIGO

Phosphate treatment promotes VSMC senescence

SAbGDay 0 + P Day 4 + P

KDIGO

Shanahan, C. M. (2013)

Senescence Associated Secretory Phenotype (SASP)

KDIGO

A.

IL6

NEG POS

POS NEG

B.GRO, GROα

OPG

SASP factors

Interleukins and chemokines IL6,IL8, GRO,GROα, MCP-1,-2,-3, ENA78, GCP-2

Growth factors and regulators Angiogenin, IGFBP-4,-6,VEGF, BMP2

Proteases TIMP-1,-2

Soluble or shed receptors or ligands

OPG, Fas, uPAR, ICAM-1

Array analysis shows VSMCs secrete pro-osteogeniccytokines in response to prelamin A accumulation

Drugs that block the DDR reduce secretion of SASP factors and calcification.

KDIGO

pATM/ATR

gH2AX

b-actin

+veControl Pre-dialysis Dialysis

P6 P9 P6 P8 P9 P8 P7

VSMCs from children on dialysis have increased levels of DNA damage and osteogenic differentiation in vitro

Pilar Sanchis et al, KI 2019

BMP2

b-actin

Runx2

P6 P9 P9 P8 P7

Control Dialysis+ve

Baseline

Ca/P

Control Dialysis

Elevated BMP2

KDIGO

ATM inhibition blocks calcification and Inflammationin VSMCs from Children on Dialysis

Control

Dialysis

Baseline Ca/P Ca/P + iATM

KDIGO

BMP2, IL6 and OPG are elevated in children on dialysis and correlate with vascular stiffness

N=490 50 100 150 200 250

3.54.04.55.05.56.06.57.07.58.08.5 p = 0.037

r = 0.38

OPG [pg/ml]

PWV

(m/s

ec)

0 50 100 150 200 2503.54.04.55.05.56.06.57.07.58.08.5

p = 0.013r = 0.44

BMP2 (pg/ml)

PW

V (

m/s

ec)

0 100 200 300 400 5000

10

20

30

40

50

60p = 0.0166r = 0.23

BMP2 (pg/ml)

IL6

(pg/

ml)

0 10 20 30 40 50 600

50

100

150

200

250

300

350

p = 0.016 r = 0.48

IL6 (pg/ml)

OP

G (

pg/m

l)

KDIGO

OPG IL6 BMP2 OPG IL6 BMP22

4

8

16

32

64

128

256

p < 0.0001

Calcification No calcification

(log

axi

s)

Circulating cytokines were significantly increased in children with calcification

KDIGO

Tissue ageing is driven by DNA damage and inflammatory mediators released from senescent tissues

KlothoFGF23

KDIGO

E-Mail karger@karger.com

Research Paper

J Vasc Res 2018;55:35–46 DOI: 10.1159/000484088

Progressive Development of Aberrant Smooth Muscle Cell Phenotype in Abdominal Aortic Aneurysm Disease

Kirsten Riches a, e Emily Clark b, c Rebecca J. Helliwell a Timothy G. Angelini f Karen E. Hemmings a, c Marc A. Bailey a, c, d Katherine I. Bridge a, c, d D. Julian A. Scott a, c, d Karen E. Porter a, c

a Leeds Institute of Cardiovascular and Metabolic Medicine (LICAMM), b Institute of Medical and Biological Engineering, School of Mechanical Engineering, and c Multidisciplinary Cardiovascular Research Centre (MCRC), University of Leeds, and d Leeds Vascular Institute, Leeds General Infirmary, Leeds , e Faculty of Life Sciences, University of Bradford, Bradford , and f Royal London Hospital, London , UK

exhibited higher levels of the DNA damage marker !H2AX (3.9-fold, p < 0.01, vs. control SMC). These features did not correlate with patients’ chronological age and are therefore potential markers for pathological premature vascular ag-ing. Early-stage PCA-SMC (control and aneurysmal) were in-distinguishable from one another across all parameters. The principal limitation of human studies is tissue availability only at the end stage of the disease. Refinement of a porcine bioreactor model would facilitate the study of temporal modulation of SMC behaviour during aneurysm develop-ment and potentially identify therapeutic targets to limit AAA progression. © 2017 S. Karger AG, Basel

Introduction

Abdominal aortic aneurysm (AAA) is a progressive, generally asymptomatic dilatation of the aorta affecting ̌ 5.5% of the population. Risk factors for AAA include male gender, increasing age, and smoking [1] . Rupture is

Keywords Abdominal aortic aneurysm, human · Smooth muscle cells · Aging · DNA damage · Senescence

Abstract Abdominal aortic aneurysm (AAA) is a silent, progressive dis-ease with a high mortality and an increasing prevalence with aging. Smooth muscle cell (SMC) dysfunction contributes to gradual dilatation and eventual rupture of the aorta. Here we studied phenotypic characteristics in SMC cultured from end-stage human AAA ( ˰ 5 cm) and cells cultured from a por-cine carotid artery (PCA) model of early and end-stage aneu-rysm. Human AAA-SMC presented a secretory phenotype and expressed elevated levels of the differentiation marker miR-145 (2.2-fold, p < 0.001) and the senescence marker SIRT-1 (1.3-fold, p < 0.05), features not recapitulated in aneu-rysmal PCA-SMC. Human and end-stage porcine aneurysmal cells were frequently multi-nucleated (3.9-fold, p < 0.001, and 1.8-fold, p < 0.01, respectively, vs. control cells) and dis-played an aberrant nuclear morphology. Human AAA-SMC

Received: September 4, 2017 Accepted: October 10, 2017 Published online: December 13, 2017

Dr. Karen E. Porter Leeds Institute of Cardiovascular and Metabolic Medicine LIGHT Laboratories, Clarendon Way, University of Leeds Leeds LS2 9JT (UK) E-Mail k.e.porter ! @ ! leeds.ac.uk

© 2017 S. Karger AG, Basel

www.karger.com/jvr

Dow

nloa

ded

by:

King

's C

olle

ge L

ondo

n

193.

61.2

07.1

79 -

2/19

/202

0 8:

49:0

5 PM

Increased DNA Damage and Senescence in AAA

Age-Associated Sirtuin 1 Reduction in Vascular Smooth Muscle Links Vascular Senescence and Inflammation to Abdominal Aortic Aneurysm

Hou-Zao Chen*, Fang Wang*, Peng Gao*, Jian-Fei Pei*, Yue Liu, Ting-Ting Xu, Xiaoqiang Tang, Wen-Yan Fu, Jie Lu, Yun-Fei Yan, Xiao-Man Wang, Lei Han, Zhu-Qin Zhang, Ran Zhang, Ming-Hui Zou, and De-Pei LiuDepartment of Biochemistry and Molecular Biology, State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China

AbstractRationale: Uncontrolled growth of abdominal aortic aneurysms (AAAs) is a life-threatening vascular disease without an effective pharmaceutical treatment. AAA incidence dramatically increases with advancing age in men. However, the molecular mechanisms by which aging predisposes individuals to AAAs remain unknown.

Objective: In this study, we investigated the role of SIRT1 (Sirtuin 1), a class III histone deacetylase, in AAA formation and the underlying mechanisms linking vascular senescence and inflammation.

Methods and Results: The expression and activity of SIRT1 were significantly decreased in human AAA samples. SIRT1 in vascular smooth muscle cells was remarkably downregulated in the suprarenal aortas of aged mice, in which AAAs induced by angiotensin II infusion were significantly elevated. Moreover, vascular smooth muscle cell–specific knockout of SIRT1 accelerated angiotensin II–induced formation and rupture of AAAs and AAA-related pathological changes, whereas vascular smooth muscle cell–specific overexpression of SIRT1 suppressed angiotensin II–induced AAA formation and progression in Apoe−/− mice. Furthermore, the inhibitory effect of SIRT1 on AAA formation was also proved in a calcium chloride (CaCl2)–induced AAA model. Mechanistically, the reduction of SIRT1 was shown to increase vascular cell senescence and upregulate p21 expression, as well as enhance vascular inflammation. Notably, inhibition of p21-dependent vascular cell senescence by SIRT1 blocked angiotensin II–induced

Correspondence to De-Pei Liu, PhD, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, No. 5 Dong Dan San Tiao, Beijing 100005, P.R. China. liudp@pumc.edu.cn; or Ming-Hui Zou, MD, PhD, Center for Molecular and Translational Medicine, Georgia State University, Atlanta, GA 30303. mzou@gsu.edu.Current address for F.W.: Department of Cardiology, China-Japan Friendship Hospital, Beijing 100029, China.Current address for P.G.: Department of Hypertension and Endocrinology, Center for Hypertension and Metabolic Diseases, Daping Hospital, Third Military Medical University, Chongqing Institute of Hypertension, Chongqing 400042, China.Current address for M.-H.Z.: Center for Molecular and Translational Medicine, Georgia State University, Atlanta, GA.*These authors contributed equally to this article.DisclosuresNone.The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.116.308895/-/DC1.

HHS Public AccessAuthor manuscriptCirc Res. Author manuscript; available in PMC 2019 June 03.

Published in final edited form as:Circ Res. 2016 October 28; 119(10): 1076–1088. doi:10.1161/CIRCRESAHA.116.308895.

Author Manuscript

Author Manuscript

Author Manuscript

Author Manuscript

KDIGO

Association of relative telomere length with cardiovascular disease ina large chronic kidney disease cohort: The GCKD study

Julia Raschenberger a, 1, Barbara Kollerits a, 1, Stephanie Titze b, Anna K!ottgen c,Barbara B!arthlein d, Arif B. Ekici e, Lukas Forer a, Sebastian Sch!onherr a,Hansi Weissensteiner a, Margot Haun a, Christoph Wanner f, Kai-Uwe Eckardt b,Florian Kronenberg a, *, for the GCKD study Investigatorsa Division of Genetic Epidemiology, Department of Medical Genetics, Molecular and Clinical Pharmacology, Medical University of Innsbruck, Innsbruck,Austriab Department of Nephrology and Hypertension, University of Erlangen-Nürnberg, Erlangen, Germanyc Division of Nephrology, Department of Medicine, University Medical Center Freiburg, Freiburg, Germanyd Chair of Medical Informatics, University of Erlangen-Nürnberg, Erlangen, Germanye Institute of Human Genetics, University of Erlangen-Nürnberg, Erlangen, Germanyf Division of Nephrology, Department of Medicine, University of Würzburg, Würzburg, Germany

a r t i c l e i n f o

Article history:Received 9 July 2015Received in revised form10 August 2015Accepted 11 August 2015Available online 14 August 2015

Keywords:Telomere lengthCardiovascular diseaseChronic kidney diseaseHigh-risk population

a b s t r a c t

Background: Chronic kidney disease (CKD) affects 10e15% of the general population and affected in-dividuals are at an increased risk for cardiovascular disease (CVD). Since telomere length is considered tobe involved in biological aging, we tested whether relative telomere length (RTL) might be a marker forthese two diseases.Methods: The German Chronic Kidney Disease (GCKD) study is an ongoing prospective cohort studyincluding patients with CKD of moderate severity. RTL was measured by qPCR in 4955 out of 5217 GCKDpatients at baseline.Results: RTL was distributed in the cohort with a mean ± SD of 0.95 ± 0.19. CVD was present in 1266patients. Each decrease of RTL by 0.1 unit was associated with a higher probability for prevalent CVD:OR ! 1.06, 95% CI 1.02e1.11, p ! 0.007 (adjusted for age, sex, eGFR, BMI, ln-CRP, smoking, hypertension,diabetes, and lipids). Similar !ndings were observed for history of speci!c CVD entities, such as coronaryartery disease (OR ! 1.05, p ! 0.025), myocardial infarction (OR ! 1.08, p ! 0.013) and percutaneoustransluminal coronary angioplasty (OR ! 1.06, p ! 0.032). The strongest associations were found forinterventions at the carotid arteries (OR ! 1.25, p ! 0.001) as well as aortic aneurysms (OR ! 1.22,p ! 0.001).Conclusions: In the presence of CKD there is a signi!cant association between shorter RTL and CVDmanifestations. RTL appears to be a marker re"ecting changes in homeostasis associated with CKD thatmay contribute to the excess CVD risk.

© 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Chronic kidney disease (CKD) is a highly prevalent disease. Datafrom the United States and Europe report about 10e15% of the

general population have signs of impaired kidney function orstructure. This prevalence increases even to 50% in high-risk sub-populations [1,2]. Patients with CKD are at high risk for cardio-vascular disease (CVD). This risk increases with decreasingglomerular !ltration rate (GFR) and increasing albuminuria. Inpatients with advanced CKD the risk for total and cardiovascularmortality [3,4] is higher than the risk for kidney failure requiringdialysis. The high prevalence of CVD in CKD patients can only partlybe attributed to traditional CVD risk factors. The CKD phenotype ischaracterized by age-related characteristics such as atherosclerosis,

* Corresponding author. Division of Genetic Epidemiology, Department of Med-ical Genetics, Molecular and Clinical Pharmacology, Medical University of Inns-bruck, Sch!opfstr. 41, A-6020 Innsbruck, Austria.

E-mail address: Florian.Kronenberg@i-med.ac.at (F. Kronenberg).1 These authors contributed equally.

Contents lists available at ScienceDirect

Atherosclerosis

journal homepage: www.elsevier .com/locate/atherosclerosis

http://dx.doi.org/10.1016/j.atherosclerosis.2015.08.0200021-9150/© 2015 Elsevier Ireland Ltd. All rights reserved.

Atherosclerosis 242 (2015) 529e534

KDIGO

Diabetes is Associated with a high Prevalence of Vascular Calcification in Peripheral Arteries

Peripheral Artery Calcification in Diabetes

Associated with increased CV mortality, amputation and ulcers, surgical complications

KDIGO

Mechanisms of Calcification in Diabetes/Charcot Foot

Premature Ageing

Oxidative stress

NeuropathyVascular Calcification

Diabetes

AGE/ RAGEInflammation

Osteogenesis Matrix change +osteogenesis

KDIGO

Neuropathy as a Novel Cause of Vascular Calcification in the Extremities

Mechanisms Unknown:

Loss of Trophic Factors?

Calcium Metabolism?

Edmonds, ME et al 1982

KDIGO

Shanahan Lab

Alex KapustinAlly SantuChin Yee HoSerena TskaliAndy DurhamAndrew CobbRobert HaywardMengxi SunSadia AhmadMeredith Whitehead

Maastricht UniversityLeon Schurgers

Aachen UniversityWilli Jahnen-Dechent

Great Ormond Street HospitalRukshana ShroffLesley ReesJohn Deanfield

Cambridge UniversityDepartment of ChemistryMelinda DuerDave ReidKarin Muller

Multi-Imaging Centre, AnatomyJeremy SkepperSergio Garcia-Manyes (Physics)

Ralph Sinkus (Imaging)Franca Fraternalli (Randal)Anne Ridley (Randal)

KDIGO