CV disease: US prevalence. American Heart Association. ..

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Leading the Way in Cardiovascular Regenerative
Medicine
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CV disease US prevalence
Myocardial ischemia 37 million
Acute MI 865,000/year
Heart failure 5 million
Chest Pain 4.2 million emergency visits/year6.4
million outpatient visits/year
Peripheral vascular disease8 million
Stroke5.7 million
American Heart Association. Heart Disease and
Stroke Statistics2007 Update.
Symptomatic coronary artery disease (CAD) or
angina pectoris.
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New paradigm for CV disease

• Human heart can regenerate
• Bone marrow derived stem cells (BMCs)
• Circulating progenitor cells (CEPCs)
• Circulating hematopoietic stem cells
• Resident stem cells
• With certain risk conditions (eg, hypertension,
  diabetes, hypercholesterolemia, aging) and
  diseases (eg, ischemic heart disease) stem cells
  are inadequate (number/quality/time)
• Can stem cell therapy correct/regenerate blood
  vessels and/or myocardium?

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Cell therapy

• Embryonic stem cells
• Cord blood stem cells
• Adult stem cells
• Circulating
• Bone marrow (BM)
• Hematopoietic
• Mesenchymal
• Tissue specific
• Fat, muscle, etc

Gulati R, Simari RD et al. Med Clin N Am.
200791769-85.
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CV disease targets for cell therapy clinical
trials

• CAD
• Refractory angina (no other options)
• Acute myocardial infarction with left ventricular
  dysfunction (early vs late)
• Heart failure (reversible ischemia vs scar)
• Peripheral arterial disease
• Claudication and critical limb ischemia
• Abdominal aortic aneurysm
• Ischemic stroke
• Nonischemic cardiomyopathy

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Some examples of CV disease targets in cell
therapy trials in the US

• Refractory angina
• Baxter CD 34 cells post G-CSF (Phase 1 2)
• Acute myocardial infarction
• Osiris IV mesenchymal cells (Phase 1)
• Neuronyx IM mesenchymal cells
• NHLBI-CCTRN IC BM mononuclear cells (TIME and
  late TIME)
• Heart failure
• Bioheart skeletal myoblasts (MARVEL)
• NHLBI-CCTRN BM mononuclear cells (FOCUS)
• Peripheral arterial disease
• Baxter CD34 cells post G-CSF for claudication
  and CLI

Courtesy of Timothy Henry, MD.
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Cell transplantation for cardiac repair and/or
inadequate blood supply Rationale
Chronic heart diseases are characterized
byirreversible loss of myocytes
Although some mitotic activity can be
identified, proliferative capacity is inadequate
Permanent deficits in number of viable,
functioning myocytes promotes development and
progression of HF
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Damaged myocardium repair New paradigm
Traditional view no new heart muscle cell formed
Usual Outcome Replacement of heart muscle with
SCAR TISSUE
New view replacement of damaged heart cells by
new cardiomyocytes
Strategy (1) Replicationof endogenous
cardiomyocytes
Strategy (2) Conversionof stem cells into new
cardiomyocytes
Grounds MD et al. J Histochem Cytochem.
200250589-610.
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Why use adult stem cells?

• Readily available
• Easy to isolate
• Autologous
• May be altered to increase gene expression
• No ethical concerns

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Role of the cell in cardiac regeneration therapy
As a cell
As a factory
As a courier
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Cell-mediated CV repair
Angiogenesis and re-endothelialization
Apoptotic bodies, cell-cell contact
(?),adhesion (?)
Exercise, VEGF,Estrogen, G-CSFEpo,
Statins,SDF-1
SDF-1, VEGF
Mobilization
Differentiation
Homing
CV risk factors
Re-endothelialization
Angiogenesis
Werner N, Nickenig G. Arterioscler Thromb Vasc
Biol. 200626(2)257-66.
VEGF vascular endothelial growth factor.
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Stem-cell homing Chemoattractive hypothesis
Adult stemcells
Chemokinereceptors
Circulating stem cellsattracted to injury
Heart withmyocardialinfarction
Area of injurysecretes chemokines
Rosenthal N. N Engl J Med. 2003349267-74.
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Possible routes for cell therapy to the heart
RCA
CFX
Balloon catheter
Intracoronary
LAD
Intravenous
Intramyocardial
Transendocardial
Strauer BE, Kornowski R. Circulation
2003107929-34.
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Endothelial Progenitor Cells (EPCs)
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EPCs in CV diseases
EPCs
Therapeutics
Pathophysiology
Atherosclerosis Heart disease Peripheral vascular
disease
Courtesy of Arshed A. Quyyumi, MD.
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Circulating EPCs aid in cardiac repair

• CD34, CD133, and VEGF2R
• Circulate in blood stream
• Contribute to repair of vascular or myocardial
  injury and collateral formation

Asahara T et al. Science. 1997275964-7. Takahash
i T et al. Nature Med. 19995434-8.
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EPC physiology

• Originate in bone marrow
• Circulate in blood stream
• Number and function (proliferation, migration,
  homing) modulated by age, CV risk factors, and
  disease
• Release stimulated by organ and vascular injury
• Participate in vascular repair (collateralization)
  and re-endothelialization, partly by paracrine
  effects
• Circulating numbers ?by exercise and drugs
  (statins and ACE inhibitors)
• Independent predictors of endothelial dysfunction
  and long-term prognosis in patients with CAD

Hill JM et al. N Engl J Med. 2003348593-600.
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EPC number has prognostic importance
N 519 males with CAD, mean age 66 y
1.00
Group 3 (high EPC level)
0.98
0.96
Group 2 (medium EPC level)
Event-free survival
0.94
Group 1 (low EPC level)
0.92
0.90
0
100
200
300
365
0
Days
Werner N et al. N Engl J Med. 2005353999-1007.
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Association between CV risk factors and EPC
colony counts
N 45 males without CAD, gt 21 years (mean age
50.3)
70
r 47.0 P 0.001
60
50
40
EPC colony-forming units
30
20
10
0
-5 0 5 10 15 20
Framingham risk score
Hill JM et al. N Engl J Med. 2003348593-600.
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Mobilization of EPCs after myocardial infarction
N 16 patients with AMI, 8 controls
P lt 0.001 P lt 0.001
P lt 0.001 P lt 0.05
300
200
MNCCD34 (/106WBCs)
100
0
1 3 7 14 28
Day
Time after onset
Shintani S et al. Circulation. 20011032776-9.
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VEGF levels correlate with increase in EPCs
450
r 0.35 P 0.01
400
350
300
250
200
MNCCD34 (cells/106 WBCs)
150
100
50
0
0
50
100
150
200
250
300
350
400
450
Plasma VEGF (pg/mL)
Shintani S et al. Circulation. 20011032776-9.
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EPC activity and coronary collaterals

• 30 patients with isolated left anterior
  descending disease
• Divided into groups with (0.33) and without
  (0.09) adequate Collateral Flow Index (CFI)

B
R 0.75P lt 0.0001
0.8
0.6
CD34/CD133 Dual PositiveCells ( of total
lymphocytes)
0.4
0.2
0
0 0.1 0.2 0.3 0.4 0.5
CFI
Inadequate coronary collateral development
associated with numbers of circulating EPCs and
impaired chemotactic and pro-angiogenic activity
Lambiase PD et al. Circulation. 20041092986-92.
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Decrease in EPCs associated with CV disease
Endothelial Progenitor Cells
Vasculoprotective agents
CV risk factors
Atherosclerosis
Disease Regression?
Disease Progression
Improvement of endothelial function Enhanced
re-endothelialization Reduced plaque
size Improved angiogenesis
Myocardial infarction Ischemic stroke Erectile
dysfunction Renal insufficiency Peripheral artery
disease
Werner N, Nickenig G. Arterioscler Thromb Vasc
Biol. 200626257-66.
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Bone Marrow Stem Cells in Cardiac Repair
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Stem cells in cardiac repair Proposed
mechanisms of action
Cell homing and tissue integration
EC differentiation SMC differentiation
Cardiac differentiation fusion
Paracrine Effects
Attraction/ Activation of CSC
Angiogenesis
Arteriogenesis
Cardiomyocyte proliferation
Vasculo-genesis
Cardio-myogenesis
Cardiomyocyte apoptosis
Scar remodeling
Modulation of inflammation
FUNCTIONAL IMPROVEMENT
Dimmeler S et al. Arterioscler Thromb Vasc Biol.
2007Oct. 19 epub.
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Bone marrow cells promote myocardial
regeneration Postulated mechanism
Infarcted myocardium
Transplanted Cells
Unknown molecular signal(s)
Cell migration to damaged area
Proliferation and differentiation
Cytoplasmic proteins
Nuclear proteins
Cardian myosin a-Sarcomeric actin Connexin 43
Csx/Nkx2.5 MEF2 GATA-4
Functional competence
Orlic D et al. Nature. 2001410701-5.
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Cardiac stem cells are derived, in part, from
bone marrow
Post-mortem analysis of 4 hearts of female
recipients of male BM transplants
Demonstration of Y-chromosomes in up to 23 of
cardiomyocytes
Blue, green arrow Y chromosomepositive true
nucleus of BM Red Derived cardiomyocyte
cytoplasm (sarcomeric actin) surrounded by
basement membrane laminin (green, arrowhead)
Deb A et al. Circulation. 20031071247-9.
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Communication between heart and bone marrow
signals in repair
Heart
endosteum
Blood vessel endothelium
SDF-1 SDF-1 transport CXCR4 Cell
Recruitment Stem/progenitor cell Maturing
leukocyte
Blood vessel endothelium
Bone marrow
Bone
Courtesy of Carl J. Pepine, MD
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BMCs regenerate infarcted myocardium in mice
Ventricular function
LVEDP
LVDP
40 30 20 10 0
120 100 80 60 40 20 0



mm Hg
mm Hg

LV dP/dt
LV dP/dt
12000 8000 4000 0
12000 8000 4000 0


mm Hg s-1
mm Hg s-1


SO
MI BM
MI
SO
MI BM
MI
Orlic D et al. Nature. 2001410701-5.
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BMCs reduce perfusion defect in ischemic pig
hearts
Kamihata H et al. Circulation. 20011041046-52.
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BMCs enhance collaterals to infarct region
LAD Ligation
BM-MNC after 3 weeks
Kamihata H et al. Circulation. 20011041046-52.
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BMC therapy increases angiogenesis in ischemic
pig hearts
BM-MNC (Factor-VII)
Control Medium (Factor-VIII)
In part via enhanced synthesis of angiogenic
factors in the infarcted region (ie, VEGF)
Kamihata H et al. Circulation. 20011041046-52.
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Infarcted myocardium repair via autologous
intracoronary mononuclear BMC transplantation
Human model
Strauer BE, et al. Circulation. 20021061913-8.
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BMCs minimize infarcted myocardium region
25 20 15 10 5 0
P 0.04
Infarct region ()
Cell therapy
Standard therapy

• At 3 months
• SV index 49 ? 56, P 0.01
• Left ventricular end-systolic volume 82?67, P
  0.01
• Thallium scintigraphy, cm2 174?128

Strauer BE et al. Circulation. 20021061913-8.
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Assessment of intracoronary cell therapy in AMI
PMC peripheral mononuclear cells RCT
randomized controlled trial WMSI wall motion
score index.
Lipinsky MJ et al. J Am Coll Cardiol.
2007501761-7.
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Effects of intracoronary cell therapy on LVEF

• Study EF change (random) EF change or
  sub-category 95 CI 95 CI
• ASTAMI, 2005 -1.49 (-2.81, 0.01)
• Bartunek, 2005 -1.10 (-9.14, 2.94)
• BOOST, 2004 -2.83 (-3.00, -0.60)
• Jannsens, 2004 -1.10 (-2.68, 0.68)
• MAGIC-3, 2006 -2.20 (-7.18, 1.23)
• Meluzin, 2006 -2.03 (-2.94, -1.04)
• REPAIR-AM, 2006 -2.59 (-1.54, -1.44)
• Strauer, 2002 -1.03 (-4.06, 2.04)
• TCT-STAMI, 2006 -6.70 (-1.89, -3.51)
• Zhan-Quan, 2006 -5.50 (-3.19, -2.83)
• Total -2.97 (-1.04, -1.22)

-10 -5 0 5 10
Favors cell therapy Favors control
Test for heterogenicity, Chi2 33.62, af 9 (P
0.0001), P 73.2Test for overall effect Z
5.35 (P 0.00001)
Lipinsky MJ et al. J Am Coll Cardiol.
2007501761-7.
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Autologous CD34 cells for intractable angina

• N 24 patients with CCS class 3/4 angina
• G-CSF 5 µg/kg/day x 5 days
• Leukapheresis performed on Day 5
• CD34 cell selection
• NOGA-guided transplantation to zones of
  myocardial ischemia
• Phase I/IIa double-blind, 31 randomization, with
  crossover of placebo patients using frozen cells

Losordo DW et al. Circulation. 20071153165-72.
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Decrease in angina frequency with CD34 cell
therapy
3
6
Months
Losordo DW et al. Circulation. 20071153165-72.
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Skeletal Myoblast Cells
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Skeletal myoblasts

• Derived from satellite cells in skeletal muscle
• With appropriate stimulus, satellite cells
  differentiate into muscle fibers
• Highly resistant to ischemia
• Do not contract spontaneously
• Do not differentiate into cardiomyocytes

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Skeletal myoblasts
2-3 cm biopsy sample of thigh vastus lateralis
(12-18 g) explanted under local anesthesia
Human skeletal myoblasts after 3-wk in vitro
culture period (magnification 40)
Courtesy of Arshed A. Quyyumi, MD.
42
Skeletal myoblast transplantation in post-MI HF
patients
Before surgery After surgery
Menasché P et al. Lancet. 2001357279-80.
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Autologous skeletal myoblast injection for
ischemic cardiomyopathy trial (MAGIC)

• Patients
• Moderate to severe LVSD referred for CABG
• Cells
• Muscle Bx from thigh
• Skeletal muscle myoblasts cultured
• Delivery
• Direct injection into scar at surgery
• Results
• Stopped early by DSMB due to low enrollment rate
• Adverse event rate similar (25 cells vs 20
  controls)
• ICD Therapy in 15
• No improvement in LVEF by TTE (primary outcome)
• LVEF improved by SPECT
• Highly significant dose-dependent LV size decrease

Cleland JGF et al. Eur J Heart Failure
2007992-97.
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Cell Therapy in CV DiseaseNewest Evidence
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Induction of pluripotent stem cells from human
fibroblasts
Transcription factors introduced via retroviral
transduction
Takahashi K et al. Cell. 2007.Yu J et al.
Science. 2007.
iPS induced pluripotent stem cells
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Evidence for successful reprogramming of human
fibroblasts

• Morphologic similarities with human embryonic
  stem (ES) cells
• Expression of surface antigens found in ES
• Telomerase activity
• Ability to sustain continuous culture
• Expression of pluripotency-associated genes
• Pluripotency demonstrated in vivo via teratoma
  formation in mice

Takahashi K et al. Cell. 2007.Yu J et al.
Science. 2007.
Demonstrated in both studies.
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Effects of composite transcription factor
modulation of human fibroblasts Conclusions

• Induced pluripotent stem cells can be generated
  from human fibroblasts by retroviral transduction
  of transcription factors
• Use of vectors that may integrate into genome,
  introducing mutations, precludes any clinical
  application at present

Takahashi K et al. Cell. 2007.Yu J et al.
Science. 2007.
48
Cell Therapy in CV DiseaseFuture Directions
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Cardiac renewal Is the goal in sight?

• Remaining young at heart is a desirable but
  elusive goal. Myocyte regeneration may accomplish
  just that.
• Continuous cell renewal in adult myocardium was
  thought to be impossible multipotent stem cells
  may be able to renew myocardium and, under
  certain circumstances, can be coaxed to repair
  the broken heart after infarction.

Anversa P, Nadal-Ginard B et al. Nature.
2002415240-3.
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Stem cell therapy More questions than answers?
Routes of delivery Surgical vs percutaneous
Timing of delivery Acute vs chronic
?
Cell type and marker Myoblast vs BM CD 34/
AC133/ SP
Target patient population Best criteria
Cell origin Embryonic vs adult BM vs
peripheral Culture expanded vs fresh
Dose Dose response
Courtesy of Timothy Henry, MD.
51
CCTRN Addressing gaps in the knowledge base
NHLBI Cardiovascular Cell Therapy Research
Network, established January 2007
Universityof Florida
MHIU of Minn
VanderbiltUniversity
Network DCC Universityof Texas
ClevelandClinic
Texas Heart Institute
DCC Data Coordinating CenterMHI Minneapolis
Heart Institute
Nat Clin Pract Cardiovasc Med. 20074403.
52
CCTRN Goal and initial phase I/II clinical trials
GoalAccelerate research into use of cell-based
therapies in management of CV diseases
TIME Effect of timing of post-MI BMC
administration on measures of LV function
Late TIME Effect of late (2-3 weeks) post-MI BMC
administration on measures of LV function
FOCUS Effect of transmyocardial BMC
administration on measures of LV function in
patients with chronic IHD
Nat Clin Pract Cardiovasc Med. 20074403.
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CCTRN Timeline of trials
Year 02
Year 01
Year 03
Year 05
Year 04
Protocol 1 Development
PRC DSMB Review
Protocol 1
Protocol 2 Development
PRC DSMB Review
Protocol 2
Protocol 3 Development
PRC DSMB Review
Protocol 3
Courtesy Timothy Henry, MD.
54
Summary

• Current clinical efforts have focused in three
  overlapping areas
• Repair of myocardium after MI
• Reconstitution of myocardium in setting of
  chronic HF
• Therapeutic angiogenesis
• Promising early clinical experience with BMC,
  endothelial cells, and skeletal myoblasts
• Numerous questions regarding techniques and
  mechanism of benefit remain
• Ongoing trials by the NHLBI CCTRN and other
  research groups should provide insight