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Stem Cells in the Vascular System. Kristina Boström May 2005. Stem Cell - Definition. Cells that undergo asymmetric division resulting in self-renewal of the parent stem cell as well as a daughter cell capable of differentiating down specific lineages.

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slide1

Stem Cells in the Vascular System

Kristina Boström

May 2005

slide2

Stem Cell - Definition

Cells that undergo asymmetric division resulting in self-renewal of the parent stem cell as well as a daughter cell capable of differentiating down specific lineages.

The tissue specific, committed stem cells provide a supply of terminally differentiated cells for physiologic tissue turnover for the life of the individual.

slide4

Stem Cell Plasticity

A stem cell, which is committed to give rise to the expected tissues, may differentiate into cells other than these expected tissues, the so-called unexpected tissues. Termed TRANSDIFFERENTIATION.

However, it is equally plausible that uncommitted stem cells exist within the tissue and have the potential of differentiating into many or all tissues. It is hard to generate convincing data (Wagers & Weissman, Cell 2004).

Committed stem cell

Uncommitted stem cell

Horwitz. Arch Med Res, 2004

slide6

Endothelial Progenitor Cells (EPC)

Mesenchymal Stem Cells (MSC)

slide7

Layers of the Vascular Wall

Endothelium

Internal elastic lamina

Media

External elastic lamina

Adventitia

Differentiation between the arterial and venous side of the vasculature.

slide8

Normal and Diseased Vessel Wall

Hillebrands et al. ATVB 2003

slide9

“The Vascular Tree”

Tree-like three-dimensional structure with branches and branch points.

slide11

Formation of Endothelial Tubes

VASCULOGENESIS

ANGIOGENESIS

Drake. Birth Defects Res 2003

Goumans et al. Trends Cardiovasc Med 2003

slide12

Embryonic Vasculogenesis

Drake. Birth Defects Res 2003

slide13

Adult Vasculogenesis

Iwami et al. J Cell Mol Med 2004

slide14

Areas of Potential Adult Vasculogenesis

Atherosclerotic plaques

Tumor formation

Bone disorders

Inflammatory diseases

Drake. Birth Defects Res 2003

slide15

ARTERIOGENESIS

Recruitment of SMC precursors and SMC differentiation

Stenmark & Abman. Annu Rev Physiol 2005

slide16

The tenet of stem cell biology is that the cells only differentiate into cell types associated with the tissue from which they were isolated.

Called into question! - more plastic than thought.

slide17

Hemangioblasts - Cells with hematopoietic and endothelial potential

Were believed to exists only in embryos

Endothelial progenitor cells (EPC) can be isolated from peripheral blood mononuclear cells (PBMNC) by flow cytometry by e.g. CD34 which previously was associated with hematopoietic stem cells.

Overturned Dogma!!

slide18

ENDOTHELIAL PROGENITOR CELLS (EPC)

  • 0.0001 - 0.02% of peripheral blood cells
  • CD34+ AC133+ VEGFR2+ lin- cells
  • Study vasculogenesis
  • Determine progenitor state in patients
  • Clinical trials of expanded cell populations
slide20

Asahara et al. Science 1997

Isolated putative ECP from human peripheral blood.

Two antigens shared by angioblasts and HSC: CD34 and Flk-1 (= VEGFR-2 and KDR).

Tested for incorporation of EPC in three animal models

Human MBC34+ cells into athymic mice with hindlimb ischemia- (heterologous transplantation).

ß-Gal overexpressing mice MB, MBFlk1+ or MBFlk1- injected into mice on same background but without ß-Gal. Incorporation in hind limb ischemia. - (homologous transplantation).

Injected DiI-labeled rabbit CD34+ or CD34- MB into rabbits with hindlimb ischemia. Found DiI labeling 1-6 weeks afterwards in ischemic limb. - (autologous transplantation).

slide21

EPC Isolated from Human Blood

Asahara et al. Science 1997;275:964

slide22

Incorportion on EPC from Peripheral Blood into Ischemic Hindlimb

Autologous Rabbit Model

Asahara et al. Science 1997;275:964

slide24

Evidence of a CD34+ cells from BM and circulation differentiate into EPC

Grown in presence of bFGF, IGF-1 and VEGF

Stained positive for CD34 and VEGFR2.

Stained for vWF and took up Ac-LDL.

Tested in

Canine BM transplant model with genetically distinct donor and recipient.

6-8 months after BM transplant, Dacron graft impervious to in-growth of vessels was implanted in the descending aorta.

12 weeks later, only donor cells covered the Dacron graft.

Shi et al. Blood 1998;92:362

slide25

J. Clin. Invest. 2002;109:337

Multipotent adults progenitor cells (MAPC) from human BM

Murine Lewis lung carcinoma spheroids in NOD-SCID mice

Studied tumor angiogenesis

slide26

Anti

Mouse

CD31

Anti

Human

ß2-microglobulin

Anti-

vWF

Reyes et al. J. Clin. Invest. 2002;109:337

slide27

(Jiang et al. PNAS 2004)

To determine the EC potential of human BM and PBC, blood vessels in sex-matched transplant recipients were evaluated

None of the >4,000 ECs examined had more than two sex chromosomes, consistent with an absence of cell fusion.

Y chromosome signals were not detected in sex-matched female recipients, excluding the vertical transmission of male cells.

None of the recipients evaluated before hematopoietic engraftment demonstrated donor-derived ECs, indicating a close linkage between the recovery of hematopoiesis and EC outcomes.

slide28

BM-Derived Endothelial Cells

Jiang et al. PNAS 2004

slide29

Putative Cascade and Expressional Profiles of Human

BM-derived EPC Differentiation

Iwami et al. J Cell Mol Med 2004

slide30

Endothelial Progenitor Cells

Positive for CD34 and VEGFR2 expression.

Sometimes CD133 (AC133, prominin) - more likely to reflect immature progenitor cells.

CD34+/VEGFR2+ cells may also represent shedded EC of the vessel wall.

Proof of EC characteristics after outgrowth and differentiation in vitro.

May also be isolated from fetal liver or umbilical cord blood.

No data on lifetime in vivo of EPC under physiological or pathological conditions.

slide31

EPC - From Bone Marrow to Vasculature

Iwami et al. J Cell Mol Med 2004

slide32

Important factors for mobilization and proliferation of EPC:

  • Physiological
  • Age
  • Gender (estrogen)
  • Embryonal development
  • Exercise
  • Pathologic
  • Smoking
  • Stable coronary artery disease
  • Myocardial infarction (tissue ischemia)
  • Vascular trauma
  • Drugs
  • Statins
  • Growth factors
  • VEGF
  • G-CSF/GM-CSF
      • SDF-1
      • Erythropoietin
      • PPARgamma
slide33

Growth factors may

Enhance population

Compensate for decline in population

Explain aging in EPC

slide34

EPC dependent on what environment it enters into.

Poor endothelialization usually leads to enhanced vascular disease.

Diabetes mellitus: decreased proliferation capacity, reduced adhesiveness and ability to form capillary tubes in vitro. Diabetics shed more EC into circulation.

Hypercholesterolemia - dysfunction in mature EC.

slide35

Chemotaxis, Adhesion, Migration

SDF-1 attracts progenitors to ischemic tissues

CXCR4

VEGF

b2-integrins and a4b1-integrins are capable of mediating cell-cell interactions important for adhesion.

slide36

Differentiation of EPC

Regulation largely unknown, but the entire VEGF response system is critical

slide37

Role in Physiology vs Pathology

Therapeutic Potential

Urbich & Dimmeler Circ Res 2004

slide38

Neovascularization

  • Circulating mature EC do not improve
  • neovascularization.
  • Tissue injury stimulate EPC incorporation.
  • Incorporation varies in the literature, between 0>50%.
  • The >50% predominantly detected in models of tumor
  • angiogenesis.
  • Even if low incorporation, EPC may have other
  • characteristics that promote neovascularization such as
  • release of proangiogenic factors.
slide40

Endothelial Regeneration

  • Dacron vascular grafts and ventricular assist devices
  • covered by endothelial progenitors.
  • Denudation of artery after balloon injury re-endothelialized.
  • Rapid re-endothelialization may improve atherosclerosis
  • and prevent restenosis.
slide41

EPC Contribute to Re-Endothelialization after Vascular Injury

Carotid Injury Model

EC visualized using FITC-labeled lection

Werner et al. Circ. Res. 2003

slide42

Therapeutic Applications of EPC

Iwami et al. J Cell Mol Med 2004

slide43

Potential for therapy of EPC

Critical limb ischemia

Myocardial infarction

Vascular grafts

Stroke

Pulmonary hypertension

Diabetic retinopathy

Neoplasm

slide44

Necessity to develop standardized methods to isolates,

phenotype, and evaluate quality of cells.

The number in the circulation may limit therapeutic use.

slide46

Layers of the Vascular Wall

Endothelium

Internal elastic lamina

Media

External elastic lamina

Adventitia

Differentiation between the arterial and venous side of the vasculature.

slide47

The bone marrow contains two apparently discrete populations of stem cells. In addition to the HSC/EPC, there are also bone marrow stromal cells or mesenchymal stem cells (MSC).

Less characterized than the HSC/EPC and its exact location within the bone marrow is less clear.

Low density in bone marrow aspirate.

slide48

Markers for MSC

Adherent cells

WGA binding and Sca-1 (null mice, late osteoporosis)

Enriched population: Sca-1+Lin-CD31-CD45-

30% plating efficiency

STRO-1+ : Includes all CFU-F

CD105 (endoglin)

Negative for CD34, CD45, CD11b

slide49

Location of MSC in bone marrow

Most likely in the vessel wall in the bone marrow.

Would be similar to vascular smooth muscle cells and pericytes, or endosteal cells.

Cultured MSC

Express alpha-SMC (70%)

H-caldesmon

Metavinculin

Calponin

SM-MHC

Proteins constituting basal lamina

Similar response to PDGF as pericytes

STRO-1+

Potential for differentiation into a variety of cell types

slide50

Bone Marrow

Pericytes

slide51

The MSC may be guided into specific, single-lineage differentiation by culture in serum-free “induction media” containing growth factors and specific treatments.

slide52

MSC in the Artery Wall

SMC precursors in adults: concept of a continuous replacement of connective tissue with e.g. marrow cells, analogously to continuous replacement of blood cells.

However, they may also be the source of ectopic tissue formation commonly seen in diseased vascular wall.

slide54

Potential sources of adult SMC precursor cells

(Liu et al. Trends Cardiovasc Med 2004)

slide55

a-SM-actin Staining of SMC Grown from PBMNC

(Liu et al. Trends Cardiovasc Med 2004)

slide56

Chimeric male a-SM-actin and calponin positive cell in neointima in female after sex-mismatched BM transplantation

(Liu et al. Trends Cardiovasc Med 2004)

slide57

MSC in Circulation and Artery Wall

Artery wall may function as a recipient and a donor of MSC.

May enter circulation and engraft elsewhere

Cells in circulation may be derived from marrow or other places

Vasculature and microvasculature present in all organs and tissues

All adult stem cells may be vascular stem cells

slide58

Calcifying Vascular Cells (CVC) - A Cloned Subpopulation of SMC

Form condensations and nodules.

Osteoblastic differentiation and

calcification occur in the nodules.

CVC express MGP and BMP-2.

slide59

Regulation of Vascular Stem Cell Lineage

TGF-ß superfamily of growth factors

BMPs

TGF-ßs

Microenvironment, matrix

Hemodynamic factors

Grafts

Pulmonary hypertension

slide60

MSC in Cardiovascular Disease

Vascular disease and injury

Cardiac disease and injury

slide61

Transplant Atherosclerosis

Hillebrands et al. ATVB 2003

slide63

Adventitial Cells Contribute to Neointima Formation in Irradiated Vein Graft

Hu et al. J Clin Invest 2004

slide66

Type and Extent of Injury May Determine from Where SMC Progenitors Come

5-100% of SMC cells in arterial injury from bone marrow in transplant models

INJURY ORIGIN OF SMC

Limited medial-VSMC damage Medial/intimal VSCM

Severe medial-VSMC damage Ingrowth from adjoining vessels

Full medial-VSMC disrupture Recruitment from non-BM sources

Recruitment from BM

slide67

Factors Affecting Engraftment of MSC

FOR

Inflammation

Injury

Ischemic injury

Atherosclerosis, neointima

Vascular graft

Cancer

Exercise

AGAINST

Healed vascular injury

External vs internal vascular injury

Hyperlipidemia

Low estrogen

Low erythropoietin

Diabetes

slide68

Potential for Therapy of MSC

Paralysis

Stroke

Heart attack

Neurodegenerative diseases

Osteogenesis imperfecta

slide69

Regeneration of Injured Myocardium

Rafii et al. Semin Cell Dev Biol 2002

slide73

Acute Myocardial Infarction and Heart Failure

In AMI, homing factors appears to be up-regulated in the injury area

Thus, EPC and MSC will enter that area

SDF-1 is a homing factor for EPC; MSC do not react

Once the injury area has healed - no homing of cells

G-CSF may be used to increase EPC pool

Improvement from vascularization, increased ejection fraction

Improvement from actual cardiac muscle regeneration