Stem Cells in the Vascular System

Kristina BoströmMay 2005

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.

Stem Cell - Definition

Horwitz. Arch Med Res, 2004

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).

Stem Cell Plasticity

Horwitz. Arch Med Res, 2004

Committed stem cell Uncommitted stem cell

Endothelial Progenitor Cells (EPC)

Mesenchymal Stem Cells (MSC)

EndotheliumInternal elastic lamina

MediaExternal elastic lamina

Adventitia

Differentiation between the arterial and venous

side of the vasculature.

Layers of the Vascular Wall

Hillebrands et al. ATVB 2003

Normal and Diseased Vessel Wall

“The Vascular Tree”

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

Endothelial Progenitor Cells

ANGIOGENESIS

Goumans et al. Trends Cardiovasc Med 2003

Formation of Endothelial Tubes

VASCULOGENESIS

Drake. Birth Defects Res 2003

Embryonic Vasculogenesis

Drake. Birth Defects Res 2003

Adult Vasculogenesis

Iwami et al. J Cell Mol Med 2004

Areas of Potential Adult Vasculogenesis

Atherosclerotic plaquesTumor formationBone disordersInflammatory diseases

Drake. Birth Defects Res 2003

ARTERIOGENESISRecruitment of SMC precursors and SMC differentiation

Stenmark & Abman. Annu Rev Physiol 2005

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.

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!!

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

Science 1997;275:964

Asahara et al. Science 1997

1. Isolated putative ECP from human peripheral blood.2. Two antigens shared by angioblasts and HSC: CD34 and Flk-1 (=

VEGFR-2 and KDR).

Tested for incorporation of EPC in three animal models

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

B. 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).

C. 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).

Asahara et al. Science 1997;275:964

EPC Isolated from Human Blood

Asahara et al. Science 1997;275:964

Incorportion on EPC from Peripheral Blood into Ischemic HindlimbAutologous Rabbit Model

Blood 1998;92:362

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

Grown in presence of bFGF, IGF-1 and VEGFStained positive for CD34 and VEGFR2.Stained for vWF and took up Ac-LDL.

Tested in 1. Canine BM transplant model with genetically distinct donor and

recipient. 2. 6-8 months after BM transplant, Dacron graft impervious to in-growth

of vessels was implanted in the descending aorta.3. 12 weeks later, only donor cells covered the Dacron graft.

Shi et al. Blood 1998;92:362

Multipotent adults progenitor cells (MAPC) from human BM

Murine Lewis lung carcinoma spheroids in NOD-SCID mice

Studied tumor angiogenesis

J. Clin. Invest. 2002;109:337

AntiHuman

ß2-microglobulin

AntiMouseCD31

Anti-vWF

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

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

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

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

(Jiang et al. PNAS 2004)

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

Jiang et al. PNAS 2004

BM-Derived Endothelial Cells

Iwami et al. J Cell Mol Med 2004

Putative Cascade and Expressional Profiles of Human BM-derived EPC Differentiation

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.

Iwami et al. J Cell Mol Med 2004

EPC - From Bone Marrow to Vasculature

PhysiologicalAge Gender (estrogen)Embryonal developmentExercise

PathologicSmokingStable coronary artery diseaseMyocardial infarction (tissue ischemia)Vascular trauma

DrugsStatins

Growth factorsVEGFG-CSF/GM-CSFSDF-1ErythropoietinPPARgamma

Important factors for mobilization and proliferation of EPC:

Growth factors may

Enhance populationCompensate for decline in populationExplain aging in EPC

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.

SDF-1 attracts progenitors to ischemic tissuesCXCR4VEGF

2-integrins and 41-integrins are capable of mediating cell-cell interactions important for adhesion.

Chemotaxis, Adhesion, Migration

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

Differentiation of EPC

Role in Physiology vs Pathology

Urbich & Dimmeler Circ Res 2004

Therapeutic Potential

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.

High recruitment of BM-derived EPC into Growing Tumors

Lyden et al. Nat. Med. 2001

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.

Werner et al. Circ. Res. 2003

Carotid Injury Model

EPC Contribute to Re-Endothelialization after Vascular Injury

EC visualized using FITC-labeled lection

Iwami et al. J Cell Mol Med 2004

Therapeutic Applications of EPC

Potential for therapy of EPC

Critical limb ischemiaMyocardial infarctionVascular graftsStrokePulmonary hypertensionDiabetic retinopathyNeoplasm

Necessity to develop standardized methods to isolates, phenotype, and evaluate quality of cells.

The number in the circulation may limit therapeutic use.

Mesenchymal Stem Cells (MSC)

EndotheliumInternal elastic lamina

MediaExternal elastic lamina

Adventitia

Differentiation between the arterial and venous

side of the vasculature.

Layers of the Vascular Wall

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.

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

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 MSCExpress alpha-SMC (70%)H-caldesmonMetavinculinCalponinSM-MHCProteins constituting basal laminaSimilar response to PDGF as pericytesSTRO-1+

Potential for differentiation into a variety of cell types

Bone Marrow Pericytes

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

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.

(Liu et al. Trends Cardiovasc Med 2004)

Potential sources of adult SMC precursor cells

(Liu et al. Trends Cardiovasc Med 2004)

-SM-actin Staining of SMC Grown from PBMNC

(Liu et al. Trends Cardiovasc Med 2004)

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

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 tissuesAll adult stem cells may be vascular stem cells

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.

Regulation of Vascular Stem Cell Lineage

TGF-ß superfamily of growth factorsBMPsTGF-ßs

Microenvironment, matrix

Hemodynamic factorsGraftsPulmonary hypertension

MSC in Cardiovascular Disease

Vascular disease and injury

Cardiac disease and injury

Hillebrands et al. ATVB 2003

Transplant Atherosclerosis

Han et al. J Vasc Res 2000

Bone Marrow Derived SMC after Large Mechanical Vascular Injury

Hu et al. J Clin Invest 2004

Adventitial Cells Contribute to Neointima Formation in Irradiated Vein Graft

Drake. Birth Defects Red 2003

Re-Endothelialization of Vascular Grafts and Vascular Stents

Sata el al. Nat Med 2002;8:403

Bone Marrow-Derived Cells in Atherosclerotic Plaque

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

INJURY ORIGIN OF SMCLimited medial-VSMC damage Medial/intimal VSCMSevere medial-VSMC damageIngrowth from adjoining vesselsFull medial-VSMC disrupture Recruitment from non-BM sources

Recruitment from BM

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

Factors Affecting Engraftment of MSC

AGAINSTHealed vascular injuryExternal vs internal vascular injuryHyperlipidemiaLow estrogenLow erythropoietinDiabetes

FORInflammationInjury Ischemic injuryAtherosclerosis, neointimaVascular graftCancerExercise

Potential for Therapy of MSC

ParalysisStrokeHeart attackNeurodegenerative diseasesOsteogenesis imperfecta

Rafii et al. Semin Cell Dev Biol 2002

Regeneration of Injured Myocardium

Kocker et al. Nat. Med. 2001

Neoangiogenesis in Infarct Zone after Injection of CD34+ Cells

Kocker et al. Nat. Med. 2001

Cardiac Improvement after Injection of CD34+ Cells

Orlic et al. Nature 2001

Bone Marrow Cells and Myocardial Regeneration

Acute Myocardial Infarction and Heart Failure

In AMI, homing factors appears to be up-regulated in the injury areaThus, 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 fractionImprovement from actual cardiac muscle regeneration