Stem Cells in the Vascular System
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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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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.

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 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


Endothelial Progenitor Cells (EPC)

Mesenchymal Stem Cells (MSC)


Layers of the Vascular Wall

Endothelium

Internal elastic lamina

Media

External elastic lamina

Adventitia

Differentiation between the arterial and venous side of the vasculature.


Normal and Diseased Vessel Wall

Hillebrands et al. ATVB 2003


“The Vascular Tree”

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



Formation of Endothelial Tubes

VASCULOGENESIS

ANGIOGENESIS

Drake. Birth Defects Res 2003

Goumans et al. Trends Cardiovasc Med 2003


Embryonic Vasculogenesis

Drake. Birth Defects Res 2003


Adult Vasculogenesis

Iwami et al. J Cell Mol Med 2004


Areas of Potential Adult Vasculogenesis

Atherosclerotic plaques

Tumor formation

Bone disorders

Inflammatory diseases

Drake. Birth Defects Res 2003


ARTERIOGENESIS

Recruitment 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) potential

  • 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



Asahara et al. potentialScience 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).


EPC Isolated from Human Blood potential

Asahara et al. Science 1997;275:964


Incorportion on EPC from Peripheral Blood into Ischemic Hindlimb

Autologous Rabbit Model

Asahara et al. Science 1997;275:964



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


J. Clin. Invest. 2002;109:337 differentiate into EPC

Multipotent adults progenitor cells (MAPC) from human BM

Murine Lewis lung carcinoma spheroids in NOD-SCID mice

Studied tumor angiogenesis


Anti differentiate into EPC

Mouse

CD31

Anti

Human

ß2-microglobulin

Anti-

vWF

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


(Jiang et al. PNAS 2004) differentiate into EPC

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.


BM-Derived Endothelial Cells differentiate into EPC

Jiang et al. PNAS 2004


Putative Cascade and Expressional Profiles of Human differentiate into EPC

BM-derived EPC Differentiation

Iwami et al. J Cell Mol Med 2004


Endothelial Progenitor Cells differentiate into EPC

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.


EPC - From Bone Marrow to Vasculature differentiate into EPC

Iwami et al. J Cell Mol Med 2004


Important factors for mobilization and proliferation of EPC: differentiate into 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


Growth factors may differentiate into EPC

Enhance population

Compensate for decline in population

Explain aging in EPC


EPC dependent on what environment it enters into. differentiate into EPC

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.


Chemotaxis, Adhesion, Migration differentiate into EPC

SDF-1 attracts progenitors to ischemic tissues

CXCR4

VEGF

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


Differentiation of EPC differentiate into EPC

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


Role in Physiology vs Pathology differentiate into EPC

Therapeutic Potential

Urbich & Dimmeler Circ Res 2004


Neovascularization differentiate into EPC

  • 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 differentiate into EPC

Lyden et al. Nat. Med. 2001


Endothelial Regeneration differentiate into EPC

  • 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.


EPC Contribute to Re-Endothelialization after Vascular Injury

Carotid Injury Model

EC visualized using FITC-labeled lection

Werner et al. Circ. Res. 2003


Therapeutic Applications of EPC Injury

Iwami et al. J Cell Mol Med 2004


Potential for therapy of EPC Injury

Critical limb ischemia

Myocardial infarction

Vascular grafts

Stroke

Pulmonary hypertension

Diabetic retinopathy

Neoplasm


Necessity to develop standardized methods to isolates, Injury

phenotype, and evaluate quality of cells.

The number in the circulation may limit therapeutic use.



Layers of the Vascular Wall Injury

Endothelium

Internal elastic lamina

Media

External elastic lamina

Adventitia

Differentiation between the arterial and venous side of the vasculature.


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 of stem cells. In addition to the HSC/EPC, there are also bone marrow stromal cells or mesenchymal stem cells (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 of stem cells. In addition to the HSC/EPC, there are also bone marrow stromal cells or mesenchymal stem cells (MSC).

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


Bone Marrow of stem cells. In addition to the HSC/EPC, there are also bone marrow stromal cells or mesenchymal stem cells (MSC).

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 differentiation by culture in serum-free “induction media” containing growth factors and specific treatments.

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.


Potential sources of adult SMC precursor cells differentiation by culture in serum-free “induction media” containing growth factors and specific treatments.

(Liu et al. Trends Cardiovasc Med 2004)


a differentiation by culture in serum-free “induction media” containing growth factors and specific treatments.-SM-actin Staining of SMC Grown from PBMNC

(Liu et al. Trends Cardiovasc Med 2004)


Chimeric male differentiation by culture in serum-free “induction media” containing growth factors and specific treatments.a-SM-actin and calponin positive cell in neointima in female after sex-mismatched BM transplantation

(Liu et al. Trends Cardiovasc Med 2004)


MSC in Circulation and Artery Wall differentiation by culture in serum-free “induction media” containing growth factors and specific treatments.

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


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 SMC

TGF-ß superfamily of growth factors

BMPs

TGF-ßs

Microenvironment, matrix

Hemodynamic factors

Grafts

Pulmonary hypertension


MSC in Cardiovascular Disease SMC

Vascular disease and injury

Cardiac disease and injury


Transplant Atherosclerosis SMC

Hillebrands et al. ATVB 2003



Adventitial Cells Contribute to Neointima Formation in Irradiated Vein Graft

Hu et al. J Clin Invest 2004


Re-Endothelialization of Vascular Grafts and Vascular Stents Irradiated Vein Graft

Drake. Birth Defects Red 2003


Bone Marrow-Derived Cells in Atherosclerotic Plaque Irradiated Vein Graft

Sata el al. Nat Med 2002;8:403


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


Factors Affecting Engraftment of MSC Progenitors Come

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


Potential for Therapy of MSC Progenitors Come

Paralysis

Stroke

Heart attack

Neurodegenerative diseases

Osteogenesis imperfecta


Regeneration of Injured Myocardium Progenitors Come

Rafii et al. Semin Cell Dev Biol 2002



Cardiac Improvement after Injection of CD34+ Cells Cells

Kocker et al. Nat. Med. 2001


Bone Marrow Cells and Myocardial Regeneration Cells

Orlic et al. Nature 2001


Acute Myocardial Infarction and Heart Failure Cells

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


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