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Stem Cells in the Vascular System Kristina Boström May 2005.

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1 Stem Cells in the Vascular System Kristina Boström May 2005

2 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

3 Horwitz. Arch Med Res, 2004

4 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 cellUncommitted stem cell


6 Endothelial Progenitor Cells (EPC) Mesenchymal Stem Cells (MSC)

7 Endothelium Internal elastic lamina Media External elastic lamina Adventitia Differentiation between the arterial and venous side of the vasculature. Layers of the Vascular Wall

8 Hillebrands et al. ATVB 2003 Normal and Diseased Vessel Wall

9 “The Vascular Tree” Tree-like three-dimensional structure with branches and branch points.

10 Endothelial Progenitor Cells

11 ANGIOGENESIS Goumans et al. Trends Cardiovasc Med 2003 Formation of Endothelial Tubes VASCULOGENESIS Drake. Birth Defects Res 2003

12 Embryonic Vasculogenesis Drake. Birth Defects Res 2003

13 Adult Vasculogenesis Iwami et al. J Cell Mol Med 2004

14 Areas of Potential Adult Vasculogenesis Atherosclerotic plaques Tumor formation Bone disorders Inflammatory diseases Drake. Birth Defects Res 2003

15 ARTERIOGENESIS Recruitment of SMC precursors and SMC differentiation Stenmark & Abman. Annu Rev Physiol 2005

16 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.

17 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!!

18 ENDOTHELIAL PROGENITOR CELLS (EPC) % of peripheral blood cells - CD34+ AC133+ VEGFR2+ lin- cells - Study vasculogenesis - Determine progenitor state in patients - Clinical trials of expanded cell populations

19 Science 1997;275:964

20 Asahara et al. Science 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).

21 Asahara et al. Science 1997;275:964 EPC Isolated from Human Blood

22 Asahara et al. Science 1997;275:964 Incorportion on EPC from Peripheral Blood into Ischemic Hindlimb Autologous Rabbit Model

23 Blood 1998;92:362

24 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 1.Canine BM transplant model with genetically distinct donor and recipient months after BM transplant, Dacron graft impervious to in-growth of vessels was implanted in the descending aorta weeks later, only donor cells covered the Dacron graft. Shi et al. Blood 1998;92:362

25 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

26 Anti Human ß2-microglobulin Anti Mouse CD31 Anti- vWF Reyes et al. J. Clin. Invest. 2002;109:337

27 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

28 Jiang et al. PNAS 2004 BM-Derived Endothelial Cells

29 Iwami et al. J Cell Mol Med 2004 Putative Cascade and Expressional Profiles of Human BM-derived EPC Differentiation

30 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.

31 Iwami et al. J Cell Mol Med 2004 EPC - From Bone Marrow to Vasculature

32 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 Important factors for mobilization and proliferation of EPC:

33 Growth factors may Enhance population Compensate for decline in population Explain aging in EPC

34 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.

35 SDF-1 attracts progenitors to ischemic tissues CXCR4 VEGF  2-integrins and  4  1-integrins are capable of mediating cell-cell interactions important for adhesion. Chemotaxis, Adhesion, Migration

36 Regulation largely unknown, but the entire VEGF response system is critical Differentiation of EPC

37 Role in Physiology vs Pathology Urbich & Dimmeler Circ Res 2004 Therapeutic Potential

38 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.

39 High recruitment of BM-derived EPC into Growing Tumors Lyden et al. Nat. Med. 2001

40 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.

41 Werner et al. Circ. Res Carotid Injury Model EPC Contribute to Re-Endothelialization after Vascular Injury EC visualized using FITC- labeled lection

42 Iwami et al. J Cell Mol Med 2004 Therapeutic Applications of EPC

43 Potential for therapy of EPC Critical limb ischemia Myocardial infarction Vascular grafts Stroke Pulmonary hypertension Diabetic retinopathy Neoplasm

44 Necessity to develop standardized methods to isolates, phenotype, and evaluate quality of cells. The number in the circulation may limit therapeutic use.

45 Mesenchymal Stem Cells (MSC)

46 Endothelium Internal elastic lamina Media External elastic lamina Adventitia Differentiation between the arterial and venous side of the vasculature. Layers of the Vascular Wall

47 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.

48 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

49 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

50 Bone MarrowPericytes

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

52 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.


54 (Liu et al. Trends Cardiovasc Med 2004) Potential sources of adult SMC precursor cells

55 (Liu et al. Trends Cardiovasc Med 2004)  -SM-actin Staining of SMC Grown from PBMNC

56 (Liu et al. Trends Cardiovasc Med 2004) Chimeric male  -SM-actin and calponin positive cell in neointima in female after sex-mismatched BM transplantation

57 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

58 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.

59 Regulation of Vascular Stem Cell Lineage TGF-ß superfamily of growth factors BMPs TGF-ßs Microenvironment, matrix Hemodynamic factors Grafts Pulmonary hypertension

60 MSC in Cardiovascular Disease Vascular disease and injury Cardiac disease and injury

61 Hillebrands et al. ATVB 2003 Transplant Atherosclerosis

62 Han et al. J Vasc Res 2000 Bone Marrow Derived SMC after Large Mechanical Vascular Injury

63 Hu et al. J Clin Invest 2004 Adventitial Cells Contribute to Neointima Formation in Irradiated Vein Graft

64 Drake. Birth Defects Red 2003 Re-Endothelialization of Vascular Grafts and Vascular Stents

65 Sata el al. Nat Med 2002;8:403 Bone Marrow-Derived Cells in Atherosclerotic Plaque

66 Type and Extent of Injury May Determine from Where SMC Progenitors Come INJURYORIGIN OF SMC Limited medial-VSMC damageMedial/intimal VSCM Severe medial-VSMC damageIngrowth from adjoining vessels Full medial-VSMC disruptureRecruitment from non-BM sources Recruitment from BM 5-100% of SMC cells in arterial injury from bone marrow in transplant models

67 Factors Affecting Engraftment of MSC AGAINST Healed vascular injury External vs internal vascular injury Hyperlipidemia Low estrogen Low erythropoietin Diabetes FOR Inflammation Injury Ischemic injury Atherosclerosis, neointima Vascular graft Cancer Exercise

68 Potential for Therapy of MSC Paralysis Stroke Heart attack Neurodegenerative diseases Osteogenesis imperfecta

69 Rafii et al. Semin Cell Dev Biol 2002 Regeneration of Injured Myocardium

70 Kocker et al. Nat. Med Neoangiogenesis in Infarct Zone after Injection of CD34+ Cells

71 Kocker et al. Nat. Med Cardiac Improvement after Injection of CD34+ Cells

72 Orlic et al. Nature 2001 Bone Marrow Cells and Myocardial Regeneration

73 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

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