sign in sign up
<<
Home , Angiogenesis, Vasculogenesis

Lecture I

Vasculogenesis and formation

a) : de novo blood vessel generation from vascular progenitor cells b) angiogenesis: formation of new blood vessels via extension or remodeling from existing and blood vessels BloodBlood vesselsvessels

Cleaver O & Melton DA, Nature Med., June 2003 The vascular wall

StructureStructure ofof bloodblood vesselsvessels andand lymphaticslymphatics

large vessel

Jain R, Nature Med. June 2003 SHORT HISTORY OF THE DISCOVERIES IN THE FIELD OF BLOOD VESSELS

William Harvey

Marcello Malpighi

Henryk Hoyer

Jaffe & Gimbrone Before Harvey

• People believed in Galen’s theory that blood was produced in the liver. William Harvey - 1628 the first systematic description of circulatory system (1578-1657) Harvey’s Methods

Harvey was very scientific in his methods. These included:

• Dissecting cold blooded animals (e.g. reptiles) to observe the movement of muscle around the .

• Dissected humans to gain a knowledge of the heart.

• Used iron rods to prove that blood was pumped through veins in one direction.

• He accurately calculated the amount of blood in the body. After Harvey

• Harvey proved that blood flows around the body. He stated that blood was carried away from the heart by arteries and returns to the heart through veins.

• Harvey proved that the heart is a pump that recirculates the blood chicken – observed capillaries in 1661 MarcelloMalpighi Wilhelm His – 1865 – coined the term „”, to differentiate the inner lining of body cavities from „epithelium”

Die Häute und Höhlen des Körpers. Basel, Schwighauser, 1865. A new classification of tissues based on histogenesis. In the present work, His put forth the basic concepts of tissue . Using serial sections and three-dimensional models to illustrate his theories, he showed that the serous spaces in the embryo are mesodermal in origin and that they are lined by the special layer which he was the first to term "endothelial" End of XIX/begining XX century – description of lymphatic system – Henryk Hoyer – Jagiellonian University

Distribution of the surface lymphatic veins on the head of trout (Salmo trutta).

Diagram of the organization of lymphatic and vein systems in chordate 1, general schema; 2, lamprey; 3, dogfish; 4, fish; 5, newt and salamander; 6, frog and toad; 7, lizard; 8, crocodile; 9, bird; 10, mammals. 1973 & 1974 – Jaffe et al. & Gimbrone et al. – isolation of human endothelial cells (EC) from the

Jaffe EA , Nachman RL , Becker CG , Minick CR Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest. 1973 Nov;52(11):2745-56 Endothelial cells were isolated from freshly obtained human umbilical cords by collagenase digestion of the interior of the umbilical vein. The cells were grown in tissue culture as a homogeneous population for periods up to 5 mo and some lines were subcultured for 10 serial passages. During the logarithmic phase of , cell-doubling time was 92 h. Light, phase contrast, and scanning electron microscopy demonstrated that cultured human endothelial cells grew as monolavers of closely opposed, polygonal large cells whereas both cultured human fibroblasts and human cells grew as overlapping layers of parallel arrays of slender, spindle-shaped cells. By transmission electron microscopy, cultured endothelial cells were seen to contain cytoplasmic inclusions (Weibel-Palade bodies) characteristic of in situ endothelial cells. These inclusions were also found in endothelial cells lining umbilical veins but were not seen in smooth muscle cells or fibroblasts in culture or in situ. Cultured endothelial cells contained abundant quantities of smooth muscle actomyosin. Cultured endothelial cells also contained ABH antigens appropriate to the tissue donor's blood type; these antigens were not detectable on cultured smooth muscle cells or fibroblasts. These studies demonstrate that it is possible to culture morphologically and immunologically identifiable human endothelial cells for periods up to 5 mo.

Gimbrone MA Jr , Cotran RS , Folkman J. Endothelial regeneration: studies with human endothelial cells in culture. Ser Haematol. 1973;6(4):453-5.Links

Ades EW, Candal FJ, Swerlick RA, George VG, Summers S, Bosse DC, Lawley TJ. HMEC-1: establishment of an immortalized human microvascular endothelial cell line. J Invest Dermatol. 1992 Dec;99(6):683-90. Endothelial cells

The crucial player in blood vessel formation

Endothelial cells in culture – cobblestone appearance VariousVarious typestypes ofof endothelialendothelial cellscells

Human umbilical vein endothelial cell line (HUVEC)

• macrovascular cells • produce very small amount of VEGF • dependent on growth factors • limited life span

Human microvascular endothelial cells (HMEC-1)

• main players in angiogenesis • immortalized cell line • generate detectable amount of VEGF • extended life span Endothelial cells

- one of the most quiescent and genetically stable cells of the body – turnover time is usually hundred of days

- proliferation is inhibited due to the contact with the is an auxiliary for the transforming -beta family of and is required for angiogenesis and .

vWF - Endothelial cell markers used to identify microvasculature in tissues Marker

CD31 (PECAM-1) CD31 on endothelial cells, leukocytes; glycosaminoglycans CD34 L-selectin CD54 (ICAM-1) LFA-1 CD62E (E-selectin) Sialyl-Lewis-X antigen and other carbohydrates CD105 (endoglin) TGF-β1 and –β3 CD106 (VCAM-1) VLA-4 integrin CD141 (thrombomodulin) Thrombin CD144 (VE-cadherin) CD144 homotypic interaction receptor B (ETBR) ET-1 B2 EphB4 EphB4 Tie-2 VEGFR-2 VEGF von Willebrand Factor (vWF) Factor VIII Uptake of Ac-LDL Scavenger receptor MorphologicalMorphological differentiationdifferentiation ofof endothelialendothelial cellscells

Skeletal muscle, heart, Endocrine and exocrine organs lung, brain

Cleaver O & Melton DA, Nature Med., June 2003 CAPILLARIES Continous

CONTINUOUS = UNFENESTRATED Basement membrane

Junctions Endothelial Occluding cell Gap Adherens

Vesicles CAPILLARIES Fenestrated

Basement Fenestrae/pore membrane

Junctions Occluding Gap Vesicles / caveolae Adherens Loose

Endothelial cell CAPILLARIES discontinous

Lack of basement membrane !

Endothelial cell CONTINOUS CAPILLARIES Transport

4. MEMBRANE TRANSPORTERS

Basal lamina 3. INTER-CELLULAR PASSAGE

Junctions Endothelial cell

Caveolae & Vesicles Typical of 2. TRANSCYTOSIS 1. DIFFUSION muscle & skin FENESTRATED CAPILLARIES Transport

fenestrae/pore 4. MEMBRANE TRANSPORTERS

5. THROUGH PORES for transport 3. INTER- of high M CELLULAR r PASSAGE ? materials

1. DIFFUSION

Typical of gut, kidney, & 2. TRANSCYTOSIS endocrine glands How the blood vessels are formed? Blood vessel formation a) vasculogenesis: de novo blood vessel generation from vascular progenitor cells b) angiogenesis: formation of new blood vessels via extension or remodeling of existing blood vessels c) : maturation of blood vessels via increasing the lumen of vessels Blood vessel formation

• Vasculogenesis: a.) during ; b.) during adulthood associated with circulating progenitor cells • Angiogenesis: a.) embryonic development b.) adulthood: , menstrual cycle, tumour-angiogenesis… PhysiologicalPhysiological angiogenesisangiogenesis inin adultsadults isis restrictedrestricted

uterus

Hair growth Wound healing New capillary formation in response to wounding TumorsTumors Rheumathoid AIDS Arthritis complications

Sight loss ExagerratedExagerrated Psoriasis angiogenesis

Stroke InsufficientInsufficient Infertility

Ulcers Sclerodermia CardiovascularCardiovascular diseasesdiseases ThreeThree waysways ofof formationformation ofof bloodblood vesselsvessels

bFGF Vasculogenesis capillaries are formed from VEGF vascular progenitor cells angioblast capillary Angiogenesis VEGF formation of new blood vessels from pre-existing vessels Ang-2

Arteriogenesis Ang1, bFGF formation of mature blood vessels; differentiation into MCP-1, PDGF veins and arteries Major growth factors and receptors involved in blood vessels formation

VEGF – vascular endothelial growth factors VEGF-A – crucial mediator of angiogenesis

VEGF-R – receptors for vascular endothelial growth factors

Angiopoietins (Ang-1, 2) Tie- 2 – receptor for Ang-1, -2

FGFs – fibroblast growth factors

PDGF – platelet-derived growth factor Mechanisms of new blood vessels formation

Vasculogenesis P

EC Endothelial Capillary Progenitor blood Cell vessel

P EC – endothelial cell P P Angiogenesis P – EC EC VEGF EC F – fibroblast Capillary EC VEGF – vascular endothelial blood vessel Network of capillaries growth factor PDGF – platelet-derived growth factor Ang-1 – angiopoetin-1 SMC SMC P Arteriogenesis F

EC increased blood flow F SMC EC Primary VEGF + PDGF SMC blood vessel VEGF + Ang-1 Mature artery Model organisms in vascular research Transgenic allow analysis of endothelial cells in living

Dorsal Longitudinal Anastomotic Vessel Intersegmental Vessels (DLAV) (Se)

Posterior Cardinal Vein Dorsal (PCV) Caudal Vein (DA) Capillary Plexus VEGF-A is required for vasculogenesis in zebrafish

Nasevicius et al., 2000

Microangiography allows high resolution mapping of mature vessels Vasculogenesis capillaries are formed from vascular progenitor cells

Vasculogenesis begins very early after the initiation of gastrulation in the mammalian embryo, with the formation of the in the and angioblast precursors in the head mesenchyme Vasculogenesis

1. First phase • Initiated from the generation of hemangioblasts ; 2. Second phase • Angioblasts proliferate and differentiate into endothelial cells 3. Third phase • Endothelial cells form primary capillary plexus VASCULOGENESIS- Blood islands YOLK SAC HEMANGIOBLASTS

ANGIOBLASTS HEMATOPOIETIC CELLS

Developing blood cells Endothelium for vessels VASCULOGENESIS - initial vessel formation

MESENCHYME

ADVENTITIAL Endothelium FIBROBLASTS

MEDIAL SMCs

Endothelium ADVENTITIAL FIBROBLASTS VASCULOGENESIS – later steps

Endothelium

MEDIAL SMCs

VASCULOGENESIS is prominent in establishing the vascular beds of liver, lung, & heart Hemangioblast is a multipotent cell, common precursor to hematopoietic and endothelial cells. Hemangioblast was first hypothesized in 1900 by Wilhelm His. Hematopoiesis in zebrafish (Danio rerio )

Expression of the flk-1 represents the earliest marker of the developing endothelial lineage during vasculogenesis SCL transcription factor is crucial for the development of blood cells and blood vessels Formation of a vascular network CommonCommon vascularvascular progenitorprogenitor cellscells forfor endothelialendothelial cellscells andand vascularvascular smoothsmooth musclemuscle cellscells Origin of endothelial and mural cells from different progenitor cells Hemangioblast

Blood cells Skeletal muscles Angioblast VEGF

Venous Arterial endothelium endothelium VE GF Common vascular F -BB progenitor /smooth muscles PDG cells (flk-1+) PDGF-BB progenitor cells of vascular smooth muscles (various forms) Origin of endothelial and mural cells from common vascular progenitor cell

Flk1+ cell differentiation in serum-free culture.

CD31 (purple)/SMA(brown) immunostaining a, Vehicle-treated cells. b, PDGF-BB. c, VEGF. d, Simultaneous administration of VEGF and PDGF-BB. Vasculogenesis in adult VasculogenesisVasculogenesis occursoccurs alsoalso inin adultadult organismorganism

Previously, postnatal vascularization was thought to occur exclusively due to angiogenesis. However, recent investigations have shown that vasculogenesis is involved in blood vessel formation also during postnatal live and the cells responsible for that are EPCs.

• Classic Paradigm: Angiogenesis – Mature ECs migrate and proliferate to form new vessels

• New Model: Angiogenesis + Vasculogenesis – Bone Marrow derived EPCs circulate to sites of

JCI 1999;103:1231-1236 Stem cells

Undifferentiated cells that can develop into any type of cell in the body Progenitor cells

Ancestor cells that can form mature cells to restore function in tissues

Endothelial progenitor cells

A primitive cell made in the bone marrow (or other organs) that can enter the bloodstream and go to areas of blood-vessel injury to help repair the damage Plasticity of adult stem cells

the ability to form specialized cell types of other tissues

(also called transdifferentiation) 14 Adult progenitor and stem cells – sources and transdifferentiation Differentiation pathways for pluripotent bone marrow stromal cells Adult progenitor cells Endothelial Progenitor Cells

• 1997 Asahara et al. reported putative EPCs or angioblasts

– Cells display progenitor phenotype – Differentiate into mature ECs in vitro – Participate in neovascularization when injected into ischemic animal model

Science 1997;275:964-967 Endothelial Progenitor Cells DiI AcLDL uptake

EPC

1:200 2h

Anna Grochot-Przeczek BODIPY AcLDL uptake EPC

Anna Grochot-Przeczek Isolectin binding by EPC Lectin from Bandeira simplicifolia

control

isolectin

Anna Grochot-Przeczek Immunohistochemical characterisation of culture-expanded EPCs

2 weeks culture Cytoplasmic factor VIII (von Willebrand factor)

tube formation Uptake on Matrigel of acetylated low density lipoproteins

Dzau et al., Hypertension 2005, Markers of endothelial progenitor cells

CD34 VEGFR2 CD133 (prominin, AC133)

CD133 is absent on mature endothelial cells and monocyti c cells NumerousNumerous originsorigins ofof endothelialendothelial progenitorprogenitor cellscells

Urbich & Dimmeler, Trends Cardiovasc Med. 2004 Mechanisms of EPC homing and differentiation

Urbich & Dimmeler, Circ Res 2004 Mobilization of vascular progenitors • Tissue results in expression of cytokines like VEGF • Recruites progenitor cells • Steady state 0.01% MNCs in blood are CEPs • Amount of circulating progenitors are increased after trauma, infectious injuries or tumour growth • 24 h after injury: 12% Mobilization of vascular progenitors • Mobilization mediated through: metalloproteinases, adhesion molecules, VEGF and PLGF ()

• Induction of MMP9 causes release of stem- cell active soluble kit ligand Blood vessel formation a) vasculogenesis: de novo blood vessel generation from vascular progenitor cells b) angiogenesis: formation of new blood vessels via extension or remodeling of existing blood vessels; c) arteriogenesis: maturation of blood vessels via increasing the lumen of vessels Angiogenesis

• Majority of vascular development occurs via angiogenesis • Growth of new blood vessels from existing vessels • Two distinct mechanisms available a.) sprouting angiogenesis b.) intussusceptive angiogenesis Sprouting angiogenesis

• Sprouting: invasion of new capillaries into unvascularized tissue from existing mature vasculature

- degradation of matrix proteins - detachment and migration of ECs - proliferation Sprouting angiogenesis Intussusceptive or non sprouting angiogenesis - remodelling of existing vessels - interendothelial contact is needed - splits into two vessels Scanning electron micrographs of chicken (CAM) vessels showing intussusceptive branching remodeling:

a: Remodeling: A pillar appears in close proximity of the branching angle b,c: From the pillar a fold develops toward the tissue of the branching angle and finally the pillar tissue merges with the interstitium of the branching angle

d: As a result, the branching angle is relocated proximalad and the angle is altered e: Pruning: A column of pillars arises close to the branching angle The pillars can merge along a line indicated by the arrows. The eccentric location of the pillars can lead to alterations in the diameter of a vascular branch. f: Ultimately, a branch can be completely pruned by repetition of the process (arrows along a Burri, PH Dev Dyn. 2004 theoretical line). StagesStages ofof angiogenesisangiogenesis

1. increase in vessel permeability

2. loosening of pericyte contact

3. proteinase release from endothelial cells

4. digestion of basement membrane and

5. migration and proliferation of endothelial cells StagesStages ofof angiogenesisangiogenesis

6. formation of vascular structures

7. fusion of new vessels

8. initiation of blood flow - inhibition of endothelial cell proliferation - inhibition of the migration of endothelial cells

9. formation of basement membrane Summary of the mechanisms of angiogenesis

arterio/ venous differentiation (/Eph) Angiogenesis - Basement Membrane Breakdown

Angiogenic Stimulus (VEGF)

Smooth Muscle Cells Matrix metalloproteinases Basement Membrane

Endothelium Angiogenesis - Endothelial Cell Migration

VEGF

Smooth Muscle Nascent Cells Vascular Sprouts

Basement Membrane

Endothelium Angiogenesis - Endothelial Cell Proliferation

VEGF

Sprout Smooth Muscle Elongation Cells

Basement Membrane

Endothelium Angiogenesis - Capillary Morphogenesis

VEGF

New Lumen Smooth Muscle Formation Cells

Basement Membrane

Endothelium Angiogenesis - Vascular Maturation

VEGF

SMC, pericyte recruitment Smooth Muscle Cells

Basement Membrane

Endothelium Crucial role of metalloproteinases in angiogenesis

MMP-2 – gelatinase A MMP-9 – MMPs are pro and anti-angiogenic

PRO-ANGIOGENIC

• Degradation of basement membrane and ECM to allow for cell detachment and migration • Cleavage of VE-cadherin cell-cell adhesion • Localization of MMP-2 to the migrating edge of the endothelial cell via binding to integrin aVb3 • Exposure of cryptic integrin binding sites by cleavage of ECM • Release of active VEGF from ECM stores • Cleavage of basement membrane to release bFGF and to release and activate TGF β MMPs are pro and anti-angiogenic

ANTI-ANGIOGENIC

• Cleavage of ligand-binding domains of FGFR1 and uPAR (inhibits FGFR signaling and uPA localization)

•Inhibition of MMP-2 binding to αVβ3 integrin by release of MMP-2 PEX domain

• Generation of antiangiogenic factors - from plaminogen - , tumostatin, arrestin, and canstatin from type XVIII and IV Matrix metalloproteinases

PRCGxPD

Zn 2+ Collagenase family

gelatin 2+ Gelatinase family binding Zn

Zn 2+ Stromelysin family

Zn 2+ MMP-7, MMP-23, MMP-26

Zn 2+ MT-MMPs

Hinge Transmembrane region Prodomain Catalytic domain Hemopexin MMP family MMP Name ECM Substrate Collagenases Interstitial Collagenase (MMP-1) fibrillar Neutrophil Collagenase (MMP-8) Collagenase-3 (MMP-13) MMP-18 (Collagenase 4, xenopus collagenase) Stromelysins Stromelysin-1 (MMP-3) laminin, fibronectin, non-fibrillar collagens Stromelysin-2 (MMP-10) Stromelysin-3 (MMP-11) Matrilysins Matrilysin (MMP-7) PUMP laminin, fibronectin, non-fibrillar collagens MMP-26 (Matrilysin-2, endometase) The membrane-bound MT1-MMP (MMP-14) gelatinase A, fibrillar MMPs collagens, proteoglycans, ECM glycoproteins MT2-MMP (MMP-15) gelatinase A MT3-MMP (MMP-16) gelatinase A MT4-MMP (MMP-17)

Gelatinases Gelatinase A (MMP-2) type I, IV, V and fibrillar collagens; gelatin Gelatinase B (MMP-9) type IV, V collagen, gelatin Others MMP MMP-19 (RASI-1) gelatin (examples) Enamelysin (MMP-20) type V collagen metalloelastase (MMP-12) elastin Proteolytic

Matrix metalloproteinases (MMPs) Serine proteinase zinc-dependent endopeptidases (plasminogen activators)

Tissue inhibitors of metalloproteinases (TIMPs) Vessel maturation and regression VesselVessel maintainancemaintainance versusversus vesselvessel regressionregression

Carmeliet, Nature Med. 2003 FormationFormation ,, maturationmaturation andand regressionregression ofof vesselsvessels Take home messages

1. Three mechanisms of formation of blood vessels

2. Common origin of blood cells and endothelial cells

3. Endothelial progenitor cells play a role in blood vessel formation also in adults

4. Numerous mediators are involved in blood vessels formation

5. Physiological angiogenesis in adults is restricted, but it is a significant component of numerous diseases, such as or atherosclerosis