Lecture I

Vasculogenesis and angiogenesis

Interesting web pagesInteresting web pages... ...

http://biotka.mol.uj.edu.pl/~hemeoxygenase

http://www.angiogenese.de/

Circulatory system and blood vessels

Blood vessels Blood vessels

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

The vascular wall

Structure of blood vessels and lymphatics Structure of blood vessels and lymphatics

Jain R, Nature Med. June 2003

large vessel

Endothelial cells

The crucial player in blood vessel formation

Endothelial cells in culture – cobblestone appearance

Morphological differentiation of endothelial cells Morphological differentiation of endothelial cells

Skeletal muscle, heart,lung, brain

Endocrine and exocrine organsintestinal villi

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

angioblast capillary

bFGF

VEGF

VEGF, Ang-2

Ang1, bFGF, MCP-1, PDGF

Vasculogenesiscapillaries are formed from

vascular progenitor cells

Angiogenesis formation of new blood vessels from pre-existing vessels

Arteriogenesisformation of mature blood vessels; differentiation into

veins and arteries

Three ways of formation of blood vessels Three ways of formation of blood vessels

Angiogenesis in numbers and dates

TotalTotal surfacesurface ofof endotheliumendothelium ofof adultadult humanhuman beingbeing isis ~ 1000 ~ 1000 mm22 (~ (~ tennistennis yard), yard), and and totaltotal cellcell massmass isis 1.5 kg 1.5 kg

TotalTotal lengthlength ofof bloodblood vesselvessel ofof adultadult humanhuman beingbeing isis twicetwice thethe EarthEarth circumferencecircumference..

ThereThere areare atat leastleast 20 20 variousvarious propro--angiogenic angiogenic andand 30 30 antianti--angiogenic angiogenic mediatorsmediatorsproducedproduced by by mammalianmammalian tissuestissues. .

FirstFirst inhibitor inhibitor ofof angiogenesisangiogenesis (CDI) (CDI) hashas beenbeen discovereddiscovered by by FolkmanFolkman andand Brem Brem inin1975, 1975, whenwhen theythey studiedstudied thethe cartilage cartilage

FirstFirst propro--angiogenic angiogenic factorfactor ((bFGFbFGF) was ) was purifiedpurified inin 1984 by 1984 by SchingSching andand KlagsbrunKlagsbrunfrom tumor cells from tumor cells

In 1972 In 1972 JudahJudah FolkmanFolkman hashas suggestedsuggested thatthat tumortumor growthgrowth isis dependent on dependent on thetheformationformation ofof newnew bloodblood vesselsvessels. .

Major growth factors and receptors involvedin blood vessels formation

VEGF – vascular endothelial growth factorsVEGF-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 – fibroblasts growth factors

PDGF – platelet-derived growth factor

Vasculogenesis

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

The human yolk sac is a membrane outside the embryo that is connected by a tube(the yolk stalk) though the umbilical opening to the embryo's midgut. The yolk sac

serves as an early site for the formation of blood and in time is incorporated into the primitive gut of the embryo

Progenitor cellsAncestor cells that can form mature cells to restore function in tissues

Endothelial progenitor cells A primitive cell made in the bone marrow that can enter

the bloodstream and go to areas of blood-vessel injury to help repair the damage

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

www.bioscience.org

Live zebrafish (Danio rerio) embryos 4 days after fertilization, with those in the middle ring stained for hemoglobin to detect blood cells. Zebrafish studies have advanced our understanding of heart and blood development. [Image: Alan J. Davidson]

Hematopoiesis in zebrafish (Danio rerio)

Expression of the flk-1 represents the earliest marker of the developing endothelial lineasge during vasculogenesis

SCL transcription factor is crucial for the developmentof blood cells and blood vessels

Formation of a vascular network

Carmeliet P.

Genes and receptors involved in different stages Genes and receptors involved in different stages of blood vessel formation of blood vessel formation

Carmeliet P.

Vessel wall assembly

Cleaver O & Melton DA, Nature Med. June 2003

Common vascular progenitor cells Common vascular progenitor cells for for endothelial cells and endothelial cells and

vascular smooth muscle cells vascular smooth muscle cells

Hemangioblast

Angioblast

Venous ArterialEndothelium endothelium

VEGF

Commonvascular

progenitor Cells

(flk-1+)

VEGF

PDGF-BBPericytes/smooth muscles

progenitor cells of vascular smooth muscles

(various forms)

Blood cells Skeletal muscles

PDGF-BB

Origin of endothelial and mural cells from different progenitor cells

PrePre-- and and postpost--natal origin of mural cells natal origin of mural cells

Jain R, Nature Med. June 2003

Vasculogenesis in adult

Progenitor cellsAncestor cells that can form mature cells to restore function in tissues

Endothelial progenitor cells A primitive cell made in the bone marrow that can enter

the bloodstream and go to areas of blood-vessel injury to help repair the damage

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

Differentiation patwhays for pluripotent bone marrow stromal cells

Differentiation pathway for EPCs within the adult marrow. The effect of aging in the presence of risk factors could be taking place at several steps in the differentiation pathway of EPCs, resulting in obsolescence. Several steps probably require the reorganization of the chromatine of progenitors via epigenetic mechanisms. The close interaction between progenitors, stromal cells, and extracellular matrix proteins (inset) has a modifier impact on the effect of growth factors and cytokines on the production, differentiation, and release of the vascular progenitors, particularly EPCs. A better understanding of these mechanisms creates a new opening for therapeutic intervention onbone marrow senescence and consequent atherosclerosis

Differentiation pathway for EPCs within the adult bone marrow

Origin and differentiation of endothelial progenitor cells from hematopoietic and non-hematopietic sources

Urbich & Dimmeler, Circ Res 2004

Markers of endothelial progenitor cells

CD34VEGFR2CD133 (prominin, AC133)

CD133 is absent on mature endothelial cells and monocytic cells

Immunohistochemical characterisation of culture-expanded EPCs

Cytoplasmic factor VIII (von Willebrand factor)

Uptake of acetylated low density lipoproteins

2 weeks culture

tube formation on Matrigel

Dzau et al., Hypertension 2005,

Numerous origins of endothelial progenitor cells

Urbich & Dimmeler, Trends Cardiovasc Med. 2004

Mechanisms of EPC homing and differentiation

Urbich & Dimmeler, Circ Res 2004

Figure 3-18 Angiogenesis by mobilization of endothelial precursor cells (EPCs) from the bone marrow and from pre-existing vessels (capillary growth). EPCs are mobilized from the bone marrow

and may migrate to a site of injury or tumor growth (upper panel). The homing mechanisms havenot yet been defined. At these sites, EPCs differentiate and form a mature network by linking with existing vessels. In angiogenesis from pre-existing vessels, endothelial cells from these vessels become motile and proliferate to form capillary sprouts (lower panel). Regardless of the initiating mechanism, vessel maturation (stabilization) involves the recruitment of pericytes and smooth

muscle cells to form the periendothelial layer. (Modified from Conway EM, Collen D, Carmeliet P:Molecular mechanisms of blood vessel growth. Cardiovasc Res 49:507, 2001.)

Angiogenesis by mobilisation of EPCs from the bone marrow

Angiogenesis in the embryo

Formation of blood vessels in embryoFormation of blood vessels in embryo

Carmeliet, Nature Med. 2000

Formation of blood vessels in adultsFormation of blood vessels in adults

Carmeliet, Nature Med. 2000

Vessel maintainance versus vessel regression Vessel maintainance versus vessel regression

Carmeliet, Nature Med. 2003

FormationFormation, , maturation and regression of vessels maturation and regression of vessels

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 capillary basement membrane

Human microvascular endothelial cells (HMEC-1)

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

Human umbilical vein endothelial cell line (HUVEC)

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

Various types of endothelial cells Various types of endothelial cells

Stages of angiogenesis Stages of angiogenesis

increase in vessel permeability and thrombin deposition loosening of pericyte contact proteinase release from endothelial cells digestion of basement membrane and extracellular matrix migration and proliferation of endothelial cells formation of vascular structures fusion of new vessels initiation of blood flow

- inhibition of endothelial cell proliferation - inhibition of the migration of endothelial cells

formation of basement membrane

Endoglin is an auxiliary receptor for the transforming growth factor-beta family of cytokines and is required for angiogenesis and heart development.

Błona podstawna(basement membrane)

Specialised form of extracellular matrix

Basement membrane in various organs

Basement membrane of the blood vessels

-collagen IV -collagen XV-collagen XVIII- laminin - heparan-sulphate proteoglycans- perlecans - nidogen/entactin -SPARC/BM-40/osteopontin

Crucial role of metalloproteinases in angiogenesis

MMP-2 – gelatinase AMMP-9 – gelatinase B

Metalloproteinases (MMPs)

Tissue inhibitors of metalloporoteinases (TIMPs)

Proteolytic enzymes

Serine proteinase (plasminogen activators)

Differentiation of endothelial cells Differentiation of endothelial cells on on basement membrane basement membrane

Physiological and pathological angiogenesis Physiological and pathological angiogenesis

Physiological angiogenesis in adults is restricted Physiological angiogenesis in adults is restricted

placenta uterus

Hair growth Wound healing

Enhanced hair growth and follicle size in VEGF transgenic mice

- hair re-growth after depilation accelerated, - hair more dense

Detmar et al., JCI

Hair growth and angiogenesis – VEGF

Hair follicle involution – TSP-1 (an inhibitor of angiogenesis)

Hair growth is Hair growth is dependent on dependent on angiogenesis angiogenesis

1 – small vesicular follicle 2 – large preovulatory follicle3 – developing corpus luteum

(~ 8 hours after ovulation)4 - non-productive follicle

undergoing atretic regression

Maisonpierre et al, Science 277: 55-60, 1997

Vascular remodeling in the Vascular remodeling in the ratrat ovary ovary

At maturation, the follicle ruptures, expels the ovum, and then undergoes reorganizationinto a cell-dense secretory structure known as the corpus luteum. This process includes awave of vascular sprouting and ingrowth that hypervascularizes the corpus luteum; these vessels eventually regress as the corpus luteum ages.

New capillary formation in response to wounding

Angiogenesis during wound healing

Robins & Cotran - Pathologic Basis of Disease, 2005

AngiogenicAngiogenic mediators mediators

RheumathoidRheumathoidArthritis Arthritis

TumorsTumorsAIDS AIDS

complicationscomplications

Psoriasis Psoriasis

Infertility Infertility

SclerodersmiaSclerodersmiaCardiovascular Cardiovascular

diseasesdiseases

UlcersUlcers

StrokeStroke

Sight lossSight loss

angiogeneza

Exagerrated Exagerrated

InsufficientInsufficient

Diseases characterized or caused by insuffienct angiogenesis or vessel regression

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

3. 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 cancer or atherosclerosis