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