Lecture I Vasculogenesis and angiogenesis 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 from existing capillaries Circulatory system 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 heart. • 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 Marcello Malpighi – observed capillaries in 1661 chicken embryo Wilhelm His – 1865 – coined the term „endothelium”, 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 embryology. 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 umbilical vein 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 growth, 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 smooth muscle 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 capillary basement membrane Endoglin is an auxiliary receptor for the transforming growth factor-beta family of cytokines and is required for angiogenesis and heart development. vWF - Von Willebrand factor Endothelial cell markers used to identify microvasculature in tissues Marker Ligand CD31 (PECAM-1) CD31 on endothelial cells, leukocytes; glycosaminoglycans CD34 L-selectin CD54 (ICAM-1) LFA-1 integrin 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 Endothelin receptor B (ETBR) ET-1 Ephrin B2 EphB4 EphB4 Ephrin B2 Tie-2 Angiopoietins 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) arteriogenesis: maturation of blood vessels via increasing the lumen of vessels Blood vessel formation • Vasculogenesis: a.) during embryonic development; b.) during adulthood associated with circulating progenitor cells • Angiogenesis: a.) embryonic development b.) adulthood: wound healing, menstrual cycle, tumour-angiogenesis… PhysiologicalPhysiological angiogenesisangiogenesis inin adultsadults isis restrictedrestricted placenta 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 – pericyte 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 zebrafish allow analysis of endothelial cells in living embryos Dorsal Longitudinal Anastomotic Vessel Intersegmental Vessels (DLAV) (Se) Posterior Cardinal Vein Dorsal Aorta (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