lecture 27: vascular progenitors

Question 1

What is the origin of blood vessels?

Answer 1

• mesoderm
  
  • hemangioblasts (CD38-, CD34-, g-kit+, CD123+, CD45+)
    
    • heamatopoietic progenitor cells
    • endothelial progenitor cells (CD31+, CD34+, VE-Cad, vWF, pNOS)
      
      • blood vessel
  • cardiovascular progenitor cells
    
    • cardiomyocytes
    • vascular progenitor cells (CD133+, CD34+/-, CD31+, CD45-, ckit+, Flk1+ (VEGFR2+))
      
      • smooth muscle cells (pericytes)
      • endothelial progenitor cells → blood vessel

Question 2

What are the two principal processes by which development of new blood vessels occurs?

Answer 2

• angiogenesis
• vasculogenesis

Question 3

What is angiogenesis?

Answer 3

• the formation of new blood vessels from pre-existing ones
• pericytes detach from the vessel wall, allowing the release of endothelial derived proteases which break down the basement membrane
• endothelial cells migrate into the surrounding interstitium to form buds
• sprouts elongate and endothelial cells proliferate, branch and interconnect
• vessel walls are stabilised by the recruitment of pericytes and smooth muscle cells and the production of extracellular matrix

Question 4

What is vasculogenesis?

Answer 4

• the formation of new blood vessels from circulating endothelial progenitor cells
• endothelial progenitor cells are recruited from the bone marrow
• endothelial progenitor cells attach and incorporate in to the endothelium
• endothelial cells proliferate and differentiate into mature endothelium
• vessel walls are stabilised by the recruitment of pericytes and smooth muscle cells and the production of ECM

Question 5

What is vascular homeostasis?

Answer 5

• tissue hypoxoia
  
  • wound
  • tissue trauma
  • infection
  • ischemia
• production of angiogeneic growth factors and chemokines (VEGF-A, bFGF, angiogenesis, SDF-1)
• proliferation of endothelial progenitor cells in bone marrow
• chemotaxis (SDF-1/CXCR4)

Question 6

What are key regulators of angiogenesis and vasculogenesis?

Answer 6

• activators
  
  • angiogenin
  • angiopoietin 1
  • basic fibroblast GF
  • hepatocyte GF
  • interleukin 8
  • placental GF
  • vascular endothelial GF
• inhibitors
  
  • angiostatin
  • endostatin
  • 2-methoxyoestradiol
  • interferon
  • interleukin 12
  • TIMP1
  • thrombospondin 1

Question 7

What is the VEGF signalling cascade and its effects?

Answer 7

• VEGFR-1
• endothelial cell motility
• endothelial cell proliferation and migration
• vascular permeability
• endothelial cell survival
• acts via a number of different pathways and proteins
• tyrosine kinase receptor
• VEGF largely produced by pericytes

Question 8

What is physiology versus pathology?

Answer 8

• maintenance and repair of the vasculature
• vascular remodelling
• restoration of blood flow to injured tissue
• neovascularization and tumour vascularisation

Question 9

What is neovascularisation?

Answer 9

• the formation of new blood vessels in tissues or areas not normally containing them
• tumour vascularisation
  
  • hypoxia induced expression of VEGF by tumour cells
  • stimulation of angiogenesis by VEGF
  • rapid tumour growth and metastasis
• choroidal neovascularisation
  
  • normal eye
  • VEGF
  • CNV-associated blood vessel growth
  • chroidal neovascularisation

Question 10

What is the therapeutic target of neovascularisation?

Answer 10

• can target many different aspects of this process
• lots of drugs e.g. mABs
• e.g. angiogenic factor production, release, binding to endothelial cell receptor
• endothelial cell proliferation
• directional migration
• tube formation
• a5b1 integrin
• intracellular signalling vascular stabilisation

Question 11

What are methods of endothelial progenitor cell (EPC) isolation?

Answer 11

• different groups have established that by plating peripheral blood on different ECM you can enhance the/select for the proliferation of different types of EPCs
• e.g. fibronectin or collagen
• fibronectin → replate non-adherent cells → colonies appear at d5-9 → early EPCs
• fibronectin → cells enumerated at d4 → no colony formation → circulating angiogenic cells (CACs)
• collagen → discard non-adherent cells daily → colonies appear at d7-21 → outgrowth endothelial cells (OECs)
• matrigel tube formation assay (OECs)
• many of the different cells express different integrins → good way of sub-typing
• EPCs are highly proliferative

Question 12

What are late outgrowth endothelial cells?

Answer 12

• resemble mature endothelial cells by morphology and phenotype
• enriched in the CD34+CD133-CD146+ blood cell fraction
• VEGFR expressed

Question 13

What are the multiple sources of EPCs when thinking about clinical application?

Answer 13

• FACS analyisis
  
  • CD31 PECAM1
  • CD105 Endoglin
  • CD147 Muc 18
  • CD144 Vascular endothelial cadherin
  • vWF von willebrand factor
  • Flk1 VEGFR
• uptake of dil-acetylated low-density lipoprotein (Dil-Ac-LDL)
• formation of capillary-like structures in matrigel
• adult peripheral blood
• umbilical cord blood

Question 14

What is the immunophenotype of human endothelial progenitor cells?

Answer 14

• endothelial progenitor cells have been characterised by the expression of multiple markers which are:
  
  • not unique to EPC; and are
  • commonly shared by multiple cell types and cell lineages
• e.g. CD34 is expressed in haematopoietic progenitor cells; and diverse muscle; mesenchymal; epithelial; neural cell types
• CD45 is also expressed on haematopoietic cells
• CD146 is on other mesenchymal stem cells → two neighbouring cells expressing the same markers
• CD14 → macrophages, dendritic cells

Question 15

What are confounding factors in identifying and isolating EPCs?

Answer 15

• endothelial cell heterogeneity
  
  • endothelial cells in arteries, arterioles, capillaries, venules, and veins have different properties and proliferative characteristics
  • vascular networks in different organs comprise specialised, functionally different endothelial cells
• lack of specific endothelial progenitor cell (EPC) biomarkers
  
  • EPC biomarkers are shared by multiple cell lineages
  • compromises enrichment and resolution of EPC from other cell types, and from their more differentiated progeny
  • compromises discrimination of resident and circulating endothelial progenitors recruited to repair injury
• lack of standardization of assays used to measure EPC proliferative potential and regenerative capacity

Question 16

What is the potential for endothelial progenitor cell therapy?

Answer 16

• critical limb ischemia
• myocardial infarction
• vascular grafts
• stroke
• pulmonary hypertension
• diabetic retinopathy
• neoplasm (bad)

Question 17

What are clinical trials of EPC for vascular diseases?

Answer 17

• coronary heart disease: 19 registered trials
• pulmonary hypertension: 4
• stroke: 2

Question 18

What are the take home messages?

Answer 18

• angiogenesis
  
  • the formation of new blood vessels from mature endothelial cells of the pre-existing vascular network
• vasculogenesis
  
  • the de novo formation of embryonic blood vessels involving the migration and differentiation of mesoderm-derived endothelial progenitors to create a vascular network