European Review for Medical and Pharmacological Sciences 1999; 3: 149-153 Kidney vasculogenesis and : role of Vascular Endothelial

F. DEL PORTO, A. MARIOTTI, M. ILARDI, F.R. MESSINA, A. AFELTRA, A. AMOROSO

Department of Medicina Clinica, University of Rome “La Sapienza” - Rome (Italy)

Abstract. – Vascular Endothelial Growth formed by vasculogenesis; later in gestation Factor (VEGF) plays a crucial role in the estabil- probably both vasculogenesis and angiogene- ishment of the vascular tree pattern. sis participate to vascular growth3. New vessels can be formed by two different ways; in the development of kidney both vascu- Many citokines partecipate to the develop- logenesis and angiogenesis partecipate to mi- ment of vascular system, of them Vascular crovessel assembly. VEGF and its receptor Endothelial Growth Factor (VEGF) play a (VEGF-R) are co-expressed during kidney crucial role in the estabilishment of the vas- organogenesis and stimulate renal blood ves- cular tree pattern4 (Figure 1). sels development, induce and mantain the fen- estred phenotype in and regulate vascular permeability. VEGF and vascular tree development VEGF and many other growth factors parteci- VEGF, a dimeric glycoprotein, member of pate to the development of embrionic glomeru- the platelet derived growth factor family5, is lar microvasculature. an important regulator of endothelial cell We believe that therapeutical use of VEGF or (EC) proliferation and migration and plays a anti-VEGF antibodies may be performed in the crucial role in regulation of microvessels per- treatment of many disorders. meability and in inducing vasodilation4-6. Key Words: VEGF m-RNA is expressed by a wide vari- Vascular Endothelial Growth Factor, Kidney, ety of non endothelial cells in location where Angiogenesis, Vasculogenesis. endothelia are proliferating: smooth muscle cells, fibroblasts, epithelial cells (podocytes in kidney); macrophages and tumor cells may produce VEGF, indicating, for this cytokine, a role in the angiogenesis of and tumour growth7. Introduction Hypoxia and some peptide growth factors induce VEGF expression: fibroblast growth After the implantation vascular and factor (FGF), epidermal growth factor hematopoietic tissues develop together1. (EGF), transforming growth factor beta In the absence of any pre-existing vessels, (TGF-b), platelet-derived growth factor mesodermally-derived cells differentiate into (PDGF), growth factor (PlGF), in- either endothelial progenitor cells (an- terleukin 6 (IL-6) and interleukin 8 (IL-8)8. gioblasts) or into primitive hematopoietic lin- bFGF is the most potent angiogenic factor eages. Differentiation of precursor cells into known. Recent reports have demonstated endothelia and assembly of endothelial prog- that vascular development requires interac- enitor into vessels occur during vasculogenesis. tion of VEGF and bFGF, indeed both parte- In contrast, during angiogenesis pre-exist- cipate to differentiation of mesenchymal cells ing vessels branch, sprout and migrate to into endothelial cells9. form new capillaries2. Many evidences lead to the hypotesis that New vessels can be formed by two differ- TGF-β plays a role in mediating cell-extracel- ent ways: vasculogenesis and angiogenesis. lular matrix interaction and that also extra- Early in gestation first endothelial cells are cellular matrix may regulate the formation of

149 F. Del Porto, A. Mariotti, M. Ilardi, F.R. Messina, A. Afeltra, A. Amoroso

Embrionic Blood Island

Hematopoietic

Angioblast

VEGF-R1 VEGF (flt-1) VEGF β

TCF- VEGF-R2 Activated (flt-1)

Mesenchymal cells Endothelial cells Tie-2 Tie-2 Ang 1

Ang 2 Ang 1

PDGF Endothelial cell-cell interaction HB-EGF and formation of tubal structures Maintenance of Mature vessels

VEGF+ Ang 2C

Angiogenesis

VEGF (-Ang 2)

Regression

VASCULOGENESIS ANGIOGENESIS Figure 1. The vascular tree development: coordinate and subsequent actions of VEGF and other cytokines in vascu- logenesis and angiogenesis. Role in the manteinance of mature vessels by reclutation of mesenchymal cells, inihibi- tion of EC proliferation and accumulation of extracellular matrix. Role in vascular regression by loss of structure and matrix contact and decrease of growth signals.

150 Kidney vasculogenesis and angiogenesis: role of vascular endothelial growth factor -like tubles. Moreover lower concen- capillary bed16. Vasculogenesis and angiogen- tration of TGF-β potentiates the effect of esis probably partecipate to microvessel as- bFGF and VEGF10. sembly in the development of kidney17. There are two different tirosine kinase re- VEGF and VEGF-R are co-expressed dur- ceptors for VEGF (VEGF-R): Flt-1 or ing kidney organogenesis and stimulate renal VEGF receptor 1 and Flk-1 or VEGF recep- blood vessels development: epithelia of tor 2. ureteric bud and the avascular renal mes- Studies on mice demonstrate that Flk-1 is enchyme express VEGF mRNA throughout the first marker of endothelial progenitors; nephrogenesis; podocytes and collecting duct during embrionic development VEGF may epithelial cells express VEGF mRNA later in support differentiation of flk-1 positive cells nephrogenesis18. into endothelial cells. Flt-1 endothelial cells At the onset of nephrogenesis, flk-1 is ex- assemblate in vascular channels; VEGF sup- pressed by undifferentiated renal mesenchy- ports cell-cell and cell-matrix interactions and mal cells and provides to vasculogenesis, later thus provide to embrionic vasculature organi- both flk-1 and flt-1 are expressed by endothe- zation. Flk-1 may interact with other ligands lial cells in glomeruli and around tubules to other than VEGF and all play a role in early support angiogenesis19. steps of endothelial differentiation11, flt-1 ex- The epithelial cells adiacent to fenestred pression on endothelial cells occurs later in endothelium in the glomerulus show an high embrionic development and its interaction constitutive expression of VEGF; the en- with VEGF permits vessels organization12. dothelial cells express VEGF-R. Continous Some authors have reported another tiro- expression of VEGF in epithelial cell sine-kinase receptor for VEGF: VEGF-R3 podocytes and expression of VEGF-R in that is later expressed only on the surface of glomerular endothelium, induce and mantain the ECs that will become vein or lymphatic the fenestrated fenotype in endothelium15. vessels3. The presence of fenestred endothelium in Many studies demonstrate the importance kidney permits filtration, secretion, and ab- of the interaction VEGF/VEGF-R for en- sorbition20. dothelial differentiation in the mouse em- Although high levels of VEGF are found bryo: flk-1 deficient mice die in utero because during embrionic development in the brain of an early defect in the development of and kidney, VEGF levels decrease only in hematopoietic cells and endothelial cells. Flt- adult brain; glomerular podocytes continue to 1 null mutant mice die later in utero. In these express VEGF, at the same manner VEGF-R endothelial differentiation is not blocked, but levels are high in the brain and kidney during there is a disorganization of the early embri- embriogenesis, but only glomerular endothe- onic vasculature13. lium express VEGF-R into adulthood. All VEGF levels are high during embriogene- these evidence confirme the hypotesis that sis and fetal development and in adulthood VEGF levels are related to vessels perme- only in hypervascularised tisses such as ovari- ability: high premebility of fenestred an corpus lutheum and in proliferating en- glomerular endothelium in adults is related to dometrium during the menstrual cycle14.The high levels of VEGF, in contrast low perme- continous expression of both VEGF in ep- ability of blood brain barrier in adult is relat- ithelial cells, such as podocytes and of ed to low VEGF levels20. VEGF-R on glomerular endothelium, in- Hypoxia seems to play a crucial role in stim- duces and manteins the fenestred phenotype ulating VEGF production by glomerular ep- in endothelium,and also regulates vascular ithelial mass and VEGF-R expression in en- permeability15. dothelial precursor cells. Some studies demon- strate the induction of VEGF and VEGF-R VEGF and kidney expression in culture of metanephric kidney Kidney is formed by an intricate system of under hypoxic conditions and indicate a role microvascular units. Each nephron microvas- for VEGF mediation of vasculogenesis by re- cular unit is formed by an afferent glomerular cruitment of flk-1 expressing angioblasts21. arteriole, a glomerular capillary tuft, an effer- Hypoxia is a rapid and potent inducer of ent glomerular arteriole and a peritubular VEGF mRNA expression. Hypoxia Inducer

151 F. Del Porto, A. Mariotti, M. Ilardi, F.R. Messina, A. Afeltra, A. Amoroso

Factor (HIF) binds the VEGF enhancer and lar endothelial cells. Some studies indicate thus induces VEGF mRNA trascription. that ephrin B1 and its receptor EphB1 are ex- HIF-1 is a helix-loop-helix (HLH) PAS fami- pressed in developing and mature murine ly of trascriptor factors that regulate the glomeruli. In the adult mouse kidney, mes- trascription of many hypoxia inducible fac- enchymal and interstitial cells do not express tors, such as erythropoietin. Distruction of ephrinB1 and its receptor, while arteriolar in- HIF binding site inhibits hypoxia inducibility timal cells and glomerular capillary endothe- of VEGF22. lial cells express them. EphrinB1/EphB1 sys- Hypoxia inducing factor Related Factor tem induces cultured human renal microvas- (HRF), another HLH Pas family member, is cular endothelial cells to assemblate into cap- expressed by endothelial cells of brain capil- illary like structures. These observations lead lary, kidney glomeruli and chorioid plexus in to the hypotesis that ephrin/Eph interaction the embryo and adult. Moreover podocytes partecipates in the cell-cell recognition express HRF that involve a role in the regula- processes required for glomerular capillary tion of vascular permeability by manteinance assembly26. of VEGF/VEGF-R high levels23 Several growth factors and their receptors partecipate to the development of embrionic glomerular microvasculature through cooper- Conclusions ative effects: VEGF, FGF, PlGF, TGF-β, PDGF, HGF (Hepatocyte growth factor) and Neoangiogenesis or its inhibition are the angiopoietin 1 family24. most mechanism leading to many patholo- The vascular branching and vessel remod- gies. elling is regulated by angiopoietin 1 and 2. VEGF levels increase during neoangiogen- Specific tirosine kinase receptors for an- esis related to many diseases such as wound giopoietin family are tie1 and tie2. Tie2 may healing, tumour, proliferative retinopathy bind both angiopoietin 1 and angiopoietin 2: and cutaneous disease7. angiopoietin 1 stimulates the differentiation In glomerular injury models, repair is me- of surrounding mesenchym into pericyte or diated by angiogenesis and it has been hypo- smooth muscle. Angiopoietin 2 levels in- tised that angiogenesis recapitulates develop- crease following angiopoietin 1 expression mental models27. and only at sites of active vessel remodelling. Neutralizing antibodies against VEGF and The interaction of angiopoietin 2 with tie2 in- bFGF reduce the endothelial proliferation, hibits receptors activation24-25. this observation leads to the hypotesis that Tie2 and its ligands angiopoietin 1 and 2 local production of VEGF and bFGF are in- interaction seems to accelerate more than in- creased where ECs proliferate to repair hibit the vascular development. This observa- glomerular damage24. tion suggests a mayor role of angiopoietin 1 Use of monoclonal antibodies in therapy in vessels remodelling25. has been ipotised to block neoangiogenesis. PDGF and angiopoietins also reclutate Some studies demonstrate that anti VEGF perycites cells to their endothelial partners. antibodies inhibit tumoral growth, riduce Another ligand-receptor system has been number and size of metastases; we can thrust hypotised to mediate cell-cell recognition: this observation to each other hypervascu- ephrin A, ephrin B and their receptors Eph larised disease, such as proliferative A and B play a role during embrionic devel- retinopathies and rheumtoid arthritis28. In opment in definig the vessels organizational contast intramuscolar or intra-arterial admin- plan. istration of VEGF augments perfusion and Interaction between ephrins and their re- collateral vessels development as it has been ceptors causes cell aggregation and promotes evidencied in a rabbit model of chronic organizational responses. EphA2 receptors hindlimb ischemia29. We ipotise that this cy- family partecipate to angiogenesis and tokine may be used in organs transplantation chemotactic responses. EphB1 receptors fam- or in other pathologies characterized by an ily is most expressed in human renal mi- impair in tree vascular development such as crovascular endothelial cells and in glomeru- congenital kidney malformation.

152 Kidney vasculogenesis and angiogenesis: role of vascular endothelial growth factor

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