title: Synergistic effect of angiotensin II on vascular endothelial growth factor-A-mediated differentiation of bone marrow-derived mesenchymal stem cells into endothelial cells.
abstract: BACKGROUND
  Increased levels of angiotensin II (Ang II) and activity of Ang II receptor type 1 (AT1R) elicit detrimental effects in cardiovascular disease. However, the role of Ang II receptor type 2 (AT2R) remains poorly defined. Mesenchymal stem cells (MSCs) replenish and repair endothelial cells in the cardiovascular system. Herein, we investigated a novel role of angiotensin signaling in enhancing vascular endothelial growth factor (VEGF)-A-mediated differentiation of MSCs into endothelial cells (ECs).
  
  
  METHODS
  Bone marrow was aspirated from the femurs of Yucatan microswine. MSCs were extracted via ficoll density centrifugation technique and were strongly immunopositive for MSC markers, CD44, CD90, and CD105, but negative for hematopoietic markers, CD14 and CD45. Subsequently, naïve MSCs were differentiated for 10 days in varying concentrations and combinations of VEGF-A, Ang II, and AT1R or AT2R antagonists. Markers specific to ECs were determined by FACS analysis.
  
  
  RESULTS
  AT1R and AT2R expression and cellular localization was demonstrated in MSCs stimulated with VEGF-A and Ang II via quantitative RT-PCR and immunofluorescence, respectively. Differentiation of naïve MSCs in media containing Ang II (2 ng/ml) plus low-dose VEGF-A (2 ng/ml) produced a significantly higher percentage of cells that were positive for expression of EC markers (for example, platelet endothelial cell adhesion molecule, vascular endothelial Cadherin and von Willebrand factor) compared to VEGF-A alone. Ang II alone failed to induce EC marker expression. MSCs differentiated with the combination of Ang II and VEGF-A were capable of forming capillary tubes using an in vitro angiogenesis assay. Induction of EC marker expression was greatly attenuated by co-treatment of Ang II/VEGF-A with the AT2R antagonist PD123319, but not the AT1R antagonist telmisartan.
  
  
  CONCLUSIONS
  We report the presence of functional AT2R receptor on porcine bone marrow-derived MSCs, where it positively regulates EC differentiation. These findings have significant implications toward therapeutic approaches based on activation of AT2R, which could be a means to stimulate regeneration of damaged endothelium and prevent vascular thrombosis.
authors.affiliation: Department of Medical Microbiology and Immunology, Creighton School of Medicine, 2500 California Plaza, Omaha, NE, 68178-0405, USA. IzuagieAttairu@creighton.edu.
authors.forename: Izuagie Attairu
authors.initials: IA
authors.lastname: Ikhapoh
authors.affiliation: Department of Biomedical Sciences, Creighton School of Medicine, 2500 California Plaza, Omaha, NE, 68178, USA. ChristopherPelham@creighton.edu.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Pelham
authors.affiliation: Department of Medical Microbiology and Immunology, Creighton School of Medicine, 2500 California Plaza, Omaha, NE, 68178-0405, USA. dkagr@creighton.edu.|Department of Biomedical Sciences, Creighton School of Medicine, 2500 California Plaza, Omaha, NE, 68178, USA. dkagr@creighton.edu.|Center for Clinical and Translational Science, CRISS II Room 510, Creighton School of Medicine, 2500 California Plaza, Omaha, NE, 68178-0405, USA. dkagr@creighton.edu.
authors.forename: Devendra K
authors.initials: DK
authors.lastname: Agrawal
mesh_terms: D000804:Angiotensin II / Q000494:pharmacology*; D047228:Angiotensin II Type 1 Receptor Blockers / Q000494:pharmacology; D057912:Angiotensin II Type 2 Receptor Blockers / Q000494:pharmacology; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D001562:Benzimidazoles / Q000494:pharmacology; D001565:Benzoates / Q000494:pharmacology; D001854:Bone Marrow Cells / Q000166:cytology; D015820:Cadherins / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects*; D004357:Drug Synergism; D042783:Endothelial Cells / Q000166:cytology* / Q000378:metabolism; D005269:Femur / Q000166:cytology; D007093:Imidazoles / Q000494:pharmacology; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D008856:Microscopy, Fluorescence; D011725:Pyridines / Q000494:pharmacology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D044140:Receptor, Angiotensin, Type 1 / Q000235:genetics / Q000378:metabolism; D044139:Receptor, Angiotensin, Type 2 / Q000235:genetics / Q000378:metabolism; D013552:Swine; D000077333:Telmisartan; D042461:Vascular Endothelial Growth Factor A / Q000037:antagonists & inhibitors / Q000235:genetics / Q000494:pharmacology*; D014841:von Willebrand Factor / Q000378:metabolism; D000099152:Cadherin 5
chemical_list: D000804:Angiotensin II; D047228:Angiotensin II Type 1 Receptor Blockers; D057912:Angiotensin II Type 2 Receptor Blockers; D015703:Antigens, CD; D001562:Benzimidazoles; D001565:Benzoates; D015820:Cadherins; D007093:Imidazoles; D011725:Pyridines; D034741:RNA, Small Interfering; D044140:Receptor, Angiotensin, Type 1; D044139:Receptor, Angiotensin, Type 2; D000077333:Telmisartan; D042461:Vascular Endothelial Growth Factor A; D014841:von Willebrand Factor; D000099152:Cadherin 5; C073402:PD 123319