ALTERATIONS IN DOUBLECORTIN EXPRESSION IN HUMAN NEURONAL STEM CELLS IN RESPONSE TO ANGIOTENSINERGIC STIMULATION IN PROLIFERATION AND DIFFERENTIATION
Abstract
Objective. To determine if selective stimulation of AT1 and AT2 subytpes in neuronal stem cells affects doublecortin expression under differentiation versus proliferation conditions. Background. Angiotensin II (Ang II) has been reported to affect cell proliferation and differentiation in cultured neuronal cells. Methods. Neural stem cells (H-9) obtained from Life Technologies were grown in a cell culture medium that was supplemented with epidermal growth factor and fibroblast growth factor (proliferation medium) or without added growth factors (differentiation medium). The cells were treated with a nonselective agonist of Ang II receptors (Sar1, Ang II, 100 μM once daily) in the presence of the AT2 receptor blocker PD 123319 (10 μM once daily) to selectively stimulate AT1 receptors, CGP42112 a selective AT2 receptor agonist (100 μM once daily), PD123319 a selective AT2 receptor antagonist (10 μM, once daily), or saline for two weeks. The cells were fixed and stained with doublecortin antibody and the cell nuclei were stained with dapi. Cells were counted for number of dapi labeled nuclei and for doublecortin (DCX) immunoreactive material. The proportion of cells positive for the neuroblast marker DCX was determined in all 8 conditions and analyzed by a two-way ANOVA. Results. Doublecortin expression was reduced by AT1 selective stimulation in differentiation conditions relative to control (Control 70.9 ± 17% and AT1 14.0 ± 6.2%, p<0.01), AT2 stimulated (AT2 86.7 ± 0.6%, p<0.01) and AT2 antagonized with PD123319 (PD123319 64.4 ± 9.3%, p<0.05). In proliferation conditions, doublecortin expression was reduced by AT2 selective stimulation and AT2 antagonism compared to control and AT1 selectively stimulated cells (p<0.01: Control 96.5 ± 1.1% ; AT1 88.7 ± 1.6% ; AT2 9.1 ± 6.1% ; PD123319 2.6 ± 1.0%). Conclusion. These results suggest that angiotensinergic inhibition of doublecortin expression is dependent upon the presence of the growth factors EGF and FGF-2. Grants. R.C. Speth: Research Grant; Significant; NIH HL-113905; Cardiovascular Neuroscience Research Fund, Nova Southeastern University. J. Munoz: President’s Faculty Research Development Grant, Nova Southeastern University.
ALTERATIONS IN DOUBLECORTIN EXPRESSION IN HUMAN NEURONAL STEM CELLS IN RESPONSE TO ANGIOTENSINERGIC STIMULATION IN PROLIFERATION AND DIFFERENTIATION
Melnick Auditorium
Objective. To determine if selective stimulation of AT1 and AT2 subytpes in neuronal stem cells affects doublecortin expression under differentiation versus proliferation conditions. Background. Angiotensin II (Ang II) has been reported to affect cell proliferation and differentiation in cultured neuronal cells. Methods. Neural stem cells (H-9) obtained from Life Technologies were grown in a cell culture medium that was supplemented with epidermal growth factor and fibroblast growth factor (proliferation medium) or without added growth factors (differentiation medium). The cells were treated with a nonselective agonist of Ang II receptors (Sar1, Ang II, 100 μM once daily) in the presence of the AT2 receptor blocker PD 123319 (10 μM once daily) to selectively stimulate AT1 receptors, CGP42112 a selective AT2 receptor agonist (100 μM once daily), PD123319 a selective AT2 receptor antagonist (10 μM, once daily), or saline for two weeks. The cells were fixed and stained with doublecortin antibody and the cell nuclei were stained with dapi. Cells were counted for number of dapi labeled nuclei and for doublecortin (DCX) immunoreactive material. The proportion of cells positive for the neuroblast marker DCX was determined in all 8 conditions and analyzed by a two-way ANOVA. Results. Doublecortin expression was reduced by AT1 selective stimulation in differentiation conditions relative to control (Control 70.9 ± 17% and AT1 14.0 ± 6.2%, p<0.01), AT2 stimulated (AT2 86.7 ± 0.6%, p<0.01) and AT2 antagonized with PD123319 (PD123319 64.4 ± 9.3%, p<0.05). In proliferation conditions, doublecortin expression was reduced by AT2 selective stimulation and AT2 antagonism compared to control and AT1 selectively stimulated cells (p<0.01: Control 96.5 ± 1.1% ; AT1 88.7 ± 1.6% ; AT2 9.1 ± 6.1% ; PD123319 2.6 ± 1.0%). Conclusion. These results suggest that angiotensinergic inhibition of doublecortin expression is dependent upon the presence of the growth factors EGF and FGF-2. Grants. R.C. Speth: Research Grant; Significant; NIH HL-113905; Cardiovascular Neuroscience Research Fund, Nova Southeastern University. J. Munoz: President’s Faculty Research Development Grant, Nova Southeastern University.