The enhancement in HUVEC migration induced by L-NAME is reverted by the NO donor DETA-NO and is independent of the cGMP pathway.

<p>(A) HUVECs were treated for 48 h with 5 mM L-NAME in the absence or in the presence of 500 nM DETA/NO for the last 24 h, as indicated. Chemotaxis experiments were then performed using 25 ng/ml VEGF as attractants. Results are expressed as the number of migrating cells. #p<0.001 <i>vs</i> basal migration in control cells (CTRL); §p<0.01 <i>vs</i> VEGF-induced migration in control cells; ***p<0.001 <i>vs</i> basal migration in L-NAME treated cells; °°°p<0.001 <i>vs</i> VEGF-induced migration in L-NAME treated cells; no significant differences between control and DETA/NO treated cells (One-way ANOVA with Bonferroni's test, n = 15). (B) HUVECs were treated for 48 h with 5 mM L-NAME or 1 µM ODQ, and chemotaxis experiments were performed as described in (A). Results are expressed as the number of migrating cells in the different experimental conditions. #p<0.001 <i>vs</i> basal migration in control cells (CTRL); §p<0.001 <i>vs</i> VEGF-induced migration in control cells; no significant differences between control and ODQ treated cells (One-way ANOVA with Bonferroni's test, n = 3). (C) cGMP accumulation in HUVECs treated for 48 h with L-NAME or ODQ was evaluated by EIA and expressed as pmol of cGMP normalized to the cell protein content (pmol/mg protein). ***p<0.001; One-way ANOVA with Bonferroni's test; n = 3.</p

Effect of eNOS silencing on HIF-1α accumulation, VEGF secretion, mtDNA and ATP levels.

<p>(A) Characterization of HUVECs transfected with eNOS siRNA: densitometric analysis of eNOS protein expression where eNOS protein levels were normalized to β-actin protein. ***p<0.001; <i>t</i> test; n = 4. Inset: representative blots of eNOS protein in cells transfected with control (ctrl) or eNOS siRNA. (B) HUVECs were transfected with control (lane 2) or eNOS siRNA (lane 3), and HIF-1α protein was detected by western blotting on the corresponding nuclear extracts. In lane 1, nuclear extracts from untransfected cells. An aliquot of total cell lysates was immunoblotted with anti eNOS antibodies to check silencing, and with anti β-actin antibodies as loading control. A representative blot of 2 comparable experiments is shown. (C) VEGF protein levels were detected by ELISA measurement in conditioned media collected from HUVECs 48 h after transfection with control or eNOS siRNA. Results are expressed as pg of VEGF normalized to the cell protein content (pg/mg protein). *p<0.05; <i>t</i> test; n = 3. (D) MtDNA (left axis) and total cellular ATP content (right axis) were measured in HUVECs transfected for 48 h with control or eNOS siRNA. In silenced cells, mtDNA and ATP were reduced by 36±0.4 and 45±9.7% respectively. **p<0.01 and ***p<0.001; <i>t</i> test; n = 3.</p

Immunofluorescence staining on primary neuronal cultures.

<p>Immunofluorescence staining with GHS-R of either cortical (CN) or hippocampal (HN) neurons at different stages of development. Both neuronal cultures show correct maturation in vitro, as assayed by positive staining of vesicular marker for excitatory neurotransmitter transporter Vglut which shows punctuated expression along neuritis at mature stages of development.</p

Evaluation of anti-GHS-R Mab specificity.

<p>A. Western blot of analysis of a selected brain region (hypothalamus) and a non-brain GHS-R bearing tissue (heart) showing a single band of the predicted molecular weight for GHSR at 48 kDa B. Immunoprecipitation of rat cortical (C) and hippocampal (H) primary neurons lysates with Mab 1D8B2 and commercial polyclonal antibody. Lysates from rat hippocampal and cortical neurons after nine days in vitro were immunoprecipitated with Mab (8 µg) or polyclonal antibody (CTRL), resolved and transferred to nitrocellulose membranes. The western blot analysis were performed with the polyclonal antibody when the monoclonal was used for immunoprecipitation and the other way around, both immunoprecipitation analysis clearly showed a 48 kD band corresponding to the predicted size of GHS-R. β-actin was used as a loading control. Sizes (kD) of molecular mass markers are indicated on the right.</p

Production and characterization of Mab anti-GHS-R.

<p>A. Immunoprecipitation of 22RV1 cell lysates with Mab 1D8B2 and commercial polyclonal antibody (CTRL). Lysates from cells were immunoprecipitated with Mab (8 µg) or polyclonal antibody, resolved and transferred to nitrocellulose membranes. The western blot analysis were performed with the polyclonal antibody when the monoclonal was used for immunoprecipitation and the other way around, both immunoprecipitation analysis clearly showed a 48 kD band corresponding to the predicted size of GHS-R. Sizes (kD) of molecular mass markers are indicated on the left. These experiments were performed independently at least twice with similar results. B. Binding analysis of Mab 1D8B2 to 22RV1 cells by flow cytometry. Purified monoclonal antibody (6 µg) were analyzed with a flow cytofluorimetric analysis to assess the binding of antibodies to the GHSR on the surface of the cells. The control consisted of an anti-GHSR purified polyclonal antibody. Experiments were performed four times with reproducible results C. Representative image of 22VR1 cells cotransfected with GHS-R siRNA and eGFP. eGFP (green) expressing cells do not show positivity for GHSR (red). D. Relative mRNA expression of GHS-R in transfected 22VR1 normalized on GAPDH. GHS-R expression is significantly lower in transfected cells(siRNA GHSR) than in non-transfected cells (GHSR UT).</p

Effects of L-NAME treatment on eNOS, VEGF and KDR expression.

<p>(A) Densitometric analysis of eNOS protein expression. ***p<0.001; <i>t</i> test; n = 11. Inset: a representative blot out of eleven is shown. Total eNOS protein was evaluated by western blotting on lysates prepared from control cells (lane 1) or from 48 h L-NAME treated cells (lane 2). β-actin was used as a loading control. (B) eNOS RNA levels were measured by RT-qPCR and normalized to the level of the housekeeping gene 18S. No significant differences between control and L-NAME treated cells (<i>t</i> test, n = 3). (C) VEGF and KDR RNA levels were measured by RT-qPCR and normalized to the level of the housekeeping gene 18S. **p<0.01 <i>vs</i> control cells (CTRL); <i>t</i> test; n = 6−4 for VEGF and KDR, respectively. (D) VEGF protein levels were detected by ELISA measurement in conditioned media collected from control or 48 h L-NAME treated cells. Results are expressed as pg of VEGF normalized to the cell protein content (pg/mg protein). **p<0.01; <i>t</i> test; n = 3. (E) KDR protein was visualized by western blot after immunoprecipitation with KDR antibodies of HUVEC lysates obtained from control (lane 1) or from 48 h L-NAME treated cells (lane 2). An aliquot of total cell lysates was immunoblotted with β-actin antibodies as a control (input). Shown is a representative blot of 2 comparable experiments. (F) Control cells (lanes 1 and 2) or 48 h L-NAME treated cells (lanes 3 and 4) were stimulated for 5 min with 25 ng/ml VEGF. Aliquots of cell lysates were separated by 10% SDS-PAGE and immunoblotted with the indicated antibodies. Actin was used as a loading control. Shown is a representative blot of 4 comparable experiments.</p

Effect of chronic NO deprivation on HUVEC vitality and mitochondrial mass and function.

<p>(A) Lysates of HUVECs treated for 48 h with 5 mM L-NAME or 30 mM glucose (high glucose, h-Glc) were separated by 12% SDS-PAGE and immunoblotted with an anti-caspase 3 antibody which recognized full length caspase-3 (35 kDa) and its large fragment resulting from cleavage (17 kDa). β-actin was used as a loading control. Shown is a representative blot of 2 comparable experiments. (B) Total cell lysates prepared as described in (A) were separated by SDS-PAGE and immunoblotted with anti Bcl–2 or anti Bax antibodies. β-actin was used as a loading control. Shown is a representative blot of 2 comparable experiments. (C) Mitochondrial DNA (mtDNA) was quantified by RT-qPCR from control cells (CTRL) or from cells treated with L-NAME for 48 h, and normalized to the level of the housekeeping gene 18S. **p<0.01; <i>t</i> test; n = 3. (D) Mitochondrial activity of control and L-NAME treated cells was evaluated by means of MTS. In parallel samples, the total cell number was measured by crystal violet staining. ***p<0.001 <i>vs</i> control cells (CTRL, set at 100%); <i>t</i> test; n = 7. (E) After L-NAME treatment, oxygen consumption was reduced by 25±6% in comparison to control cells. The values were normalized to the cell protein content. *p<0.05; <i>t</i> test, n = 3. (F) Total cellular ATP levels were reduced by 25±7% after 48 h of 5 =  mM L-NAME treatment. **p<0.01 <i>vs</i> control cells (CTRL, set at 100%); <i>t</i> test; n = 8.</p

Comparison of GHS-R levels in cortical and hippocampal neurons.

<p>A. GHS-R mRNA expression levels in cortical (red) and hippocampal (blue) primary neurons normalized on expression levels at 4 div. GHS-R show a significant increase in expression levels at 9 and 16 div, with a significant reduction at later stages (21 div) in both neuronal populations. B. Relative mRNA expression of GHS-R at different developmental stages of hippocampal (red) and cortical (blue) neuronal cells normalized on GAPDH. GHS-R is significantly more expressed in hippocampal rather than cortical neurons at 9 and 16 div.</p

L-NAME treatment induces HIF-1α nuclear accumulation.

<p>(A) HIF-1α protein levels were detected by western blotting of nuclear extracts from control HUVECs (lane 1) or from HUVECs treated with L-NAME for 48 h (lane 2). Shown is a representative blot of 4 comparable experiments. HIF-1α migrates as a doublet with apparent molecular weight of 118 and 120 kDa. (B) Densitometric analysis of nuclear HIF-1α protein levels. *p<0.05; <i>t</i> test; n = 4. (C) HIF-1α RNA levels were measured by RT-qPCR and normalized to the level of the housekeeping gene 18S. No significant differences between control and L-NAME treated cells (<i>t</i> test; n = 3). (D) VEGF protein levels were detected by ELISA measurement in conditioned media collected from HUVECs transfected with the empty vector (pcDNA3) or with the expression vector ΔARNT, and treated with L-NAME for the 48 h following transfection. Results are expressed as pg of VEGF normalized to the cell protein content (pg/mg protein). **p<0.01 <i>vs</i> untreated cells transfected with pcDNA3; ***p<0.001 <i>vs</i> L-NAME treated cells transfected with pcDNA3 (One-way ANOVA with Bonferroni's test; n = 3). (E) HUVECs were transfected with pcDNA3 or ΔARNT, and treated with L-NAME for the 48 h following transfection when indicated. Chemotaxis experiments were then performed using 25 ng/ml VEGF as attractant. Results are expressed as the number of migrating cells. #p<0.001 <i>vs</i> basal migration in untreated pcDNA3 cells; §p<0.001 <i>vs</i> VEGF-induced migration in untreated pcDNA3 cells; ***p<0.001 <i>vs</i> basal migration in pcDNA3 cells treated with L-NAME; °°°p<0.001 <i>vs</i> VEGF-induced migration in pcDNA3 cells treated with L-NAME; no significant differences between untreated pcDNA3 and ΔARNT transfected cells and between untreated and L-NAME treated ΔARNT tranfected cells (One-way ANOVA with Bonferroni's test, n = 10).</p