Differences in activation of intracellular signaling in primary human retinal endothelial cells between isoforms of VEGFA 165

Abstract

Purpose: There are reports that a b-isoform of vascular endothelial growth factor-A 165 (VEGFA₁₆₅b) is predominant in normal human vitreous, switching to the a-isoform (VEGFA₁₆₅a) in the vitreous of some diseased eyes. Although these isoforms appear to have a different ability to activate the VEGF receptor 2 (VEGFR2) in various endothelial cells, the nature of their ability to activate intracellular signaling pathways is not fully characterized, especially in retinal endothelial cells. We determined their activation potential for two key intracellular signaling pathways (MAPK, AKT) over complete dose-response curves and compared potential effects on the expression of several VEGFA₁₆₅ target genes in primary human retinal microvascular endothelial cells (HRMECs).

Methods: To determine full dose-response curves for the activation of MAPK (ERK1/2), AKT, and VEGFR2, direct in-cell western assays were developed using primary HRMECs. Potential differences in dose-response effects on gene expression markers related to endothelial cell and leukocyte adhesion (ICAM1, VCAM1, and SELE) and tight junctions (CLDN5 and OCLN) were tested with quantitative PCR.

Results: Activation dose-response analysis revealed much stronger activation of MAPK, AKT, and VEGFR2 by the a-isoform at lower doses. MAPK activation in primary HRMECs displayed a sigmoidal dose-response to a range of VEGFA ₁₆₅ a concentrations spanning 10-250 pM, which shifted higher into the 100-5,000 pM range with VEGFA ₁₆₅ b. Similar maximum activation of MAPK was achieved by both isoforms at high concentrations. Maximum activation of AKT by VEGFA ₁₆₅ b was only half of the maximum activation from VEGFA ₁₆₅ a. At a lower intermediate dose, where VEGFA ₁₆₅ a activated intracellular signaling stronger than VEGFA ₁₆₅ b, the changes in VEGFA target gene expression were generally greater with VEGFA ₁₆₅ a.

Conclusions: In primary HRMECs, VEGFA ₁₆₅ a could maximally activate MAPK and AKT at lower concentrations where VEGFA ₁₆₅ b had relatively little effect. The timing for maximum activation of MAPK was similar for the isoforms, which is different from that reported for non-retinal endothelial cells. Although differences in VEGFA ₁₆₅ a and VEGFA ₁₆₅ b are limited to the sequence of their six C-terminal six amino acids, this results in a large difference in their ability to activate at least two key intracellular signaling pathways and VEGF-target gene expression in primary human retinal endothelial cells.

Figure 1

MAPK and AKT are activated in HRMECs by VEGFA₁₆₅a and VEGFA₁₆₅b. A: Immunoblots of total MAPK and activated MAPK (phospho-MAPK) 10 min after treatment with VEGFA₁₆₅a (Va), VEGFA₁₆₅b (Vb), or media control (C). Two representative experiments are shown. B: Immunoblots of total AKT and activated AKT (phospho-AKT) 10 min after treatment with VEGFA₁₆₅a (Va), VEGFA₁₆₅b (Vb), or media control (C). Two representative experiments are shown. C: Fold activation of MAPK and AKT from three immunoblotting experiments. P values are shown, t test relative to media controls (onefold).

Figure 2

VEGFA₁₆₅a strongly activates MAPK in primary HRMECs at doses where VEGFA₁₆₅b has little effect. Activation of MAPK was measured with in situ immunofluorescence with a phosphospecific antibody (phospho-Thr202/Tyr204) for p44/42. A: Activation of MAPK at three time points after the addition of 1,000 pM VEGFA₁₆₅a or VEGFA₁₆₅b. Bar shows standard deviation. (t test, ***p<0.001 relative to untreated control, n=8 biologic replicates.) B: Dose–response curves for activation of MAPK. The median effective dose (ED₅₀) was 73 pM for VEGFA₁₆₅a and 1,015 pM for VEGFA₁₆₅b. Bars indicate the 95% confidence interval for data fit to the four-parameter log-logistic function. n=4 biologic replicates per dose.

Figure 3

VEGFA₁₆₅b is a weaker activator of AKT in primary HRMECs compared to VEGFA₁₆₅a. A: Activation of AKT was measured with in situ immunofluorescence with a phosphospecific antibody for Ser473-AKT at five time points after the addition of 20 ng/ml (1,050 pM) VEGFA₁₆₅a or VEGFA₁₆₅b. Bar shows standard deviation. (t test, ***p<0.001 relative to untreated control, n=4 biologic replicates.) B: Dose–response curves for activation of AKT. The median effective dose (ED₅₀) value for AKT activation by VEGFA₁₆₅a was 53 pM compared to 126 pM for VEGFA₁₆₅b. Bars indicate the 95% confidence interval for data fit to the four-parameter log-logistic function. n=4 biologic replicates per dose.

Figure 4

Differences in the activation of intracellular signaling between VEGFA₁₆₅a and VEGFA₁₆₅b in primary HRMECs begin at the receptor VEGFR2. VEGFA₁₆₅a was a stronger activator of VEGFR2 compared to VEGFA₁₆₅b. The median effective dose (ED₅₀) for activation by VEGFA₁₆₅a was 254 pM compared to 1,192 pM for VEGFA₁₆₅b. Bars indicate the 95% confidence interval for data fit to the four-parameter log-logistic function. n=4 biologic replicates per dose.

Figure 5

VEGFA₁₆₅a has a stronger effect on leukocyte–endothelial cell adhesion gene expression in primary HRMECs than VEGFA₁₆₅b. Relative gene expression was measured with quantitative PCR (qPCR). A: Expression of the ICAM1 gene at 1, 3, 6, and 24 h after treatment with VEGFA₁₆₅. Confluent human retinal microvascular endothelial cells (HRMECs) were treated with a saturating high dose of VEGFA₁₆₅a or VEGFA₁₆₅b (100 ng/ml, 5,300 pM). B: Expression of the ICAM1 gene comparing VEGFA₁₆₅a and VEGFA₁₆₅b at low, intermediate, and high doses measured after 6 h. C: Expression of the SELE gene at 1, 3, 6, and 24 h after treatment with VEGFA₁₆₅. D: Expression of the SELE gene comparing VEGFA₁₆₅a and VEGFA₁₆₅b at low, intermediate, and high doses measured after 6 h. E: Expression of the VCAM1 gene at 1, 3, 6, and 24 h after treatment with VEGFA₁₆₅. F: Expression of the VCAM1 gene comparing VEGFA₁₆₅a and VEGFA₁₆₅b at low, intermediate, and high doses measured after 6 h. (Triplicate assays, error bars show standard deviation. ANOVA (ANOVA): compared to media control *p<0.05, **p<0.01; compared to VEGFA₁₆₅a ^†p<0.05, ^††p<0.01).

Figure 6

VEGFA₁₆₅a has a stronger effect on the expression of tight-junction-protein genes in primary HRMECs than VEGFA₁₆₅b. Relative gene expression measured with quantitative PCR (qPCR). A: Expression of the CLDN5 gene at 1, 3, 6, and 24 h after treatment with VEGFA₁₆₅. Confluent human retinal microvascular endothelial cells (HRMECs) were treated with a saturating high dose of VEGFA₁₆₅a or VEGFA₁₆₅b (100 ng/ml, 5,300 pM). B: Expression of the CLDN5 gene comparing VEGFA₁₆₅a and VEGFA₁₆₅b at low, intermediate, and high doses measured after 6 h. C: Expression of the OCLN gene at 1, 3, 6, and 24 h after treatment with VEGFA₁₆₅. D: Expression of the OCLN gene comparing VEGFA₁₆₅a and VEGFA₁₆₅b at low, intermediate, and high doses measured after 6 h. (Triplicate assays, error bars show standard deviation. ANOVA (ANOVA): compared to media control *p<0.05, **p<0.01; compared to VEGFA₁₆₅a ^†† p<0.01).