Catecholamines facilitate VEGF-dependent angiogenesis via β2-adrenoceptor-induced Epac1 and PKA activation

Abstract

Chronic stress has been associated with the progression of cancer and antagonists for β-adrenoceptors (βAR) are regarded as therapeutic option. As they are also used to treat hemangiomas as well as retinopathy of prematurity, a role of endothelial β2AR in angiogenesis can be envisioned. We therefore investigated the role of β2AR-induced cAMP formation by analyzing the role of the cAMP effector molecules exchange factor directly activated by cAMP 1 (Epac1) and protein kinase A (PKA) in endothelial cells (EC). Epac1-deficient mice showed a reduced amount of pre-retinal neovascularizations in the model of oxygen-induced retinopathy, which is predominantly driven by vascular endothelial growth factor (VEGF). siRNA-mediated knockdown of Epac1 in human umbilical vein EC (HUVEC) decreased angiogenic sprouting by lowering the expression of the endothelial VEGF-receptor-2 (VEGFR-2). Conversely, Epac1 activation by β2AR stimulation or the Epac-selective activator cAMP analog 8-p-CPT-2'-O-Me-cAMP (8-pCPT) increased VEGFR-2 levels and VEGF-dependent sprouting. Similar to Epac1 knockdown, depletion of the monomeric GTPase Rac1 decreased VEGFR-2 expression. As Epac1 stimulation induces Rac1 activation, Epac1 might regulate VEGFR-2 expression through Rac1. In addition, we found that PKA was also involved in the regulation of angiogenesis in EC since the adenylyl cyclase (AC) activator forskolin (Fsk), but not 8-pCPT, increased sprouting in Epac1-depleted HUVEC and this increase was sensitive to a selective synthetic peptide PKA inhibitor. In accordance, β2AR- and AC-activation, but not Epac1 stimulation increased VEGF secretion in HUVEC.Our data indicate that high levels of catecholamines, which occur during chronic stress, prime the endothelium for angiogenesis through a β2AR-mediated increase in endothelial VEGFR-2 expression and VEGF secretion.

Keywords: EPAC; PKA; VEGF; angiogenesis; cAMP.

Figure 1. Retinae of Epac1^−/− mice are protected from oxygen-induced vascular damage

New born pups at p7 along with their nursing mothers were put into 75% oxygen for 5 days and then returned to room air for another 5 days to induce relative hypoxia. At p17, mice were sacrificed, eyes enucleated and then fixed. (A) After embedding the eyes into paraffin, serial retinal sections were cut and stained with periodic acid and Schiff's base followed by hematoxylin. Representative pictures of one such retinal section from WT and its littermate Epac1^−/− mouse are shown. White arrowheads show the neovascular tufts. ILM: inner limiting membrane, GCL: ganglion cell layer, INL: inner nuclear layer. A quantification of neovascular endothelial nuclei originating from the ILM towards the vitreous per section per eye in WT and Epac1^−/− mice is shown below (n=13, Student's unpaired t-test, *p<0.01 vs. WT). (B) Retinae were dissected and subsequently stained with collagen IV antibody followed by staining with FITC-conjugated secondary antibody to visualize the retinal vasculature. Area of avascular zone relative to total retinal area in WT and Epac1^−/− mice from (B) to compare the levels of hypoxia present in the retina at p17 (n=7). Scale bar in (A): 20 μm, in (B): 200 μm.

Figure 2. siRNA-mediated depletion of Epac1 in HUVEC impairs basal and VEGF-induced angiogenic responses

(A) Sub-confluent HUVEC were either transfected with control siRNA or with siRNA against Epac1 (siEpac1-1). A representative Western blot is shown to detect the efficiency of knockdown 48 h post transfection (above). γ-tubulin was used as a loading control. Quantification of Epac1 bands relative to γ-tubulin using densitometric analysis is shown below (n=10, Student's paired t-test, *p<0.001 vs. siControl). (B) 24 h after transfection with the indicated siRNAs, sprouting assay was performed and spheroids were stimulated with or without 50 ng/ml of VEGF. Representative pictures are shown above. The mean of the cumulative sprout length measured from about 10 spheroids is shown below (n=5, Repeated measures of two-way ANOVA with Bonferroni's multiple comparison post-test, *p<0.05 vs. Basal siControl, ^#p<0.05 vs. VEGF siControl). (C) is similar to A except that a different siRNA sequence against Epac1 (siEpac1-2) (n=39, Student's paired t-test, *p<0.001 vs. siControl) was used. (D) 48 h post transfection with the indicated siRNAs, a ‘scratch’ was created in HUVEC monolayer followed by stimulation with or without 50 ng/ml VEGF. The data show the migrated distance 24 h post stimulation (n=5, repeated measures of two-way ANOVA with Bonferroni's multiple comparison post-test, *p<0.05 vs. siControl Basal, #p<0.05 vs. VEGF siControl).

Figure 3. Activation of β₂AR increases sprouting angiogenesis by cAMP-dependent activation of Epac1

(A) In the sprouting assay, HUVEC spheroids were stimulated with either 30 μM of the Epac activator (8-pCPT) or by 10 μM forskolin (Fsk) for 24 h. Subsequently, the cumulative sprout length was measured (n=5, one-way ANOVA with Bonferroni's multiple comparison post-test, *p<0.05 vs. Basal, ^#p<0.05 vs. 8-pCPT). (B) Serum starved HUVEC were stimulated with either 30 μM 8-pCPT or 10 μM Fsk for 10 min, followed by an active Rap1-pull down assay. Subsequently, total and GTP-bound Rap1 were detected using Western blot analysis. A representative blot is shown in the above panel. For the quantification of the Rap1-GTP bands relative to total Rap1, densitometric analyses from the Western blots were performed (n=3, one-way ANOVA with Bonferroni's multiple comparison post-test, *p<0.05 vs. Basal). (C) After 20 min pre-treatment with 75 μM of IBMX, HUVEC were stimulated for 10 min with either 5 μM of isoproterenol (Iso) or 10 μM of Fsk. Cells were lysed and the cAMP content was measured by ELISA (n=5, one-way ANOVA with Bonferroni's multiple comparison test *p<0.05 vs. Basal, ^#p<0.05 vs. Iso). (D) An active Rap1-pull down assay was performed as in B, after 10 min stimulation with 5 μM Iso. A representative blot and the quantification are shown (n=5, Student's paired t-test *p<0.05 vs. Basal). (E) Shows the effect of 5 μM Iso on the cumulative sprout length in the presence or absence of 10 μM atenolol (n=4-8, one-way ANOVA with Bonferroni's multiple comparison test, *p<0.05 vs. Basal without atenolol). (F) Shows the effect of 10 μM salbutamol on the cumulative sprout length (n=4, Student's paired t-test *p<0.05 vs. Basal).

Figure 4. PKA contributes to cAMP-induced increase in sprouting angiogenesis

(A) 24 h after transfecting HUVEC with either control siRNA or siRNA against Epac1, sprouting assays were performed in the presence or absence of either 30 μM of the Epac activator (8-pCPT) or 10 μM forskolin (Fsk). Subsequently, the cumulative sprout length was measured (n=10, repeated measures of two-way ANOVA with Bonferroni's multiple comparison post-test; *p<0.05 vs. siControl Basal, §p<0.05 vs. siControl 8-pCPT, ^#p<0.05 vs. siEpac1 Basal). (B) In the sprouting assay, HUVEC spheroids were pre-treated with or without 1 μM of myristoylated-PKI (PKI) for 20 min followed by stimulation with or without 10 μM Fsk for 24 h. The cumulative sprout length was analyzed subsequently (n=4, two-way ANOVA with Bonferroni's multiple comparison test, *p<0.001 vs. Control Basal, ^#p<0.05 vs. Fsk Control). (C) In the sprouting assay, after 20 min of pre-treatment with or without 1 μM of PKI, spheroids were stimulated with 30 μM 8-pCPT (n=5, one-way ANOVA with Bonferroni's multiple comparison test, *p<0.05 vs. Basal). (D) Serum starved HUVEC were stimulated with either 30 μM of 8-pCPT or 10 μM of Fsk. 8h post stimulation, the amount of VEGF mRNA was quantified relative to RPL10 mRNA by qPCR (n=6, one-way ANOVA with Bonferroni's multiple comparison test, *p<0.05 vs. Basal, ^#p<0.05 vs. 8-pCPT (E) Serum starved HUVEC were stimulated with either 30 μM 8-pCPT, 10 μM Fsk or 5 μM Iso for 24 h. Amount of VEGF secreted in the media was then measured from the supernatants using ELISA (n=6, one-way ANOVA with Bonferroni's multiple comparison post-test, *p<0.05 vs. Basal).

Figure 5. Epac1 regulates VEGFR-2 expression

(A) HUVEC were transfected with control siRNA or siRNA against Epac1. 72 h post transfection, cells were lysed and VEGFR-2 and Epac1 protein levels were detected using Western blot analysis. γ-tubulin was used as a loading control (above). Densitometric analysis for the quantification of VEGFR-2 expression relative to γ-tubulin is shown below (n=6, Student's paired t-test, *p<0.01 vs. siControl). (B) 48 h post transfection with siRNAs, VEGFR-2 mRNA was quantified relative to RPL10 mRNA (n=4, Student's paired t-test, *p<0.05 vs. siControl). (C) After transfecting HUVEC either with control siRNA or siRNA against Epac1 for 48 h, the content of VEGF secreted in the media was determined using ELISA (n=3, Student's paired t-test). (D) HUVEC were transfected with control pre-miRNA or with pre-miRNA 92b. 48 h post transfection, cells were lysed and miRNA-92b levels were detected relative to U6 miRNA by qPCR to determine the efficiency of transfection (left). Right panel shows the Western blot analysis to detect Epac1 and VEGFR-2 protein levels. γ-tubulin was used as loading control (n=5, Student's paired t-test, *p<0.05 vs. miControl) (E) Serum starved HUVEC were stimulated for 24 h with either 30 μM 8-pCPT or 10 μM forskolin (Fsk) or 5 μM isoproterenol (Iso). Cells were lysed and Western blot analysis was performed to determine VEGFR-2 protein levels using anti-VEGFR-2 antibody. γ-tubulin was used as a house keeping protein. A representative blot is shown above. Densitometric analysis for the quantification of VEGFR-2 bands relative to γ-tubulin is shown below (n=7, one-way ANOVA with Bonferroni's multiple comparison post-test, *p<0.05 vs. Basal).

Figure 6. Rac1 as a mediator of Epac1-dependent VEGFR-2 expression

(A) Serum starved HUVEC were stimulated with 30 μM 8-pCPT for 10 min. Subsequently, an active Rac1 pull-down assay was performed followed by Western blot analysis to detect GTP-bound and total Rac1. A representative blot is shown above. Densitometric analyses to quantify GTP-Rac1 band relative to total Rac1 are shown below (n=9, Student's paired t-test, *p<0.01 vs. Basal). (B) HUVEC were transfected with siControl or siRac1. 72 h post transfection, cell lysates were investigated for Rac1, VEGFR-2 and Epac1 proteins by Western blot analysis. γ-tubulin was used as a loading control. A representative blot is shown above. The protein content of Rac1, VEGFR-2 and Epac1 relative to γ-tubulin analysis was quantified by densitometry (n=4, Student's paired t-test, *p<0.05 vs. siControl, ns, p>0.05 vs. siControl). (C) HUVEC were transfected with either control siRNA or siRNA against Rac1. 48 h after transfection, cell lysates were analyzed for VEGFR-2 mRNA levels by qPCR. RPL10 mRNA was used as an endogenous control (n=4, Student's paired t-test, *p<0.05 vs. siControl) (D) 72 h after transfecting HUVEC with the indicated siRNAs, VEGF concentrations in the culture media were measured by ELISA (n=4, Student's paired t-test, ns, p>0.05 vs. siControl).

Figure 7. The Epac1-induced upregulation of VEGFR-2 is functionally relevant

(A) 24 h after transfection with either siControl or siEpac1-2, sprouting assays were performed and the resultant spheroids were stimulated with or without 50 ng/ml VEGF. The data show the cumulative sprout length (n=8, repeated measures of two-way ANOVA with Bonferroni's multiple comparison post-test, *p<0.05 vs. Basal siControl, ^#p<0.05 vs. VEGF siControl). (B) Spheroids were stimulated for 24 h with either 30 μM 8-pCPT or 10 μM forskolin (Fsk) in the absence or presence of 50 ng/ml VEGF. The cumulative sprout length was measured subsequently (n=8, one-way ANOVA with Bonferroni's multiple comparison test, *p<0.05 vs. Basal without VEGF, ^#p<0.05 vs. Basal with VEGF, ^§p<0.05 vs. 8-pCPT without VEGF).