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An antiangiogenic isoform of VEGF-A contributes to impaired vascularization in peripheral artery disease.

Kikuchi R, Nakamura K, MacLauchlan S, Ngo DT, Shimizu I, Fuster JJ, Katanasaka Y, Yoshida S, Qiu Y, Yamaguchi TP, Matsushita T, Murohara T, Gokce N, Bates DO, Hamburg NM, Walsh K - Nat. Med. (2014)

Bottom Line: Here we show that clinical PAD is associated with elevated levels of an antiangiogenic VEGF-A splice isoform (VEGF-A165b) and a corresponding reduction in levels of the proangiogenic VEGF-A165a splice isoform.In mice, VEGF-A165b expression was upregulated by conditions associated with impaired limb revascularization, including leptin deficiency, diet-induced obesity, genetic ablation of the secreted frizzled-related protein 5 (Sfrp5) adipokine and transgenic overexpression of Wnt5a in myeloid cells.These results indicate that inflammation-driven expression of the antiangiogenic VEGF-A isoform can contribute to impaired collateralization in ischemic cardiovascular disease.

View Article: PubMed Central - PubMed

Affiliation: 1] Molecular Cardiology and Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, Massachusetts, USA. [2] Department of Medical Technique, Nagoya University Hospital, Nagoya, Aichi, Japan.

ABSTRACT
Peripheral artery disease (PAD) generates tissue ischemia through arterial occlusions and insufficient collateral vessel formation. Vascular insufficiency in PAD occurs despite higher circulating levels of vascular endothelial growth factor A (VEGF-A), a key regulator of angiogenesis. Here we show that clinical PAD is associated with elevated levels of an antiangiogenic VEGF-A splice isoform (VEGF-A165b) and a corresponding reduction in levels of the proangiogenic VEGF-A165a splice isoform. In mice, VEGF-A165b expression was upregulated by conditions associated with impaired limb revascularization, including leptin deficiency, diet-induced obesity, genetic ablation of the secreted frizzled-related protein 5 (Sfrp5) adipokine and transgenic overexpression of Wnt5a in myeloid cells. In a mouse model of PAD, delivery of VEGF-A165b inhibited revascularization of ischemic hind limbs, whereas treatment with an isoform-specific neutralizing antibody reversed impaired revascularization caused by metabolic dysfunction or perturbations in the Wnt5a-Sfrp5 regulatory system. These results indicate that inflammation-driven expression of the antiangiogenic VEGF-A isoform can contribute to impaired collateralization in ischemic cardiovascular disease.

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Wnt5a regulate VEGF-A165b expression via Ror2/JNK/SC35-dependent mechanism in macrophagesa. VEGF-A165b protein expression was measured by western immunoblot analysis in the presence or absence of recombinant Wnt5a in cultured RAW264.7 cells after treatment for 24 hours. b.Vegfa165b mRNA expression was measured by qRT-PCR 8 hours after treatment with Wnt5a (500 μg/ml) or vehicle in RAW264.7 cells that had been pre-incubated with vehicle, sh-control or sh-Ror2. ANOVA with post-hoc Tukey HSD. c. Phosphorylation of JNK (p-JNK) was measured by western blot analysis in cells treated with or without recombinant Wnt5a for 30 minutes. Relative phosphorylation levels of JNK were quantified by using ImageJ. Results are shown as the mean ± S.E. (n = 3/group). ANOVA with post-hoc Tukey HSD. d. The effects of JNK inhibition and Sfrp5 treatment or Wnt5a-induced Vegfa165b expression. Vegfa165b mRNA expression was measured by qRT-PCR. SP600125 was added 10 minutes prior to the addition of recombinant Wnt5a (500 ng/ml). Recombinant Sfrp5 was added at 1 μg/ml. Results are shown as the mean ± S.E. (n = 3/group). ANOVA with post-hoc Tukey HSD. e. SC35 protein expression was measured by western blot analysis in the presence or absence of stimulation with recombinant Wnt5a. The effects of the JNK inhibitor SP600125 or Sfrp5 on Wnt5a-induced SC35 expression were evaluated at 1 hour. Relative levels of SC35 were quantified by using ImageJ. Immunoblots were normalized to LaminA/C signal. Results are shown as the mean ± S.E. (n = 3/group). ANOVA with post-hoc Tukey HSD. f. SC35 is essential for Vegfa165b expression. Vegfa165b mRNA expression was measured by qRT-PCR. Cells were treated with sh-control and sh-SC35 for 8 hours and treated with or without recombinant Wnt5a. ANOVA with post-hoc Tukey HSD. g. Graphic scheme of proposed mechanism. *P < 0.05, **P < 0.01, ***P < 0.001.
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Figure 4: Wnt5a regulate VEGF-A165b expression via Ror2/JNK/SC35-dependent mechanism in macrophagesa. VEGF-A165b protein expression was measured by western immunoblot analysis in the presence or absence of recombinant Wnt5a in cultured RAW264.7 cells after treatment for 24 hours. b.Vegfa165b mRNA expression was measured by qRT-PCR 8 hours after treatment with Wnt5a (500 μg/ml) or vehicle in RAW264.7 cells that had been pre-incubated with vehicle, sh-control or sh-Ror2. ANOVA with post-hoc Tukey HSD. c. Phosphorylation of JNK (p-JNK) was measured by western blot analysis in cells treated with or without recombinant Wnt5a for 30 minutes. Relative phosphorylation levels of JNK were quantified by using ImageJ. Results are shown as the mean ± S.E. (n = 3/group). ANOVA with post-hoc Tukey HSD. d. The effects of JNK inhibition and Sfrp5 treatment or Wnt5a-induced Vegfa165b expression. Vegfa165b mRNA expression was measured by qRT-PCR. SP600125 was added 10 minutes prior to the addition of recombinant Wnt5a (500 ng/ml). Recombinant Sfrp5 was added at 1 μg/ml. Results are shown as the mean ± S.E. (n = 3/group). ANOVA with post-hoc Tukey HSD. e. SC35 protein expression was measured by western blot analysis in the presence or absence of stimulation with recombinant Wnt5a. The effects of the JNK inhibitor SP600125 or Sfrp5 on Wnt5a-induced SC35 expression were evaluated at 1 hour. Relative levels of SC35 were quantified by using ImageJ. Immunoblots were normalized to LaminA/C signal. Results are shown as the mean ± S.E. (n = 3/group). ANOVA with post-hoc Tukey HSD. f. SC35 is essential for Vegfa165b expression. Vegfa165b mRNA expression was measured by qRT-PCR. Cells were treated with sh-control and sh-SC35 for 8 hours and treated with or without recombinant Wnt5a. ANOVA with post-hoc Tukey HSD. g. Graphic scheme of proposed mechanism. *P < 0.05, **P < 0.01, ***P < 0.001.

Mentions: To examine the regulation of VEGF-A165b at a mechanistic level, cultured RAW264.7 cells, a mouse macrophage line, were treated with recombinant Wnt5a. Wnt5a-stimulation led to a dose-dependent increase in VEGF-A165b (Fig. 4a), and the upregulation of this Vegfa splice variant could also be detected at the level of mRNA (Fig. 4b). Consistent with the finding that Wnt5a signals in a non-canonical manner,26 knockdown of Ror2, a Frizzled co-receptor protein, abolished Wnt5a-mediated Vegfa165b induction (Fig. 4b). Also consistent with a non-canonical signaling mechanism, Wnt5a treatment led to the activation of JNK (Fig. 4c), and pretreatment with the JNK inhibitor SP600125 abolished Wnt5a-mediated Vegfa165b expression (Fig. 4d). Similarly, treatment with recombinant Sfrp5 protein blocked VEGF-A165b expression under these conditions. Further investigation revealed that Wnt5a upregulated the expression of SC35, a pre-mRNA splicing factor implicated in the alternative processing of Vegfa at exon 8,27 in a JNK- and Sfrp5-dependent manner (Fig. 4e). Knockdown of SC35 blocked the Wnt5a-mediated induction of Vegfa165b (Fig. 4f). Collectively, these data suggest that Wnt5a promotes the processing of Vegfa to the exon 8b-containing isoform, at least in part, via an Ror2/JNK/SC35-dependent mechanism (Fig. 4g).


An antiangiogenic isoform of VEGF-A contributes to impaired vascularization in peripheral artery disease.

Kikuchi R, Nakamura K, MacLauchlan S, Ngo DT, Shimizu I, Fuster JJ, Katanasaka Y, Yoshida S, Qiu Y, Yamaguchi TP, Matsushita T, Murohara T, Gokce N, Bates DO, Hamburg NM, Walsh K - Nat. Med. (2014)

Wnt5a regulate VEGF-A165b expression via Ror2/JNK/SC35-dependent mechanism in macrophagesa. VEGF-A165b protein expression was measured by western immunoblot analysis in the presence or absence of recombinant Wnt5a in cultured RAW264.7 cells after treatment for 24 hours. b.Vegfa165b mRNA expression was measured by qRT-PCR 8 hours after treatment with Wnt5a (500 μg/ml) or vehicle in RAW264.7 cells that had been pre-incubated with vehicle, sh-control or sh-Ror2. ANOVA with post-hoc Tukey HSD. c. Phosphorylation of JNK (p-JNK) was measured by western blot analysis in cells treated with or without recombinant Wnt5a for 30 minutes. Relative phosphorylation levels of JNK were quantified by using ImageJ. Results are shown as the mean ± S.E. (n = 3/group). ANOVA with post-hoc Tukey HSD. d. The effects of JNK inhibition and Sfrp5 treatment or Wnt5a-induced Vegfa165b expression. Vegfa165b mRNA expression was measured by qRT-PCR. SP600125 was added 10 minutes prior to the addition of recombinant Wnt5a (500 ng/ml). Recombinant Sfrp5 was added at 1 μg/ml. Results are shown as the mean ± S.E. (n = 3/group). ANOVA with post-hoc Tukey HSD. e. SC35 protein expression was measured by western blot analysis in the presence or absence of stimulation with recombinant Wnt5a. The effects of the JNK inhibitor SP600125 or Sfrp5 on Wnt5a-induced SC35 expression were evaluated at 1 hour. Relative levels of SC35 were quantified by using ImageJ. Immunoblots were normalized to LaminA/C signal. Results are shown as the mean ± S.E. (n = 3/group). ANOVA with post-hoc Tukey HSD. f. SC35 is essential for Vegfa165b expression. Vegfa165b mRNA expression was measured by qRT-PCR. Cells were treated with sh-control and sh-SC35 for 8 hours and treated with or without recombinant Wnt5a. ANOVA with post-hoc Tukey HSD. g. Graphic scheme of proposed mechanism. *P < 0.05, **P < 0.01, ***P < 0.001.
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Figure 4: Wnt5a regulate VEGF-A165b expression via Ror2/JNK/SC35-dependent mechanism in macrophagesa. VEGF-A165b protein expression was measured by western immunoblot analysis in the presence or absence of recombinant Wnt5a in cultured RAW264.7 cells after treatment for 24 hours. b.Vegfa165b mRNA expression was measured by qRT-PCR 8 hours after treatment with Wnt5a (500 μg/ml) or vehicle in RAW264.7 cells that had been pre-incubated with vehicle, sh-control or sh-Ror2. ANOVA with post-hoc Tukey HSD. c. Phosphorylation of JNK (p-JNK) was measured by western blot analysis in cells treated with or without recombinant Wnt5a for 30 minutes. Relative phosphorylation levels of JNK were quantified by using ImageJ. Results are shown as the mean ± S.E. (n = 3/group). ANOVA with post-hoc Tukey HSD. d. The effects of JNK inhibition and Sfrp5 treatment or Wnt5a-induced Vegfa165b expression. Vegfa165b mRNA expression was measured by qRT-PCR. SP600125 was added 10 minutes prior to the addition of recombinant Wnt5a (500 ng/ml). Recombinant Sfrp5 was added at 1 μg/ml. Results are shown as the mean ± S.E. (n = 3/group). ANOVA with post-hoc Tukey HSD. e. SC35 protein expression was measured by western blot analysis in the presence or absence of stimulation with recombinant Wnt5a. The effects of the JNK inhibitor SP600125 or Sfrp5 on Wnt5a-induced SC35 expression were evaluated at 1 hour. Relative levels of SC35 were quantified by using ImageJ. Immunoblots were normalized to LaminA/C signal. Results are shown as the mean ± S.E. (n = 3/group). ANOVA with post-hoc Tukey HSD. f. SC35 is essential for Vegfa165b expression. Vegfa165b mRNA expression was measured by qRT-PCR. Cells were treated with sh-control and sh-SC35 for 8 hours and treated with or without recombinant Wnt5a. ANOVA with post-hoc Tukey HSD. g. Graphic scheme of proposed mechanism. *P < 0.05, **P < 0.01, ***P < 0.001.
Mentions: To examine the regulation of VEGF-A165b at a mechanistic level, cultured RAW264.7 cells, a mouse macrophage line, were treated with recombinant Wnt5a. Wnt5a-stimulation led to a dose-dependent increase in VEGF-A165b (Fig. 4a), and the upregulation of this Vegfa splice variant could also be detected at the level of mRNA (Fig. 4b). Consistent with the finding that Wnt5a signals in a non-canonical manner,26 knockdown of Ror2, a Frizzled co-receptor protein, abolished Wnt5a-mediated Vegfa165b induction (Fig. 4b). Also consistent with a non-canonical signaling mechanism, Wnt5a treatment led to the activation of JNK (Fig. 4c), and pretreatment with the JNK inhibitor SP600125 abolished Wnt5a-mediated Vegfa165b expression (Fig. 4d). Similarly, treatment with recombinant Sfrp5 protein blocked VEGF-A165b expression under these conditions. Further investigation revealed that Wnt5a upregulated the expression of SC35, a pre-mRNA splicing factor implicated in the alternative processing of Vegfa at exon 8,27 in a JNK- and Sfrp5-dependent manner (Fig. 4e). Knockdown of SC35 blocked the Wnt5a-mediated induction of Vegfa165b (Fig. 4f). Collectively, these data suggest that Wnt5a promotes the processing of Vegfa to the exon 8b-containing isoform, at least in part, via an Ror2/JNK/SC35-dependent mechanism (Fig. 4g).

Bottom Line: Here we show that clinical PAD is associated with elevated levels of an antiangiogenic VEGF-A splice isoform (VEGF-A165b) and a corresponding reduction in levels of the proangiogenic VEGF-A165a splice isoform.In mice, VEGF-A165b expression was upregulated by conditions associated with impaired limb revascularization, including leptin deficiency, diet-induced obesity, genetic ablation of the secreted frizzled-related protein 5 (Sfrp5) adipokine and transgenic overexpression of Wnt5a in myeloid cells.These results indicate that inflammation-driven expression of the antiangiogenic VEGF-A isoform can contribute to impaired collateralization in ischemic cardiovascular disease.

View Article: PubMed Central - PubMed

Affiliation: 1] Molecular Cardiology and Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, Massachusetts, USA. [2] Department of Medical Technique, Nagoya University Hospital, Nagoya, Aichi, Japan.

ABSTRACT
Peripheral artery disease (PAD) generates tissue ischemia through arterial occlusions and insufficient collateral vessel formation. Vascular insufficiency in PAD occurs despite higher circulating levels of vascular endothelial growth factor A (VEGF-A), a key regulator of angiogenesis. Here we show that clinical PAD is associated with elevated levels of an antiangiogenic VEGF-A splice isoform (VEGF-A165b) and a corresponding reduction in levels of the proangiogenic VEGF-A165a splice isoform. In mice, VEGF-A165b expression was upregulated by conditions associated with impaired limb revascularization, including leptin deficiency, diet-induced obesity, genetic ablation of the secreted frizzled-related protein 5 (Sfrp5) adipokine and transgenic overexpression of Wnt5a in myeloid cells. In a mouse model of PAD, delivery of VEGF-A165b inhibited revascularization of ischemic hind limbs, whereas treatment with an isoform-specific neutralizing antibody reversed impaired revascularization caused by metabolic dysfunction or perturbations in the Wnt5a-Sfrp5 regulatory system. These results indicate that inflammation-driven expression of the antiangiogenic VEGF-A isoform can contribute to impaired collateralization in ischemic cardiovascular disease.

Show MeSH
Related in: MedlinePlus