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Attenuation of choroidal neovascularization by histone deacetylase inhibitor.

Chan N, He S, Spee CK, Ishikawa K, Hinton DR - PLoS ONE (2015)

Bottom Line: Most strikingly, TSA markedly down-regulates the expression of VEGF receptor-2 in human vascular endothelial cells and, thus, can knock down pro-angiogenic cell signaling.Additionally, TSA suppresses CNV-associated wound healing response and RPE epithelial-mesenchymal transdifferentiation.This report suggests that TSA, and possibly HDACi's in general, should be further evaluated for their therapeutic potential for the treatment of CNV.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Keck School of Medicine of the University of Southern California, Los Angeles, CA, United States of America; Doheny Eye Institute, Los Angeles, CA, United States of America.

ABSTRACT
Choroidal neovascularization (CNV) is a blinding complication of age-related macular degeneration that manifests as the growth of immature choroidal blood vessels through Bruch's membrane, where they can leak fluid or hemorrhage under the retina. Here, we demonstrate that the histone deacetylase inhibitor (HDACi) trichostatin A (TSA) can down-regulate the pro-angiogenic hypoxia-inducible factor-1α and vascular endothelial growth factor (VEGF), and up-regulate the anti-angiogenic and neuro-protective pigment epithelium derived factor in human retinal pigment epithelial (RPE) cells. Most strikingly, TSA markedly down-regulates the expression of VEGF receptor-2 in human vascular endothelial cells and, thus, can knock down pro-angiogenic cell signaling. Additionally, TSA suppresses CNV-associated wound healing response and RPE epithelial-mesenchymal transdifferentiation. In the laser-induced model of CNV using C57Bl/6 mice, systemic administration of TSA significantly reduces fluorescein leakage and the size of CNV lesions at post-laser days 7 and 14 as well as the immunohistochemical expression of VEGF, VEGFR2, and smooth muscle actin in CNV lesions at post-laser day 7. This report suggests that TSA, and possibly HDACi's in general, should be further evaluated for their therapeutic potential for the treatment of CNV.

No MeSH data available.


Related in: MedlinePlus

TSA inhibits VEGFR2 in BCECs and HUVECs.Real-time PCR (A) and Western blot (B) were performed on BCECs (left panel) and HUVECs (right panel) treated with 0–0.7 μM TSA for 24h or 48 h. (A-B) TSA exerts a dose-dependent reduction effect on the mRNA and protein levels of VEGFR2. (C) Densitometry data for Western blot result of VEGFR2 normalized by GAPDH levels for BCECs (left panel) and HUVECs (right panel). (*: t test p<0.05, **: t test p<0.01) (D) HUVECs were treated with 0–0.7 μM TSA for 48 h, and then stimulated with 25 ng/mL of human recombinant VEGF for 10 min, followed by Western blot analysis (left panel). VEGF significantly induces the phosphorylation of VEGFR2, but the phosphorylation was attenuated by TSA at 0.7 μM, concomitant with a down-regulation of VEGFR2 total protein level. Densitometry data (right panel) for Western blot result of VEGFR2 and phospho-VEGFR2 normalized by GAPDH levels. (*: t test p<0.05) (E) ChIP assay was performed as described in Fig. 3 on HUVECs treated with 0.5 μM TSA for 48 h. Released chromatin was then amplified by PCR using primers targeting VEGFR2 encompassing the region from 200 bp upstream of the transcription start site to 200 bp downstream of the transcription start site. Amplified chromatin was then run on a 1% agarose gel. Less promoter opening was found in TSA-treated cells than in untreated cells. Densitometry of ChIP assay result normalized by input levels. (*: t test p<0.05).
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pone.0120587.g010: TSA inhibits VEGFR2 in BCECs and HUVECs.Real-time PCR (A) and Western blot (B) were performed on BCECs (left panel) and HUVECs (right panel) treated with 0–0.7 μM TSA for 24h or 48 h. (A-B) TSA exerts a dose-dependent reduction effect on the mRNA and protein levels of VEGFR2. (C) Densitometry data for Western blot result of VEGFR2 normalized by GAPDH levels for BCECs (left panel) and HUVECs (right panel). (*: t test p<0.05, **: t test p<0.01) (D) HUVECs were treated with 0–0.7 μM TSA for 48 h, and then stimulated with 25 ng/mL of human recombinant VEGF for 10 min, followed by Western blot analysis (left panel). VEGF significantly induces the phosphorylation of VEGFR2, but the phosphorylation was attenuated by TSA at 0.7 μM, concomitant with a down-regulation of VEGFR2 total protein level. Densitometry data (right panel) for Western blot result of VEGFR2 and phospho-VEGFR2 normalized by GAPDH levels. (*: t test p<0.05) (E) ChIP assay was performed as described in Fig. 3 on HUVECs treated with 0.5 μM TSA for 48 h. Released chromatin was then amplified by PCR using primers targeting VEGFR2 encompassing the region from 200 bp upstream of the transcription start site to 200 bp downstream of the transcription start site. Amplified chromatin was then run on a 1% agarose gel. Less promoter opening was found in TSA-treated cells than in untreated cells. Densitometry of ChIP assay result normalized by input levels. (*: t test p<0.05).

Mentions: To study whether TSA can inhibit VEGFR2 expression, BCECs were treated in EBM containing 1% FBS with 0–0.7 μM TSA for 24 h, and the cell lysates were analyzed by real-time PCR and Western blot. TSA down-regulated VEGFR2 mRNA and protein expression in a dose-dependent manner; both isoforms were prominently down-regulated in the same decreasing pattern in Western blot. (Fig. 10A-C, left panels; t test: *: p<0.05, **: p<0.01; ANOVA: real-time PCR: p<0.01, Western blot: p<0.0001) Interestingly, the mature, higher molecular weight VEGFR2 was the dominant isoform expressed in BCECs. (Fig. 10B) However, the supply of BCECs was limited, and thus the effect of TSA on VEGFR2 was also examined in HUVECs. HUVECs were treated in EBM containing 1% FBS with 0–0.7 μM TSA for 48 h, and the cell lysates were analyzed by real-time PCR and Western blot. A similar dose-dependently reduced expression in VEGFR2 mRNA and protein levels was also observed in HUVECs. (Fig. 10A-C, right panels; t test: p<0.05; ANOVA; real-time PCR: p<0.01, Western blot: p<0.01)


Attenuation of choroidal neovascularization by histone deacetylase inhibitor.

Chan N, He S, Spee CK, Ishikawa K, Hinton DR - PLoS ONE (2015)

TSA inhibits VEGFR2 in BCECs and HUVECs.Real-time PCR (A) and Western blot (B) were performed on BCECs (left panel) and HUVECs (right panel) treated with 0–0.7 μM TSA for 24h or 48 h. (A-B) TSA exerts a dose-dependent reduction effect on the mRNA and protein levels of VEGFR2. (C) Densitometry data for Western blot result of VEGFR2 normalized by GAPDH levels for BCECs (left panel) and HUVECs (right panel). (*: t test p<0.05, **: t test p<0.01) (D) HUVECs were treated with 0–0.7 μM TSA for 48 h, and then stimulated with 25 ng/mL of human recombinant VEGF for 10 min, followed by Western blot analysis (left panel). VEGF significantly induces the phosphorylation of VEGFR2, but the phosphorylation was attenuated by TSA at 0.7 μM, concomitant with a down-regulation of VEGFR2 total protein level. Densitometry data (right panel) for Western blot result of VEGFR2 and phospho-VEGFR2 normalized by GAPDH levels. (*: t test p<0.05) (E) ChIP assay was performed as described in Fig. 3 on HUVECs treated with 0.5 μM TSA for 48 h. Released chromatin was then amplified by PCR using primers targeting VEGFR2 encompassing the region from 200 bp upstream of the transcription start site to 200 bp downstream of the transcription start site. Amplified chromatin was then run on a 1% agarose gel. Less promoter opening was found in TSA-treated cells than in untreated cells. Densitometry of ChIP assay result normalized by input levels. (*: t test p<0.05).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
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pone.0120587.g010: TSA inhibits VEGFR2 in BCECs and HUVECs.Real-time PCR (A) and Western blot (B) were performed on BCECs (left panel) and HUVECs (right panel) treated with 0–0.7 μM TSA for 24h or 48 h. (A-B) TSA exerts a dose-dependent reduction effect on the mRNA and protein levels of VEGFR2. (C) Densitometry data for Western blot result of VEGFR2 normalized by GAPDH levels for BCECs (left panel) and HUVECs (right panel). (*: t test p<0.05, **: t test p<0.01) (D) HUVECs were treated with 0–0.7 μM TSA for 48 h, and then stimulated with 25 ng/mL of human recombinant VEGF for 10 min, followed by Western blot analysis (left panel). VEGF significantly induces the phosphorylation of VEGFR2, but the phosphorylation was attenuated by TSA at 0.7 μM, concomitant with a down-regulation of VEGFR2 total protein level. Densitometry data (right panel) for Western blot result of VEGFR2 and phospho-VEGFR2 normalized by GAPDH levels. (*: t test p<0.05) (E) ChIP assay was performed as described in Fig. 3 on HUVECs treated with 0.5 μM TSA for 48 h. Released chromatin was then amplified by PCR using primers targeting VEGFR2 encompassing the region from 200 bp upstream of the transcription start site to 200 bp downstream of the transcription start site. Amplified chromatin was then run on a 1% agarose gel. Less promoter opening was found in TSA-treated cells than in untreated cells. Densitometry of ChIP assay result normalized by input levels. (*: t test p<0.05).
Mentions: To study whether TSA can inhibit VEGFR2 expression, BCECs were treated in EBM containing 1% FBS with 0–0.7 μM TSA for 24 h, and the cell lysates were analyzed by real-time PCR and Western blot. TSA down-regulated VEGFR2 mRNA and protein expression in a dose-dependent manner; both isoforms were prominently down-regulated in the same decreasing pattern in Western blot. (Fig. 10A-C, left panels; t test: *: p<0.05, **: p<0.01; ANOVA: real-time PCR: p<0.01, Western blot: p<0.0001) Interestingly, the mature, higher molecular weight VEGFR2 was the dominant isoform expressed in BCECs. (Fig. 10B) However, the supply of BCECs was limited, and thus the effect of TSA on VEGFR2 was also examined in HUVECs. HUVECs were treated in EBM containing 1% FBS with 0–0.7 μM TSA for 48 h, and the cell lysates were analyzed by real-time PCR and Western blot. A similar dose-dependently reduced expression in VEGFR2 mRNA and protein levels was also observed in HUVECs. (Fig. 10A-C, right panels; t test: p<0.05; ANOVA; real-time PCR: p<0.01, Western blot: p<0.01)

Bottom Line: Most strikingly, TSA markedly down-regulates the expression of VEGF receptor-2 in human vascular endothelial cells and, thus, can knock down pro-angiogenic cell signaling.Additionally, TSA suppresses CNV-associated wound healing response and RPE epithelial-mesenchymal transdifferentiation.This report suggests that TSA, and possibly HDACi's in general, should be further evaluated for their therapeutic potential for the treatment of CNV.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Keck School of Medicine of the University of Southern California, Los Angeles, CA, United States of America; Doheny Eye Institute, Los Angeles, CA, United States of America.

ABSTRACT
Choroidal neovascularization (CNV) is a blinding complication of age-related macular degeneration that manifests as the growth of immature choroidal blood vessels through Bruch's membrane, where they can leak fluid or hemorrhage under the retina. Here, we demonstrate that the histone deacetylase inhibitor (HDACi) trichostatin A (TSA) can down-regulate the pro-angiogenic hypoxia-inducible factor-1α and vascular endothelial growth factor (VEGF), and up-regulate the anti-angiogenic and neuro-protective pigment epithelium derived factor in human retinal pigment epithelial (RPE) cells. Most strikingly, TSA markedly down-regulates the expression of VEGF receptor-2 in human vascular endothelial cells and, thus, can knock down pro-angiogenic cell signaling. Additionally, TSA suppresses CNV-associated wound healing response and RPE epithelial-mesenchymal transdifferentiation. In the laser-induced model of CNV using C57Bl/6 mice, systemic administration of TSA significantly reduces fluorescein leakage and the size of CNV lesions at post-laser days 7 and 14 as well as the immunohistochemical expression of VEGF, VEGFR2, and smooth muscle actin in CNV lesions at post-laser day 7. This report suggests that TSA, and possibly HDACi's in general, should be further evaluated for their therapeutic potential for the treatment of CNV.

No MeSH data available.


Related in: MedlinePlus