Valproic Acid Induces Endothelial-to-Mesenchymal Transition-Like Phenotypic Switching - PubMed (original) (raw)

doi: 10.3389/fphar.2018.00737. eCollection 2018.

Antoinette Bugyei-Twum 2 3, Pratiek N Matkar 2 3, Husain Al-Mubarak 3, Hao H Chen 2 3, Mohamed Adam 2 3, Shubha Jain 4, Tanya Narang 1, Rawand M Abdin 5, Mohammad Qadura 3 4, Kim A Connelly 2 3, Howard Leong-Poi 2 3, Krishna K Singh 3 4 6 7 8

Affiliations

Valproic Acid Induces Endothelial-to-Mesenchymal Transition-Like Phenotypic Switching

Shamini Murugavel et al. Front Pharmacol. 2018.

Abstract

Valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, is a widely used anticonvulsant drug that is currently undergoing clinical evaluation for anticancer therapy due to its anti-angiogenic potential. Endothelial cells (ECs) can transition into mesenchymal cells and this form of EC plasticity is called endothelial-to-mesenchymal transition (EndMT), which is widely implicated in several pathologies including cancer and organ fibrosis. However, the effect of VPA on EC plasticity and EndMT remains completely unknown. We report herein that VPA-treatment significantly inhibits tube formation, migration, nitric oxide production, proliferation and migration in ECs. A microscopic evaluation revealed, and qPCR, immunofluorescence and immunoblotting data confirmed EndMT-like phenotypic switching as well as an increased expression of pro-fibrotic genes in VPA-treated ECs. Furthermore, our data confirmed important and regulatory role played by TGFβ-signaling in VPA-induced EndMT. Our qPCR array data performed for 84 endothelial genes further supported our findings and demonstrated 28 significantly and differentially regulated genes mainly implicated in angiogenesis, endothelial function, EndMT and fibrosis. We, for the first time report that VPA-treatment associated EndMT contributes to the VPA-associated loss of endothelial function. Our data also suggest that VPA based therapeutics may exacerbate endothelial dysfunction and EndMT-related phenotype in patients undergoing anticonvulsant or anticancer therapy, warranting further investigation.

Keywords: endothelial cell; endothelial dysfunction; endothelial-to-mesenchymal transition; fibrosis; valproic acid.

PubMed Disclaimer

Figures

FIGURE 1

FIGURE 1

Valproic acid (VPA) impairs angiogenesis and negatively regulates eNOS/AKT expression and nitric oxide production in HUVECs. (A) Cultured HUVECs were transferred to a Matrigel-coated plate (BD Biosciences, San Jose, CA, United States) and treated with VPA or its diluent. Representative micrographs showing capillary-like tube formation in HUVECs 5 h after treatment with VPA. Tubular structures in 4 fields per group were semi-quantitatively analyzed. N = 4–5 in triplicate. ∗∗p < 0.01 vs. control group. (B) Bar graph demonstrating the migratory potential of cultured HUVECs after 12 h of VPA treatment. N = 4 in triplicate. ∗p < 0.05 vs. control group. (C) qPCR for eNOS and (D) immunoblot for eNOS, phospho (p)eNOS and quantification of eNOS on total RNA and protein extracted from HUVECs after 24 h of VPA treatment. N = 3 in triplicate. ∗p < 0.05, ∗∗∗p < 0.001 vs. control group. GAPDH was used as a loading control for immunoblot and internal control for qPCR. (E) Immunoblot for AKT, (p)AKT and quantification for AKT on total protein extracted from HUVECs after 24 h of VPA treatment. N = 3 in triplicate. ∗p < 0.05 vs. control group. (F) Bar graph demonstrating the nitric oxide production in cultured HUVECs after 6 h of VPA treatment. N = 10 in triplicate. ∗p < 0.05 vs. control group.

FIGURE 2

FIGURE 2

Valproic acid causes marked morphological and ultrastructural changes, and promotes EndMT-like phenotypic switching in HUVECs. (A) Diluent-treated control HUVECs, cultured on a two-dimensional plate, formed a confluent monolayer with the typical EC ‘cobblestone’ morphology (left panel). VPA treatment resulted in marked morphological changes whereby HUVECs took on an enlarged spindle-shaped appearance with smooth surfaces (right panel). Both micrographs were taken at the same magnification (10X). (B) Immunofluorescent micrographs demonstrating cytoskeletal protein re-organization in HUVECs following VPA treatment. α-Actinin positivity is indicated in green and nuclei were stained with DAPI (blue); scale bar = 10 μm. (C) HUVECs were treated with diluent control or VPA. Total RNA and protein were extracted at 24 and 48 h, respectively. Differential (C) transcript (qPCR) ∗∗p < 0.01, ∗∗∗p < 0.001 and (D) protein (immunoblotting) levels of key endothelial and mesenchymal markers as well as (E) VE-Cadherin and _N_-Cadherin immunofluorescent staining in control- and VPA-treated HUVECs indicate EndMT with VPA treatment. Nuclei were stained with DAPI (blue). Micrographs are representative images of HUVECs taken 48 h post-treatment; scale bar = 20 μm.

FIGURE 3

FIGURE 3

Valproic acid-associated EndMT is not specific to HUVECs. Cultured (A) HCAECs and (B) HMVECs were treated with 5 mM of VPA for 24 h and total RNA was extracted to perform the qPCR for EndMT markers. ∗∗p < 0.01, ∗∗∗p < 0.001 vs. corresponding control group. (C) Cultured HUVECs were treated with 5 mM of VPA for 24 h and total RNA was extracted to perform the qPCR for Snail1. ∗∗p < 0.01 vs. corresponding control group.

FIGURE 4

FIGURE 4

Valproic acid-associated EndMT occurs via a Tgfβ-dependent signaling pathways. HUVECs were treated with either diluent or VPA and total RNA and protein were extracted 24 h post-treatment to measure the TGFβ1 transcript (A) and phosphorylation of SMAD2/3/5 (B). These changes were accompanied by up-regulation of (C) CTGF and (D) collagen I transcript levels ∗p < 0.05 and ∗∗∗p < 0.001. (E) VPA also induced SLUG expression in VPA-treated HUVECs. Cultured HUVECs were pre-treated with TGFβ-inhibitor SIS3 and then with VPA for 24 h. Later protein and RNA were extracted to confirm the TGFβ-inhibition via measuring pSMAD3 protein levels (F) and qPCR for EndMT marker levels (G). In the bargraph the SIS3 + VPA group is shown by beaded bargraph. N = 3 in triplicates. ∗p < 0.05 vs. corresponding VPA group.

FIGURE 5

FIGURE 5

Valproic acid inhibits cell proliferation and up-regulates cyclin-dependent kinase inhibitor p21 expression in HUVECs. (A) Cultured HUVECs were treated with diluent or VPA and cell proliferation was evaluated at 0, 24, and 48 h post-VPA treatment. N = 6 in triplicates. ∗∗∗p < 0.001 vs. corresponding control group. (B) Bar graph representing the qPCR data for p21 performed on total RNA extracted from cultured HUVECs after 24 h of diluent or VPA treatment. N = 3 in triplicates. ∗∗∗p < 0.001 vs. corresponding control group.

References

    1. Arciniegas E., Sutton A. B., Allen T. D., Schor A. M. (1992). Transforming growth factor beta 1 promotes the differentiation of endothelial cells into smooth muscle-like cells in vitro. J. Cell Sci. 103(Pt 2) 521–529. - PubMed
    1. Atisha D., Bhalla M. A., Morrison L. K., Felicio L., Clopton P., Gardetto N., et al. (2004). A prospective study in search of an optimal B-natriuretic peptide level to screen patients for cardiac dysfunction. Am. Heart J. 148 518–523. 10.1016/j.ahj.2004.03.014 - DOI - PubMed
    1. Bai H., Gao Y., Hoyle D. L., Cheng T., Wang Z. Z. (2017). Suppression of transforming growth factor-beta signaling delays cellular senescence and preserves the function of endothelial cells derived from human pluripotent stem cells. Stem Cells Transl. Med. 6 589–600. 10.5966/sctm.2016-0089 - DOI - PMC - PubMed
    1. Bergers G., Benjamin L. E. (2003). Tumorigenesis and the angiogenic switch. Nat. Rev. Cancer 3 401–410. 10.1038/nrc1093 - DOI - PubMed
    1. Bezecny P. (2014). Histone deacetylase inhibitors in glioblastoma: pre-clinical and clinical experience. Med. Oncol. 31:985. 10.1007/s12032-014-0985-5 - DOI - PubMed

LinkOut - more resources