Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 15 (6), 3644-3650

Palmitate Impairs Angiogenesis via Suppression of Cathepsin Activity

Affiliations

Palmitate Impairs Angiogenesis via Suppression of Cathepsin Activity

Jin Zhang et al. Mol Med Rep.

Abstract

Angiogenesis requires the interaction of multiple variable factors to promote endothelial cell adhesion, migration and survival. Palmitate, a free fatty acid, exhibits an anti‑angiogenic effect via interference with endothelial cell function, whereas cysteine proteases are important in protein turnover and are termed positive modulators of neovascularization. However, the association between these two factors regarding the regulation of human endothelial cell function remains to be elucidated. By using cell counting kit‑8, the Transwell method and an annexin V‑fluorescein isothiocyanate/propidium iodide apoptosis detection kit, the present study reported that high levels of palmitate result in a significant decrease in endothelial cell proliferation and invasion, and induced cell apoptosis; cathepsin L and S inhibitors may suppress palmitate‑induced apoptosis. Conversely, the results of the cathepsin L and S activity assay and reverse-transcription-quantitative polymerase chain reaction indicated that palmitate inhibited cathepsin‑induced endothelial cell invasion, partially via suppressing the expression and activity of cathepsin L and S. The findings of the present study suggested that the potent anti‑angiogenic properties of palmitate may be mediated by cysteine proteases.

Figures

Figure 1.
Figure 1.
Effect of palmitate and cathepsin inhibitors on cell proliferation. (A) Identification of human umbilical vein endothelial cells using fluorescence microscopy. Cells were treated with an anti-CD31 antibody for 2 h followed by a rhodamine-conjugated secondary antibody, indicated in red. Nuclei were stained with DAPI, indicated in blue. Scale bar=100 µM. (B) Cells were suspended in M199/fetal bovine serum medium supplemented with Cell Counting kit-8 solution. Absorbance values were determined at a wavelength of 450 nm. Cells were treated with specific cathepsin inhibitors against (C) cathepsin L and (D) cathepsin S for 1 h prior to exposure to palmitate for 24 h. Data are expressed as the mean ± standard deviation from triplicate experiments. *P<0.001 vs. 0 mM. CD, cluster of differentiation.
Figure 2.
Figure 2.
Effect of palmitate and cathepsin inhibitors on cell apoptosis. Cell apoptosis was analyzed by flow cytometry. (A) A total of 1×106 cells suspended in 200 µl binding buffer containing 2.5 µl annexin V-FITC and 2.5 µl PI were analyzed following incubation in the dark. Percentage of cells in (B) early (lower right quadrant, Annexin V+PI) and (C) late (upper right quandrant, annexin V+PI+) apoptotic stages. Three separate experiments were performed. Data are expressed as the mean ± standard deviation. *P<0.05 vs. 0 mM. FITC, fluorescein isothiocyanate; PI, propidium iodide.
Figure 3.
Figure 3.
Effect of palmitate and cathepsin inhibitors on cell invasion. (A) Images represent alteration of cell invasion as assessed by a Transwell assay. Scale bar=100 µM. (B) Analysis of Transwell assay data. Data are expressed as the mean ± standard deviation from at least three separate experiments. *P<0.05 vs. 0 mM palmitate+medium; P<0.05 vs. 0 mM palmitate+Z-FF-FMK; #P<0.05 vs. 0 mM palmitate+Z-FL-COCHO.H2O.
Figure 4.
Figure 4.
Inhibition of cathepsins S and L by palmitate. (A) Cell lysates were incubated with the fluorogenic substrate detecting cathepsin L or S activity. Fluorescence was measured at an excitation wavelength of 400 nm and an emission wavelength of 505 nm. *P<0.05 vs. 0 mM. Cathepsin L and S and cystatin C mRNA expression levels were (B) detected and (C) quantified using reverse transcription-quantitative polymerase chain reaction. Cathepsin L and S and cystatin C protein expression levels were (D) detected and (E) quantified using western blot. Tubulin served as loading control. Blots are representative of at least 3 independent experiments. Data are presented as the mean ± standard deviation from triplicate experiments. *P<0.05 vs. 0 mM palmitate, cathepsin L; #P<0.05 vs. 0 mM palmitate, cathepsin S; P<0.05 vs. 0 mM palmitate, cystatin C.

Similar articles

See all similar articles

Cited by 1 PubMed Central articles

References

    1. Binet F, Sapieha P. ER stress and angiogenesis. Cell Metab. 2015;22:560–575. doi: 10.1016/j.cmet.2015.07.010. - DOI - PubMed
    1. Pourrajab F, Zarch A Vakili, Hekmatimoghaddam S, Zare-Khormizi MR. MicroRNAs; easy and potent targets in optimizing therapeutic methods in reparative angiogenesis. J Cell Mol Med. 2015;19:2702–2714. doi: 10.1111/jcmm.12669. - DOI - PMC - PubMed
    1. Urbich C, Heeschen C, Aicher A, Sasaki K, Bruhl T, Farhadi MR, Vajkoczy P, Hofmann WK, Peters C, Pennacchio LA, et al. Cathepsin L is required for endothelial progenitor cell-induced neovascularization. Nat Med. 2005;11:206–213. doi: 10.1038/nm1182. - DOI - PubMed
    1. Shi GP, Sukhova GK, Kuzuya M, Ye Q, Du J, Zhang Y, Pan JH, Lu ML, Cheng XW, Iguchi A, et al. Deficiency of the cysteine protease cathepsin S impairs microvessel growth. Circ Res. 2003;92:493–500. doi: 10.1161/01.RES.0000060485.20318.96. - DOI - PubMed
    1. Conus S, Simon HU. Cathepsins: Key modulators of cell death and inflammatory responses. Biochem Pharmacol. 2008;76:1374–1382. doi: 10.1016/j.bcp.2008.07.041. - DOI - PubMed

MeSH terms

Feedback