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Plasminogen K5 activates mitochondrial apoptosis pathway in endothelial cells by regulating Bak and Bcl-xL subcellular distribution

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Abstract

Plasminogen Kringle 5(K5) is a proteolytic fragment of plasminogen, which displays potent anti-angiogenic activities. K5 has been shown to induce apoptosis in proliferating endothelial cells; however the exact mechanism has not been well explored. The present study was designed to elucidate the possible molecular mechanism of K5-induced endothelial cell apoptosis. Our results showed that K5 inhibited basic fibroblast growth factors activated in human umbilical vein endothelial cells (HUVECs), indicating proliferation in a dose-dependent manner and induced endothelial cell death via apoptosis. K5 exposure activated caspase 7, 8 and 9. These results suggested that both the intrinsic mitochondrial apoptosis pathway and extrinsic pathway might be involved in K5-induced apoptosis. K5 reduced mitochondrial membrane potential (MMP) of HUVECs, demonstrating mitochondrial depolarization in HUVECs. K5 increased the ratio of Bak to Bcl-xL on mitochondria, decreased the ratio in cytosol, and had no effect on the total amounts of these proteins. K5 also did not effect on Bax/Bcl-2 distribution. K5 increased the ratio of Bak to Bcl-xL on mitochondrial that resulted in mitochondrial depolarization, cytochrome c release and consequently the cleavage of caspase 9. These results suggested that K5 induces endothelial cell apoptosis at least in part via activating mitochondrial apoptosis pathway. The regulation of K5 on Bak and Bcl-xL distribution may play an important role in endothelial cell apoptosis. These results provide further insight into the anti-angiogenesis roles of K5 in angiogenesis-related ocular diseases and solid tumors.

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Abbreviations

K5:

Human plasminogen kringle 5

HUVECs:

Human umbilical vein endothelial cells

bFGF:

Basic fibroblast growth factor

MMP:

Mitochondrial membrane potential

MOM:

Mitochondrial outer membrane

Cyto c:

Cytochrome c

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Acknowledgments

This study was supported by National Nature Science Foundation of China, Grant Number: 30600724, 30700120, 30872980, 30971208, 30973449, 81070746, 81001014; National Key Sci-Tech Special Project of China, Grant Number: 2008ZX10002-019, 2009ZX09103-642; Key Project of Nature Science Foundation of Guangdong Province, China, Grant Number: 10251008901000009; Guangdong natural science fund, Grant Number: 7002329, 10151008901000007; Program for Young Teacher in University, Grant Number: 09YKPY73, 10YKPY28; Department of health of Guangdong Province medical research fund, Grant Number A2008548; Program for Bureau of Health of Guangzhou Municipality fund, Grant Number: 2007-YB-061.

Conflict of interest

The author has declared that no competing interests exist.

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Authors and Affiliations

  1. Department of Biochemistry, Zhongshan Medical School, Sun Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou, 510080, Guangdong Province, China

    Xiaoqiong Gu, Yachao Yao, Rui Cheng, Yang Zhang, Zhiyu Dai, Zhonghan Yang, Weibin Cai, Guoquan Gao & Xia Yang

  2. Department of Laboratory, Guangzhou Women and Children’s Medical Center, Guangzhou, 510623, Guangdong Province, China

    Xiaoqiong Gu & Genping Wan

  3. China Key Laboratory of Tropical Disease Control (Sun Yat-sen University), Ministry of Education, Guangzhou, 510080, China

    Guoquan Gao

  4. Department of Education of Guangdong Province, Key Laboratory of Functional Molecules from Marine Microorganisms (Sun Yat-sen University), Guangzhou, 510080, Guangdong Province, China

    Xia Yang

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  1. Xiaoqiong Gu
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Correspondence to Guoquan Gao or Xia Yang.

Additional information

X. Gu and Y. Yao have contributed equally to this study.

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10495_2011_618_MOESM1_ESM.tif

Supplement Fig. 1 Effects of caspases inhibitors on the apoptosis of HUVECs induced by K5. a Cells were starved overnight and then treated with K5 at concentration of 640 nmol/l in the presence of 20 ng/ml bFGF and 0.2% FBS for 48 h. The irreversible caspases inhibitors Z-IETD-FMK (caspase8 inhibitor), Z-LEHD-FMK (caspase9 inhibitor), and Z-VAD-FMK (Pan-caspase inhibitor) were used at a final concentration of 10μmol/l dissolved in DMSO and were added 30 min prior to K5 treatment. Cells were stained with Annexin V and PI, respectively, and then quantified by flow cytometry analysis. PBS-treated cells were used as negative control. b All values of statistical analysis represent the mean of three determinations (\( \bar{x} \pm s \),n = 3). (*P<0.05 vs control). The same concentration of 0.1% DMSO as in the caspase inhibitors was added in all other groups to eliminate the interference of DMSO. (TIFF 442 kb)

10495_2011_618_MOESM2_ESM.tif

Supplement Fig. 2 Effect of Z-IETD-FMK on K5-induced distribution of Bak. HUVECs were treated with or without 640 nmol/l K5 and Z-IETD-FMK for 24 h in the presence of 20 ng/ml bFGF and 0.2% FBS. lane1, control; lane 2, treatment by 640 nmol/l K5; lane 3, treatment by Z-IETD-FMK, the inhibitor of caspase8 dissolved in 0.1% DMSO; lane 4, treatment by 10 μmol/l Z-IETD-FMK dissolved in 0.1% DMSO and 640 nmol/l K5. (TIFF 294 kb)

10495_2011_618_MOESM3_ESM.tif

Supplement Fig. 3 Effects of K5 on Bax and Bcl-2 subcellular distribution in HUVECs. a HUVECs were treated with 640nmol/l K5 for 24 h in the presence of 20 ng/ml bFGF and 0.2% FBS. Cells were fractionated to mitochondria and cytosol subfraction. Bax and Bcl-2 proteins in total cell lysate, mitochondria and cytosol subtraction were detected by Western blotting assay. β-actin was loaded as a control to total cell lysate and cytosol subfraction, Cox-IV was loaded as a control to mitochondria subfraction. b Statistics of Bax and Bcl-2 proteins relatively expressed. Signals of each protein were normalized to their control. c Ratios of Bax to Bcl-2 in all kinds of protein were normalized to their control. Each bar in B and C represents a mean±s of three separate experiments. (TIFF 452 kb)

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Gu, X., Yao, Y., Cheng, R. et al. Plasminogen K5 activates mitochondrial apoptosis pathway in endothelial cells by regulating Bak and Bcl-xL subcellular distribution. Apoptosis 16, 846–855 (2011). https://doi.org/10.1007/s10495-011-0618-9

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