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Apoptosis of bone marrow mesenchymal stem cells caused by homocysteine via activating JNK signal.

Cai B, Li X, Wang Y, Liu Y, Yang F, Chen H, Yin K, Tan X, Zhu J, Pan Z, Wang B, Lu Y - PLoS ONE (2013)

Bottom Line: Apoptosis of BMSCs in response to various pathological stimuli leads to the attenuation of healing ability of BMSCs.Furthermore, apoptotic appearance and mitochondrial membrane potential depolarization in homocysteine-treated BMSCs was significantly reversed by JNK inhibitor but not p38 MAPK and ERK inhibitors.Homocysteine treatment caused a significant reduction of BMSCs-secreted VEGF and IGF-1 in the culture medium.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, State-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Harbin Medical University, Harbin, Heilongjiang Province, China.

ABSTRACT
Bone marrow mesenchymal stem cells (BMSCs) are capable of homing to and repair damaged myocardial tissues. Apoptosis of BMSCs in response to various pathological stimuli leads to the attenuation of healing ability of BMSCs. Plenty of evidence has shown that elevated homocysteine level is a novel independent risk factor of cardiovascular diseases. The present study was aimed to investigate whether homocysteine may induce apoptosis of BMSCs and its underlying mechanisms. Here we uncovered that homocysteine significantly inhibited the cellular viability of BMSCs. Furthermore, TUNEL, AO/EB, Hoechst 333342 and Live/Death staining demonstrated the apoptotic morphological appearance of BMSCs after homocysteine treatment. A distinct increase of ROS level was also observed in homocysteine-treated BMSCs. The blockage of ROS by DMTU and NAC prevented the apoptosis of BMSCs induced by homocysteine, indicating ROS was involved in the apoptosis of BMSCs. Moreover, homocysteine also caused the depolarization of mitochondrial membrane potential of BMSCs. Furthermore, apoptotic appearance and mitochondrial membrane potential depolarization in homocysteine-treated BMSCs was significantly reversed by JNK inhibitor but not p38 MAPK and ERK inhibitors. Western blot also confirmed that p-JNK was significantly activated after exposing BMSCs to homocysteine. Homocysteine treatment caused a significant reduction of BMSCs-secreted VEGF and IGF-1 in the culture medium. Collectively, elevated homocysteine induced the apoptosis of BMSCs via ROS-induced the activation of JNK signal, which provides more insight into the molecular mechanisms of hyperhomocysteinemia-related cardiovascular diseases.

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Homocysteine induced apoptotic cellular changes of BMSCs.(a) AO/EB double staining demonstrated the effects of difference concentrations of homocysteine on the apoptosis of BMSCs. BMSCs were incubated with homocysteine for 24 h. (b) Hoeschest33342 staining detected the changes in the nucleus of BMSCs after treatment with homocysteine 30, 100 and 300 µM (scale bar, 20 µm). (c, e) The inhibitory effects of homocysteine on BMSCs were determined by Live/Dead staining. (d, f) TUNEL was used to determine the effects of homocysteine on BMSCs apoptosis (n = 3 independent experiments). * p<0.05 versus Control.
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pone-0063561-g002: Homocysteine induced apoptotic cellular changes of BMSCs.(a) AO/EB double staining demonstrated the effects of difference concentrations of homocysteine on the apoptosis of BMSCs. BMSCs were incubated with homocysteine for 24 h. (b) Hoeschest33342 staining detected the changes in the nucleus of BMSCs after treatment with homocysteine 30, 100 and 300 µM (scale bar, 20 µm). (c, e) The inhibitory effects of homocysteine on BMSCs were determined by Live/Dead staining. (d, f) TUNEL was used to determine the effects of homocysteine on BMSCs apoptosis (n = 3 independent experiments). * p<0.05 versus Control.

Mentions: As displayed in Figure 2a, AO/EB double staining demonstrated that treatment with homocysteine 100 and 300 µM for 24 h induced apoptosis of BMSCs characterized by the distinctive red-orange fluorescence. Hoechest33342 staining also showed that BMSCs after exposing to different concentrations of homocysteine for 24 h displayed apoptotic morphological changes such as nucleus condensation (Figure 2b). Likewise, Live/Dead staining also showed that the percentage of staining-positive BMSCs was significantly increased from 5.5% to 28.3% and 48.7% after incubation with homocysteine 100 and 300 µM for 24 h, respectively (Figure 2c, e). We also performed TUNEL assay to observe if homocysteine induced BMSCs apoptosis. As shown in Figure 2d, treatment with homocysteine 100 and 30 0µM for 24 h increased the positive apoptotic cell percentage from 2.3% to 19.8% and 41.4% in BMSCs, respectively (p<0.05, n = 3 independent experiments, Figure 2f). These studies suggest that homocysteine plays a proapoptotic role in BMSCs.


Apoptosis of bone marrow mesenchymal stem cells caused by homocysteine via activating JNK signal.

Cai B, Li X, Wang Y, Liu Y, Yang F, Chen H, Yin K, Tan X, Zhu J, Pan Z, Wang B, Lu Y - PLoS ONE (2013)

Homocysteine induced apoptotic cellular changes of BMSCs.(a) AO/EB double staining demonstrated the effects of difference concentrations of homocysteine on the apoptosis of BMSCs. BMSCs were incubated with homocysteine for 24 h. (b) Hoeschest33342 staining detected the changes in the nucleus of BMSCs after treatment with homocysteine 30, 100 and 300 µM (scale bar, 20 µm). (c, e) The inhibitory effects of homocysteine on BMSCs were determined by Live/Dead staining. (d, f) TUNEL was used to determine the effects of homocysteine on BMSCs apoptosis (n = 3 independent experiments). * p<0.05 versus Control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3646804&req=5

pone-0063561-g002: Homocysteine induced apoptotic cellular changes of BMSCs.(a) AO/EB double staining demonstrated the effects of difference concentrations of homocysteine on the apoptosis of BMSCs. BMSCs were incubated with homocysteine for 24 h. (b) Hoeschest33342 staining detected the changes in the nucleus of BMSCs after treatment with homocysteine 30, 100 and 300 µM (scale bar, 20 µm). (c, e) The inhibitory effects of homocysteine on BMSCs were determined by Live/Dead staining. (d, f) TUNEL was used to determine the effects of homocysteine on BMSCs apoptosis (n = 3 independent experiments). * p<0.05 versus Control.
Mentions: As displayed in Figure 2a, AO/EB double staining demonstrated that treatment with homocysteine 100 and 300 µM for 24 h induced apoptosis of BMSCs characterized by the distinctive red-orange fluorescence. Hoechest33342 staining also showed that BMSCs after exposing to different concentrations of homocysteine for 24 h displayed apoptotic morphological changes such as nucleus condensation (Figure 2b). Likewise, Live/Dead staining also showed that the percentage of staining-positive BMSCs was significantly increased from 5.5% to 28.3% and 48.7% after incubation with homocysteine 100 and 300 µM for 24 h, respectively (Figure 2c, e). We also performed TUNEL assay to observe if homocysteine induced BMSCs apoptosis. As shown in Figure 2d, treatment with homocysteine 100 and 30 0µM for 24 h increased the positive apoptotic cell percentage from 2.3% to 19.8% and 41.4% in BMSCs, respectively (p<0.05, n = 3 independent experiments, Figure 2f). These studies suggest that homocysteine plays a proapoptotic role in BMSCs.

Bottom Line: Apoptosis of BMSCs in response to various pathological stimuli leads to the attenuation of healing ability of BMSCs.Furthermore, apoptotic appearance and mitochondrial membrane potential depolarization in homocysteine-treated BMSCs was significantly reversed by JNK inhibitor but not p38 MAPK and ERK inhibitors.Homocysteine treatment caused a significant reduction of BMSCs-secreted VEGF and IGF-1 in the culture medium.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, State-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Harbin Medical University, Harbin, Heilongjiang Province, China.

ABSTRACT
Bone marrow mesenchymal stem cells (BMSCs) are capable of homing to and repair damaged myocardial tissues. Apoptosis of BMSCs in response to various pathological stimuli leads to the attenuation of healing ability of BMSCs. Plenty of evidence has shown that elevated homocysteine level is a novel independent risk factor of cardiovascular diseases. The present study was aimed to investigate whether homocysteine may induce apoptosis of BMSCs and its underlying mechanisms. Here we uncovered that homocysteine significantly inhibited the cellular viability of BMSCs. Furthermore, TUNEL, AO/EB, Hoechst 333342 and Live/Death staining demonstrated the apoptotic morphological appearance of BMSCs after homocysteine treatment. A distinct increase of ROS level was also observed in homocysteine-treated BMSCs. The blockage of ROS by DMTU and NAC prevented the apoptosis of BMSCs induced by homocysteine, indicating ROS was involved in the apoptosis of BMSCs. Moreover, homocysteine also caused the depolarization of mitochondrial membrane potential of BMSCs. Furthermore, apoptotic appearance and mitochondrial membrane potential depolarization in homocysteine-treated BMSCs was significantly reversed by JNK inhibitor but not p38 MAPK and ERK inhibitors. Western blot also confirmed that p-JNK was significantly activated after exposing BMSCs to homocysteine. Homocysteine treatment caused a significant reduction of BMSCs-secreted VEGF and IGF-1 in the culture medium. Collectively, elevated homocysteine induced the apoptosis of BMSCs via ROS-induced the activation of JNK signal, which provides more insight into the molecular mechanisms of hyperhomocysteinemia-related cardiovascular diseases.

Show MeSH
Related in: MedlinePlus