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Lovastatin exerts protective effects on endothelial cells via upregulation of PTK2B

View Article: PubMed Central - PubMed

ABSTRACT

Statins are HMG-CoA reductase inhibitors that are used to decrease the blood levels of low-density lipoprotein (LDL). In addition, they have been shown to exert pleiotropic protective effects in the absence of LDL-lowering activity. The present study investigated the effects of lovastatin on global gene expression in human umbilical vein endothelial cells (HUVECs), in order to further explore its ability to protect against oxidized (ox)-LDL-induced cytotoxicity. HUVECs were treated with lovastatin for 2–24 h, and gene expression patterns were analyzed using cDNA microarrays. The results suggested that numerous genes were regulated by lovastatin, including certain genes associated with cell survival, such as PTK2B, BCL2 and MAP3K3. In particular, PTK2B, which has been shown to exert anti-apoptotic effects against ox-LDL-induced cell injury, was upregulated by lovastatin. Knockdown of PTK2B was able to attenuate ox-LDL-induced cell injury, and this was associated with decreased levels of phosphorylated-AKT and eNOS, and inhibition of mitochondrial-dependent apoptosis. In conclusion, the results of the present study suggested that lovastatin protects against ox-LDL-induced cell injury, potentially via the upregulation of PTK2B, which regulates the anti-apoptosis signaling pathway.

No MeSH data available.


Knock-down of PTK2B expression attenuated ox-LDL-induced HUVEC injury. HUVECs were transiently transfected with PTK2B siRNA or scramble siRNA for 48 h, followed by treatment with 20 µg/ml ox-LDL for 24 h. (A) Cell viability was determined using a cell counting kit-8 assay. (B) Cell apoptosis was determined by Annexin V and PI staining, followed by flow cytometry. (C) Percentage of apoptotic cells. (D) Protein expression was analyzed by western blotting and band intensities were determined using ImageJ software following normalization to a control (β-actin). Data are presented as the mean ± standard deviation of three independent experiments. *P<0.05 vs. the control cells. ox-LDL, oxidized low-density lipoprotein; HUVECs, human umbilical vein endothelial cells; siRNA, small interfering RNA; PI, propidium iodide.
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f4-etm-0-0-3547: Knock-down of PTK2B expression attenuated ox-LDL-induced HUVEC injury. HUVECs were transiently transfected with PTK2B siRNA or scramble siRNA for 48 h, followed by treatment with 20 µg/ml ox-LDL for 24 h. (A) Cell viability was determined using a cell counting kit-8 assay. (B) Cell apoptosis was determined by Annexin V and PI staining, followed by flow cytometry. (C) Percentage of apoptotic cells. (D) Protein expression was analyzed by western blotting and band intensities were determined using ImageJ software following normalization to a control (β-actin). Data are presented as the mean ± standard deviation of three independent experiments. *P<0.05 vs. the control cells. ox-LDL, oxidized low-density lipoprotein; HUVECs, human umbilical vein endothelial cells; siRNA, small interfering RNA; PI, propidium iodide.

Mentions: AKT is known to promote cell survival by upregulating the expression of BCL2 and downregulating the expression of BAX (35). In addition, PTK2B has been shown to be an upstream protein of AKT and eNOS (36). Therefore, it may be hypothesized that PTK2B promotes the AKT/BCL2 signaling cascade. To test this hypothesis, PTK2B expression in HUVECs was transiently knocked down for 48 h using siRNA, after which HUVECs were treated with ox-LDL for 24 h to observe the effect of PTK2B on cell viability and apoptosis. The results demonstrated that silencing of PTK2B expression significantly decreased cell viability, thus indicating its importance for cell survival in HUVECs (Fig. 4A). Notably, knockdown of PTK2B was able to markedly reduce ox-LDL-induced cytotoxicity. Subsequently, the ox-LDL-induced apoptosis of HUVECs following knockdown of PTK2B was analyzed. The ability of ox-LDL to induce apoptosis of HUVECs was markedly compromised following knockdown of PTK2B (Fig. 4B and C). Furthermore, we examined the signaling cascade involved in this pathway. As is shown in Fig. 4D, PTK2B was downregulated following treatment of HUVECs with ox-LDL and siRNA, although the extent of ox-LDL-induced PTK2B downregulation was markedly reduced in PTK2B knockdown cells. Accordingly, the ox-LDL-induced inhibition of p-AKT and eNOS, downregulation of BCL2 and upregulation of BAX and cleaved caspase-3 were greatly compromised following knockdown of PTK2B. These results suggest that the PTK2B-mediated signaling pathway is upregulated by lovastatin and serves an important role in the lovastatin-mediated protection against ox-LDL-induced cytotoxicity.


Lovastatin exerts protective effects on endothelial cells via upregulation of PTK2B
Knock-down of PTK2B expression attenuated ox-LDL-induced HUVEC injury. HUVECs were transiently transfected with PTK2B siRNA or scramble siRNA for 48 h, followed by treatment with 20 µg/ml ox-LDL for 24 h. (A) Cell viability was determined using a cell counting kit-8 assay. (B) Cell apoptosis was determined by Annexin V and PI staining, followed by flow cytometry. (C) Percentage of apoptotic cells. (D) Protein expression was analyzed by western blotting and band intensities were determined using ImageJ software following normalization to a control (β-actin). Data are presented as the mean ± standard deviation of three independent experiments. *P<0.05 vs. the control cells. ox-LDL, oxidized low-density lipoprotein; HUVECs, human umbilical vein endothelial cells; siRNA, small interfering RNA; PI, propidium iodide.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f4-etm-0-0-3547: Knock-down of PTK2B expression attenuated ox-LDL-induced HUVEC injury. HUVECs were transiently transfected with PTK2B siRNA or scramble siRNA for 48 h, followed by treatment with 20 µg/ml ox-LDL for 24 h. (A) Cell viability was determined using a cell counting kit-8 assay. (B) Cell apoptosis was determined by Annexin V and PI staining, followed by flow cytometry. (C) Percentage of apoptotic cells. (D) Protein expression was analyzed by western blotting and band intensities were determined using ImageJ software following normalization to a control (β-actin). Data are presented as the mean ± standard deviation of three independent experiments. *P<0.05 vs. the control cells. ox-LDL, oxidized low-density lipoprotein; HUVECs, human umbilical vein endothelial cells; siRNA, small interfering RNA; PI, propidium iodide.
Mentions: AKT is known to promote cell survival by upregulating the expression of BCL2 and downregulating the expression of BAX (35). In addition, PTK2B has been shown to be an upstream protein of AKT and eNOS (36). Therefore, it may be hypothesized that PTK2B promotes the AKT/BCL2 signaling cascade. To test this hypothesis, PTK2B expression in HUVECs was transiently knocked down for 48 h using siRNA, after which HUVECs were treated with ox-LDL for 24 h to observe the effect of PTK2B on cell viability and apoptosis. The results demonstrated that silencing of PTK2B expression significantly decreased cell viability, thus indicating its importance for cell survival in HUVECs (Fig. 4A). Notably, knockdown of PTK2B was able to markedly reduce ox-LDL-induced cytotoxicity. Subsequently, the ox-LDL-induced apoptosis of HUVECs following knockdown of PTK2B was analyzed. The ability of ox-LDL to induce apoptosis of HUVECs was markedly compromised following knockdown of PTK2B (Fig. 4B and C). Furthermore, we examined the signaling cascade involved in this pathway. As is shown in Fig. 4D, PTK2B was downregulated following treatment of HUVECs with ox-LDL and siRNA, although the extent of ox-LDL-induced PTK2B downregulation was markedly reduced in PTK2B knockdown cells. Accordingly, the ox-LDL-induced inhibition of p-AKT and eNOS, downregulation of BCL2 and upregulation of BAX and cleaved caspase-3 were greatly compromised following knockdown of PTK2B. These results suggest that the PTK2B-mediated signaling pathway is upregulated by lovastatin and serves an important role in the lovastatin-mediated protection against ox-LDL-induced cytotoxicity.

View Article: PubMed Central - PubMed

ABSTRACT

Statins are HMG-CoA reductase inhibitors that are used to decrease the blood levels of low-density lipoprotein (LDL). In addition, they have been shown to exert pleiotropic protective effects in the absence of LDL-lowering activity. The present study investigated the effects of lovastatin on global gene expression in human umbilical vein endothelial cells (HUVECs), in order to further explore its ability to protect against oxidized (ox)-LDL-induced cytotoxicity. HUVECs were treated with lovastatin for 2&ndash;24 h, and gene expression patterns were analyzed using cDNA microarrays. The results suggested that numerous genes were regulated by lovastatin, including certain genes associated with cell survival, such as PTK2B, BCL2 and MAP3K3. In particular, PTK2B, which has been shown to exert anti-apoptotic effects against ox-LDL-induced cell injury, was upregulated by lovastatin. Knockdown of PTK2B was able to attenuate ox-LDL-induced cell injury, and this was associated with decreased levels of phosphorylated-AKT and eNOS, and inhibition of mitochondrial-dependent apoptosis. In conclusion, the results of the present study suggested that lovastatin protects against ox-LDL-induced cell injury, potentially via the upregulation of PTK2B, which regulates the anti-apoptosis signaling pathway.

No MeSH data available.