Limits...
Lonafarnib is a potential inhibitor for neovascularization.

Sun L, Xie S, Peng G, Wang J, Li Y, Qin J, Zhong D - PLoS ONE (2015)

Bottom Line: In this study, we found that lonafarnib, a specific inhibitor of farnesyl transferase, elicits inhibitory effect on vascular endothelial capillary assembly in vitro in a dose-dependent manner.In addition, we showed that lonafarnib treatment led to a dose-dependent decrease in scratch wound closure in vitro, whereas it had little effect on endothelial cell proliferation.Mechanistically, we found that the catalytic β subunit of farnesyl transferase associated with a cytoskeletal protein important for the establishment and maintenance of cell polarity.

View Article: PubMed Central - PubMed

Affiliation: Lung Cancer Institute, Tianjin Medical University General Hospital, Tianjin, China.

ABSTRACT
Atherosclerosis is a common cardiovascular disease that involves the build-up of plaque on the inner walls of the arteries. Intraplaque neovacularization has been shown to be essential in the pathogenesis of atherosclerosis. Previous studies showed that small-molecule compounds targeting farnesyl transferase have the ability to prevent atherosclerosis in apolipoprotein E-deficient mice, but the underlying mechanism remains to be elucidated. In this study, we found that lonafarnib, a specific inhibitor of farnesyl transferase, elicits inhibitory effect on vascular endothelial capillary assembly in vitro in a dose-dependent manner. In addition, we showed that lonafarnib treatment led to a dose-dependent decrease in scratch wound closure in vitro, whereas it had little effect on endothelial cell proliferation. These data indicate that lonafarnib inhibits neovascularization via directly targeting endothelial cells and disturbing their motility. Moreover, we demonstrated that pharmacological inhibition of farnesyl transferase by lonafarnib significantly impaired centrosome reorientation toward the leading edge of endothelial cells. Mechanistically, we found that the catalytic β subunit of farnesyl transferase associated with a cytoskeletal protein important for the establishment and maintenance of cell polarity. Additionally, we showed that lonafarnib remarkably inhibited the expression of the cytoskeletal protein and interrupted its interaction with farnesyl transferase. Our findings thus offer novel mechanistic insight into the protective effect of farnesyl transferase inhibitors on atherosclerosis and provide encouraging evidence for the potential use of this group of agents in inhibiting plaque neovascularization.

No MeSH data available.


Related in: MedlinePlus

Characterization of the interaction between MAPRE1 and FTβ.(A) Schematic representations of full length (FL) and truncated forms of MAPRE1 were shown. (B) HUVECs were transfected with pCMV-HA-FTβand plasmids that express various truncated forms of MAPRE1 tagged with GST. GST pull-down and Western blot were then performed to characterize the FTβbinding region on MAPRE1. The expressions of the MAPRE1 variants were monitored and comparable amounts of cell lysates were loaded in the pull-down assay (lower panel). (C) Schematic representations of full length (FL) and truncated forms of FTβwere shown. (D) HUVECs were transfected with GST-MAPRE1 and plasmids that express various truncated forms of FTβtagged with HA. GST pull-down (PD) and Western blot were then performed to examine their interaction and characterize the MAPRE1 interacting region on FTβ. The expressions of the FTβvariants were monitored and comparable amounts of cell lysates were loaded in the pull-down assay (middle panel).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4390146&req=5

pone.0122830.g005: Characterization of the interaction between MAPRE1 and FTβ.(A) Schematic representations of full length (FL) and truncated forms of MAPRE1 were shown. (B) HUVECs were transfected with pCMV-HA-FTβand plasmids that express various truncated forms of MAPRE1 tagged with GST. GST pull-down and Western blot were then performed to characterize the FTβbinding region on MAPRE1. The expressions of the MAPRE1 variants were monitored and comparable amounts of cell lysates were loaded in the pull-down assay (lower panel). (C) Schematic representations of full length (FL) and truncated forms of FTβwere shown. (D) HUVECs were transfected with GST-MAPRE1 and plasmids that express various truncated forms of FTβtagged with HA. GST pull-down (PD) and Western blot were then performed to examine their interaction and characterize the MAPRE1 interacting region on FTβ. The expressions of the FTβvariants were monitored and comparable amounts of cell lysates were loaded in the pull-down assay (middle panel).

Mentions: The findings that pharmacological inhibition of farnesyl transferase by lonafarnib impaired the position of centrosome suggest that the protein might function in the process of centrosome reorientation. In an effort to elucidate the underlying molecular mechanism, we found that the catalytic β subunit of farnesyl transferase appeared to associate with a cytoskeletal protein named microtubule-associated protein RP/EB family member 1(MAPRE1), a key regulator of cell polarization[8]. To confirm our observation, a series of truncated forms of MAPRE1 tagged with GST were constructed, and the representative truncations were depicted in Fig 5A. HUVECs were then transfected with these plasmids together with the HA-FTβ-expressing plasmid, and cell lysates were analyzed by GST pull-down assay. As shown in Fig 5B, GST-MAPRE1 was able to pull down HA-FTβ, validating the association of farnesyl transferaseβ with MAPRE1. In addition, the full length of MAPRE1 (1–268) was required for the interaction with FTβ, and other truncations of MAPRE1 (amino acids 1–115, 116–208, 209–268, 1–208, 116–268) abrogated the binding with FTβ. Similarly, by constructing various truncated forms of FTβ(Fig 5C), we sought to identify the MAPRE1 binding region in FTβ. GST pull-down assay revealed that amino acids 1–373, 76–437 and 76–373 of FTβwere able to interact with GST-MAPRE1 but that amino acids 1–138 abrogated the association with MAPRE1 (Fig 5D), indicating that amino acids 139–373 of FTβwere essential for their interaction. These data thus identified MAPRE1 as a binding partner of farnesyl transferase in vascular endothelial cells.


Lonafarnib is a potential inhibitor for neovascularization.

Sun L, Xie S, Peng G, Wang J, Li Y, Qin J, Zhong D - PLoS ONE (2015)

Characterization of the interaction between MAPRE1 and FTβ.(A) Schematic representations of full length (FL) and truncated forms of MAPRE1 were shown. (B) HUVECs were transfected with pCMV-HA-FTβand plasmids that express various truncated forms of MAPRE1 tagged with GST. GST pull-down and Western blot were then performed to characterize the FTβbinding region on MAPRE1. The expressions of the MAPRE1 variants were monitored and comparable amounts of cell lysates were loaded in the pull-down assay (lower panel). (C) Schematic representations of full length (FL) and truncated forms of FTβwere shown. (D) HUVECs were transfected with GST-MAPRE1 and plasmids that express various truncated forms of FTβtagged with HA. GST pull-down (PD) and Western blot were then performed to examine their interaction and characterize the MAPRE1 interacting region on FTβ. The expressions of the FTβvariants were monitored and comparable amounts of cell lysates were loaded in the pull-down assay (middle panel).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4390146&req=5

pone.0122830.g005: Characterization of the interaction between MAPRE1 and FTβ.(A) Schematic representations of full length (FL) and truncated forms of MAPRE1 were shown. (B) HUVECs were transfected with pCMV-HA-FTβand plasmids that express various truncated forms of MAPRE1 tagged with GST. GST pull-down and Western blot were then performed to characterize the FTβbinding region on MAPRE1. The expressions of the MAPRE1 variants were monitored and comparable amounts of cell lysates were loaded in the pull-down assay (lower panel). (C) Schematic representations of full length (FL) and truncated forms of FTβwere shown. (D) HUVECs were transfected with GST-MAPRE1 and plasmids that express various truncated forms of FTβtagged with HA. GST pull-down (PD) and Western blot were then performed to examine their interaction and characterize the MAPRE1 interacting region on FTβ. The expressions of the FTβvariants were monitored and comparable amounts of cell lysates were loaded in the pull-down assay (middle panel).
Mentions: The findings that pharmacological inhibition of farnesyl transferase by lonafarnib impaired the position of centrosome suggest that the protein might function in the process of centrosome reorientation. In an effort to elucidate the underlying molecular mechanism, we found that the catalytic β subunit of farnesyl transferase appeared to associate with a cytoskeletal protein named microtubule-associated protein RP/EB family member 1(MAPRE1), a key regulator of cell polarization[8]. To confirm our observation, a series of truncated forms of MAPRE1 tagged with GST were constructed, and the representative truncations were depicted in Fig 5A. HUVECs were then transfected with these plasmids together with the HA-FTβ-expressing plasmid, and cell lysates were analyzed by GST pull-down assay. As shown in Fig 5B, GST-MAPRE1 was able to pull down HA-FTβ, validating the association of farnesyl transferaseβ with MAPRE1. In addition, the full length of MAPRE1 (1–268) was required for the interaction with FTβ, and other truncations of MAPRE1 (amino acids 1–115, 116–208, 209–268, 1–208, 116–268) abrogated the binding with FTβ. Similarly, by constructing various truncated forms of FTβ(Fig 5C), we sought to identify the MAPRE1 binding region in FTβ. GST pull-down assay revealed that amino acids 1–373, 76–437 and 76–373 of FTβwere able to interact with GST-MAPRE1 but that amino acids 1–138 abrogated the association with MAPRE1 (Fig 5D), indicating that amino acids 139–373 of FTβwere essential for their interaction. These data thus identified MAPRE1 as a binding partner of farnesyl transferase in vascular endothelial cells.

Bottom Line: In this study, we found that lonafarnib, a specific inhibitor of farnesyl transferase, elicits inhibitory effect on vascular endothelial capillary assembly in vitro in a dose-dependent manner.In addition, we showed that lonafarnib treatment led to a dose-dependent decrease in scratch wound closure in vitro, whereas it had little effect on endothelial cell proliferation.Mechanistically, we found that the catalytic β subunit of farnesyl transferase associated with a cytoskeletal protein important for the establishment and maintenance of cell polarity.

View Article: PubMed Central - PubMed

Affiliation: Lung Cancer Institute, Tianjin Medical University General Hospital, Tianjin, China.

ABSTRACT
Atherosclerosis is a common cardiovascular disease that involves the build-up of plaque on the inner walls of the arteries. Intraplaque neovacularization has been shown to be essential in the pathogenesis of atherosclerosis. Previous studies showed that small-molecule compounds targeting farnesyl transferase have the ability to prevent atherosclerosis in apolipoprotein E-deficient mice, but the underlying mechanism remains to be elucidated. In this study, we found that lonafarnib, a specific inhibitor of farnesyl transferase, elicits inhibitory effect on vascular endothelial capillary assembly in vitro in a dose-dependent manner. In addition, we showed that lonafarnib treatment led to a dose-dependent decrease in scratch wound closure in vitro, whereas it had little effect on endothelial cell proliferation. These data indicate that lonafarnib inhibits neovascularization via directly targeting endothelial cells and disturbing their motility. Moreover, we demonstrated that pharmacological inhibition of farnesyl transferase by lonafarnib significantly impaired centrosome reorientation toward the leading edge of endothelial cells. Mechanistically, we found that the catalytic β subunit of farnesyl transferase associated with a cytoskeletal protein important for the establishment and maintenance of cell polarity. Additionally, we showed that lonafarnib remarkably inhibited the expression of the cytoskeletal protein and interrupted its interaction with farnesyl transferase. Our findings thus offer novel mechanistic insight into the protective effect of farnesyl transferase inhibitors on atherosclerosis and provide encouraging evidence for the potential use of this group of agents in inhibiting plaque neovascularization.

No MeSH data available.


Related in: MedlinePlus