Diabetes is associated with impairment of angiogenesis such as reduction of myocardial capillary formation. Our previous studies demonstrate that disruption of Angiopoietin-1 (Ang-1)/Tie-2 signaling pathway contributes to the diabetes-associated impairment of angiogenesis. Protein tyrosine phosphatase (PTP) has a critical role in the regulation of insulin signal by inhibition of tyrosine kinase phosphorylation. In present study, we examined the role of protein tyrosine phosphatase-1 (SHP-1) in diabetes-associated impairment of Ang-1/Tie-2 angiogenic signaling and angiogenesis. SHP-1 expression was significantly increased in diabetic db/db mouse hearts. Furthermore, SHP-1 bond to Tie-2 receptor and stimulation with Ang-1 led to SHP-1 dissociation from Tie-2 in mouse heart microvascular endothelial cell (MHMEC). Exposure of MHMEC to high glucose (HG, 30 mmol/L) increased SHP-1/Tie-2 association accompanied by a significant reduction of Tie-2 phosphorylation. Exposure of MHMEC to HG also blunted Ang-1-mediated SHP-1/Tie-2 dissociation. Knockdown of SHP-1 significantly attenuated HG-induced caspase-3 activation and apoptosis in MHMEC. Treatment with PTP inhibitors restored Ang-1-induced Akt/eNOS phosphorylation and angiogenesis. Our data implicate a critical role of SHP-1 in diabetes-associated vascular complications, and that upregulation of Ang-1/Tie-2 signaling by targeting SHP-1 should be considered as a new therapeutic strategy for the treatment of diabetes-associated impairment of angiogenesis.
Angiogenesis is mainly regulated by the vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) and the angiopoietins/Tie-2 system. Receptor tyrosine kinases (RTKs) represent a major class of cell-surface molecules that regulate angiogenesis. VEGFR and the Tie-2 receptor are the principal RTK families and play critical roles in the regulation of angiogenesis [
Protein tyrosine phosphatase (PTP) has been shown to negatively regulate insulin signaling by dephosphorylation of insulin receptor tyrosine kinase [
In our present study, we hypothesize that hyperglycemia and diabetes impair Ang-1/Tie-2 signaling and angiogenesis by a mechanism involving upregulation of SHP-1 expression and SHP-1/Tie-2 interaction. Our data suggest that increased SHP-1 has a crucial role in the diabetes-associated impairment of angiogenesis by interfering with the Ang-1/Tie-2 angiogenic signaling.
MHMECs was isolated from C57BL/6J mouse hearts and cultured as previously described [
To induce apoptosis, MHMEC were exposed to serum-free medium for 72 hours under high glucose (HG, 30 mmol/L) or normal glucose (NG, 5 mmol/L) conditions. Endothelial cell apoptosis was measured by counting TUNEL positive cells per 100 endothelial cells following the manufacturer’s instructions (Promega, WI). Caspase-3 activity was measured using the caspase-3 kit (Sigma, MO).
MHMEC lysates were immunoprecipitated with anti-mouseTie-2 antibody followed by incubation with a 1 : 1 protein A: protein G-sepharose slurry. The immunoprecipitates were then subjected to SDS-PAGE gels and transferred to nitrocellulose membranes. The membranes were immunoblotting anti-SHP-1 (1 : 1000, Santa Cruz, CA) or anti-phospho-tyrosine (4G10, 1 : 1000 Upstate Biotech, NY). The membranes were washed and incubated with a secondary antibody coupled to horseradish peroxidase.
Fifty micrograms of total protein of myocardial tissue or MHMEC lysates were separated using SDS-gel electrophoresis. The membranes were immunoblotted with SHP-1 (1 : 1000), eNOS and Tie-2 (1 : 1000, Cell Signaling Technology, MA) antibodies. For eNOS and Akt phosphorylation, the membranes were immunoblotted with rabbit anti-phospho-Akt and anti-phospho-eNOS (1 : 1000, Cell Signaling, MA).
MHMEC (approx. 80% confluent) was treated with SHP-1 siRNA (mouse, Santa Cruz, CA) for 24 hours to inhibition of SHP-1 expression according to the manufacturer’s instructions. Knockdown of SHP-1 was confirmed by Western blot analysis of SHP-1 protein expression (data not shown).
Cell survival was assayed using the MTT assay kit (Roche Diagnostic Corp., Indianapolis, IN).
The C57BL/6J mice and db/db mice were purchased from Jackson Laboratory (Bar Harbor, Maine). Sixteen male db/db mice at 12 weeks of age were divided into two groups: [
The experimental mouse hearts were harvested and immediately flash frozen. Tissue sections were incubated with fluorescein-labeled antibodies against Griffonia Bandeiraea Simplicifolia Isolectin B4 (IB4, 1 : 200, Sigma Co) to label the endothelial cells, and myocardial capillary density was measured using image analysis software (Image J, NIH). The number of capillaries (IB4) was counted and expressed as capillary density per mm2 [
Mouse aortae were isolated and collected from C57BL/6J and db/db mice, placed in the middle of organ culture dishes and overlaid with 300
All results were expressed as mean ± SD. Statistical analysis was performed using unpaired student
Western blot analysis showed that SHP-1 protein was expressed both in C57BL/6J mouse and diabetic db/db mouse hearts. Intriguingly, the expression of SHP-1 protein was significantly increased in db/db mouse hearts in comparison to C57BL/6J controls (Figure
Expression of SHP-1 in the control C57BL/6J and db/db mouse hearts. Western blot showing SHP-1 expression in control C57BL/6J and diabetic db/db mouse hearts. Densitometric data shows that SHP-1 protein expression was significantly increased in diabetic db/db mouse hearts in comparison with control C57BL/6J mice (
To examine whether SHP-1 binds to Tie-2, MHMEC lysates were immunoprecipitated with Tie-2 antibody and blotted with SHP-1 antibody. As shown in Figure
Immunoprecipitation and Western blot analysis showing that SHP-1 associates with Tie-2 receptor and high glucose alters the SHP-1/Tie-2 association in MHMEC. (a) SHP-1 binds to the Tie-2 receptor under normal glucose (NG) conditions; exposure of MHMEC to high glucose (HG) leads to a significant increase in the SHP-1/Tie-2 association (
To determine whether SHP-1 was involved in Ang-1-mediated Tie-2 activation, the effect of Ang-1 on Tie-2/SHP-1 association was examined. As shown in Figure
Next, the functional role of SHP-1 in high glucose-induced endothelial dysfunction was investigated. Treatment of MHMEC with SHP-1 siRNA significantly suppressed caspase-3 activity under normal glucose (NG) and HG conditions (Figure
Knockdown of SHP-1 by siRNA blunts HG-induced cell apoptosis in MHMEC. (a) Caspase-3 ELISA analysis showing that transfection of MHMEC with SHP-1 siRNA significantly attenuated caspase-3 activation under NG or HG conditions (
To further investigate the role of PTP in the Ang-1/Tie-2 signaling, the PTP inhibitor on Ang-1-induced Akt/eNOS phosphorylation was examined in MHMEC under HG conditions. MHMEC was pretreated with PTP inhibitor sodium orthovanadate (OV, 5
Inhibition of PTP enhances Ang-1-induced Akt and eNOS phosphorylation under HG conditions. (a) Western blot showing that pretreatment of MHMEC with the PTP inhibitor orthovanadate (OV, 5
Treatment of MHMEC with Ang-1 (250 ng/mL) significantly attenuated caspase-3 activity. The inhibitory effect of Ang-1 on caspase-3 activity was further enhanced in the presence of OV (Figure
Inhibition of PTP promotes Ang-1-induced suppression of caspase-3 activation and increases cell survival under HG conditions. (a) Caspase-3 activity assay showing that exposure of MHMEC to Ang-1 significantly attenuates HG-induced caspase-3 activation; pretreatment of MHMEC with OV before the Ang-1 treatment further enhances the effect of Ang-1 on caspase-3 activity under HG conditions (
As shown in Figure
Treatment with PTP inhibitor restores the angiogenic response to Ang-1 in diabetic db/db mouse. (a) Representative images of Ang-1-induced aortic explants sprouting in C57BL/6J and diabetic db/db mice. Stimulation of C57BL/6J mouse aortic explants with Ang-1 (250 ng/mL) leads to robust vessel outgrowth. Ang-1-induced vessel outgrowth was blunted in the db/db mouse aortic explants. (b) Representative images of Ang-1-induced vessel outgrowth in the db/db mouse aortic explants in the presence and absence of PTP inhibitor OV. (c) Quantitative areas of vessel outgrowth in db/db mouse aortic explants showing Ang-1-induced vessel outgrowth were significantly increased in the presence of PTP inhibitor OV (
To examine whether inhibition of PTP augments myocardial angiogenesis in diabetic hearts, an orally bioavailable PTP inhibitor bis-(maltolato)oxovanadium (IV) (BMOV) was given to the experimental db/db mice. Treatment of db/db mice with BMOV for 2 weeks resulted in a significant decrease in SHP-1 expression (Figure
Systemic treatment with the PTP inhibitor attenuates SHP-1 expression and increases eNOS and myocardial capillary density in db/db mice. (a) Western blot analysis showing that treatment with BMOV for 2 weeks leads to a significant suppression of SHP-1 expression in the db/db mice compared to db/db control without BMOV (
The current study demonstrates that SHP-1 binds to the Tie-2 to form a SHP-1/Tie-2 complex and that the Ang-1, an agonist of Tie-2, causes SHP-1 dissociation from Tie-2. This finding implicates a potential role of SHP-1 in Ang-1-induced Tie-2 phosphorylation. Intriguingly, high glucose increases formation of the SHP-1/Tie-2 complex and this is accompanied by Tie-2 dephosphorylation. Ang-1 failed to cause SHP-1 dissociation from Tie-2 under HG conditions. Suppression of SHP-1 expression significantly attenuated endothelial apoptosis and improved diabetes-associated impairment of angiogenesis. These data strongly suggest a critical role for SHP-1 and the SHP-1/Tie-2 association in diabetes-associated impairment of angiogenesis.
The Src-homology-domain-2- (SH2-) containing tyrosine phosphatases (SHP-1 and SHP-2) have been shown to interact with multiple growth factor receptors including Tie-2 [
Illustrating our working hypothesis for the SHP-1-induced disruption of Ang-1/Tie-2 signaling under HG conditions and in diabetes. (a) In a resting state, SHP-1 maintains Tie-2 inactivation. (b) Stimulation with Ang-1 causes a dissociation of SHP-1 and Tie-2, thus leading to Tie-2 tyrosine phosphorylation, and its downstream signaling Akt and eNOS activation. (c) Stimulation with Ang-1 fails to lead to the dissociation of SHP-1 and Tie-2, thus resulting in a disruption of Ang-1/Tie-2 signaling under hyperglycemic conditions and in diabetes. NG: normal glucose; HG: high glucose; SHP-1: protein tyrosine phosphatase-1.
Our data also demonstrated that knockdown of SHP-1 by siRNA significantly prevented HG-induced caspase-3 activation and endothelial apoptosis. Our study further demonstrates that inhibition of PTP augmented Ang-1-induced cell survival under HG conditions and restored angiogenic responses in diabetic vessel explants. Inhibition of PTP has been shown to enhance angiogenic signaling and promote VEGF-induced angiogenesis [
Other PTPs, including PTP1B, SHP-2, PTP-
In summary, our present study demonstrates that hyperglycemia and diabetes impair angiogenesis by a mechanism involving upregulation of SHP-1 and SHP-1/Tie-2 association. Our study also shows that pharmacological inhibition of PTP or genetic deletion of SHP-1 enhances Ang-1/Tie-2 signaling and improves angiogenesis in diabetes. Our data implicate that restoration of Ang-1/Tie-2 signaling by PTP inhibitors should be considered as a new therapeutic strategy for the treatment or prevention of diabetic impaired angiogenesis.
This work was supported by grant from HL102042 to J. X. Chen.