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 [27
]. SHP-2 is primarily associated with enhanced cell growth, whereas SHP-1 has been shown to play a negative regulatory role in endothelial cell proliferation [18
]. SHP-1 suppresses VEGF and EGF-induced endothelial proliferation, whereas knockdown of SHP-1 augments VEGF- and FGF-2-induced angiogenic responses [20
]. SHP-1 also showed to negatively modulate glucose homeostasis via de-phosphorylation of insulin RTK signaling [32
]. Our study demonstrated that SHP-1 expression was significantly increased whereas SHP-2 expression remained unchanged in diabetic db/db mouse hearts. Our present study also demonstrated that SHP-1 works as a novel client protein for Tie-2, and stimulation with Ang-1 led to SHP-1 dissociation from Tie-2, implicating a potential interaction between SHP-1 and Ang-1-induced Tie-2 phosphorylation. This notion was further validated by our finding that exposure of MHMEC to HG increased SHP-1/Tie-2 association but decreased Tie-2 phosphorylation. This was consistent with our previous studies that Ang-1-induced Tie-2 phosphorylation was damped under HG conditions [33
]. Taken together, the present study reveals a potential novel mechanism for the disruption of Ang-1/Tie-2 signaling by SHP-1 in diabetes. We speculate that protein tyrosine phosphatases, including SHP-1, maintain Tie-2 inactivation by de-phosphorylation, whereas stimulation with Ang-1 leads to dissociation of SHP-1 from Tie-2 and results in Tie-2 phosphorylation and its downstream signaling Akt and eNOS activation. Under hyperglycemic conditions and in diabetes, stimulation with Ang-1 fails to cause the dissociation of SHP-1 from Tie-2, resulting in disruption of Ang-1/Tie-2 signaling ().
Figure 8 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 (more ...)
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 [34
]. Inhibition of PTP also promoted collateral blood vessel formation and increased blood flow in a rat model of hind-limb ischemia [14
]. Inhibition of PTP has been shown to attenuate endothelial dysfunction via upregulation of eNOS in the mouse model of chronic heart failure [35
] and treatment with the nonselective PTP inhibitors such as vanadate and BMOV-enhanced insulin receptor activation and restored insulin signaling in diabetic rats [36
]. The protective effect of PTP inhibitors on endothelial cell dysfunction was mediated by the enhancement of Akt/eNOS phosphorylation in diabetic rats [36
]. Consistent with these findings, our data showed that pretreatment of MHMEC with a PTP inhibitor enhanced Ang-1-induced Akt/eNOS phosphorylation. Our present study also demonstrated that systemic treatment of diabetic db/db mouse with the PTP inhibitor BMOV significantly suppressed SHP-1 expression and increased eNOS expression. This was accompanied by increase in myocardial capillary density. Our study provides new evidence that diabetes may impede angiogenesis by a mechanism involving upregulation of PTP activity which negatively regulates angiogenesis by inhibition of angiogenic growth factor phosphorylation such as Ang-1/Tie-2 system.
4.1. Limitation of This Study
Other PTPs, including PTP1B, SHP-2, PTP-ε, VE-PTP, CD148, may also play key roles in the regulation of myocardial angiogenesis in diabetes. Further elucidation of the intracellular mechanisms of PTP with, such as, PTPB1 on diabetes-associated impairment of angiogenic signaling and angiogenesis is needed. We acknowledge that it is technically impossible to examine all PTPs enzymes in a similar manner since specific inhibitors are lacking for each individual isoform of the PTPs. We also acknowledge the potential integrated effects of SHP-1 and PKC beta signaling. Identification of all the mechanisms involved will require additional experiments to evaluate the roles of PTPs and PKC signaling pathways in diabetes-associated impairment of angiogenesis.
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.