Protein tyrosine phosphatase 1B inhibition promotes diabetic wound healing via activation of the antioxidant enzyme heme oxygenase 1

Background and aims: Diabetic foot ulcers are a leading cause of hospital
admissions for people with diabetes in the developed world. Protein tyrosine
phosphatase 1B (PTP1B) is a negative regulator of the insulin signaling
pathway and it is upregulated in diabetes and diabetic foot ulcers. Evidence
has emerged that PTP1B is involved in the pathology of diabetic ulcers which
indicates that its inhibition may be a potential therapeutic approach. Our main
aim was to evaluate the role of PTP1B inhibition in diabetic wound healing
and study the underlying mechanisms.
Materials and methods: Diabetes in male C57BL/6 mice was induced with
streptozotocin (STZ), i.p. injections (50 mg/Kg), for 5 consecutive days. After
6 weeks of diabetes, two 6 mm excision wounds per mouse were created in
the dorsum and the wounds were treated topically, twice a day, up to day 3
with: vehicle (control); 1 μg of Trodusquemine (PTP1B inhibitor); 1 μg of
Protoporphyrin IX zinc (II) (heme oxygenase 1 inhibitor - HO1i) and
PTP1Bi+HO1i. The wounds were measured every day up to day 10 post
wounding. The skin was collected at day 3 and 10 post wounding.
Immunohistochemistry was used for the detection of M1 macrophages, with
CD68 and TNF-alpha, or M2 macrophages, with CD68 and CD206.
Immunohistochemistry was also used to evaluate the vascularization of the
skin with the endothelial cell marker CD31, the proliferation with ki67, and
the levels of heme oxygenase 1 (HO1). The levels of PTP1B were measured
by western blot and the activity of HO1 was evaluated by the production of
bilirubin. The production of reactive oxygen species (ROS) was measured
with dihydroethidium (DHE).
Results: PTP1B levels were significantly increased in unwounded (474
±150 % of control, p<0.01) and wounded skin (day 3 post wounding, 398
±68 % of control, p<0.01) of diabetic mice when compared to healthy
animals. The treatment with PTP1B inhibitor improved diabetic wound
healing progression when compared to non-treated wounds (day 10, 1.1
±0.2% and 7.2±1.4% of original wound, p<0.01). Moreover, PTP1B
inhibition decreased the inflammatory environment in the wounds with
a decrease in M1/M2 ratio (day 3, 0.7±0.1 and 1.6±0.2, p<0.01). In
addition, the increase in angiogenesis (147.4±5.5 % of control, p<0.01)
and cell proliferation (141.1±5.9 % of control, p<0.01), after 10 days of
wound induction, was also observed. The oxidative stress, in diabetic
wounds after 3 days post wounding, was significantly decreased by
PTP1B inhibition (68.1±6.1 % of control, p<0.01) and the increase in
HO1 levels (219.4±20.7 % of control, p<0.01) and activity (170.4±15.7
% of control, p<0.01) was also observed. These effects of the inhibition of
PTP1B were reverted by HO1 inhibition.
Conclusion: PTP1B inhibition promotes wound healing in diabetes with
decrease in the inflammatory environment and the oxidative stress which
improves the regenerative capacity of the skin with an enhance in angiogenesis and proliferation, through an increase of the antioxidant defense
HO1. This study suggests that PTP1B is a target of interest for the treatment of diabetic foot ulcers.