Effects of the superoxide dismutase-mimetic compound tempol on endothelial dysfunction in streptozotocin-induced diabetic rats.

Evidence exists to support the beneficial effects of superoxide dismutase on endothelial dysfunction induced by hyperglycemia in vitro. In vivo, however, studies of the effects of native superoxide dismutase preparations on the vascular complications accompanying diabetes are limited, and their therapeutic application potential has so far been disappointing. The objective of this study was to evaluate, for the first time in vivo, the effects of long-term administration of tempol, a stable superoxide dismutase-mimic compound, on diabetes-induced endothelial dysfunction in rats. Diabetes was induced by streptozotocin and rats were monitored for 8 weeks with or without treatment with tempol (100 mg/kg, s.c., b.i.d). Diabetic rats showed increased vascular levels of superoxide, which was accompanied by increased levels of the oxidative stress markers malondialdehyde and 8-epi-prostaglandin F(2alpha). In addition, the vasorelaxant as well as the cGMP-producing effects of acetylcholine and glyceryl trinitrate were reduced in diabetic rats. Treatment with tempol abolished not only the differences in the vascular content of superoxide, malondialdehyde and 8-epi-prostaglandin F(2alpha), but also the differences in the relaxation and cGMP responses of aortic rings to both acetylcholine and glyceryl trinitrate between control and diabetic rats. These results support the involvement of reactive oxygen species in mediation of hyperglycemia-induced endothelial dysfunction in vivo, and provide the rationale for potential utilization of stable superoxide dismutase-mimic nitroxides for the prevention of the vascular complications accompanying diabetes.

Effects of the SOD mimic nitroxide 3-carbamoyl-PROXYL on oxidative stress markers and endothelial dysfunction in streptozotocin-induced diabetic rats.

OBJECTIVE: To evaluate the effects of 3-carbamoyl-PROXYL (CP), a stable superoxide dismutase (SOD) mimic compound, on oxidative stress markers and endothelial dysfunction in diabetic rats. ANIMALS AND METHODS: Rats were made diabetic by a single vein injection of streptozotocin (65 mg/kg) and diabetes was verified by the existence of excessive hyperglycemia a week after the treatment. Control and diabetic rats received vehicle or drug for eight weeks, after which the vascular tissue was examined for relaxation and oxidative stress markers. RESULTS: Diabetic rats showed increased vascular levels of superoxide that were accompanied by increased tissue levels of the oxidative stress markers malondialdehyde (MDA) and 8-iso-prostaglandin F2alpha (8-ISO). The vasorelaxant as well as the cyclic guanosine 5'-monophosphate (cGMP)-producing effects of acetylcholine (ACh) and nitroglycerine were reduced in diabetic rats. Treatment of diabetic rats with CP (50 mg/kg intraperitoneally, bid) abolished not only the differences in superoxide, MDA and 8-ISO levels, but also the differences in the relaxation and cGMP responses of vascular tissue between control and diabetic rats to both ACh and nitroglycerine. CONCLUSIONS: These results support the involvement of reactive oxygen species in mediation of diabetes-induced endothelial dysfunction in vivo, and provide the rationale for the potential use of SOD mimics in the treatment of diabetes.

Human alpha-defensin regulates smooth muscle cell contraction: a role for low-density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor.

We have previously identified alpha-defensin in association with medial smooth muscle cells (SMCs) in human coronary arteries. In the present paper we report that alpha-defensin, at concentrations below those found in pathological conditions, inhibits phenylephrine (PE)-induced contraction of rat aortic rings. Addition of 1 microM alpha-defensin increased the half-maximal effective concentration (EC(50)) of PE on denuded aortic rings from 32 to 630 nM. The effect of alpha-defensin was dose dependent and saturable, with a half-maximal effect at 1 microM. alpha-Defensin binds to human umbilical vein SMCs in a specific manner. The presence of 1 microM alpha-defensin inhibited the PE-mediated Ca(++) mobilization in SMCs by more than 80%. The inhibitory effect of alpha-defensin on contraction of aortic rings and Ca(++) mobilization was completely abolished by anti-low-density lipoprotein receptor-related protein/alpha(2-)macroglobulin receptor (LRP) antibodies as well as by the antagonist receptor-associated protein (RAP). alpha-Defensin binds directly to isolated LRP in a specific and dose-dependent manner; the binding was inhibited by RAP as well as by anti-LRP antibodies. alpha-Defensin is internalized by SMCs and interacts with 2 intracellular subtypes of protein kinase C (PKC) involved in muscle contraction, alpha and beta. RAP and anti-LRP antibodies inhibited the binding and internalization of alpha-defensin by SMCs and its interaction with intracellular PKCs. These observations suggest that binding of alpha-defensin to LRP expressed in SMCs leads to its internalization; internalized alpha-defensin binds to PKC and inhibits its enzymatic activity, leading to decreased Ca(++) mobilization and SMC contraction in response to PE.

In vitro and in vivo effects of tPA and PAI-1 on blood vessel tone.

Tissue type plasminogen activator (tPA) is a key enzyme in the fibrinolytic cascade. In this paper we report that tPA contains 2 independent epitopes that exert opposite effects on blood vessel tone. Low concentrations of tPA (1 nM) inhibit the phenylephrine (PE)-induced contraction of isolated aorta rings. In contrast, higher concentrations (20 nM) stimulate the contractile effect of PE. The 2 putative vasoactive epitopes of tPA are regulated by the plasminogen activator inhibitor-1 (PAI-1) and by a PAI-1-derived hexapeptide that binds tPA. TNK-tPA, a tPA variant in which the PAI-1 docking site has been mutated, stimulates PE-induced vasoconstriction at all concentrations used. The stimulatory, but not the inhibitory, effect of tPA on the contraction of isolated aorta rings was abolished by anti-low-density lipoprotein receptor-related protein/alpha(2)-macroglobulin receptor (LRP) antibodies. Administering tPA or TNK-tPA to rats regulates blood pressure and cerebral vascular resistance in a dose-dependent mode. In other in vivo experiments we found that the vasopressor effect of PE is more pronounced in tPA knockout than in wild-type mice. Our findings draw attention to a novel role of tPA and PAI-1 in the regulation of blood vessel tone that may affect the course of ischemic diseases.

Binding of urokinase to low density lipoprotein-related receptor (LRP) regulates vascular smooth muscle cell contraction.

Urokinase plasminogen activator (uPA) is a multifunctional protein that has been implicated in several physiological and pathological processes involving cell adhesion and migration in addition to fibrinolysis. In a previous study we found that two-chain urokinase plasminogen activator (tcuPA) stimulates phenylephrine-induced vasoconstriction of isolated rat aortic rings. In the present paper we report that uPA(-/-) mice have a significantly lower mean arterial blood pressure than do wild type mice and that aortic rings from uPA(-/-) mice show an attenuated contractile response to phenylephrine. In contrast, the blood pressure of urokinase receptor knockout (uPAR(-/-)) mice and the response of their isolated aortic rings to phenylephrine were normal, indicating that the effect of uPA on vascular contraction is independent of uPAR. Addition of mouse and human uPA almost completely reversed both the impaired vascular contractility and the lower arterial blood pressure in vivo. The in vitro and in vivo effects of infused uPA on aortic contractility and the restoration of normal blood pressure in uPA(-/-) mice were prevented by antibody to low-density lipoprotein receptor-related protein/alpha(2)-macroglobulin receptor (LRP). A modified form of uPA that lacks the kringle failed to restore the blood pressure in uPA(-/-) mice, notwithstanding having a longer half-life in the circulation. Ligands that regulate the interaction of uPA with LRP, such as PAI-1 or the PAI-1-derived peptide (EEIIMD), abolished the vasoactivity of tcuPA in vitro and in vivo. These studies identify a novel signal transducing cellular receptor pathway involved in the regulation of vascular contractility.