Endothelin antagonism normalizes VEGF signaling and cardiac function in STZ-induced diabetic rat hearts.

Abnormal alterations in cardiac expression of vascular endothelial growth factor (VEGF) as well as its receptors and impairment in the development of coronary collaterals have recently been reported in diabetic subjects. However, the presence of pharmacological intervention on these defects in diabetes remains unsettled. Here, we studied the effect of endothelin (ET) receptor blockade on cardiac VEGF signaling pathways and cardiac function in Sprague-Dawley rats 5 wk after induction of type I diabetes with streptozotocin (65 mg/kg ip) in comparison with age-matched control rats. After streptozotocin (1 wk), some diabetic rats were treated with the ET receptor antagonist SB-209670 (1 mg/day) for 4 wk. VEGF, its receptors, and its angiogenic signaling molecules [phosphorylated Akt and endothelial nitric-oxide synthase (eNOS)] were analyzed by Western blot, ELISA, real-time PCR, and immunohistochemistry, and cardiac function was evaluated by echocardiography. Coronary capillary morphology was assessed by lectin and enzymatic double staining. We found significant decreases in cardiac expression of VEGF, its receptors, phosphorylation of Akt and eNOS, and coronary capillary density in diabetic rats compared with controls. Treatment of diabetic rats with SB-209670 reversed these alterations to the control levels and ameliorated impairment of cardiac function. From a molecular point of view, the present study is the first to indicate the potential usefulness of an ET receptor antagonist in the treatment of cardiac dysfunction in type I diabetes.

Differential effects of selective endothelin type a receptor antagonist on the gene expression of vascular endothelial growth factor and its receptors in streptozotocin-induced diabetic heart.

Cardiovascular complications are an important feature of diabetes mellitus (DM). Abnormal and decreased coronary collateral development has been implicated in the pathogenesis of cardiac complications in DM. More recently, decreased expression of vascular endothelial growth factor (VEGF) and its receptors has been found in diabetic heart. To our knowledge, no study has focused on the therapeutic improvement associated with VEGF in diabetic heart. DM was induced by intraperitoneal injection of streptozotocin (65 mg/kg) in Sprague-Dawley rats, while control rats received only citrate buffer. After 1 week, the streptozotocin-treated rats were randomly divided into two groups: one group received the selective endothelin (ET) type A receptor antagonist TA-0201 at a dose of 1 mg/kg/day for 2 weeks by osmotic mini-pump, and the vehicle group received saline only. The plasma glucose level was 504 +/- 75 mg/dl in the diabetic rats and was unchanged by treatment with ET antagonist. The body weight was decreased in the diabetic rats compared with the control rats, but the left ventricular (LV)-body weight ratio was increased in the diabetic group and was unaffected by treatment with ET antagonist. mRNA expression of VEGF and its receptors (Flt-1 and Flk-1) in the LV tissues was assessed using real-time polymerase chain reaction. VEGF expression was significantly decreased in diabetic heart and was greatly improved by treatment with ET antagonist. The expression of VEGF receptors was down-regulated in early diabetic heart but was not recovered by treatment with ET antagonist. ET and its receptor A might have differential regulation on the gene expressions of VEGF and its receptors in early diabetic heart.

A model of retinal ischemia-reperfusion injury in rats by subconjunctival injection of endothelin-1.

The retinal ischemia-reperfusion model is used in the study of transient ischemia-related diseases, such as central retinal artery occlusion, angle-closure glaucoma, and others. There are two methods for experimentally producing an ischemia-reperfusion model in the rat retina: (i) the intraocular pressure is greatly raised by increasing the height of the infusion bottle connected with the needle in the anterior chamber; or (ii) the blood vessel that accompanies the optic nerve in retina is ligated. However, each method has some drawbacks. For example, in the first method, the needle must be fixed in the anterior chamber for 1 hr, thus, the technique is not stable and mechanical damage to ocular structures sometimes occurs. In the second method, because of the unavoidable involvement of the optic nerve, damage to the nerve induces retinal changes unrelated to ischemia. In this study, we injected endothelin (ET)-1 under the conjunctiva of the eyeball (subconjunctival injection), and evaluated whether a retinal ischemia-reperfusion model could be generated by this method, simply and noninvasively. We injected 4 x 10(-5) M ET-1 solution into the right eye of the rat and injected a control vehicle (artificial tears) into the left eye. From 5-60 mins after the injection, 50 mg/ml fluorescein isothiocyanate (FITC)-dextran was injected to the left ventricle of heart. Then, the retina was removed and flat mounted. We compared the perfusion conditions of the FITC-dextran to each retina in the right and left eye. There was a complete perfusion of FITC-dextran in the retinal main artery, vein, and the capillary vessels in all of the control eyes. However, perfusion could not be completely observed in the ET-1 injected eye from 5-35 mins after injection; afterwards, the flow was returned. This method of subconjunctival injection of ET-1 is, thus, a feasible technical option for producing a retinal ischemia-reperfusion model in rat.

Effect of endothelin dual receptor antagonist on VEGF levels in streptozotocin-induced diabetic rat retina.

Diabetic retinopathy (DR), one of the most serious causes of blindness, is often associated with the upregulation of vascular endothelial growth factor (VEGF) in retina. Recently, leukocyte adhesion (leukostasis) is blamed for the occlusion of retinal capillary vascularity, which ultimately contributes to the progression of diabetic retinopathy. In addition, intercellular adhesion molecule-1 (ICAM-1), a representative factor for leukostasis, is increased in the diabetic retina. Endothelin (ET)-1, a potent vasoconstrictor peptide, is deeply linked to the pathogenesis of diabetic retinopathy. Different therapeutic interventions concerning VEGF have already been proposed to prevent diabetic retinopathy. However, no study yet has reported whether ET-1 dual receptor antagonist could alter the upregulated VEGF and ICAM-1 levels in the diabetic retina. The present study investigated the effect of ET(A/B) dual receptor antagonist (SB209670; 1 mg/rat/day) on the expression of VEGF and ICAM-1 in the diabetic rat retina. Diabetes was induced by intraperitoneal injection of streptozotocin (STZ; 65 mg/kg) in Sprague-Dawley rats, whereas control rats (non-DM control) received only citrate buffer. After 1 week, the STZ-administered rats were randomly divided into two groups: one group (DM+SB209670) received ET(A/B) dual receptor antagonist for 2 weeks, and a vehicle group (DM+vehicle) was treated only with saline. After the treatment period, the retinas were removed from the eyeballs. In DM+vehicle group, the VEGF expression of the retinas was significantly increased (32.8 pg/mg) in comparison to that in the non-DM control group (26.2 pg/mg); this upregulation of VEGF was reversed in the DM+SB209670 group (28.6 pg/mg). The expression of retinal ICAM-1 was increased in the DM+vehicle group (152.2 pg/mg) compared with the non-DM control group (121.6 pg/mg). However, SB209670 treatment did not alter the expression of retinal ICAM-1 level (154.8 pg/ml) in DM rats. Thus we conclude that an ET(A/B) dual receptor antagonist could reverse the expression level of VEGF in the diabetic retina while failing to normalize the upregulated ICAM-1 expression.

An endothelin type A receptor antagonist reverses upregulated VEGF and ICAM-1 levels in streptozotocin-induced diabetic rat retina.

Diabetic retinopathy, a cause of blindness, is often associated with the upregulation of vascular endothelial growth factor (VEGF) in the retina. Recently, leukocyte adhesion (leukostasis) is claimed for the occlusion of retinal capillary vascularity, which ultimately assists in the progression of diabetic retinopathy. In addition, intercellular adhesion molecule-1 (ICAM-1), a representative factor for leukostasis, is increased in diabetic retina. Endothelin (ET)-1, a potent vasoconstrictor peptide, is closely linked to the pathogenesis of diabetic retinopathy. Different therapeutic interventions concerning VEGF have already been proposed to prevent diabetic retinopathy. However, no study has yet reported concerning the effects of ET-1 receptor antagonist on the upregulated VEGF and ICAM-1 in morphologically intact diabetic retina. The current study investigated the effect of ET(A) receptor antagonist (TA-0201; 1 mg kg(-1) day(-1)) on the expressions of VEGF and ICAM-1 in rat diabetic retina. Diabetes was induced by intraperitoneal injection of streptozotocin (70 mg/kg) in Sprague-Dawley rats, whereas control rats (Cont) received only citrate buffer. After 1 week, the streptozotocin-administered rats were randomly divided into two groups: ET(A) receptor antagonist-treated group (DM+TA-0201) and saline-treated group (DM+vehicle). After the treatment for 4 weeks, the retina was removed from the eyeball. In DM+vehicle group, the VEGF expression of retina was significantly increased (33.5 pg/mg) in comparison with that in the Cont group (25.1 pg/mg), and the upregulation of VEGF was reversed in DM+TA-0201 group (26.9 pg/mg), a phenomenon consistent with the change in VEGF mRNA levels. The expression of retinal ICAM-1 was increased in DM+vehicle group (55.1 pg/mg) compared with Cont group (43.8 pg/mg), and ET antagonism completely blocked this increase (43.8 pg/mg). Moreover, an increased leukostasis by 3.3-fold in DM+vehicle retina was returned to the control level by ET antagonism. In the current study, there was no obvious retinal morphological alteration from both the hematoxylin and eosin staining and the FITC-dextran angiography. Thus, ET(A) receptor antagonist might be useful in preventing the progression of diabetic retinopathy, as evidenced by suppressing the increase in VEGF and ICAM-1 levels as well as leukostasis in morphologically intact diabetic retina.

Effects of endothelin-1 eyedrops on the retina in rats.

Eye disorder accompanied with chronic retinal microvascular obstruction, such as diabetic retinopathy, exists in many diseases. However, it is difficult to produce this model experimentally in the animal eye. Endothelin-1 eyedrops were prepared in order to examine whether the eyedrops affect the rat retina and whether we can produce an obstruction model. Endothelin-1 eyedrops diluted by artificial tears in seven stages from 4 x 10(-5) M to 4 x 10(-11) M were arranged. We administered this solution three times a day in the left eye of male Sprague-Dawley rats. Artificial tears alone were applied to the right eye as a control vehicle. After 2 weeks, rats were sacrificed under anesthesia and the retinal tissues were isolated. As an index to the action of endothelin- 1 eyedrops to the retina, the expressions of endothelin-A (ETA) and endothelin-B (ETB) receptors in the retina were compared in both eyes. Frozen sections of the retina were immunostained to reveal the distribution of the ETA and ETB receptors. We also examined ETA and ETB mRNA expression by quantitative real-time polymerase chain reaction. As a result, the expressions of ETA and ETB receptors are reduced with both immunostaining and the mRNA levels in the left eye, in which endothelin-1 eyedrops were applied at 4 x 10(-5) M. It is suggested that endothelin-1 eyedrops affected the retina and the possibility of producing the experimental model of chronic microvascular obstruction in the rat retina.

A case of congenital oblique retraction syndrome with upshoot in adduction.

We treated a 27-year-old man who suffered from a congenital eye movement abnormality. The patient could not adduct the left eye, and showed exotropia, hypertropia and excyclotropia of the left eye. The range of abduction was also limited. The left eye showed marked upshoot on right gaze and eye globe retraction on right down gaze under conditions of right-eye viewing. The superior rectus muscle of the left eye was recessed and transposed nasally. The insertion of the superior oblique muscle was found to be defective. The upshoot and face turn improved after surgery. The globe retraction on right down gaze was no longer evident. We believe that the superior rectus muscle was innervated by an anomalous branch of the oculomotor nerve, which was originally destined to innervate the medial rectus, and that simultaneous contraction of the superior and inferior rectus muscles caused the globe retraction on right down gaze. This case also suggests the possibility that some part of the upshoot in adduction in Duane's syndrome is due to the co-contraction of the superior and medial rectus muscles due to this innervation anomaly.