Evaluation of an Intravitreal Rho-Associated Kinase Inhibitor Depot Formulation in a Rat Model of Diabetic Retinopathy.

Rho-associated kinase (ROCK) activation was shown to contribute to microvascular closure, retinal hypoxia, and to retinal pigment epithelium (RPE) barrier disruption in a rat model of diabetic retinopathy. Fasudil, a clinically approved ROCK inhibitor, improved retinal perfusion and reduced edema in this model, indicating that ROCK inhibition could be a promising new therapeutic approach for the treatment of diabetic retinopathy. However, due to its short intravitreal half-life, fasudil is not suitable for long-term treatment. In this study, we evaluated a very potent ROCK1/2 inhibitor (BIRKI) in a depot formulation administered as a single intravitreal injection providing a slow release for at least four weeks. Following BIRKI intravitreal injection in old Goto-Kakizaki (GK) type 2 diabetic rats, we observed a significant reduction in ROCK1 activity in the retinal pigment epithelium/choroid complex after 8 days and relocation of ROCK1 to the cytoplasm and nucleus in retinal pigment epithelium cells after 28 days. The chronic ROCK inhibition by the BIRKI depot formulation restored retinal pigment epithelial cell morphology and distribution, favored retinal capillaries dilation, and reduced hypoxia and inner blood barrier leakage observed in the diabetic retina. No functional or morphological negative effects were observed, indicating suitable tolerability of BIRKI after intravitreous injection. In conclusion, our data suggest that sustained ROCK inhibition, provided by BIRKI slow-release formulation, could be a valuable treatment option for diabetic retinopathy, especially with regard to the improvement of retinal vascular infusion and protection of the outer retinal barrier.

A small-molecule inhibitor of lectin-like oxidized LDL receptor-1 acts by stabilizing an inactive receptor tetramer state.

The C-type lectin family member lectin-like oxidized LDL receptor-1 (LOX-1) has been object of intensive research. Its modulation may offer a broad spectrum of therapeutic interventions ranging from cardiovascular diseases to cancer. LOX-1 mediates uptake of oxLDL by vascular cells and plays an important role in the initiation of endothelial dysfunction and its progression to atherosclerosis. So far only a few compounds targeting oxLDL-LOX-1 interaction are reported with a limited level of characterization. Here we describe the identification and characterization of BI-0115, a selective small molecule inhibitor of LOX-1 that blocks cellular uptake of oxLDL. Identified by a high throughput screening campaign, biophysical analysis shows that BI-0115 binding triggers receptor inhibition by formation of dimers of the homodimeric ligand binding domain. The structure of LOX-1 bound to BI-0115 shows that inter-ligand interactions at the receptor interfaces are key to the formation of the receptor tetramer thereby blocking oxLDL binding.

LAMP-2 deficient mice show depressed cardiac contractile function without significant changes in calcium handling.

OBJECTIVE: Mutations in the highly glycosylated lysosome associated membrane protein-2 (LAMP-2) cause, as recently shown, familial Danon disease with mental retardation, mild myopathy and fatal cardiomyopathy. Extent and basis of the contractile dysfunction is not completely understood. METHODS: In LAMP-2 deficient mice, we investigated cardiac function in vivo using Doppler-echocardiography and contractile function in vitro in isolated myocardial trabeculae. RESULTS: LAMP-2 deficient mice displayed reduced ejection fraction (EF) (58.9+/-3.4 vs. 80.7+/-5.1%, P<0.05) and reduced cardiac output (8.3+/-3.1 vs. 14.7+/-3.6 ml/min, P<0.05) as compared to wild-type controls. Isolated multicellular muscle preparations from LAMP-2 deficient mice confirmed depressed force development (3.2+/-0.6 vs. 8.4+/-0.9 mN/mm2, P<0.01). All groups showed similar force-frequency behaviour when normalised to baseline force. Post-rest potentiation was significantly depressed at intervals>15 s in LAMP-2 deficient mice (P<0.05). Although attenuated in absolute force development, the normalised inotropic response to increased calcium and beta-adrenoreceptor stimulation was unaltered. Electron microscopic analysis revealed autophagic vacuoles in LAMP-2 deficient cardiomyocytes. Protein analysis showed unaltered levels of SERCA2a, calsequestrin and phospholamban. CONCLUSIONS: Cardiac contractile function in LAMP-2 deficient mice as a model for Danon disease is significantly attenuated. The occurrence of autophagic vacuoles in LAMP-2 deficient myocytes is likely to be causal for the depressed contractile function resulting in an attenuated cardiac pump reserve.

FKBP12.6 overexpression decreases Ca2+ spark amplitude but enhances [Ca2+]i transient in rat cardiac myocytes.

Ryanodine receptors/Ca2+-release channels (RyR2) from the sarcoplasmic reticulum (SR) provide the Ca2+ required for contraction at each cardiac twitch. RyR2 are regulated by a variety of proteins, including the immunophilin FK506 binding protein (FKBP12.6). FKBP12.6 seems to be important for coupled gating of RyR2 and its deficit and alteration may be involved in heart failure. The role of FKBP12.6 on Ca2+ release has not been analyzed directly, but rather it was inferred from the effects of immunophilins, such us FK506 and rapamycin, which, among other effects, dissociates FKBP12.6 from the RyR2. Here, we investigated directly the effects of FKBP12.6 on local (Ca2+ sparks) and global [intracellular Ca2+ concentration ([Ca2+]i) transients] Ca2+ release in single rat cardiac myocytes. The FKBP12.6 gene was transfected in single myocytes using the adenovirus technique with a reporter gene strategy based on green fluorescent protein (GFP) to check out the success of transfections. Control myocytes were transfected with only GFP (Ad-GFP). Rhod-2 was used as the Ca2+ indicator, and cells were viewed with a confocal microscope. We found that overexpression of FKBP12.6 decreases the occurrence, amplitude, duration, and width of spontaneous Ca2+ sparks. FK506 had diametrically opposed effects. However, overexpression of FKBP12.6 increased the [Ca2+]i transient amplitude and accelerated its decay in field-stimulated cells. The associated cell shortening was increased. SR Ca2+ load, estimated by rapid caffeine application, was increased. In conclusion, FKBP12.6 overexpression decreases spontaneous Ca2+ sparks but increases [Ca2+]i transients, in relation with enhanced SR Ca2+ load, therefore improving excitation-contraction coupling.