A drug-tunable Flt23k gene therapy for controlled intervention in retinal neovascularization.

Gene therapies that chronically suppress vascular endothelial growth factor (VEGF) represent a new approach for managing retinal vascular leakage and neovascularization. However, constitutive suppression of VEGF in the eye may have deleterious side effects. Here, we developed a novel strategy to introduce Flt23k, a decoy receptor that binds intracellular VEGF, fused to the destabilizing domain (DD) of Escherichia coli dihydrofolate reductase (DHFR) into the retina. The expressed DHFR(DD)-Flt23k fusion protein is degraded unless "switched on" by administering a stabilizer; in this case, the antibiotic trimethoprim (TMP). Cells transfected with the DHFR(DD)-Flt23k construct expressed the fusion protein at levels correlated with the TMP dose. Stabilization of the DHFR(DD)-Flt23k fusion protein by TMP was able to inhibit intracellular VEGF in hypoxic cells. Intravitreal injection of self-complementary adeno-associated viral vector (scAAV)-DHFR(DD)-Flt23k and subsequent administration of TMP resulted in tunable suppression of ischemia-induced retinal neovascularization in a rat model of oxygen-induced retinopathy (OIR). Hence, our study suggests a promising novel approach for the treatment of retinal neovascularization. Schematic diagram of the tunable system utilizing the DHFR(DD)-Flt23k approach to reduce VEGF secretion. a The schematic shows normal VEGF secretion. b Without the ligand TMP, the DHFR(DD)-Flt23k protein is destabilized and degraded by the proteasome. c In the presence of the ligand TMP, DHFR(DD)-Flt23k is stabilized and sequestered in the ER, thereby conditionally inhibiting VEGF. Green lines indicate the intracellular and extracellular distributions of VEGF. Blue lines indicate proteasomal degradation of the DHFR(DD)-Flt23k protein. Orange lines indicate the uptake of cell-permeable TMP. TMP, trimethoprim; VEGF, vascular endothelial growth factor; ER, endoplasmic reticulum.

AAV-mediated gene delivery of the calreticulin anti-angiogenic domain inhibits ocular neovascularization.

Ocular neovascularization is a common pathological feature in diabetic retinopathy and neovascular age-related macular degeneration that can lead to severe vision loss. We evaluated the therapeutic efficacy of a novel endogenous inhibitor of angiogenesis, the calreticulin anti-angiogenic domain (CAD180), and its functional 112-residue fragment, CAD-like peptide 112 (CAD112), delivered using a self-complementary adeno-associated virus serotype 2 (scAAV2) in rodent models of oxygen-induced retinopathy and laser-induced choroidal neovascularization. The expression of CAD180 and CAD112 was elevated in human umbilical vein endothelial cells transduced with scAAV2-CAD180 or scAAV2-CAD112, respectively, and both inhibited angiogenic activity in vitro. Intravitreal gene delivery of scAAV2-CAD180 or scAAV2-CAD112 significantly inhibited ischemia-induced retinal neovascularization in rat eyes (CAD180: 52.7% reduction; CAD112: 49.2% reduction) compared to scAAV2-mCherry, as measured in retinal flatmounts stained with isolectin B4. Moreover, the retinal structure and function were unaffected by scAAV2-CAD180 or scAAV2-CAD112, as measured by optical coherence tomography and electroretinography. Moreover, subretinal delivery of scAAV2-CAD180 or scAAV2-CAD112 significantly attenuated laser-induced choroidal neovascularization in mouse eyes compared to scAAV2-mCherry, as measured by fundus fluorescein angiography (CAD180: 62.4% reduction; CAD112: 57.5% reduction) and choroidal flatmounts (CAD180: 40.21% reduction; CAD112: 43.03% reduction). Gene delivery using scAAV2-CAD180 or scAAV2-CAD112 has significant potential as a therapeutic option for the management of ocular neovascularization.

Prevention of aortic elastic lamina defects by losartan in apolipoprotein(E)-deficient mouse.

1. In a previous study, we identified prevalent internal elastic lamina (IEL) defects in the aorta of hyperlipidaemic apolipoprotein E (ApoE)-deficient mice that are thought to provide a structural basis for the development of atherosclerosis and intimal thickening. In the present study, we examined the effects of losartan, an angiotensin AT1 receptor antagonist, on the development of IEL defects. 2. Male 18-week-old ApoE-deficient mice (maintained on a normal diet) were treated with losartan (3 or 30 mg/kg per day) for 10 weeks via the drinking water. The IEL defects were quantified histologically by measuring the continuity of the IEL within the inner curvature of the aortic arch. 3. In untreated animals, there was an age-dependent increase in IEL defects from 7.2 ± 2.1% at 18 weeks to 13.8 ± 4.0% at 28 weeks. Treatment with the high dose of losartan significantly prevented the development of IEL defects (4.7 ± 1.3% at 28 weeks; P < 0.05 vs untreated). This effect was independent of changes in blood pressure or plasma lipid levels. Using quantitative real-time polymerase chain reaction, we found that the effects of losartan were not associated with changes in levels of matrix metalloproteinase (MMP)-2 and MMP-9, tissue inhibitor of matrix metalloproteinase-1 or inflammatory markers in the aorta. 4. The results suggest that the renin-angiotensin system may contribute to the development of aortic IEL defects in a blood pressure-independent manner.

Annexin-1 peptide Anx-1(2-26) protects adult rat cardiac myocytes from cellular injury induced by simulated ischaemia.

1 The anti-inflammatory properties of annexin-1 peptides have been largely ascribed to their powerful antineutrophil actions in vivo. We have recently reported that the N-terminal fragment of annexin-1, Anx-1(2-26), preserves contractile function of cardiac muscle in vitro. The aim of the present study was to determine if Anx-1(2-26) elicits protective actions specifically on the cardiac myocyte (in the absence of neutrophils), using a model of metabolic inhibition to simulate ischaemia. 2 Metabolic inhibition of cardiac myocytes (4 h incubation at 37 degrees C in HEPES-containing buffer supplemented with 2-deoxy-D-glucose, D,L-lactic acid and pH adjusted to 6.5) followed by 2.5 h recovery in normal medium markedly increased creatine kinase (CK) and lactate dehydrogenase (LDH) levels by 179+/-39 and 26+/-7 IU L(-1) (both n=40, P<0.001), respectively. However, cellular injury was significantly decreased when Anx-1(2-26) (0.3 microM) was present during metabolic inhibition, CK by 74+/-10% and LDH by 71+/-6% (both n=31, P<0.001), respectively. 3 Boc 2 (10 microM), a nonselective formyl peptide receptor antagonist, present during metabolic inhibition, abolished the cardioprotective effect of Anx-1(2-26). 4 Addition of chelerythrine (10 microM), 5-hydroxydecanoate (500 microM) or SB202190 (1 microM) during metabolic inhibition also abolished Anx-1(2-26)-induced cardioprotection. 5 Cellular injury induced by metabolic inhibition was also largely prevented when myocytes were incubated with Anx-1(2-26) for 5 min with 10 min recovery prior to the insult, or when Anx-1(2-26) was present only during the recovery period following drug-free metabolic inhibition. 6 In conclusion, the annexin-1 peptide Anx-1(2-26) potently prevents cardiac myocyte injury induced by metabolic inhibition, an action that was dependent at least in part on the activation of the formyl peptide receptor family of G-protein-coupled receptors, protein kinase C, p38 mitogen-activated protein kinase and ATP-sensitive potassium channels.

Natural phenolic compounds as cardiovascular therapeutics: potential role of their antiinflammatory effects.

A number of epidemiological studies have shown that diets rich in plant-derived phenolic compounds reduce the risk of coronary heart disease. The chronic antioxidant and hypolipidemic activities of these compounds have important roles in prevention of lipoprotein oxidation and atherosclerotic lesion development. In recent years, it has been recognized that inflammation is directly involved in development of cardiovascular disease and clinical events such as atherosclerotic plaque rupture (which is the trigger of acute coronary syndrome), arterial restenosis, and myocardial ischemia-reperfusion injury. Phenolic compounds have significant antiinflammatory effects, including inhibition of adhesion molecule, cytokine and chemokine gene expression; inhibition of platelet function; augmentation of endothelial nitric oxide release; suppression of smooth muscle activation; and other effects on proinflammatory factors such as endothelin and matrix metalloproteinases. However, direct evidence of acute therapeutic benefits of phenolic compounds in cardiovascular disorders remains sparse. This review attempts to integrate the inflammatory mechanisms involved in these cardiovascular diseases with recent findings on the antiinflammatory effects of phenolic compounds. Findings from the limited in vivo studies in this regard are discussed. It is suggested that searching for novel phenolic compounds with higher specificity and efficacy may represent a fruitful approach in development of new cardiovascular therapeutics.