Suppression of choroidal neovascularization by vasohibin-1, a vascular endothelium-derived angiogenic inhibitor.

PURPOSE. To determine the expression of vasohibin-1 during the development of experimentally induced choroidal neovascularization (CNV) and to investigate the effect of vasohibin-1 on the generation of CNV. METHODS. CNV lesions were induced in the eyes of wild-type (WT) and vasohibin-1 knockout (KO) mice by laser photocoagulation. The expression of vasohibin-1, vascular endothelial growth factor (VEGF), VEGF receptor-1 (VEGFR1), VEGFR2, and pigment epithelial-derived factor (PEDF) was determined by semiquantitative reverse transcription-polymerase chain reaction. The expression of vasohibin-1 was also examined by immunohistochemistry with anti-CD68, anti-alpha smooth muscle actin (αSMA), anti-cytokeratin, and anti-CD31. Vasohibin-1 was injected into the vitreous and the activity and size of the CNV were determined by fluorescein angiography and in choroidal flat mounts. RESULTS. Vasohibin-1 was detected not only in CD31-positive endothelial cells but also in CD68-positive macrophages and αSMA-positive retinal pigment epithelial cells. Strong vasohibin-1 expression was observed at day 28, when the CNV lesions had regressed by histologic examination. The vasohibin-1 level was significantly decreased at day 14 and increased at day 28 after laser application. Significantly less VEGFR2 expression was observed on day 4 after vasohibin-1. The expression of PEDF was not significantly changed by vasohibin-1 injection. Vasohibin-1 injection significantly suppressed the CNV, with no adverse side effects. The CNV lesions in the vasohibin-1-KO mice were significantly larger than those in the WT mice. CONCLUSIONS. The endogenous expression of vasohibin-1 is associated with the natural course of the development of CNV. Intravitreal injections of vasohibin-1 may be a method for inhibiting CNV.

Glycosaminoglycan modification of neuropilin-1 modulates VEGFR2 signaling.

Neuropilin-1 (NRP1) is a co-receptor for vascular endothelial growth factor (VEGF) that enhances the angiogenic signals cooperatively with VEGFR2. VEGF signaling is essential for physiological and pathological angiogenesis through its effects on vascular endothelial cells (ECs) and smooth muscle cells (SMCs), but the mechanisms coordinating this response are not well understood. Here we show that a substantial fraction of NRP1 is proteoglycan modified with either heparan sulfate or chondroitin sulfate on a single conserved Ser. The composition of the NRP1 glycosaminoglycan (GAG) chains differs between ECs and SMCs. Glycosylation increased VEGF binding in both cell types, but the differential GAG composition of NRP1 mediates opposite responsiveness to VEGF in ECs and SMCs. Finally, NRP1 expression and its GAG modification post-transcriptionally regulate VEGFR2 protein expression. These findings indicate that GAG modification of NRP1 plays a critical role in modulating VEGF signaling, and may provide new insights into physiological and pathological angiogenesis.

Opening of Ca2+-activated K+ channels is involved in ischemic preconditioning in canine hearts.

Brief periods of ischemia that precede sustained ischemia can markedly reduce infarct size (IS), a phenomenon that is known as ischemic preconditioning (IP). Several investigators have shown that elevation of the intracellular Ca(2+) level ([Ca(2+)](i)) during the antecedent brief periods of ischemia triggers the cardioprotective mechanism of IP. Since opening of Ca(2+) activated K(+) (K(Ca)) channels is reported to be cardioprotective, we hypothesized that these channels may be involved in the cardioprotective mechanism of IP. In anesthetized open-chest dogs, myocardial ischemia/reperfusion injury was created by occlusion of the left anterior descending coronary artery (LAD) for 90 min followed by 6 h of reperfusion. First, we showed that the treatment with NS1619, a K(Ca) channel opener, reduced IS (IS in NS1619 group and control group, 19.8 +/- 5.5% vs. 45.4 +/- 3.5% of the area at risk, P < 0.05). Next, four cycles coronary occlusion for 5 min and reperfusion (IP) were performed before the 90-min occlusion with or without the infusion of potent K(Ca) channel inhibitors, iberiotoxin (IbTX) and charybdotoxin (ChTX). IP markedly reduced IS (IS in the IP group was 8.2 +/- 1.8%, P < 0.01 vs. control group). Infusion of either of K(Ca) channel blockers during IP blunted the IS-limiting effect of IP (IS in the IP + IbTX and IP + ChTX groups was 30.7 +/- 7.0% and 35.5 +/- 3.7%, respectively, P < 0.05, vs. IP group). However, the cardioprotective effect of IP was not blunted by the treatment with ChTX when treated only during reperfusion (14.0 +/- 4.1%). Thus, we conclude that the opening of K(Ca) channel is involved in early trigger phase of the molecular mechanism of IP.

Protein kinase A as another mediator of ischemic preconditioning independent of protein kinase C.

BACKGROUND: We and others have reported that transient accumulation of cyclic AMP (cAMP) in the myocardium during ischemic preconditioning (IP) limits infarct size independent of protein kinase C (PKC). Accumulation of cAMP activates protein kinase A (PKA), which has been demonstrated to cause reversible inhibition of RhoA and Rho-kinase. We investigated the involvement of PKA and Rho-kinase in the infarct limitation by IP. METHODS AND RESULTS: Dogs were subjected to 90-minute ischemia and 6-hour reperfusion. We examined the effect on Rho-kinase activity during sustained ischemia and infarct size of (1) preischemic transient coronary occlusion (IP), (2) preischemic activation of PKA/PKC, (3) inhibition of PKA/PKC during IP, and (4) inhibition of Rho-kinase or actin cytoskeletal deactivation during myocardial ischemia. Either IP or dibutyryl-cAMP treatment activated PKA, which was dose-dependently inhibited by 2 PKA inhibitors (H89 and Rp-cAMP). IP and preischemic PKA activation substantially reduced infarct size, which was blunted by preischemic PKA inhibition. IP and preischemic PKA activation, but not PKC activation, caused a substantial decrease of Rho-kinase activation during sustained ischemia. These changes were cancelled by preischemic inhibition of PKA but not PKC. Furthermore, either Rho-kinase inhibition (hydroxyfasudil or Y27632) or actin cytoskeletal deactivation (cytochalasin-D) during sustained ischemia achieved the same infarct limitation as preischemic PKA activation without affecting systemic hemodynamic parameters, the area at risk, or collateral blood flow. CONCLUSIONS: Transient preischemic activation of PKA reduces infarct size through Rho-kinase inhibition and actin cytoskeletal deactivation during sustained ischemia, implicating a novel mechanism for cardioprotection by ischemic preconditioning independent of PKC and a potential new therapeutic target.

Lamr1 functional retroposon causes right ventricular dysplasia in mice.

Arrhythmogenic right ventricular dysplasia (ARVD) is a hereditary cardiomyopathy that causes sudden death in the young. We found a line of mice with inherited right ventricular dysplasia (RVD) caused by a mutation of the gene laminin receptor 1 (Lamr1). This locus contained an intron-processed retroposon that was transcribed in the mice with RVD. Introduction of a mutated Lamr1 gene into normal mice by breeding or by direct injection caused susceptibility to RVD, which was similar to that seen in the RVD mice. An in vitro study of cardiomyocytes expressing the product of mutated Lamr1 showed early cell death accompanied by alteration of the chromatin architecture. We found that heterochromatin protein 1 (HP1) bound specifically to mutant LAMR1. HP1 is a dynamic regulator of heterochromatin sites, suggesting that mutant LAMR1 impairs a crucial process of transcriptional regulation. Indeed, mutant LAMR1 caused specific changes to gene expression in cardiomyocytes, as detected by gene chip analysis. Thus, we concluded that products of the Lamr1 retroposon interact with HP1 to cause degeneration of cardiomyocytes. This mechanism may also contribute to the etiology of human ARVD.