Blockade of MMP-2 and MMP-9 inhibits corneal lymphangiogenesis.

PURPOSE: To investigate the roles of a selective MMP-2 and -9 inhibitor (SB-3CT) in corneal inflammatory lymphangiogenesis. METHODS: The expression of MMP-2 and -9 in the cornea after suture inplacement, treated with SB-3CT or negative control, was detected by real-time polymerase chain reaction (PCR). Inflammatory corneal neovascularization (NV) was induced by corneal suture placement. Mice were treated with SB-3CT eye drops (twice daily for 1 week, 5 µL per drop; 50, 100, or 200 µM). The outgrowth of blood and lymphatic vessels, and macrophage recruitment were analyzed by immunofluorescence assay. The expressions of vascular endothelial growth factor-C (VEGF-C) and its receptor VEGFR-3 were tested by real-time PCR. RESULTS: MMP-2 and -9 expression were suppressed significantly by treatment with SB-3CT. The data demonstrated, for the first time, that SB-3CT strongly reduced corneal lymphangiogenesis and macrophage infiltration during inflammation. Furthermore, expressions of VEGF-C and its receptor VEGFR-3 were significantly inhibited by SB-3CT during corneal lymphangiogenesis. CONCLUSIONS: These novel findings indicated that blockade of MMP-2 and -9 could inhibit lymphangiogenesis. Further investigation of this factor may provide novel therapies for transplant rejection and other lymphatic disorders.

Propofol increases the Ca2+ sensitivity of BKCa in the cerebral arterial smooth muscle cells of mice.

AIM: Propofol has the side effect of hypotension especially in the elderly and patients with hypertension. Previous studies suggest propofol-caused hypotension results from activation of large conductance Ca(2+)-sensitive K channels (BKCa). In this study, the effects of propofol on the Ca(2+) sensitivity of BKCa were investigated in mice cerebral arterial smooth muscle cells. METHODS: Single smooth muscle cells were prepared from the cerebral arteries of mice. Perforated whole-cell recoding was conducted to investigate the whole-cell BKCa current and spontaneous transient outward K(+) current (STOC). Inside-out patch configuration was used to record the single channel current and to study the Ca(2+)- and voltage-dependence of BKCa. RESULTS: Propofol (56 and 112 µmol/L) increased the macroscopic BKCa and STOC currents in a concentration-dependent manner. It markedly increased the total open probability (NPo) of single BKCa channel with an EC(50) value of 76 µmol/L. Furthermore, propofol significantly decreased the equilibrium dissociation constant (K(d)) of Ca(2+) for BKCa channel. The K(d) value of Ca(2+) was 0.881 µmol/L in control, and decreased to 0.694, 0.599 and 0.177 µmol/L, respectively, in the presence of propofol 28, 56 and 112 µmol/L. An analysis of the channel kinetics revealed that propofol (112 µmol/L) significantly increased the open dwell time and decreased the closed dwell time, which stabilized BKCa channel in the open state. CONCLUSION: Propofol increases the Ca(2+) sensitivity of BKCa channels, thus lowering the Ca(2+) threshold of the channel activation in arterial smooth muscle cells, which causes greater vasodilating effects.

[Gene mapping and mutation detection in a family with congenital anterior polar cataract].

OBJECTIVE: To map the gene mutation responsible for autosomal dominant inherited congenital anterior polar cataract in a Chinese family. METHODS: Peripheral blood samples were collected from the members in this congenital cataract family. DNA was extracted from the blood samples. A gene scan was performed using approximately 400 microsatellite markers (ABI). Linkage analysis was processed to define the region of mutated gene. High density primers labeled with fluorescent stain for the positive region were adopted for fine targeting and haplotype analysis was performed. Mutation detection was carried out by sequencing candidate genes. RESULTS: The maximum two-point LOD score was obtained at D21S1252, Z(max) = 3.23 (θ(max) = 0.00). After fine targeting and haplotype analysis, the mutated gene was located within a 18.47 cM region between D21S263 and D21S266 on chromosome 21q22.11-q22.3. Direct sequencing of the candidate gene revealed a G©öA transition in exon 3 of CRYAA. CONCLUSION: The present study has identified a missense mutation in CRYAA associated with congenital anterior polar cataract in a Chinese family.

Blocking neuropilin-2 enhances corneal allograft survival by selectively inhibiting lymphangiogenesis on vascularized beds.

PURPOSE: To investigate the potential inhibitory effects of RNA interference-mediated knockdown of neuropilin-2 (NP2) on inflammation-induced corneal hemangiogenesis and lymphangiogenesis, and whether selective inhibition of lymphangiogenesis on vascularized recipient beds before transplantation improves the graft survival. METHODS: Mouse lymphatic endothelial cells were transfected with the plasmid expressing artificial microRNA (amiRNA) against mouse NP2, and the down-regulation of VEGF-C-induced NP2 expression by NP2 amiRNA was evaluated by real-time PCR and western blot assays. Next, NP2 amiRNA or negative control amiRNA was injected intrastromally into BALB/c mouse model of suture-induced corneal neovascularization three days after surgery. Corneas were harvested 1 week after suture placement and the formation of lymphatic and blood vessels as well as the recruitment of macrophage was evaluated by immunohistochemical staining. The neovascularized graft beds treated by NP2 amiRNA or control then served as recipients of orthotopic corneal transplants, and age-matched C57BL/6 donors were used. Corneal allografts were examined twice a week for 8 weeks, and graft clarity was quantified by means of an opacity score. RESULTS: VEGF-C-induced NP2 expression at both mRNA and protein levels was significantly suppressed by NP2 amiRNA in mouse lymphatic endothelial cells. Intrastromal administration of NP2 amiRNA reduced corneal lymphangiogenesis by 45% versus control (p=0.015), but corneal hemangiogenesis (p=0.815) and the recruitment of CD11 antigen-like family member B (CD11b)-positive macrophage (p=0.589) were unchanged. Kaplan-Meier survival analysis revealed a better graft survival rate in the vascularized recipient beds pre-treated by NP2 amiRNA in comparison to controls (p=0.014). CONCLUSIONS: Knockdown of NP2 improves corneal graft survival by selectively inhibiting lymphangiogenesis in vascularized beds before transplantation. Thus our results open new treatment options for transplant rejection and other lymphatic disorders.

[Changes of hemodynamics during establishment of animal model of acute myocardial infarction induced by ligation of left coronary artery in goat].

OBJECTIVE: To study the changes in hemodynamics in experimental acute myocardial infarction induced by ligation of the left coronary artery in goat. METHODS: Animal model of acute myocardial infarction was reproduced in 20 goats by ligation of the left coronary artery through xyphoid process. ST segment of electrocardiogram (ECG), mean artery blood pressure (MAP), central venous pressure (CVP), and heart rate (HR) were observed before, immediately, 30 minutes, 1 hour and 2 hours after ligation of the left coronary artery. RESULTS: ECG of all goats was normal before operation. Immediately and 30 minutes after ligation, elevation of ST-segment was seen in 8 and 10 goats respectively, and in 18 goats elevation of ST-segment was observed 2 hours after ligation. Four weeks after the operation, pathological Q wave was shown in the chest leads of ECG in 18 goats. There was no significant difference in MAP, CVP and HR between before and after ligation. Frequent ventricular premature beats were found in 6 goats, but they were stopped after intravenous infusion of lidocaine. CONCLUSION: Small area of experimental acute myocardial infarction in goats shows slight effect on hemodynamics, though the production of myocardial infarction is reliable, and the life of the goats could be maintained for a long time after the ligation of the left coronary artery. The experiment provides a valuable animal model for the study of coronary heart disease.