Anton Lennikov

Tissue Kallikrein Attenuates Choroidal Neovascularization via Cleavage of Vascular Endothelial Growth Factor

PURPOSEnTo investigate the antiangiogenic properties of tissue kallikrein in a murine model of laser-induced choroidal neovascularization (CNV).nnnMETHODSnCNV was induced in male C57BL/6J mice by laser photocoagulation. The animals received daily subcutaneous injections of tissue kallikrein (50 μg/kg) or vehicle control for 2 days before the laser photocoagulation, and this treatment continued until sample collection. Seven days after laser injury, the CNV size was quantified. The levels of monocyte chemoattractant protein (MCP)-1, intercellular adhesion molecule (ICAM)-1, and interleukin (IL)-6 were assessed by enzyme-linked immunosorbent assay 3 days after laser injury. Cleavage of mouse VEGF with tissue kallikrein was assessed in vivo and in vitro. The protein levels of bradykinin were assessed in the RPE-choroid complexes and hearts.nnnRESULTSnA significant decrease in CNV size was observed in animals treated with tissue kallikrein (27,168.3 ± 2432.2 μm(2)) compared with vehicle-treated controls (36,374.6 ± 3204.1 μm(2), P < 0.05). Tissue kallikrein treatment significantly reduced MCP-1, ICAM-1, and IL-6 levels in RPE-choroid complexes. Furthermore, immunoblotting showed the bands, presumably corresponding to the fragmented VEGF(164) protein, in the samples of both mouse VEGF preincubated with tissue kallikrein and RPE-choroid complexes obtained from animals treated with tissue kallikrein. In addition, bradykinin was unchanged in the RPE-choroid complexes of animals treated with tissue kallikrein, whereas the level of bradykinin was increased in the heart obtained from these experimental animals.nnnCONCLUSIONSnThe current data indicate that kallikrein exhibits antiangiogenic properties by cleaving VEGF(164) in a laser-induced CNV model.

Induction of Heat Shock Protein 70 Ameliorates Ultraviolet-Induced Photokeratitis in Mice

Acute ultraviolet (UV) B exposure causes photokeratitis and induces apoptosis in corneal cells. Geranylgeranylacetone (GGA) is an acyclic polyisoprenoid that induces expression of heat shock protein (HSP)70, a soluble intracellular chaperone protein expressed in various tissues, protecting cells against stress conditions. We examined whether induction of HSP70 has therapeutic effects on UV-photokeratitis in mice. C57 BL/6 mice were divided into four groups, GGA-treated (500 mg/kg/mouse) and UVB-exposed (400 mJ/cm2), GGA-untreated UVB-exposed (400 mJ/cm2), GGA-treated (500 mg/kg/mouse) but not exposed and naive controls. Eyeballs were collected 24 h after irradiation, and corneas were stained with hematoxylin and eosin (H&E) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). HSP70, reactive oxygen species (ROS) production, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and protein kinase B (Akt) expression were also evaluated. Irradiated corneal epithelium was significantly thicker in the eyes of mice treated with GGA compared with those given the vehicle alone (p < 0.01). Significantly fewer TUNEL-positive cells were observed in the eyes of GGA-treated mice than controls after irradiation (p < 0.01). Corneal HSP70 levels were significantly elevated in corneas of mice treated with GGA (p < 0.05). ROS signal was not affected by GGA. NF-κB activation was reduced but phospho-(Ser/Ther) Akt substrate expression was increased in corneas after irradiation when treated with GGA. GGA-treatment induced HSP70 expression and ameliorated UV-induced corneal damage through the reduced NF-κB activation and possibly increased Akt phosphorilation.

Single center study on ethnic and clinical features of Behcet’s disease in Moscow, Russia

For the purpose of investigating Behcet’s disease (BD) in Russia, 250 consecutive patients (177 men and 73 women) diagnosed with BD between 1990 and 2010 at the Research Institute of Rheumatology, Russian Academy of Medical Sciences in Moscow were enrolled in this study. The ethnic backgrounds of the patients were reported as follows: 23.2xa0% (58 cases) from Russia, 12.8xa0% (32 cases) from Azerbaijan, 14.4xa0% (36 cases) from Armenia, 8.8xa0% (22 cases) from Chechnya, and 21.6xa0% (55 cases) from Dagestan. The remaining 19.2xa0% (48 cases) were from other regions or of unknown origin. More than half (57.6xa0%) of the Behcet’s disease patients originated from Central Asia, specifically Azerbaijan, Armenia, Chechnya, and Dagestan. The mean age at disease onset was 31.5u2009±u20099.38 (13–60)u2009years old, and the most typical initial manifestations were oral aphthous ulcers. Patients aged 20–39xa0years old were more commonly affected and displayed a wide clinical spectrum of the disease, with varieties of severe internal organ involvement. The manifestations observed throughout the course of the disease included oral aphthous ulcers (100xa0%), various cutaneous lesions (88.8xa0%), genital ulcers (81.2xa0%), and ocular lesions (54.0xa0%). Besides these, many organs/systems were implicated in patient cases, namely joint (53.2xa0%), vascular (25.2xa0%), neurological (8.0xa0%), gastrointestinal (25.2xa0%), and cardiac (5.6xa0%) systems. Involvements of ocular (pu2009<u20090.01) and skin (pu2009<u20090.01) lesions were more frequent in men than in women. HLA-B51 and HLA-A26 typing was performed in 127 patients and 508 healthy controls. HLA-B51 was found in 63.0xa0% of BD patients compared to 20.7xa0% of the healthy control subjects (pu2009<u20090.001), and HLA-A26 was present in 11.3xa0% of BD patients and 18.9xa0% of the control group. This study shows the presence of BD in Russia, and it is suggested that its prevalence in Central Asian people is much higher than that in White Russian.

Amelioration of experimental autoimmune uveoretinitis by inhibition of glyceraldehyde‐derived advanced glycation end‐product formation

AGEs are permanently modified macromolecule derivatives that form through nonenzymatic glycation of amino groups of proteins. Glycer‐AGEs are highly toxic and play an important role in the pathogenesis of chronic inflammatory diseases. However, the contribution of glycer‐AGEs to the pathogenesis of uveitis is unclear. In this study, we measured serum levels of glycer‐AGEs in 100 patients with endogenous uveitis (22 with HLA‐B27‐associated uveitis, 20 with VKH disease, 14 with Behçets disease, and 44 with sarcoidosis) and 33 healthy volunteers. We then examined the effect of the AGE inhibitor in a mouse model of human endogenous uveitis (EAU) by continuous oral administration of pyridoxamine at 200 or 400 mg/kg/day. Regardless of the etiology, serum glycer‐AGE levels were significantly higher in patients with uveitis than in healthy subjects. Treatment with 400 mg/kg pyridoxamine significantly reduced the clinical and histological severity of EAU and was accompanied by a significant decrease in serum and retinal glycer‐AGE levels and suppression of translocation of NF‐κB p65 into the nucleus of retinal cells. Serum glycer‐AGE levels may therefore serve as a biomarker of human uveitis, as well as systemic inflammation, and may contribute to the progression of uveitis, including diabetic iritis, via the activation of NF‐κB.

Increase of lysosomal phospholipase A2 in aqueous humor by uveitis

This study was conducted to elucidate pathophysiological roles of the lysosomal phospholipase A2 (LPLA2), a phospholipid-degrading enzyme, of the aqueous humor (AH) in uveitis using an animal model and clinical specimens. Endotoxin-induced uveitis (EIU) was induced by subcutaneous injections of lipopolysaccharide from Escherichia coli to seven-week-old male Lewis rats. Inflammation of the anterior chamber (AC) was evaluated by measurement of the protein concentration of rat AH. The LPLA2 activity in the AH, serum and cerebrospinal fluid obtained from EIU rats was detected using liposomes consisting of 1,2-dioleoylphosphatidylglycerol/N-acetylsphingosine as the substrate under acidic conditions. Immunohistochemical analysis was performed using antibodies against CD11b and LPLA2. Sixty-five human AH specimens, in which 11 eyes had a history of chronic uveitis, were collected during patient cataract surgeries and used to determine LPLA2 activity. The LPLA2 activity in rat AH was significantly increased by EIU induction, and was correlated to the extent of inflammation in the AC. By contrast, the LPLA2 activity in rat serum or cerebrospinal fluid was not influenced by EIU induction. According to the immunohistochemistry, LPLA2 was found in CD11b positive cells in the AC of the EIU rats. In the clinical specimens, the AH obtained from the patients with a history of uveitis possessed significantly higher LPLA2 activity than that from the senile patients with cataract but without other ocular diseases. These results demonstrate that the LPLA2 activity in the AH is augmented with the inflammation in the AC and suggest that the LPLA2 in the AH participates in the inflammation process in the AC.