The angiopoietin/Tie-2 system regulates pericyte survival and recruitment in diabetic retinopathy

PURPOSE: The angiopoietin (Ang) system plays an important role in vascular stabilization and pathologic neovascularization. The hypothesis for the study was that, in addition to modulating endothelial cell behavior, the angiopoietin/Tie-2 system also regulates the pericyte apoptosis and/or the vessel maturation associated with diabetic retinopathy. METHODS: Tie-2 expression in cultured retinal pericytes was analyzed by using ELISA, Western Blot analysis, and flow cytometry. CD13 (aminopeptidase N) expression in pericytes was determined by Western blot analysis and Ang effects verified with Tie-2 antisense treatment. Cell proliferation was monitored by crystal violet uptake, and pericyte migration was assessed in a scrape wound. Annexin V-FITC flow cytometry was used to quantify pericyte apoptosis. RESULTS: Pericytes expressed a functionally active Tie-2 receptor upregulated by both Ang-1 and -2 (P < 0.05). In pericytes undergoing apoptosis induced by either TNF-alpha or high glucose Ang-1 increased survival (P < 0.05 for TNF-alpha; P < 0.01 for high glucose), whereas Ang-2 increased apoptosis. Ang-1 enhanced CD13 expression in a dose-dependent manner (P < 0.05) and stimulated pericyte migration in a synthetic matrix wound-healing assay that was associated with a change in F-actin organization. Addition of Tie-2 antisense confirmed that angiopoietins act through Tie-2. CONCLUSIONS: These findings demonstrate that Tie-2 is functional in pericytes and may play an important role in the progression of diabetic retinopathy, by regulating pericyte loss and influencing the activation state and recruitment of pericytes.     

Herbal treatment following post-seizure induction in rat by lithium pilocarpine: Scutellaria lateriflora (Skullcap), Gelsemium sempervirens (Gelsemium) and Datura stramonium (Jimson Weed) may prevent development of spontaneous seizures

About 1 week after the induction of status epilepticus in male rats by a single systemic injection of lithium (3 mEq/kg) and pilocarpine (30 g/kg), rats were continuously administered one of three herbal treatments through the water supply for 30 days. A fourth group received colloidal minerals and diluted food grade hydrogen peroxide in tap water, while a fifth group of rats received only tap water (control). Herbal treatments were selected for their historical antiseizure activities and sedative actions on the nervous system. The numbers of spontaneous seizures per day during a 15 min observation interval were recorded for each rat during the treatment period and during an additional 30 days when only tap water was given. Rats that received a weak solution of the three herbal fluid extracts of Scutellaria lateri flora (Skullcap), Gelsemium sempervirens (Gelsemium) and Datura stramonium (Jimson Weed) displayed no seizures during treatment while all the other groups were not seizure-free. However, when this treatment was removed, the rats in this group displayed numbers of spontaneous seizures comparable to the controls. Although there is no proof that herbal remedies can control limbic or temporal lobe epilepsy, the results of this experiment strongly suggest that the appropriate combination of herbal compounds may be helpful as adjunctive interventions.     

Main partitioning criteria for the characterization of the health status in the freshwater mussel Anodonta cygnea from spontaneously polluted area in Western Ukraine

The aim of this study was to appreciate the consequences of spontaneous human activity for freshwater mollusks in the generally ecologically sustainable area in Western Ukraine. For this, bivalve mollusk, Anodonta cygnea, at three sites, with mixed agricultural and municipal activities (A), close to a municipal water inlet (F) and the cooling pond of a nuclear power plant (N), were studied in spring, summer, and autumn. The set of parameters included the characteristics of oxidative stress (activity of catalase (CAT), levels of protein carbonyls (PC)), levels of reduced and oxidized glutathione (GSH, GSSG, respectively), activities of lactate dehydrogenase (LD), cholinesterase (ChE), ethoxyresorufin‐O‐deethylase (EROD), glutathione S‐transferase (GST) in the digestive gland, and concentrations of vitellogenin‐like proteins (Vtg‐LP) in gonads and also morphological indices. Although the discriminant functional analysis confirmed the general seasonal regularities for studied groups, it allowed to discriminate between sites (P < 0.05). At site A, oxidative stress; high levels of LD, EROD, and GST; and low levels of ChE and condition factor were reflected. This demonstrated the sensitivity of mussels to constant effect of mixed pollution. At site N, oxidative injury was shown that might be explained by the constantly high temperature. At site F, abrupt elevations of Vtg‐LP and EROD levels in autumn were probably related to an emergency situation on the nearby dump. So, both chronic and temporal environmental effects were reflected by a set of markers in mollusk. The classification and regression tree (CART) algorithm selected GSH and PC in the digestive gland and Vtg‐LP as partitioning criteria for the characterization of mussel health status. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2012.     

Analytical approaches for monitoring exposure to organophosphorus and carbamate agents through analysis of protein adducts

Appropriate treatment of a poisoned patient requires knowing the identity of the poison. This review summarizes the methods for identifying poisoning by organophosphorus and carbamate poisons. Mass spectrometry methods identify the poison from the adducts they form with proteins in blood. The most sensitive method uses potassium fluoride to release the organophosphorus agent from its covalent binding to serine 198 of human butyrylcholinesterase. The released poison is identified by gas chromatography-mass spectrometry. The drawback of this method is that it does not detect exposure to agents such as soman, because butyrylcholinesterase adducts with these agents age to a non-reactivatable form. A method that detects both aged and non-aged organophosphylated adducts as well as carbamate adducts is one that digests butyrylcholinesterase with a protease and analyzes the modified peptide by mass spectrometry. This method does not distinguish between poisons that have the same mass after reaction with butyrylcholinesterase--for example, between exposure to chlorpyrifos oxon and paraoxon. Albumin forms a stable, covalent bond with organophosphates on tyrosine 411. The rate of reaction with albumin is much slower than with butyrylcholinesterase, but albumin adducts persist for a longer time in the circulation; they do not age; and they do not release the organophosphate when a patient is treated with an oxime.     

Protection from the toxicity of diisopropylfluorophosphate by adeno-associated virus expressing acetylcholinesterase

Organophosphorus esters (OP) are highly toxic chemicals used as pesticides and nerve agents. Their acute toxicity is attributed to inhibition of acetylcholinesterase (AChE, EC 3.1.1.7) in nerve synapses. Our goal was to find a new therapeutic for protection against OP toxicity. We used a gene therapy vector, adeno-associated virus serotype 2 (AAV-2), to deliver murine AChE to AChE-/- mice that have no endogenous AChE activity. The vector encoded the most abundant form of AChE: exons 2, 3, 4, and 6. Two-day old animals, with an immature immune system, were injected. AChE delivered intravenously was expressed up to 5 months in plasma, liver, heart, and lung, at 5-15% of the level in untreated wild-type mice. A few mice formed antibodies, but antibodies did not block AChE activity. The plasma AChE was a mixture of dimers and tetramers. AChE delivered intramuscularly had 40-fold higher activity levels than in wild-type muscle. None of the AChE was collagen-tailed. No retrograde transport through the motor neurons to the central nervous system was detected. AChE delivered intrastriatally assembled into tetramers. In brain, the AAV-2 vector transduced neurons, but not astrocytes and microglia. Vector-treated AChE-/- mice lived longer than saline-treated controls. AChE-/- mice were protected from diisopropylfluorophosphate-induced respiratory failure when the vector was delivered intravenously, but not intrastriatally. Since vector-treated animals had no AChE activity in diaphragm muscle, protection from respiratory failure came from AChE in other tissues. We conclude that AChE scavenged OP and in this way protected the activity of butyrylcholinesterase (BChE, EC 3.1.1.8) in motor endplates.     

Methamidophos,dichlorvos, O‐methoate and diazinon pesticides used in Turkey make a covalent bond with butyrylcholinesterase detected by mass spectrometry

Organophosphorus pesticides used most commonly in Turkey include methamidophos, dichlorvos, O‐methoate and diazinon. These toxic chemicals or their metabolites make a covalent bond with the active site serine of butyrylcholinesterase. Our goal was to identify the adducts that result from the reaction of human butyrylcholinesterase with these pesticides. Highly purified human butyrylcholinesterase was treated with a 20‐fold molar excess of pesticide. The protein was denatured by boiling and digested with trypsin. MS and MSMS spectra of HPLC‐purified peptides were acquired on a MALDI‐TOF‐TOF 4800 mass spectrometer. It was found that methamidophos added a mass of +93, consistent with addition of methoxy aminophosphate. A minor amount of adduct with an added mass of +109 was also found. Dichlorvos and O‐methoate both made dimethoxyphosphate (+108) and monomethoxyphosphate adducts (+94). Diazinon gave a novel adduct with an added mass of +152 consistent with diethoxythiophosphate. Inhibition of enzyme activity in the presence of diazinon developed slowly (15 h), concomitant with isomerization of diazinon via a thiono‐thiolo rearrangement. The isomer of diazinon yielded diethoxyphosphate and monoethoxyphosphate adducts with added masses of +136 and +108. MSMS spectra confirmed that each of the pesticides studied made a covalent bond with serine 198 of butyrylcholinesterase. These results can be used to identify the class of pesticides to which a patient was exposed.     

Lifetime exposure to a soluble TGF-beta antagonist protects mice against metastasis without adverse side effects

TGF-betas play diverse and complex roles in many biological processes. In tumorigenesis, they can function either as tumor suppressors or as pro-oncogenic factors, depending on the stage of the disease. We have developed transgenic mice expressing a TGF-beta antagonist of the soluble type II TGF-beta receptor:Fc fusion protein class, under the regulation of the mammary-selective MMTV-LTR promoter/enhancer. Biologically significant levels of antagonist were detectable in the serum and most tissues of this mouse line. The mice were resistant to the development of metastases at multiple organ sites when compared with wild-type controls, both in a tail vein metastasis assay using isogenic melanoma cells and in crosses with the MMTV-neu transgenic mouse model of metastatic breast cancer. Importantly, metastasis from endogenous mammary tumors was suppressed without any enhancement of primary tumorigenesis. Furthermore, aged transgenic mice did not exhibit the severe pathology characteristic of TGF-beta null mice, despite lifetime exposure to the antagonist. The data suggest that in vivo the antagonist may selectively neutralize the undesirable TGF-beta associated with metastasis, while sparing the regulatory roles of TGF-betas in normal tissues. Thus this soluble TGF-beta antagonist has potential for long-term clinical use in the prevention of metastasis.     

LOX family enzymes expression in vaginal tissue of premenopausal women with severe pelvic organ prolapse

Introduction and Hypothesis  

The extracellular matrix proteins collagen and elastin provide tissue strength and resilience, whereas lysyl oxidase enzymes play a major role in their stabilization. This study examines the expression and tissue localization of lysyl oxidase family proteins in the anterior vaginal wall of premenopausal women with advanced pelvic organ prolapse (POP, n = 15) and asymptomatic controls (n = 11). All women were in the proliferative phase of menstrual cycle.