Double-Stranded RNA Attenuates the Barrier Function of Human Pulmonary Artery Endothelial Cells

Circulating RNA may result from excessive cell damage or acute viral infection and can interact with vascular endothelial cells. Despite the obvious clinical implications associated with the presence of circulating RNA, its pathological effects on endothelial cells and the governing molecular mechanisms are still not fully elucidated. We analyzed the effects of double stranded RNA on primary human pulmonary artery endothelial cells (hPAECs). The effect of natural and synthetic double-stranded RNA (dsRNA) on hPAECs was investigated using trans-endothelial electric resistance, molecule trafficking, calcium (Ca2+) homeostasis, gene expression and proliferation studies. Furthermore, the morphology and mechanical changes of the cells caused by synthetic dsRNA was followed by in-situ atomic force microscopy, by vascular-endothelial cadherin and F-actin staining. Our results indicated that exposure of hPAECs to synthetic dsRNA led to functional deficits. This was reflected by morphological and mechanical changes and an increase in the permeability of the endothelial monolayer. hPAECs treated with synthetic dsRNA accumulated in the G1 phase of the cell cycle. Additionally, the proliferation rate of the cells in the presence of synthetic dsRNA was significantly decreased. Furthermore, we found that natural and synthetic dsRNA modulated Ca2+ signaling in hPAECs by inhibiting the sarco-endoplasmic Ca2+-ATPase (SERCA) which is involved in the regulation of the intracellular Ca2+ homeostasis and thus cell growth. Even upon synthetic dsRNA stimulation silencing of SERCA3 preserved the endothelial monolayer integrity. Our data identify novel mechanisms by which dsRNA can disrupt endothelial barrier function and these may be relevant in inflammatory processes

Malignant astrocyte swelling and impaired glutamate clearance drive the expansion of injurious spreading depolarization foci

Spreading depolarizations (SDs) indicate injury progression and predict worse clinical outcome in acute brain injury. We demonstrate in rodents that acute brain swelling upon cerebral ischemia impairs astroglial glutamate clearance and increases the tissue area invaded by SD. The cytotoxic extracellular glutamate accumulation (>15 mu M) predisposes an extensive bulk of tissue (4-5 mm(2)) for a yet undescribed simultaneous depolarization (SiD). We confirm in rat brain slices exposed to osmotic stress that SiD is the pathological expansion of prior punctual SD foci (0.5-1 mm(2)), is associated with astrocyte swelling, and triggers oncotic neuron death. The blockade of astrocytic aquaporin-4 channels and Na+/K+/Cl- co-transporters, or volume-regulated anion channels mitigated slice edema, extracellular glutamate accumulation (<10 mu M) and SiD occurrence. Reversal of slice swelling by hyperosmotic mannitol counteracted glutamate accumulation and prevented SiD. In contrast, inhibition of glial metabolism or inhibition of astrocyte glutamate transporters reproduced the SiD phenotype. Finally, we show in the rodent water intoxication model of cytotoxic edema that astrocyte swelling and altered astrocyte calcium waves are central in the evolution of SiD. We discuss our results in the light of evidence for SiD in the human cortex. Our results emphasize the need of preventive osmotherapy in acute brain injury

Endothelial-Mesenchymal Transition of Brain Endothelial Cells: Possible Role during Metastatic Extravasation

Cancer progression towards metastasis follows a defined sequence of events described as the metastatic cascade. For extravasation and transendothelial migration metastatic cells interact first with endothelial cells. Yet the role of endothelial cells during the process of metastasis formation and extravasation is still unclear, and the interaction between metastatic and endothelial cells during transendothelial migration is poorly understood. Since tumor cells are well known to express TGF-beta, and the compact endothelial layer undergoes a series of changes during metastatic extravasation (cell contact disruption, cytoskeletal reorganization, enhanced contractility), we hypothesized that an EndMT may be necessary for metastatic extravasation. We demonstrate that primary cultured rat brain endothelial cells (BEC) undergo EndMT upon TGF-beta 1 treatment, characterized by the loss of tight and adherens junction proteins, expression of fibronectin, beta 1-integrin, calponin and a-smooth muscle actin (SMA). B16/F10 cell line conditioned and activated medium (ACM) had similar effects: claudin-5 down-regulation, fibronectin and SMA expression. Inhibition of TGF-beta signaling during B16/F10 ACM stimulation using SB-431542 maintained claudin-5 levels and mitigated fibronectin and SMA expression. B16/F10 ACM stimulation of BECs led to phosphorylation of Smad2 and Smad3. SB-431542 prevented SMA up-regulation upon stimulation of BECs with A2058, MCF-7 and MDA-MB231 ACM as well. Moreover, B16/F10 ACM caused a reduction in trans-endothelial electrical resistance, enhanced the number of melanoma cells adhering to and transmigrating through the endothelial layer, in a TGF-beta-dependent manner. These effects were not confined to BECs: HUVECs showed TGF-beta-dependent SMA expression when stimulated with breast cancer cell line ACM. Our results indicate that an EndMT may be necessary for metastatic transendothelial migration, and this transition may be one of the potential mechanisms occurring during the complex phenomenon known as metastatic extravasation

Systemic nitroglycerin increases nNOS levels in rat trigeminal nucleus caudalis.

Systemic administration of nitroglycerin, a nitric oxide donor, triggers in migraineurs a delayed attack of unknown mechanisms. Subcutaneous nitroglycerin (10 mg/kg) produced a significant increase of nitric oxide synthase (NOS)- and c-fos-immunoreactive neurons in the cervical part of trigeminal nucleus caudalis in rats after 4 h. This effect was not observed in the thoracic dorsal horn. Similar increase of NOS and c-fos was obtained in the brain stem after a somatic nociceptive stimulus, i.e. on the side of the formalin injection in the lip. Nitric oxide is thus able to increase NOS availability in second order nociceptive trigeminal neurons, which may be relevant for central sensitization and the understanding of its effect in migraine

Detection and identification of phytoplasmas in peach based on woody indexing and molecular methods

Symptoms resembling phytoplasma disease have been observed on peach trees in a seed-source plantation of stone fruits in south Hungary quite recently. In this publication we report on the results of woody indexing of symptomatic peach trees on GF 305 indicator in the field and under greenhouse conditions as well as on molecular studies. Phytoplasma infection detected on GF 305 indicators in greenhouse and field indexing was confirmed by PCR. Nested PCR was conducted using universal primer pairs followed by group and subgroup specific primers for the second amplification. RFLP analysis of nested PCR products was performed using Rsal restriction enzyme. Based on the results of molecular studies it can be concluded that phytoplasmas, belonging to the European stone fruit yellows subgroup (16SrX-B) were identified in peach trees. Further studies on symptomatic peach trees originating from different parts of Hungary are in progress

Tomato spotted wilt virus (TSWV) in birthwort (Aristolochia clematitis L.) in Hungary

Tomato spotted wild virus (TSWV) has become an important plant pathogen during the past 10 years in Hungary. This virus belongs to the family of Bunyaviridae, genus Tospovirus. According to the latest studies, this virus has about 1090 host plant species, including crops and weeds. In the autumn of 2009, some Aristolochia clematitis plants were collected showing symptoms of TSWV infection. On the basis of electron microscopic and molecular studies, leaf samples of Aristolochia clematitis L. found in a vineyard proved to be infected with Tomato spotted wilt virus (TSWV). This is the first report on the occurrence of TSWV in Aristolochia in Hungary

Identification of phytoplasmas on poamceous fruit tree species in Hungary

In Hungary local occurrence of Apple proliferation disease(AP) on apple was described for the first time in the early sixties, since that time the disease has been found in different part of the country based on the results of biological indexing. The first suspicious sign for the presence of Pear decline disease (PD) in Hungarian pear orchard was indicated in the early 70s, but at the time there were no adequate laboratory techniques for identification. The aim of the present study was the molecular identification of phytoplasmas in symptom-showing apple trees, as well as confirmation of the occurrence of PD phytoplasma in symptomatic pear trees, testing them in parallel also on woody indicators. Symptom showing trees, indicating possible phytoplasma infection, were sampled in different growing regions in 2002 for indexing. AP was detected on Golden Delicious using root and double grafting. Typical symptoms of PD did not appear on root or double grafted Pyrus communis cv. Comice. Nucleic acids were prepared from all samples by chloroform/phenol extraction from fresh, frozen or dry phloem tissue of leaves and young branches. Nested PCR procedures were employed with different sets of primers to amplify phytoplasma 16SrDNA, or 16SrDNA plus spacer region. The identity of PCR products was confirmed by RFLP as 16SrX-A (AP) and 16SrX-C (PD). It was concluded that molecular methods were useful for identification of phytoplasmas in several of the symptomatic orchard trees of apple and pear respectively. Both greenhouse indexing and nested PCR/RFLP were suitable for detection and identification of AP from bearing trees and the phytoplasma was identified by molecular methods also in the inoculated AP indicators. PD could not be detected in greenhouse indexing