Phillip Luthert

The RhoA Activator GEF-H1/Lfc Is a Transforming Growth Factor-beta Target Gene and Effector That Regulates alpha-Smooth Muscle Actin Expression and Cell Migration

TGFβ induces various responses, including Rho signaling. How TGFβ stimulates Rho is poorly understood. Our data indicate that GEF-H1 is a target and effector of TGFβ to regulate Rho signaling, gene expression, and cell migration, suggesting that it represents a new marker and possible therapeutic target for degenerative and fibrotic diseases.

The RhoA Activator GEF-H1/Lfc Is a Transforming Growth Factor-β Target Gene and Effector That Regulates α-Smooth Muscle Actin Expression and Cell Migration

TGFβ induces various responses, including Rho signaling. How TGFβ stimulates Rho is poorly understood. Our data indicate that GEF-H1 is a target and effector of TGFβ to regulate Rho signaling, gene expression, and cell migration, suggesting that it represents a new marker and possible therapeutic target for degenerative and fibrotic diseases.

The phagocytic capacity of neurones.

Phagocytosis is defined as the ingestion of particulates over 0.5 µm in diameter and is associated with cells of the immune system such as macrophages or monocytes. Neurones are not generally recognized to be phagocytic. Using light, confocal, time‐lapse and electron microscopy, we carried out a wide range of in‐vitro and in‐vivo experiments to examine the phagocytic capacity of different neuronal cell types. We demonstrated phagocytosis of material by neurones, including cell debris and synthetic particles up to 2.8 µm in diameter. We showed phagocytosis in different neuronal types, and demonstrated that debris can be transported from neurite extremities to cell bodies and persist within neurones. Flow cytometry analysis demonstrated the lack of certain complement receptors on neurones but the presence of others, including integrin receptors known to mediate macrophage phagocytosis, indicating that a restricted set of phagocytosis receptors may mediate this process. Neuronal phagocytosis occurs in vitro and in vivo, and we propose that this is a more widespread and significant process than previously recognized. Neuronal phagocytosis may explain certain inclusions in neurones during disease, cell‐to‐cell spread of disease, neuronal death during disease progression and provide a potential mechanism for therapeutic intervention through the delivery of particulate drug carriers.

Hyperosmolar Opening of the Blood-Brain Barrier in the Energy-Depleted Rat Brain. Part 1. Permeability Studies

A simple saline perfusion system was used to investigate the effects of hyperosmolar solutions of arabinose and mannitol upon the permeability of the blood-brain barrier. The small, polar molecule [14C]mannitol and the larger, visual marker Evans blue were used as indicators of barrier integrity in the perfused energydepleted brain. One-minute perfusion of hyperosmolar solutions consistently opened the barrier suggesting that the mechanism of osmotic barrier opening is independent of energy-producing metabolism. The accumulation of radiolabel in the brain was expressed as the ratio of tissue to perfusate radioactivity (Rt/Rp) and, for cerebrum, this increased from a control value of 0.0022 ± 0.0007 (mean ± SEM; n = 4) to a value of 0.0124 ± 0.0008 (n = 4) following 0.9 M arabinose and to 0.0495 ± 0.0072 (n = 4) following 1.8 M arabinose. There was a significant reduction of water content of hyperosmolar perfused brains. These findings support the hypothesis that osmotic barrier opening is the result of the passive shrinkage of endothelial cells and the surrounding tissue.

Maintenance of the integrity of the blood-brain barrier in the rat during an in situ saline-based perfusion.

Integrity of the blood-brain barrier to the small polar tracer mannitol was maintained for up to 30 min during an in situ perfusion of the brain with a saline-based solution containing the metabolic inhibitor 2,4-dinitrophenol. The patency of the capillary bed after perfusion was demonstrated by injecting a solution of Indian ink and gelatin, and ultrastructural examination showed the microvasculature to be well preserved. These findings suggest that the blood-brain barrier can be studied under conditions that are independent of normal cerebral function and metabolism.

The effect of dexamethasone on vascular permeability of experimental brain tumours

SummaryThe vessels of experimental gliomas show an abnormally high permeability to small polar molecules, such as mannitol. To establish whether this change in vessel permeability is modified by treatment with the corticosteroid dexamethasone, the kinetics of [14C]mannitol transfer into rat astrocytomas were estimated in both steroid- and saline-treated, tumourbearing animals. This was achieved by injecting [14C]mannitol i.v., using a specially devised technique, so as to maintain a constant concentration of tracer in the blood plasma. In separate experiments steady levels of the tracer were maintained in the circulation from 1 to 30 min. Mean plasma and tumour radioactivity were measured, and the apparent transfer constant of mannitol across the vascular endothelium and the size of the extravascular extracellular mannitol space in the tumours were calculated.Despite a significant clinical improvement in the treated animals and adequate circulating levels of dexamethasone at the time of the permeability studies, no difference in either the apparent transfer constant for the movement of mannitol into the tumours or the fractional extracellular mannitol space was detected between these animals and the controls. With steroid treatment both tumour-bearing and non-tumour bearing animals lost weight, and in the latter there was no consistent change in routine biochemical or haematological parameters. It was concluded that under these conditions it is unlikely that clinical improvement with dexamethasone therapy was due to a non-specific reduction in tumour vessel permeability to polar substances.

The Effect of a Low pH Saline Perfusate Upon the Integrity of the Energy-Depleted Rat Blood-Brain Barrier

The effect of a low pH perfusate upon the integrity of the rat blood-brain barrier was studied using an in situ supravital brain perfusion technique in which high-energy phosphates are depleted. Control animals were perfused for 10 min with a Ringers salt solution containing the metabolic inhibitor 2,4-dinitrophenol (DNP) and adjusted to a pH of 7.4. In two separate experimental groups the perfusate, consisting of either the same medium as the controls or with additional buffering from Tris maleate, was switched after 5 min at a pH of 7.4, to a medium adjusted to pH 5.5 with lactic acid. Following a total perfusion time of 10 min, the integrity of the blood-brain barrier was assessed using the small molecular weight tracer [14C]mannitol. The cerebral perfusate flow rates (CPFR) after 10 min of perfusion were also determined in the three groups by perfusing for 40 s with [14C]iodoantipyrine. In each group, mannitol was excluded from the tissue of the brain to the same degree as has been previously reported in vivo, indicating an intact blood-brain barrier. There was also no significant pH-dependent change in CPFR. Ultrastructural examination of animals that had been perfusion fixed following in situ perfusion revealed no obvious differences between the cerebral endothelium of the control and low pH perfused animals. These results demonstrate that in the absence of energy-producing metabolism a perfusate pH of 5.5 is insufficient to disrupt the blood-brain barrier.

The RhoA Activator GEF-H1/Lfc Is a TGF-β Target Gene and Effector that Regulates α-Smooth Muscle Actin Expression and Cell Migration

TGFβ induces various responses, including Rho signaling. How TGFβ stimulates Rho is poorly understood. Our data indicate that GEF-H1 is a target and effector of TGFβ to regulate Rho signaling, gene expression, and cell migration, suggesting that it represents a new marker and possible therapeutic target for degenerative and fibrotic diseases.

Transport of thiamin across the blood-brain barrier of the rat in the absence of aerobic metabolism

By employing an anaerobic brain perfusion technique the dependency of thiamin transport across the blood-brain barrier upon high-energy phosphate production has been studied. Analysis of the data revealed essentially identical influx kinetics as those previously reported in vivo, the influx being considerably greater than that for the non-transported small molecule mannitol. These results provide for the first time direct evidence that, unlike at other cell surfaces, the influx of thiamin at the blood-brain barrier is independent of energy-yielding metabolism.

Selective closure of the vascular bed of an experimental glioma model during in situ saline perfusion

Flow through the vasculature of an experimental rat glioma has been investigated during in situ perfusion of the brain, via the ascending aorta, with a simple saline solution. Using such a system, it has been shown previously that the blood–brain barrier will remain intact with an adequate cerebral perfusate flow rate for at least 10 min, providing that the metabolic inhibitor 2,4–dinitrophenol is present. Cerebral perfusate flow rate was measured in both tumour and non–tumour areas using [14C] iodoantipyrine. The perfusion pump rate was set between 4.8 and 84 ml/min in different animals and the mean flow rate for cerebral hemisphere remote from the tumour was 1.03 ± 0.67 ml/min/g (mean ± sd; n= 17) whereas that for intracerebral tumour was considerably lower at 0.060 ± 0.11 ml/min/g (mean ± sd; n= 17). Linear regression of tumour flow on cerebrum flow showed a highly significant correlation (r= 0.88). Light and electron microscope examination of the tumour vessels revealed no luminal obstruction or perivascular oedema to explain the low flow. We suggest that perfusion with a low viscosity medium, at flow rates that result in a low intraluminal pressure, probably causes glioma vessels to close preferentially because they require a higher intraluminal pressure to remain patent than do normal cerebral vessels.