Approximate complex electrical potential distribution in the monodomain model with unequal conductivity and relative permittivity anisotropy ratios.

OBJECTIVE: Electrical conductivity and relative permittivity are properties that indicate muscle health and they have different values parallel and perpendicular to the direction of the myofiber, a concept known as anisotropy. When the intrinsic electrical properties of muscle have ratios of anisotropy that are different then there is no analytical solution that can describe the electrical potential distribution in the tissue. APPROACH: Here, we present approximate analytical solutions to monodomain equations with unequal anisotropy ratios. For this, we base our analysis on perturbation theory where the electrical potential is approximated by the sum of the zeroth- and first-order terms of an infinite series. MAIN RESULTS: The validity of the approach is confirmed using experimental data for healthy and diseased muscle available online. SIGNIFICANCE: A better understanding of electrical potential distribution in anisotropic skeletal muscle tissue will allow the development of improved diagnostic tools for neuromuscular diseases.

Permittivity of ex vivo healthy and diseased murine skeletal muscle from 10 kHz to 1 MHz.

A better understanding of the permittivity property of skeletal muscle is essential for the development of new diagnostic tools and approaches for neuromuscular evaluation. However, there remain important knowledge gaps in our understanding of this property in healthy and diseased skeletal muscle, which hinder its translation into clinical application. Here, we report the permittivity of gastrocnemius muscle in healthy wild type mice and murine models of spinal muscular atrophy, muscular dystrophy, diabetes, amyotrophic lateral sclerosis and in a model of myofiber hypertrophy. Data were measured ex vivo from 10 kHz to 1 MHz using the four-electrode impedance technique. Additional quantitative histology information were obtained. Ultimately, the normative data reported will offer the scientific community the opportunity to develop more accurate models for the validation and prediction of experimental observations in both pre-clinical and clinical neuromuscular disease research.

New electrical impedance methods for the in situ measurement of the complex permittivity of anisotropic skeletal muscle using multipolar needles.

This paper provides a rigorous analysis on the measurement of the permittivity of two-dimensional anisotropic biological tissues such as skeletal muscle using the four-electrode impedance technique. The state-of-the-art technique requires individual electrodes placed at the same depth in contact with the anisotropic material, e.g. using monopolar needles. In this case, the minimum of measurements in different directions needed to estimate the complex permittivity and its anisotropy direction is 3, which translates into 12 monopolar needle insertions (i.e. 3 directions × 4 electrodes in each direction). Here, we extend our previous work and equip the reader with 8 new methods for multipolar needles, where 2 or more electrodes are spaced along the needle's shaft in contact with the tissue at different depths. Using multipolar needles, the new methods presented reduce the number of needle insertions by a factor of 2 with respect to the available methods. We illustrate the methods with numerical simulations and new experiments on ex vivo ovine skeletal muscle (n = 3). Multi-frequency longitudinal and transverse permittivity data from 30 kHz to 1 MHz is made publicly available in the supplementary material. The methods presented here for multipolar needles bring closer the application of needle electrical impedance to patients with neuromuscular diseases.

New electrical impedance methods for the in situ measurement of the complex permittivity of anisotropic biological tissues.

The capability of measuring the complex permittivity of tissues has the potential to provide valuable new insights to inform medical assessment and diagnosis. However, existing electrical impedance approaches have practical limitations when aiming to measure tissues' anisotropy with accuracy. Here we present new methods that overcome the limitations of previous approaches by modeling the anisotropy in both the resistivity and reactivity of tissues measured in three or more different directions. These new methods are validated with numerical simulations and in situ experiments on healthy ovine skeletal muscle. The obtained data between 3 kHz and 1 MHz are also made publicly available in the supplementary information.

Why are tumour blood vessels abnormal and why is it important to know?

Tumour blood vessels differ from their normal counterparts for reasons that have received little attention. We report here that they are of at least six distinct types, we describe how each forms, and, looking forward, encourage the targeting of tumour vessel subsets that have lost their vascular endothelial growth factor-A (VEGF-A) dependency and so are likely unresponsive to anti-VEGF-A therapies.

Historical and ecological determinants of genetic structure in arctic canids.

Wolves (Canis lupus) and arctic foxes (Alopex lagopus) are the only canid species found throughout the mainland tundra and arctic islands of North America. Contrasting evolutionary histories, and the contemporary ecology of each species, have combined to produce their divergent population genetic characteristics. Arctic foxes are more variable than wolves, and both island and mainland fox populations possess similarly high microsatellite variation. These differences result from larger effective population sizes in arctic foxes, and the fact that, unlike wolves, foxes were not isolated in discrete refugia during the Pleistocene. Despite the large physical distances and distinct ecotypes represented, a single, panmictic population of arctic foxes was found which spans the Svalbard Archipelago and the North American range of the species. This pattern likely reflects both the absence of historical population bottlenecks and current, high levels of gene flow following frequent long-distance foraging movements. In contrast, genetic structure in wolves correlates strongly to transitions in habitat type, and is probably determined by natal habitat-biased dispersal. Nonrandom dispersal may be cued by relative levels of vegetation cover between tundra and forest habitats, but especially by wolf prey specialization on ungulate species of familiar type and behaviour (sedentary or migratory). Results presented here suggest that, through its influence on sea ice, vegetation, prey dynamics and distribution, continued arctic climate change may have effects as dramatic as those of the Pleistocene on the genetic structure of arctic canid species.

Fiber characteristics of qiviut and guard hair from wild muskoxen (Ovibos moschatus).

In response to increasing commercial interest and the high market value of qiviut (the downy underwool of the muskox), we have employed standards and measurements used in the wool and cashmere industries to describe qiviut fiber characteristics. Fleece samples (qiviut with guard hair) were shaved from the midshoulder of 299 wild muskox hides of known sex and age (1, 2, 3, and 4+ yr) during the Banks Island, Canada, muskox harvest in November 1997. Samples were analyzed for fiber diameter distribution of raw fiber and qiviut, scoured and qiviut yields, and lengths of guard hair and qiviut fiber. We found a sex x age interaction for average fiber diameter (AFD) in raw fiber (P= 0.002) and qiviut (P < 0.001) only. Adult males had significantly coarser AFD than females (21.5 microm, males vs 20.1 microm, females and 18.2 microm, males vs 17.5 microm, females) for raw fiber and qiviut, respectively. Qiviut AFD from yearlings was 1.7 microm finer than the AFD of adult qiviut. Fiber diameter distribution (SD) decreased with age in the raw sample (P < 0.003) and qiviut (P < 0.001) and qiviut SD was greater (P < 0.001) in males than in females. Qiviut theoretical yield (% mass of fibers < or = 30 microm) increased (P < 0.001) with age, and females had higher theoretical yields than males (P < 0.001). Scoured yield did not vary between sexes in any age class and averaged 93.3%. Qiviut staple length did not differ with either age or sex. In summary, differences between the sexes were small up to the 3rd yr, and these differences were not likely to be of commercial importance. However, considering that AFD is a primary commercial criterion of value, AFD changes from 16.5 microm in yearlings to 18.2 microm in adults and from 17.5 microm in adult females to 18.2 microm in adult males would be expected to result in significant differences in commercial value.

The neurotransmitter dopamine inhibits angiogenesis induced by vascular permeability factor/vascular endothelial growth factor.

Angiogenesis has an essential role in many important pathological and physiological settings. It has been shown that vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), a potent cytokine expressed by most malignant tumors, has critical roles in vasculogenesis and both physiological and pathological angiogenesis. We report here that at non-toxic levels, the neurotransmitter dopamine strongly and selectively inhibited the vascular permeabilizing and angiogenic activities of VPF/VEGF. Dopamine acted through D2 dopamine receptors to induce endocytosis of VEGF receptor 2, which is critical for promoting angiogenesis, thereby preventing VPF/VEGF binding, receptor phosphorylation and subsequent signaling steps. The action of dopamine was specific for VPF/VEGF and did not affect other mediators of microvascular permeability or endothelial-cell proliferation or migration. These results reveal a new link between the nervous system and angiogenesis and indicate that dopamine and other D2 receptors, already in clinical use for other purposes, might have value in anti-angiogenesis therapy.

Glomeruloid microvascular proliferation follows adenoviral vascular permeability factor/vascular endothelial growth factor-164 gene delivery.

Glomeruloid bodies are a defining histological feature of glioblastoma multiforme and some other tumors and vascular malformations. Little is known about their pathogenesis. We injected a nonreplicating adenoviral vector engineered to express vascular permeability factor/vascular endothelial growth factor-164 (VPF/VEGF(164)) into the ears of athymic mice. This vector infected local cells that strongly expressed VPF/VEGF(164) mRNA for 10 to 14 days, after which expression gradually declined. Locally expressed VPF/VEGF(164) induced an early increase in microvascular permeability, leading within 24 hours to edema and deposition of extravascular fibrin; in addition, many pre-existing microvessels enlarged to form thin-walled, pericyte-poor, "mother" vessels. Glomeruloid body precursors were first detected at 3 days as focal accumulations of rapidly proliferating cells in the endothelial lining of mother vessels, immediately adjacent to cells expressing VPF/VEGF(164). Initially, glomeruloid bodies were comprised of endothelial cells but subsequently pericytes and macrophages also participated. As they enlarged by endothelial cell and pericyte proliferation, glomeruloid bodies severely compromised mother vessel lumens and blood flow. Subsequently, as VPF/VEGF(164) expression declined, glomeruloid bodies devolved throughout a period of weeks by apoptosis and reorganization into normal-appearing microvessels. These results provide the first animal model for inducing glomeruloid bodies and indicate that VPF/VEGF(164) is sufficient for their induction and necessary for their maintenance.

Prey specialization may influence patterns of gene flow in wolves of the Canadian Northwest.

This study characterizes population genetic structure among grey wolves (Canis lupus) in northwestern Canada, and discusses potential physical and biological determinants of this structure. Four hundred and ninety-one grey wolves, from nine regions in the Yukon, Northwest Territories and British Columbia, were genotyped using nine microsatellite loci. Results indicate that wolf gene flow is reduced significantly across the Mackenzie River, most likely due to the north-south migration patterns of the barren-ground caribou herds that flank it. Furthermore, although Banks and Victoria Island wolves are genetically similar, they are distinct from mainland wolf populations across the Amundsen Gulf. However, low-level island-mainland wolf migration may occur in conjunction with the movements of the Dolphin-Union caribou herd. Whereas previous authors have examined isolation-by-distance in wolves, this study is the first to demonstrate correlations between genetic structure of wolf populations and the presence of topographical barriers between them. Perhaps most interesting is the possibility that these barriers reflect prey specialization by wolves in different regions.

Retinoic acid selectively inhibits the vascular permeabilizing effect of VPF/VEGF, an early step in the angiogenic cascade.

All-trans-retinoic acid (RA) and other retinoids modulate cell growth and differentiation, generally favoring terminal cell differentiation and inhibiting carcinogenesis. Retinoids are also reported to inhibit angiogenesis and endothelial cell migration, actions that are also anti-carcinogenic. Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is a multifunctional cytokine secreted by many tumors. It renders microvessels hyperpermeable to plasma and stimulates endothelial cell migration and division. To investigate further the mechanisms by which RA inhibits angiogenesis, we evaluated the effects of RA on VPF/VEGF-induced angiogenesis and microvascular permeability. RA selectively inhibited the angiogenic response induced by VPF/VEGF, but not that induced by fibroblast growth factor-2 (FGF-2), in the CAM assay. RA and two of its isomers also inhibited the vascular permeabilizing effect of VPF/VEGF but not that induced by histamine. The vascular permeabilization induced by VPF/VEGF and blocked by RA takes place within 1-15 min, too short a time frame for RA to act by modulating transcription through classic retinoid receptors. RA also inhibited VPF/VEGF-induced phosphorylation of PLC-gamma and synthesis of cGMP but actually increased VPF/VEGF binding to cultured endothelial cells. Taken together, these findings indicate that RA selectively blocks VPF/VEGF-induced microvascular permeability and angiogenesis and also identify VPF/VEGF as a major target of RA action. The selectivity of RA's action suggests that other, RA-independent pathways must exist for the angiogenesis induced by FGF-2 and the vascular permeabilizing effect of histamine.

Ultrastructural localization of the vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) receptor-2 (FLK-1, KDR) in normal mouse kidney and in the hyperpermeable vessels induced by VPF/VEGF-expressing tumors and adenoviral vectors.

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) interacts with two high-affinity tyrosine kinase receptors, VEGFR-1 and VEGFR-2, to increase microvascular permeability and induce angiogenesis. Both receptors are selectively expressed by vascular endothelial cells and are strikingly increased in tumor vessels. We used a specific antibody to localize VEGFR-2 (FLK-1, KDR) in microvascular endothelium of normal mouse kidneys and in the microvessels induced by the TA3/St mammary tumor or by infection with an adenoviral vector engineered to express VPF/VEGF. A pre-embedding method was employed at the light and electron microscopic levels using either nanogold or peroxidase as reporters. Equivalent staining was observed on both the luminal and abluminal surfaces of tumor- and adenovirus-induced vascular endothelium, but plasma membranes at interendothelial junctions were spared except at sites connected to vesiculovacuolar organelles (VVOs). VEGFR-2 was also localized to the membranes and stomatal diaphragms of some VVOs. This staining distribution is consistent with a model in which VPF/VEGF increases microvascular permeability by opening VVOs to allow the transendothelial cell passage of plasma and plasma proteins.

A comparison of heavy metal levels in the kidneys of High Arctic and mainland caribou populations in the Northwest Territories of Canada.

Aluminum, nickel, cadmium, mercury and lead levels were measured in the kidney tissue of Banks Island Peary caribou and barren-ground caribou, from the Bluenose herd, of the western Northwest Territories of Canada. Cadmium concentrations of Bluenose caribou were similar to those reported elsewhere for barren-ground caribou and showed a positive correlation with age. Cadmium concentrations of Peary caribou were significantly lower than those of Bluenose caribou regardless of age, were the lowest reported for caribou during winter, and did not show a positive correlation with age. Mercury levels, expressed on a wet weight basis, were similar to those reported for other barren-ground caribou. Mercury levels were significantly higher in Bluenose [mean 10.45 microg g(-1) (dry wt.); S.E.= 0.85; n = 20] than Peary [mean 5.43 microg g(-1) (dry wt.); S.E. = 0.31; n = 20] caribou. Aluminum concentrations for Bluenose and Peary caribou were similar [mean 1.48 microg g(-1) (dry wt.); S.E. = 0.17; n = 20 and mean 1.56 microg g(-1) (dry wt.); S.E.= 0.15; n = 20, respectively), but were considerably lower than those reported for barren-ground caribou elsewhere. Lead and nickel concentrations were low and similar between Bluenose, Peary and other reported barren-ground caribou populations. Higher cadmium and mercury concentrations in Bluenose caribou are consistent with the hypothesis that caribou with a high dietary lichen component have higher contaminant levels. It is unlikely that subsistence harvesters would consume enough kidney during a year to exceed the tolerable intake of cadmium recommended by the WHO.

Heterogeneity of the angiogenic response induced in different normal adult tissues by vascular permeability factor/vascular endothelial growth factor.

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is an angiogenic cytokine with potential for the treatment of tissue ischemia. To investigate the properties of the new blood vessels induced by VPF/VEGF, we injected an adenoviral vector engineered to express murine VPF/VEGF164 into several normal tissues of adult nude mice or rats. A dose-dependent angiogenic response was induced in all tissues studied but was more intense and persisted longer (months) in skin and fat than in heart or skeletal muscle (< or =3 weeks). The initial response (within 18 hours) was identical in all tissues studied and was characterized by microvascular hyperpermeability, edema, deposition of an extravascular fibrin gel, and the formation of enlarged, thin-walled pericyte-poor vessels ("mother" vessels). Mother vessels developed from preexisting microvessels after pericyte detachment and basement membrane degradation. Mother vessels were transient structures that evolved variably in different tissues into smaller daughter vessels, disorganized vessel tangles (glomeruloid bodies), and medium-sized muscular arteries and veins. Vascular structures closely resembling mother vessels and each mother vessel derivative have been observed in benign and malignant tumors, in other examples of pathological and physiological angiogenesis, and in vascular malformations. Together these data suggest that VPF/VEGF has a role in the pathogenesis of these entities. They also indicate that the angiogenic response induced by VPF/VEGF is heterogeneous and tissue specific. Finally, the muscular vessels that developed from mother vessels in skin and perimuscle fat have the structure of collaterals and could be useful clinically in the relief of tissue ischemia.

Different pathways of macromolecule extravasation from hyperpermeable tumor vessels.

Tumor microvessels are hyperpermeable to plasma proteins, a consequence of tumor cell-secreted vascular permeability factor/vascular endothelial growth factor (VPF/VEGF). However, the pathways by which macromolecules extravasate from tumor vessels have been little investigated. To characterize tumor vessels more precisely and to elucidate the pathways by which macromolecules extravasated from them, we studied two well-defined, VPF/VEGF-secreting murine carcinomas, MOT and TA3/St. Whether grown in ascites or solid form, MOT tumors induced large, pericyte-poor "mother" vessels whose lining endothelium developed fenestrae that involved 1.8-5.6% of the surface. Fenestrae developed in parallel with markedly reduced endothelial cell vesiculo-vacuolar organelles (VVOs). TA3/St tumors, which secreted more VPF/VEGF than MOT tumors, elicited mother vessels with unchanged VVOs and without fenestrae. In both tumors, a plasma protein tracer, ferritin, extravasated through VVOs and in MOT tumors ferritin also extravasated through fenestrae. Endothelial gaps were not observed in either tumor. Thus, not all VPF/VEGF-secreting tumors induce fenestrated endothelium. Also, VVOs provide an internal store of membrane that can be transferred to the endothelial cell surface to provide the substantial increase in plasma membrane necessary for mother vessel formation in MOT tumors. Such transfer was apparently unnecessary in TA3/St tumors in which extensive early endothelial cell division provided the increased plasma membrane necessary for forming mother vessels.

Pathways of macromolecular extravasation across microvascular endothelium in response to VPF/VEGF and other vasoactive mediators.

OBJECTIVE: The goal of these studies was to define the anatomic pathways by which circulating macromolecules extravasate from the hyperpermeable microvessels that supply tumors and from normal venules that have been rendered hyperpermeable by vasoactive mediators. METHODS: Extravasation pathways of circulating macromolecular tracers were followed by several morphological techniques: light and fluorescence microscopy, transmission electron microscopy of routine as well as ultrathin and serial sections, computer-assisted three-dimensional reconstructions, and morphometry. RESULTS AND DISCUSSION: Macromolecules extravasated across tumor microvessels or across normal venules rendered hyperpermeable by VPF/VEGF, histamine, or serotonin by three primary pathways: 1) Vesiculo-vacuolar organelles (VVOs), clusters of cytoplasmic vesicles and vacuoles that span endothelial cytoplasm from lumen to ablumen; 2) trans-endothelial cell (EC), pores, and 3) fenestrae. We also present data concerning the structure and function of VVOs as well as evidence that VVOs form as the result of linking together and fusion of caveolae-sized unit vesicles. Under suitable conditions VVOs also afforded a pathway for macromolecular transport in the reverse direction, i.e., from vascular ablumen to lumen. Finally, in addition to opening VVOs to the passage of macromolecules, mediators such as VPF/VEGF may also induce structural rearrangements of VVOs, transforming them into trans-EC pores or fenestrae.

Vascular permeability factor/vascular endothelial growth factor and the significance of microvascular hyperpermeability in angiogenesis.

This Chapter has reviewed the literature concerning VPF/VEGF as a potent vascular permeabilizing cytokine. In accord with this important role, microvessels have been found to be hyperpermeable to plasma proteins and other circulating macromolecules at sites where VPF/VEGF and its receptors are overexpressed, i.e., in tumors, healing wounds, retinopathies, many important inflammatory conditions and in certain physiological processes, such as ovulation and corpus luteum formation. Moreover, microvascular hyperpermeability to plasma proteins was shown to have an important consequence: the laying down of a fibrin-rich extracellular matrix. This provisional matrix, in turn, favors and supports the ingrowth of fibroblasts and endothelial cells which, together, transform the provisional matrix into the mature stroma characteristic of tumors and healed wounds. Finally, we have considered the pathways by which these and other circulating macromolecules cross the endothelium of normal and VPF/VEGF-permeabilized microvessels. These pathways include VVOs and trans-endothelial openings that have been variously interpreted as inter-endothelial cell gaps or trans-endothelial cell pores. At least some trans-endothelial cell pores may arise from VVOs. In conclusion, these data provide new insights into the mechanisms of angiogenesis and stroma formation, insights which are potentially applicable to a wide variety of disease states and which may lead to identification of new targets for therapeutic intervention.

Platelets exit venules by a transcellular pathway at sites of F-met peptide-induced acute inflammation in guinea pigs.

Platelets maintain the integrity of vascular endothelium, but also appear outside of blood vessels in pathological states such as acute inflammation. However, it is widely believed that platelets extravasate from blood vessels only as the result of endothelial injury and that, on contacting extravascular collagen, they undergo a morphologically defined activation sequence and release their granule contents. We here report that platelets may cross intact venular endothelium without exhibiting this release reaction or injury. Platelets became adherent to the luminal surface of venular endothelium within approximately 15 min of intradermal injection of 10(-5) M N-formyl-methionyl-leucyl-phenylalanine in guinea pig flank skin. Individual intact platelets were noted in large endothelial cell cytoplasmic vacuoles from which they subsequently migrated abluminally. They then crossed the vascular basal lamina and entered the dermis without exhibiting evidence of a release reaction. Serial electron-microscopic sections confirmed that the cytoplasmic vacuoles within which platelets crossed endothelial cells were independent of interendothelial cell junctions which remained normally closed. Platelets extended pseudopods and gave other evidence of cell motility. These findings require a paradigm shift in our thinking about platelet movement and functions.

Enhancement of the functional repertoire of the rat parietal peritoneal mesothelium in vivo: directed expression of the anticoagulant and antiinflammatory molecule thrombomodulin.

We have used our previously described ex vivo mesothelial cell (MC)-mediated gene therapy strategy (Gene Ther. 2:393-401, 1995) to modify the functional properties of the rat parietal peritoneal mesothelium in vivo by expression of a membrane-bound recombinant protein on the MC surface. Rat primary MCs were stably transfected (using strontium phosphate DNA coprecipitation) with a plasmid containing the gene for rat thrombomodulin (TM), a transmembrane glycoprotein that functions as an essential cofactor for the physiological activation of the anticoagulant protein C by the enzyme thrombin. As demonstrated by immunohistochemistry and by direct equilibrium binding with radiolabeled thrombin, genetically modified MCs expressed high levels of TM antigen on their surface in vitro. As judged by a thrombin-dependent protein C activation assay, such MC membrane-bound TM was biologically active. Once reseeded on the denuded parietal peritoneal surface of syngeneic recipients, these TM-transfected MCs continued to express TM antigen in vivo for at least 90 days. Moreover, the recombinant TM expressed on the reconstituted parietal mesothelium retained its ability to activate protein C in a thrombin-dependent manner. Our data indicate that MC-mediated expression of TM can be used to augment the anticoagulant properties of the parietal peritoneal surface. In general, our results suggest that ex vivo MC-mediated gene therapy can be used to deliver other therapeutic transmembrane proteins to the MC surface to enhance the functional repertoire of the parietal mesothelium in vivo.