Angiogenic potential of microvessel fragments established in three-dimensional collagen gels

Summary During angiogenesis, the microvasculature displays both vessel remodeling and expansion under the control of both cellular and extracellular influences. We have evaluated the role of angiogenic and angiostatic molecules on angiogenesis in anin vitro model that more appropriately duplicates the cellular and extracellular components of this process. Freshly isolated microvessel fragments from rat adipose tissue (RFMF) were cultured within three-dimensional collagen I gels. These fragments were characterized at the time of isolation and were composed of vessel segments observed in the microvasculature of fatin situ (i.e., arterioles, venules, and capillaries). Fragments also exhibited characteristic ablumenally associated cells including smooth muscle cells and pericytes. Finally, fragments were encased in an extracellular matrix composed of collagen type IV and collagen type I/III. The elongation of microvascular elements was subsequently evaluated using morphologic and immunocytochemical techniques. The proliferation, migration, and elongation of cellular elements in microvessel fragments from rat adipose tissue was dependent on initial fragment density, matrix density, and required serum. Inclusion of endothelial cell growth factors to microvessel fragments from rat adipose tissue 3-D cultures resulted in the accelerated elongation of tube structures and the expression of von Willebrand factor in cells constituting these tubes. Molecules with reported angiostatic capacity (e.g., transforming growth factor and hydrocortisone) inhibited vessel tube elongation. In vitro methods have been developed to evaluate numerous mechanisms associated with angiogenesis, including endothelial cell proliferation, migration, and phenotypic modulation. Microvascular endothelial cell fragments described in this study represent anin vitro population of cells that accurately duplicate thein vivo microcirculatory elements of fat. The proliferation of cells and elongation of microvascular elements subsequently observed in three-dimensional cultures provides anin vitro model of angiogenesis. Microvascular formation in this system results from pre-existing microvessel fragments unlike tube formation observed when cultured endothelial cells are placed in three-dimensional gels. This form of tube formation from cultured endothelium is more characteristic of vasculogenesis. Thus, the formation of microvascular elements from microvessel fragments provides the opportunity to examine the mechanisms regulating angiogenesis in anin vitro system amenable to precise experimental manipulation.     

Mechanisms for species differences in receptor-mediated carcinogenesis

Species differences resulting from a number of mechanisms are common in receptor-mediated chemical carcinogenesis. In this review, examples of possible mechanisms underlying these differences are discussed, including ligand metabolism, receptor polymorphisms, receptor isoforms, receptor levels, and crosstalk between signal transduction pathways. In addition, a number of other mechanisms also are likely to be important. The developmental state of the animal will determine the expression of receptors in different tissues. The regulatory pathways for cell proliferation and cell death and cell cycle check point controls can vary among species and tissues. Adaptation or potentiation of responses during chronic exposures to chemicals can greatly influence species differences. The mechanisms of adaptive processes are poorly understood but probably highly important for chronic toxicities such as cancer. Finally, different species may have different stem cell populations that are the targets for neoplastic transformation, and this will influence receptor-mediated carcinogenic responses. The implications of species differences in receptor-mediated responses for risk assessment are discussed.     

Towards an understanding of the relations between tree nutrition,nutrient cycling and environment

The paper contains a discussion of the interrelations between the sciences used by managers of forest land to improve their management, in particular with respect to the plant nutrient economy of the forest ecosystems. Both site studies and studies of nutrient cycling have been carried out for long periods without proper consideration of tree nutrition. Therefore these studies contributed less to the understanding of the role of nutrients as regulators of processes in ecosystems than might have been expected. This situation has improved, especially within the last decade. In addition the necessity to manage forest land for environmental values as well as for forest yield requires new interdisciplinary approaches in the study of the roles of plant nutrients in the forest. Even more branches of biological and environmental sciences than those just mentioned must be involved.     

Toxicology of vanadium compounds in diabetic rats: The action of chelating agents on vanadium accumulation

The possible use of vanadium compounds in the treatment of diabetic patients is now being evaluated. However, previously to establish the optimal maximum dose for diabetes therapy, it should be taken into account that vanadium is a highly toxic element to man and animals. The toxic effects of vanadium are here reviewed. The tissue vanadium accumulation, which would mean an additional risk of toxicity following prolonged vanadium administration is also discussed. Recently, it has been shown that coadministration of vanadate and TIRON, an effective chelator in the treatment of vanadium intoxication, reduced the tissue accumulation of this element, decreasing the possibility of toxic side effects derived from chronic vanadium administration without diminishing the hypoglycemic effect of vanadium. However, previously to assess the effectiveness of this treatment in diabetic patients, a critical reevaluation of the antidiabetic action of vanadium and its potential toxicity is clearly needed.     

Cloning and expression in Escherichia coli of the obtusifoliol 14α-demethylase of Sorghum bicolor (L.) Moench, a cytochrome P450 orthologous to the sterol 14α-demethylases (CYP51) from fungi and mammals

Obtusifoliol 14β-demethylase from Sorghum bicolor (L.) Moench has been cloned using a gene-specific probe generated using PCR primers designed from an internal 14 amino acid sequence. The sequence identifies sorghum obtusifoliol 14α-demethylase as a cytochrome P450 and it is assigned to the CYP51 family together with the sterol 14α-demethylases from fungi and mammals. The presence of highly conserved regions in the amino acid sequences, analogous substrates and the same metabolic role demonstrate that the sterol 14α-demethylases are orthologous enzymes. The sterol 14α-demethylases catalyse an essential step in sterol biosynthesis as evidenced by the absence of a 14α-methyl group in all known functional sterols. A functional sorghum obtusifoliol 14α-demethylase was expressed at high levels in Escherichia coli and purified using an efficient method based on temperature-induced Triton X-114 phase partitioning. The recombinant purified enzyme produced a type I spectrum with obtusifoliol as substrate. Reconstitution of purified recombinant enzyme with sorghum NADPH—cytochrome P450 reductase in dilaurylphosphatidylcholine micelles confirms that obtusifoliol 14α-demethylase catalyses the 14α-demethylation of obtusifoliol to 4α-methyl-5α-ergosta-8,14,24(28)-trien-3β-ol as evidenced by GC—MS. The isolation of a cDNA clone encoding the plant sterol 14α-demethylase, combined with the previously isolated cDNA clones for fungal and mammalian sterol 14α-demethylases, provides an important tool in the rational design of specific inhibitors towards the individual sterol 14α-demethylases.     

Biochemistry and pharmacology of 7α-substituted androstenediones as aromatase inhibitors

The inhibition of aromatase, the enzyme responsible for converting androgens to estrogens, is therapeutically useful for the endocrine treatment of hormone-dependent breast cancer. Research by our laboratory has focused on developing competitive and irreversible steroidal aromatase inhibitors, with an emphasis on synthesis and biochemistry of 7α-substituted androstenediones. Numerous 7α-thiosubstituted androst-4-ene-3,17-diones are potent competitive inhibitors, and several 1,4-diene analogs, such as 7α-(4′-aminophenylthio)-androsta-1,4-diene-3,17-dione (7α-APTADD), have demonstrated effective enzyme-activated irreversible inhibition of aromatase in microsomal enzyme assays. One focus of current research is to examine the effectiveness and biochemical pharmacology of 7α-APTADD in vivo. In the hormone-dependent 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary carcinoma model system, 7α-APTADD at a 50 mg/kg/day dose caused an initial decrease in mean tumor volume during the first week, and tumor volume remained unchanged throughout the remaining 5-week treatment period. This agent lowers serum estradiol levels and inhibits ovarian aromatase activity. A second research area has focused on the synthesis of more metabolically stable inhibitors by replacing the thioether linkage at the 7α position with a carbon-carbon linkage. Several 7α-arylaliphatic androst-4-ene-3,17-diones were synthesized by 1,6-conjugate additions of appropriate organocuprates to a protected androst-4,6-diene or by 1,4-conjugate additions to a seco-A-ring steroid intermediate. These compounds were all potent inhibitors of aromatase with apparent K_is ranging between 13 and 19 nM. Extension of the research on these 7α-arylaliphatic androgens includes the introduction of a C₁---C₂ double bond in the A-ring to provide enzyme-activated irreversible inhibitors. The desired 7α-arylaliphatic androsta-1,4-diene-3,17-diones were obtained from their corresponding 7α-arylaliphatic androst-4-ene-3,17-diones by oxidation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). These inhibitors demonstrated enzyme-mediated inactivation of aromatase with apparent k_inacts ranging from 4.4 × 10⁻⁴ to 1.90 x 10⁻³ s⁻¹. The best inactivator of the series was 7α-phenpropylandrosta-1,4-diene-3,17-dione, which exhibited a T_1/2 of 6.08 min. Aromatase inhibition was also observed in MCF-7 human mammary carcinoma cell cultures and in JAr human choriocarcinoma cell cultures, exhibiting IC₅₀ values of 64-328 nM. The 7α-arylaliphatic androgens thus demonstrate potent inhibition of aromatase in both microsomal incubations and in choriocarcinoma cell lines expressing aromatase enzymatic activity. Additionally, the results from these studies provide further evidence for the presence of a hydrophobic binding pocket existing near the 7α-position of the steroid in the active site of aromatase. The size of the 7α-substituent influences optimal binding of steroidal inhibitors to the active site and affects the extent of enzyme-mediated inactivation observed with androsta-1,4-diene-3,17-dione analogs.     

Role of inhibition of nitric oxide production in monocrotaline-induced pulmonary hypertension

Mathew, Rajamma, Elizabeth S. Gloster, T. Sundararajan, Carl I. Thompson, Guillermo A. Zeballos, andMichael H. Gewitz. Role of inhibition of nitric oxide productionin monocrotaline-induced pulmonary hypertension. J. Appl. Physiol. 82(5): 1493-1498, 1997.Monocrotaline (MCT)-induced pulmonary hypertension (PH) isassociated with impaired endothelium-dependent nitric oxide(NO)-mediated relaxation. To examine the role of NO in PH,Sprague-Dawley rats were given a single subcutaneous injection ofnormal saline [control (C)], 80 mg/kg MCT, or the same doseof MCT and a continuous subcutaneous infusion of 2 mg · kg¹ · day¹of molsidomine, a NO prodrug (MCT+MD). Two weeks later, plasma NO₃ levels, pulmonary arterialpressure (Ppa), ratio of right-to-left ventricular weights (RV/LV) toassess right ventricular hypertrophy, and pulmonary histology wereevaluated. The plasma NO₃ level inthe MCT group was reduced to 9.2 ± 1.5 µM(n = 12) vs. C level of 17.7 ± 1.8 µM (n = 8; P < 0.02). In the MCT+MD group,plasma NO₃ level was 12.3 ± 2.0 µM (n = 8). Ppa and RV/LV in theMCT group were increased compared with C [Ppa, 34 ± 3.4 mmHg(n = 6) vs. 19 ± 0.8 mmHg(n = 8) and 0.41 ± 0.01 (n = 9) vs. 0.25 ± 0.008 (n = 8), respectively;P < 0.001]. In the MCT+MDgroup, Ppa and RV/LV were not different when compared with C [19 ± 0.5 mmHg (n = 5) and 0.27 ± 0.01 (n = 9), respectively;P < 0.001 vs. MCT]. Medial wall thickness of lung vessels in the MCT group was increased comparedwith C [31 ± 1.5% (n = 9)vs. 13 ± 0.66% (n = 9);P < 0.001], and MDpartially prevented MCT-induced pulmonary vascular remodeling [22 ± 1.2% (n = 11);P < 0.001 vs. MCT and C].These results indicate that a defect in the availability of bioactive NO may play an important role in the pathogenesis of MCT-induced PH.

     

Intravenous vs. oral rehydration: effects on subsequent exercise-heat stress

Castellani, John W., Carl M. Maresh, Lawrence E. Armstrong,Robert W. Kenefick, Deborah Riebe, Marcos Echegaray, Douglas Casa, andV. Daniel Castracane. Intravenous vs. oral rehydration: effects onsubsequent exercise-heat stress. J. Appl.Physiol. 82(3): 799-806, 1997.This studycompared the influence of intravenous vs. oral rehydration afterexercise-induced dehydration during a subsequent 90-min exercisebout. It was hypothesized that cardiovascular, thermoregulatory, and hormonal variables would be the same between intravenous and oral rehydration because of similar restoration ofplasma volume (PV) and osmolality (Osmo). Eight non-heat-acclimated menreceived three experimental treatments (counterbalanced design) immediately after exercise-induced dehydration (33°C) to 4%body weight loss. Treatments were intravenous 0.45% NaCl (iv; 25 ml/kg), no fluid (NF), and oral saline (Oral; 25 ml/kg).After rehydration and rest (2 h total), subjects walked at 50% maximalO₂ consumption for up to 90 min at36°C. The following observations were made: 1) heart rate was higher(P < 0.05) in Oral vs. ivat minutes 45, 60, and75 of exercise;2) rectal temperature, sweat rate, percent change in PV, and change in plasma Osmo were similar between ivand Oral; 3) change in plasmanorepinephrine decreased less (P < 0.05) in Oral compared with iv at minute45; 4) changes in plasma adrenocorticotropic hormone and cortisol were similar between ivand Oral after exercise was initiated; and5) exercise time was similar betweeniv (77.4 ± 5.4 min) and Oral (84.2 ± 2.3 min). These datasuggest that after exercise-induced dehydration, iv and Oral wereequally effective as rehydration treatments. Thermoregulation, changein adrenocorticotropic hormone, and change in cortisol were notdifferent between iv and Oral after exercise began; this is likely dueto similar percent change in PV and change in Osmo.

     

Evidence for Linkage of Bipolar Disorder to Chromosome 18 with a Parent-of-Origin Effect

A susceptibility gene on chromosome 18 and a parent-of-origin effect have been suggested for bipolar affective disorder (BPAD). We have studied 28 nuclear families selected for apparent unilineal transmission of the BPAD phenotype, by using 31 polymorphic markers spanning chromosome 18. Evidence for linkage was tested with affected-sib-pair and LOD score methods under two definitions of the affected phenotype. The affected-sib-pair analyses indicated excess allele sharing for markers on 18p within the region reported previously. The greatest sharing was at D18S37: 64% in bipolar and recurrent unipolar (RUP) sib pairs (P = .0006). In addition, excess sharing of the paternally, but not maternally, transmitted alleles was observed at three markers on 18q: at D18S41, 51 bipolar and RUP sib pairs were concordant for paternally transmitted alleles, and 21 pairs were discordant (P = .0004). The evidence for linkage to loci on both 18p and 18q was strongest in the 11 paternal pedigrees, i.e., those in which the father or one of the father's sibs is affected. In these pedigrees, the greatest allele sharing (81%; P = .00002) and the highest LOD score (3.51; θ = 0.0) were observed at D18S41. Our results provide further support for linkage of BPAD to chromosome 18 and the first molecular evidence for a parent-of-origin effect operating in this disorder. The number of loci involved, and their precise location, require further study.