Anti-tumor activity of motesanib in a medullary thyroid cancer model.

BACKGROUND: Medullary thyroid cancer (MTC) is frequently associated with mutations in the tyrosine kinase Ret and with increased expression of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2). Motesanib is an investigational, orally administered small molecule antagonist of VEGFR1, 2, and 3; platelet-derived growth factor receptor (PDGFR); Kit; and possibly Ret. AIM: The aim of this study was to investigate the effects of motesanib on wildtype and mutant Ret activity in vitro and on tumor xenograft growth in a mouse model of MTC. METHODS/RESULTS: In cellular phosphorylation assays, motesanib inhibited the activity of wild-type Ret (IC(50)=66 nM), while it had limited activity against mutant Ret C634W (IC(50)=1100 nM) or Ret M918T (IC(50)>2500 nM). In vivo, motesanib significantly inhibited the growth of TT tumor cell xenografts (expressing Ret C634W) and significantly reduced tumor blood vessel area and tumor cell proliferation, compared with control. Treatment with motesanib resulted in substantial inhibition of Ret tyrosine phosphorylation in TT xenografts and, at comparable doses, in equivalent inhibition of VEGFR2 phosphorylation in both TT xenografts and in mouse lung tissue. CONCLUSIONS: The results of this study demonstrate that motesanib inhibited thyroid tumor xenograft growth predominantly through inhibition of angiogenesis and possibly via a direct inhibition of VEGFR2 and Ret expressed on tumor cells. These data suggest that targeting angiogenesis pathways and specifically the VEGF pathway may represent a novel therapeutic approach in the treatment of MTC.

The effects of keratinocyte growth factor in preclinical models of mucositis.

The epithelium of the oral cavity and small intestine of the gastrointestinal tract have a high rate of cell renewal and as such, are sensitive to cytotoxic therapies that kill rapidly dividing cells. Mucositis is a complication of cancer therapy where impairment of the regenerative capacity of the epithelium leads to atrophy, ulceration and a loss of barrier function. Keratinocyte growth factor (KGF) is an epithelial cell-specific growth and differentiation factor that is trophic for the mucosal epithelium of the gastrointestinal tract. In this study, KGF in normal animals caused epithelial thickening in the squamous epithelium of the oral cavity and increased crypt depth and villus height of the small intestine. It also appeared to regulate gene expression in these tissues including that of some antioxidant enzymes and intestinal trefoil protein. KGF has been shown to be efficacious in several preclinical models of mucositis where KGF pretreatment reduced weight loss typically seen during and after the course of therapy and significantly improved survival. At a tissue level KGF reduced atrophy, accelerated regrowth, and decreased ulcer formation of the oral epithelium after irradiation, and improved crypt survival and prevented villus atrophy in the small intestine of irradiated or chemotherapy-treated mice. Preliminary studies suggest that its efficacy may be partly a consequence of the growth and differentiation effect, and also partly due to regulation of the expression of genes that play a role in mucosal protection. These data suggest that KGF may be useful for the prevention or treatment of mucositis in patients treated with regimens of cancer therapy that have gastrointestinal toxicity.

Stimulatory effects of B7-related protein-1 on cellular and humoral immune responses in mice.

Inducible costimulator (ICOS) and B7-related protein-1 (B7RP-1) constitute a receptor-ligand pair involved in T cell costimulation. In this study, the stimulatory effects of B7RP-1 on cellular and humoral immune responses were investigated giving mice a construct with the extracellular domain of murine B7RP-1 fused with human IgG1 Fc (B7RP-1-Fc). B7RP-1-Fc stimulated contact hypersensitivity (CH) given near either the time of sensitization or challenge with oxazolone. When given near challenge time, B7RP-1-Fc stimulated CH more than a construct containing the extracellular domain of murine B7.2 and Fc (B7.2-Fc). B7RP-1-Fc increased the number of cells in lymph nodes draining the skin sensitized with oxazolone, especially activated T cells. B7RP-1-Fc also increased the ability of the cells in these lymph nodes to induce CH when transfused into naive mice. B7RP-1-Fc stimulated the production of anti-keyhole limpet hemocyanin (KLH) Ab, increasing anti-KLH IgG, IgG2a, and IgE, whereas B7.2-Fc did not affect this production. B7RP-1-Fc also increased the number of cells in lymph nodes draining the skin immunized with KLH and their production of IFN-gamma, IL-4, and IL-10 in response to KLH. Finally, B7RP-1-Fc increased the presence of eosinophils in the bronchoalveolar lavage and lungs of mice sensitized and challenged with OVA so to mount an asthmatic reaction. B7RP-1-Fc stimulates both cellular and humoral immune responses in vivo by increasing number and function of T and B cells reacting to Ag exposure.

Keratinocyte growth factor protects mice from chemotherapy and radiation-induced gastrointestinal injury and mortality.

Keratinocyte growth factor (KGF) stimulates the proliferation and differentiation of epithelial cells including those of the gastrointestinal tract. Although chemotherapeutics and radiation exposure kill rapidly proliferating tumor cells, rapidly dividing normal cells of the host's gastrointestinal tract are also frequently damaged, leading to the clinical condition broadly termed "mucositis." In this report, recombinant human KGF used as a pretreatment in several mouse models of chemotherapy and/or radiation-induced gastrointestinal injury significantly improved mouse survival. Using multiple-dose 5-fluorouracil, methotrexate, and radiation in combination and total body radiation alone models, KGF increased survival by 55% or greater. In the models that used chemotherapy with or without radiation, KGF significantly ameliorated weight loss after injury and accelerated weight gain during recovery. The basis of these systemic benefits appears to be due in part to the trophic effects of the growth factor on the intestinal epithelium because KGF pretreatment caused an increase in measures of mucosal thickness (villus height and crypt depth) that persisted during the course of 5-fluorouracil chemotherapy. Treatment with KGF also afforded a 3.5-fold improvement in crypt survival in the small intestine, suggesting that KGF also has a direct effect on the crypt stem cells. These data indicate that KGF may be therapeutically useful to lessen the intestinal side effects of current cancer therapy regimens.

Regulation of brain capillary endothelial thrombomodulin mRNA expression.

BACKGROUND AND PURPOSE: Endothelial cells regulate hemostasis in part via expression of thrombomodulin, a potent anticoagulant protein. The purpose of this study was to analyze brain capillary endothelial cell expression of thrombomodulin mRNA. METHODS: Bovine brain capillary endothelial cells were grown in a blood-brain barrier model in which endothelial cells form capillary-like structures. In situ hybridization and polymerase chain reaction (PCR) were used to examine thrombomodulin expression. Endothelial cells were then cocultured with astrocytes. We examined both coculture and monoculture preparations for gamma-glutamyl transpeptidase (GGTP), a marker of the blood-brain barrier. We then used quantitative-competitive PCR to compare thrombomodulin expression in endothelial monocultures and astrocyte-endothelial cocultures after 1 and 7 days of culture. RESULTS: Both in situ hybridization and PCR studies demonstrated thrombomodulin mRNA expression by endothelial cells. During 1 week of astrocyte-endothelial coculture, there was (1) progressive association of astrocytes with capillary-like structures and (2) expression of GGTP; endothelial monocultures did not express GGTP. There was no significant difference in thrombomodulin mRNA expression for cocultures versus monocultures after 1 day. After 1 week, however, astrocyte-endothelial cocultures had markedly decreased thrombomodulin mRNA compared with monocultures (9 +/- 2 versus 189 +/- 62 pg/mL; P < .025). This thrombomodulin mRNA decrease thus occurred when elements of the blood-brain barrier phenotype were demonstrable, ie, when astrocyte association with capillary-like structures was maximal and when GGTP was expressed in cocultures. CONCLUSIONS: These findings indicate astrocyte regulation of thrombomodulin mRNA expression in vitro and suggest an important role for the blood-brain barrier in the regulation of thrombomodulin.

Detection of platelet-derived growth factor (PDGF)-AA in actively healing human wounds treated with recombinant PDGF-BB and absence of PDGF in chronic nonhealing wounds.

Some human chronic dermal wounds treated with recombinant platelet-derived growth factor-BB (rPDGF-BB) show increased healing coupled with fibroblast activation and granulation tissue formation. To determine whether endogenous PDGF is associated with healing and nonhealing dermal ulcer phenotypes, we developed monoclonal antibodies capable of recognizing the three isoforms of PDGF, AA, AB, and BB dimers, and capable of discriminating between two alternatively spliced A chain transcripts. We detected little PDGF isoform expression in normal skin and in nonhealing dermal ulcers. In contrast, in surgically created acute wounds and chronic ulcers treated with rPDGF-BB, markedly upregulated levels of PDGF-AA (long form) were found. In both types of wounds, increased PDGF-AA was detected primarily in capillaries and fibroblasts, although in rPDGF-BB-treated chronic wounds, widespread expression of PDGF-AA was somewhat delayed. With continued treatment, the long form of PDGF-AA, which can preferentially bind extracellular matrix, was expressed only in capillaries, while fibroblasts began synthesizing the short form of PDGF-AA. Within capillaries, all endothelial cells and varying numbers of pericytes and smooth muscle cells contained PDGF-AA. In all wounds, macrophages and keratinocytes were not a major contributor. While PDGF-BB and PDGF-AB were present in a minority of healing wounds, they were usually present at lower levels than PDGF-AA. PDGF-beta receptors, which bind only PDGF-BB and not other isoforms, were found in normal skin and granulation tissue, providing a molecular basis for treating human chronic wounds with exogenous rPDGF-BB.

The antiproliferative activity of c-myb and c-myc antisense oligonucleotides in smooth muscle cells is caused by a nonantisense mechanism.

Smooth muscle cell (SMC) proliferation is thought to play a major role in vascular restenosis after angioplasty and is a serious complication of the procedure. Developing antisense (AS) oligonucleotides as therapeutics is attractive because of the potentially high specificity of binding to their targets, and several investigators have reported inhibition of SMC proliferation in vitro and in vivo by using AS strategies. We report here the results of our experiments on vascular SMCs using AS oligonucleotides directed toward c-myb and c-myc. We found that significant inhibition of SMC proliferation occurred with these specific AS sequences but that this inhibition was clearly not via a hybridization-dependent AS mechanism. Rather, inhibition was due to the presence of four contiguous guanosine residues in the oligonucleotide sequence. This was demonstrated in vitro in primary cultures of SMCs and in arteries ex vivo. The ex vivo model developed here provides a rapid and effective system in which to screen potential oligonucleotide drugs for restenosis. We have further explored the sequence requirements of this non-AS effect and determined that phosphorothioate oligonucleotides containing at least two sets of three or four consecutive guanosine residues inhibit SMC proliferation in vitro and ex vivo. These results suggest that previous AS data obtained using these and similar, contiguous guanosine-containing AS sequences be reevaluated and that there may be an additional class of nucleic acid compounds that have potential as antirestenosis therapeutics.

The uptake and distribution of phosphorothioate oligonucleotides into vascular smooth muscle cells in vitro and in rabbit arteries.

Oligonucleotides are a class of compounds with potential as therapeutics for a variety of clinical applications. Local delivery of oligonucleotides to the arterial wall is a challenging aspect of the development of these therapeutics for restenosis, and herein we report experiments characterizing the uptake and distribution of phosphorothiate oligonucleotides into vascular smooth muscle cells in primary cultures and in rabbit arteries. Primary cultures of smooth muscle cells incubated with rhodamine-oligonucleotides showed uptake only into cytoplasmic vesicles. No nuclear or cytosolic localization was detected. In normal arteries there was no visible tissue or cellular uptake of oligonucleotides after intralumenal administration. However, in balloon-injured arteries there was significant oligonucleotide uptake into the tissue with apparent cytoplasmic delivery to the medial smooth muscle cells, as evinced by intense staining of their nuclei with labeled oligonucleotides. Measurement of FITC-oligonucleotide in artery extracts showed significantly greater uptake in injured, compared with normal arteries. Light and electron microscopic studies demonstrated a correlation between the degree of damage and the amount of uptake. These results demonstrate that oligonucleotides penetrate easily into the arterial wall of balloon-injured arteries and accumulate in the medial smooth muscle cells-the target cells for antirestenosis therapeutics following balloon angioplasty.

Expression of mRNA for glial fibrillary acidic protein after experimental cerebral injury.

This study was undertaken to determine whether a mRNA for glial fibrillary acidic protein (GFAP) was present in increased amounts as a response to injury and, if so, how was its temporal expression related to the demonstration of GFAP by immunocytochemical techniques. A cerebral freeze-injury was produced in mice and at intervals thereafter the animals were anesthetized, perfused with formalin and histological sections of the brain through the injured area were prepared. A riboprobe for GFAP mRNA labeled with S35 and an immunocytochemical probe for GFAP were utilized to localize mRNA and GFAP immunoreactivity, respectively. For mRNA studies, the histological slide exposed to either sense or antisense probe was overlaid with x-ray film or dipped in photographic emulsion. The developed film was quantitated by digital image analysis. Emulsions were examined by dark-field microscopy. The results indicate that mRNA for GFAP is increased in the cortex in the environs of the injury by 6 hours, becomes maximal at 4-5 days, and is present in increased amounts up to 14 days. The message is enhanced in the adjacent cortex, the subpial region, the adjacent corpus callosum and in the ipsilateral and contralateral callosal radiations. This pattern of enhancement follows the distribution of post-injury edema. Glial fibrillary acidic protein is demonstrable at 24-48 hours after injury. Thus, there is a rapid response of the astrocyte to injury with increased mRNA expression that is followed by expression of GFAP immunoreactivity.

In vitro interaction of astrocytes and pericytes with capillary-like structures of brain microvessel endothelium.

A new method to study the interaction of astrocytes and pericytes with cerebral capillary endothelial cells in vitro is described. Endothelial cells derived from bovine brain were cultured on gelatin coated slides and covered with type 1 collagen. Endothelial cells aggregated and formed capillary-like structures (CS) within 3 days. The lining cells of the CS stained immunohistochemically for factor VIII-related antigen. Astrocytes isolated from neonatal mice or pericytes from bovine brain were added to the preparations after the formation of CS. After various periods of co-culture, the slides were fixed with methanol and examined with the immunohistochemical stain for glial fibrillary acidic protein or smooth muscle actin to demonstrate astrocytes or pericytes respectively. Five hours after addition, only 10% of astrocytes were associated with CS. However, by 24 hours, 70% of the astrocytes had assumed a position adjacent to the CS. The astrocytes then developed processes which were intimately apposed to the CS by 3 days, at which time they resembled the in vivo structural relationship between astrocytes and microvessels that occur in areas of central nervous system injury. Progressive elongation of the astrocytes or their processes at the CS was evident at 6 and 9 days of co-culture. The cross-section of CS co-cultured with astrocytes showed continuous cells surrounding a lumen, and the endothelial cells appeared to be connected by tight junctions. When pericytes were added to CS cultures they also preferentially associated with CS, but the contact occurred more rapidly than with astrocytes, 50% being associated with CS by 5 hours. The CS were almost completely covered with elongated pericytes by 24 hours. A chemotactic assay was developed that showed that there was a chemotactic attraction of pericytes to the CS. Thus an in vitro system is now available to study the interrelationships of these cell types and their interaction in development, regeneration and differentiation of the blood-brain barrier.

Astrocyte growth stimulation by a soluble factor produced by cerebral endothelial cells in vitro.

Conditioned medium from isolated cerebral capillary endothelial cells (ECCM) was found to promote DNA synthesis in astrocytes and pericytes, but not in oligodendrocytes or endothelial cells (EC) in vitro. The astrocyte was the cell of primary interest and the cell tested in the following experiments. The effect of ECCM on astrocytes was concentration and time dependent. The growth factor was released by EC into the medium in a cumulative manner for up to 72 hours. This release was not the result of a nonspecific leakage of an internal store, since the DNA synthetic activity of cell lysates was negligible. The growth factor secretion per cell was higher in sparse than in confluent EC cultures and was partially inhibited by preincubation of EC with interleukin-1. The DNA synthetic activity was due to a peptide, different from basic fibroblast growth factor, transferrin, bovine fibronectin and platelet derived growth factor, with a molecular weight greater than 50,000. The peptide derived from the cerebral capillary EC could be involved in the local signaling between cell types that control new vessel formation in development, in regeneration after brain tissue injury, or in tumor formation.

Choline uptake by cerebral capillary endothelial cells in culture.

A passage of choline from blood to brain and vice versa has been demonstrated in vivo. Because of the presence of the blood-brain barrier, such passage takes place necessarily through endothelial cells. To get a better understanding of this phenomenon, the choline transport properties of cerebral capillary endothelial cells have been studied in vitro. Bovine endothelial cells in culture were able to incorporate [3H]choline by a carrier-mediated mechanism. Nonlinear regression analysis of the uptake curves suggested the presence of two transport components in cells preincubated in the absence of choline. One component showed a Km of 7.59 +/- 0.8 microM and a maximum capacity of 142.7 +/- 9.4 pmol/2 min/mg of protein, and the other one was not saturable within the concentration range used (1-100 microM). When cells were preincubated in the presence of choline, a single saturable component was observed with a Km of 18.5 +/- 0.6 microM and a maximum capacity of 452.4 +/- 42 pmol/2 min/mg of protein. [3H]Choline uptake by endothelial cells was temperature dependent and was inhibited by the choline analogs hemicholinium-3, deanol, and AF64A. The presence of ouabain or 2,4-dinitrophenol did not affect the [3H]choline transport capacity of endothelial cells. Replacement of sodium by lithium and cell depolarization by potassium partially inhibited choline uptake. When cells had been preincubated without choline, recently transported [3H]choline was readily phosphorylated and incorporated into cytidine-5'-diphosphocholine and phospholipids; however, under steady-state conditions most (63%) accumulated [3H]choline was not metabolized within 1 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of inorganic lead on some functions of the cerebral microvessel endothelium.

The effect of inorganic lead on two functions of cerebral microvessel endothelium, cell division and glucose analog uptake, was investigated. Lead concentrations considered to be toxic in humans inhibited both functions in cultured endothelial cells. Both effects were dependent on the length of lead exposure and dose over the range of 10(-4) to 10(-6) M lead acetate. After 4 days of exposure there were 76% fewer cells in 10(-4) M lead-exposed cultures relative to control cultures. After 4 days of exposure to 10(-5) M lead there were 55% fewer cells, and after 10(-6) M lead exposure there were 15% fewer cells. Two days after 10(-4) M lead exposure [methyl-3H]thymidine incorporation into endothelial cells was inhibited by 71%. Incorporation was inhibited 47% by 10(-5) M lead but 10(-6) M lead did not inhibit incorporation after 2 days of exposure. Glucose analog uptake was inhibited in both contact-inhibited and log-phase cells; however, the latter were more sensitive to lead and this increased sensitivity correlated with a higher lead content in this cell population. Both the specific carrier-mediated and the nonspecific components of glucose analog uptake were inhibited by exposure of the endothelial cells to lead. A lead exposure of 40 min produced a significant effect on the uptake mechanism. In order to manifest its effects the lead had to be present in serum-containing medium, suggesting that some serum component was necessary to present the lead to the endothelial cells. These findings imply that the initial target of inorganic lead in the CNS may be the plasma membrane of the capillary endothelial cells, and that lead may act by altering the physiological function of these membranes.

Insulin stimulates DNA synthesis in cerebral microvessel endothelium and smooth muscle.

Experiments were performed to test the hypothesis that insulin stimulates DNA synthesis in cerebral microvessel endothelium and smooth muscle. Cultured endothelium and smooth muscle derived from isolated mouse cerebral microvessels were exposed to insulin in serum-free medium, and [3H]-thymidine incorporation in the cells was measured. Up to 40-fold stimulation of DNA synthesis in endothelium and fourfold stimulation in smooth muscle were observed. Stimulation became maximal in both cell types at an insulin concentration of approximately 10(4) ng/ml, although an effect was observed at much lower concentrations. Similar concentrations of insulin produced a less-dramatic (approximately twofold) increase in both endothelial and smooth muscle cell numbers. This effect of insulin, observed in microvessel endothelium and smooth muscle, but not in bovine aortic endothelium, emphasizes another way in which large- and small-vessel endothelia appear to differ.

Uptake of glucose analogues into cultured cerebral microvessel endothelium.

Tritiated glucose analogues 3-O-methylglucose (3-OMG) and 2-deoxyglucose (2-DG) were used to study glucose uptake properties in established lines of cultured mouse cerebral microvessel endothelium. Uptake of both analogues was similar in terms of rate and absolute amount for the first two minutes. Thereafter, intracellular accumulation of 2-DG continued at a more rapid rate because of intracellular phosphorylation of this substrate. The uptake of 3-OMG uptake was temperature-dependent, independent of Na+, and not inhibited by ouabain or 2,4-dinitrophenol. Phloretin and cytochalasin B both significantly inhibited 3-OMG uptake. Other hexoses in high concentration acted as competitive inhibitors at the endothelial cell membrane. Pre-incubation of cells with 50 mM D-glucose resulted in higher levels of 3-OMG accumulation than in control cells (counter-transport phenomenon). In contrast to findings at the blood-brain barrier in vivo, insulin was found to stimulate 3-OMG uptake. Maximal stimulation of approximately 3-fold was found at ambient insulin concentrations of 1,000 ng/ml or higher. The findings provide support at the cellular level for some components of the model of carrier-mediated glucose transport across the blood-brain barrier which has been postulated to exist in vivo. The effect of insulin is discussed in the light of new data that show stimulation of glucose analogue transport into isolated cerebral capillaries.

Spontaneous spongy degeneration of the mouse brain.

A spontaneously-occurring spongy disorder of the white matter of the central nervous system was discovered in the Charles River strain of Swiss-Webster mice and is described in this report. The disorder was transmitted with an autosomal recessive pattern of inheritance. Clinical characteristics of the affected animals included enlargement of the cranium, failure to thrive and tremor of the hind limbs when held by the tail in a suspended position. Maintenance of the colony with propagation of the disease was achieved by selective in-breeding of litter mates. Light microscopic examination of the central nervous system revealed a spongy degeneration of the white matter of the entire neuraxis. Ultrastructural studies localized the abnormality to the cell body and processes of the astrocyte which appeared distended and enlarged with dispersion of cytoplasmic organelles. Hemidesmosomes were prominent in the foot processes of astrocytes. This animal model bears a similar morphology and pattern of inheritance to Canavan's spongy degeneration of the white matter in humans and should provide a base for future investigations aimed at gaining insight into the pathogenesis of the human and this animal neurological disorder.