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Biomedical subjects

D C West

Publications and source records attributed to D C West.

At least 19 recordsLinked to original sources
PubMed2005

The inhibition of neutrophil-endothelial cell adhesion by hyaluronan independent of CD44.

OBJECTIVE: To study the effect of hyaluronan on cell adhesion and recruitment both in vitro and in vivo, since hyaluronan both inhibits restenosis and is anti-inflammatory. When administered to animals undergoing angioplasty the recruitment of cells into the restenotic plaque is inhibited, as well as into inflammatory lesions. The recent discovery that ICAM-1 binds hyaluronan and exhibits the B(X(7))B HA binding motif, led us also to investigate whether cell adhesion could be modulated by hyaluronan. MATERIALS AND METHODS: Human neutrophils were adhered to human umbilical vein (HUVEC) or Ea.hy.926 HUVEC cells stimulated with phorbol myristate acetate (PMA) or tumour necrosis factor (TNFalpha). Neutrophil binding in vivo utilized FMLP-stimulated hamster cheek pouch post-capillary venules. RESULTS: Hyaluronan inhibited human neutrophil adhesion to both PMA and TNFalpha-stimulated HUVEC. Ea.hy.926 human immortal HUVECs expressed ICAM-1 in response to TNFalpha and PMA. E-selectin was also upregulated by 6 h with TNFalpha but not significantly with PMA. TNFalpha induced CD44 expression within 4 h, but PMA not significantly up to 6 h. However, specific binding of [125I]hyaluronan to Ea.hy.926 cells was increased by PMA-stimulation at 4 h. Neutrophil adhesion to PMA-stimulated Ea.hy.926 HUVECs was inhibited in a concentration dependent fashion by both anti-ICAM-1 and hyaluronan (1 ng/ml-10 microg/ml) at 4 h. At 1 mg/ml adhesion was stimulated by hyaluronan. Hyaluronan had no effect on neutrophil adhesion to resting Ea.hy.926 cells. Hyaluronan (25 mg/kg, i.v.) inhibited cell adhesion to FMLP-stimulated post capillary venules of the hamster cheek pouch, whilst leaving cell rolling unaffected. CONCLUSIONS: These results show that hyaluronan, at concentrations below those where intra-molecular associations occur, binds selectively to stimulated endothelial cells and inhibits neutrophil adhesion in vitro and in vivo via a mechanism which may involve molecules other than CD44, such as ICAM-1.

Animals↗
PubMed2002

The role of the matrix metalloproteinases during in vitro vessel formation.

Matrix metalloproteinases (MMPs) constitute a large family of extracellular matrix degrading proteases implicated in a number of physiological and pathological processes, including angiogenesis. However, the relative importance of the individual MMPs in vessel formation is poorly understood. Using the three-dimensional rat aortic model, the role of the MMPs in angiogenesis in vitro was investigated both by the use of synthetic MMP inhibitors, and by a study of the expression of nine MMPs and three of their endogenous inhibitors (the TIMPs) during vessel formation. Inhibition of microvessel growth in this model by the MMP inhibitor Marimastat demonstrated the requirement of the MMPs for angiogenesis in both collagen and fibrin matrices (half-maximal inhibition at 5 and 80 nM, respectively). The profile of MMP expression was seen to be modified by both matrix composition and exogenous growth factors. For example, whilst the gelatinase MMP-2 and stromelysin MMP-3 were present at high levels in fibrin culture, the stromelysin MMP-11 and membrane-type-1-MMP were more highly expressed during vessel formation in collagen. The angiogenic basic fibroblast growth factor (bFGF) upregulated the expression of the gelatinases (MMP-2 and MMP-9), the stromelysins (MMP-3, MMP-10 and MMP-11) and the interstitial collagenase MMP-13, whereas vascular endothelial growth factor (VEGF) led to a marked increase in expression of MMP-2 only. Together, the environment-dependent upregulation in expression of a number of MMPs during angiogenesis, and the total inhibition of vessel growth observed at nanomolar concentrations of synthetic MMP inhibitors, suggests a major collective role of these enzymes in angiogenesis, and provides a basis for further development of MMP inhibitors for anti-angiogenic therapy.

Animals↗
PubMed2001

Human cutaneous fatty acid-binding protein induces metastasis by up-regulating the expression of vascular endothelial growth factor gene in rat Rama 37 model cells.

Human cutaneous fatty acid-binding protein (C-FABP) gene is capable of inducing the metastatic phenotype when overexpressed in nonmetastatic rat Rama 37 cells. However, the mechanism of how it induces metastasis is not clear. Northern and slot blot analyses revealed that expression of the endogenous vascular endothelial growth factor (VEGF) gene was increased by 3.8-5.2-fold in the C-FABP-transfected cells (pSV-CFABP-R37) and in their metastatic sublines (e.g., Met-1) when compared with that in the nonmetastatic control transfectant pSV-R37 cells generated by transfection of only plasmid DNA. Higher levels of VEGF immunoreactive protein were also secreted from the malignant C-FABP-expressing cells. Reverse transcription-PCR detected two VEGF transcript isoforms, VEGF(164) and VEGF(188), in both the nonmetastatic control transfectant pSV-R37 cells and the malignant metastatic Met-1 cells. Chick chorioallantoic membrane assays showed that the conditioned medium of the control pSV-R37 cells possessed only very weak angiogenic activity, whereas conditioned media from the metastatic C-FABP transfectants and their sublines were strongly angiogenic and could be inhibited by antibodies to VEGF. Transfection of VEGF(164) cDNA in an expression vector into nonmetastatic Rama 37 cells produced a cell clone (R37-VEGF-2) that expressed high levels of VEGF. Inoculation of R37-VEGF-2 cells into syngeneic Wistar Furth rats produced metastases in a significant number (Fisher's exact test, P < 0.01) of animals (18 of 31 animals), whereas the control, vector alone-transfected R37-PSV cells produced no metastases (0 of 30 animals). Immunocytochemical methods demonstrated a strong positive staining for VEGF and an increased microvessel density in the primary tumors produced from PSV-VEGF-2 cells in comparison with tumors produced from control transfectants. Immunocytochemical staining for factor VIII detected a 3.5-fold increase in microvessel density of the primary tumors produced by PSV-VEGF-2 cells when compared with that of the primary tumors developed from the control pSV-R37 cells. Therefore, we suggest that overexpression of the C-FABP gene in the original transfectants induces metastasis through up-regulation of expression of the VEGF gene in this rat Rama 37 model system, and thus VEGF may play a crucial role in this particular metastatic cascade.

Animals↗
PubMed2000

In vitro and in vivo production of vascular endothelial growth factor by chronic lymphocytic leukemia cells.

Expansion of primary solid tumors and their malignant dissemination are angiogenesis-dependent. Vascular endothelial growth factor (VEGF) is the key factor playing a pivotal role in solid tumor-induced angiogenesis. Recent studies indicate that angiogenesis may also be involved in the pathogenesis of certain hemic malignancies, including B-cell chronic lymphocytic leukemia (B-CLL). Mechanisms underlying angiogenesis in B-CLL and the role of VEGF in this process are incompletely understood. In this study, it was examined whether angiogenically functional VEGF is produced by B-CLL cells. Immunohistochemical staining with antibodies against VEGF and CD34, an endothelial cell marker, demonstrated the presence of VEGF protein and abundant blood vessels in infiltrated lymphoreticular tissues. Low levels of VEGF were detected by ELISA in the culture media of unstimulated cells; this was enhanced up to 7-fold by hypoxic stimulation. SDS-PAGE and Western blot analysis of the concentrated culture media showed 2 isoforms of VEGF protein with molecular weights of 28 and 42 kd, respectively. RNA hybridization showed that these cells expressed VEGF mRNA. Reverse transcription-polymerase chain reaction, combined with nucleotide sequence analysis, revealed that the predominantly expressed isoforms were VEGF121 and VEGF165. Moreover, (3)H-thymidine incorporation and an in vivo angiogenic assay demonstrated that the VEGF produced by CLL cells can induce angiogenesis by stimulating endothelial cell proliferation. In conclusion, this study shows that B-CLL cells produce VEGF and demonstrates the angiogenic effects of this growth factor, which may be relevant for the tissue phase of the disease.

Antigens, CD34↗
PubMed2000

Critical thinking in graduate medical education: A role for concept mapping assessment?

CONTEXT: Tools to assess the evolving conceptual framework of physicians-in-training are limited, despite their critical importance to physicians' evolving clinical expertise. Concept mapping assessment (CMA) enables teachers to view students' organization of their knowledge at various points in training. OBJECTIVE: To assess whether CMA reflects expected differences and changes in the conceptual framework of resident physicians, whether concept maps can be scored reliably, and how well CMA scores relate to the results of standard in-training examination. DESIGN, SETTING, AND PARTICIPANTS: A group of 21 resident physicians (9 first-year and 12 second- and third-year residents) from a university-based pediatric training program underwent concept map training, drew a preinstruction concept map about seizures, completed an education course on seizures, and then drew a postinstruction map. Maps were scored independently by 3 raters using a standardized method. The study was conducted in May and June 1999. MAIN OUTCOME MEASURES: Preinstruction map total scores and subscores in 4 categories compared with postinstruction map scores; map scores of second- and third-year residents compared with first-year residents; and interrater correlation of map scores. RESULTS: Total CMA scores increased after instruction from a mean (SD) preinstruction map score of 429 (119) to a mean postinstruction map score of 516 (196) (P =.03). Second- and third-year residents scored significantly higher than first-year residents before instruction (mean [SD] score of 472 [116] vs 371 [102], respectively; P =.04), but not after instruction (mean [SD] scores, 561 [203] vs 456 [179], respectively; P =.16). Second- and third-year residents had greater preinstruction map complexity as measured by cross-link score (P =.01) than first-year residents. The CMA score had a weak to no correlation with the American Board of Pediatrics In-training Examination score (r = 0.10-0.54). Interrater correlation of map scoring ranged from weak to moderate for the preinstruction map (r = 0.51-0.69) and moderate to strong for the postinstruction map (r = 0.74-0.88). CONCLUSIONS: Our data provide preliminary evidence that concept mapping assessment reflects expected differences and change in the conceptual framework of resident physicians. Concept mapping assessment and standardized testing may measure different cognitive domains. JAMA. 2000;284:1105-1110

California↗
PubMed2000

Biphenotypic sarcoma with characteristics of both a Ewing sarcoma and a desmoplastic small round cell tumor.

BACKGROUND: The EWS gene, a transcription factor of unknown function, is involved in chromosomal translocations associated with a wide variety of tumors, particularly small round blue cell tumors such as Ewing sarcoma. It has previously been reported that desmoplastic small round blue cell tumor (DSRBCT) frequently has an associated t(11;22) abnormality resulting from fusion of the EWS and WT-1 genes. PROCEDURE: We report a case of a small round blue cell tumor with characteristics of both Ewing sarcoma and DSRBCT with a t(11;22) translocation leading to fusion of the EWS and FLI1genes. RESULTS: The translocation point and fusion products were confirmed by polymerase chain reaction amplification and restriction fragment mapping of the products. CONCLUSIONS: The biphenotypic nature of this case and the apparent promiscuity of the EWS gene in tumor-associated translocations coupled with other reports of biphenotypic childhood sarcomas has potential implications for the relationship between small round blue cell tumors and the mechanism of EWS/FLI1 oncogenesis.

Carcinoma, Small Cell↗
PubMed2000

Interleukin-8 as an autocrine growth factor for human colon carcinoma cells in vitro.

Cell lines derived from human colon carcinomas secrete interleukin 8 (IL-8) in vitro and this chemokine has also been detected immunohistochemically in human colon carcinoma specimens, in which it is tumour cell associated. In these experiments, IL-8 was shown to comprise an important component of the angiogenic activity of colon carcinoma cell line supernatants. The effect of modulating IL-8 activity upon the growth of the colon carcinoma cell lines HCT116A, HT29 and CaCo2 was investigated. Supplementing endogenously produced IL-8 by recombinant chemokine led to stimulation of cell growth. Neutralization of the effect of endogenously produced IL-8, either with the specific antagonist peptide AcRRWWCR or with blocking anti-IL-8 antibody, resulted in around 50% inhibition of cell growth (P<0.05). All of the colon carcinoma cell lines tested expressed mRNA for both IL-8RA and RB when grown at confluence. At the protein level, all cell lines expressed IL-8RA. Expression of IL-8RB was weak, although increased expression was seen in HCT116A cells as they approached confluence. Antibodies to IL-8RA and RB did not affect proliferation at low cell density but were strongly inhibitory when cells were cultured at a higher density. These data suggest that IL-8 acts as an autocrine growth factor for colon carcinoma cell lines and would support the concept that a similar autocrine loop operates in vivo.

Carcinoma↗
PubMed2000

Angiogenically active vascular endothelial growth factor is over-expressed in malignant human and rat prostate carcinoma cells.

Vascular endothelial growth factor (VEGF) is one of the most potent factors for stimulating angiogenesis, an essential process required for expansion of primary tumour and dissemination of malignant cells. To investigate the possible role of VEGF in facilitating metastasis of prostate cancer via stimulating angiogenesis, we have used Northern and slot blotting, reverse transcription polymerase chain reaction, nucleotide sequence analysis and enzyme-linked immunosorbent assay to compare the VEGF expression in series of human and rat cell lines with either benign or malignant characteristics. We have also employed the chick chorioallantoic membrane (CAM) assay to measure the angiogenic activity of the VEGF derived from both benign and malignant cells. The level of VEGF mRNA expressed in the seven malignant human and rat cell lines is 3.5- to 10-fold higher than that expressed in the benign cell lines. The three metastatic variants, generated by transfection of a benign cell line with DNA extracted from prostate carcinoma cells, expressed 2.5 to 5 times more VEGF mRNA than their parental benign cells. While VEGF 121 and 165 were predominantly expressed by both the benign and malignant cells, the transcript representing VEGF 189 isoform was only detected in the malignant cells. At protein level, three human malignant cell lines produced more VEGF (2.7-7.9 ng ml(-1)) than the benign cell line (1.3 ng ml(-1)). CAM assay detected a VEGF-dependent angiogenic activity in the medium from malignant cells, but only a relatively weak VEGF-independent activity in the medium from benign cells. These results demonstrated that malignant cells did over-express VEGF and only the VEGF derived from malignant cells was angiogenically active. Thus, we suggest that the VEGF produced by malignant cells might play an important role in facilitating metastasis of prostatic cancer.

Allantois↗
PubMed2000

Ewing sarcoma family of tumors.

The Ewing sarcoma family of tumors (ESFT) shares a common neural histogenesis, tumor genetics, and a fascinating biology, all of which hold the promise of identifying new therapeutic targets. The genetic hallmark of ESFT is the presence of the t(11;22)(q24;q12), which creates the EWS/FLI1 fusion gene and results in the expression of a chimeric protein. This article reviews much of the important work that has been reported over the past year regarding the biology and therapy of ESFT. Major studies include the observation that the early region (E1A) of human adenovirus type 5 is directly linked to and may initiate production of the EWS/FLI1 fusion transcript in normal human fibroblasts and keratinocytes. In addition, there is new information regarding the function of EWS/FLI1 and downstream signals by which it acts. New clinical information continues to support the addition of ifosfamide to standard chemotherapy regimens and help further refine prognostic features, including biologic features that may someday allow better risk stratification and therapy design.

Adolescent↗
PubMed2000

Oscillations and long-lasting correlations in a model of the lateral geniculate nucleus and visual cortex.

We have previously developed a model of the corticogeniculate system to explore cortically induced synchronization of lateral geniculate nucleus (LGN) neurons. Our model was based on the experiments of Sillito et al. Recently Brody discovered that the LGN events found by Sillito et al. correlate over a much longer period of time than expected from the stimulus-driven responses and proposed a cortically induced slow covariation in LGN cell membrane potentials to account for this phenomenon. We have examined the data from our model, and we found, to our surprise, that the model shows the same long-term correlation. The model's behavior was the result of a previously unsuspected oscillatory effect, not a slow covariation. The oscillations were in the same frequency range as the well-known spindle oscillations of the thalamocortical system. In the model, the strength of feedback inhibition from the cortex and the presence of low-threshold calcium channels in LGN cells were important. We also found that by making the oscillations more pronounced, we could get a better fit to the experimental data.

Computer Simulation↗
PubMed2000

The angiogenesis inhibitor SU5416 has long-lasting effects on vascular endothelial growth factor receptor phosphorylation and function.

SU5416, a selective inhibitor of the tyrosine kinase activity of the vascular endothelial growth factor (VEGF) receptor Flk-1/KDR, is currently in Phase III clinical trials for the treatment of advanced malignancies. In cellular assays, SU5416 inhibits the VEGF-dependent mitogenic/proliferative response of human umbilical vein endothelial cells (HUVECs). In tumor xenograft models, SU5416 inhibits the growth of tumors from a variety of origins by inhibiting tumor angiogenesis. In three different human tumor xenograft models, infrequent (once or twice a week) administration of SU5416 is efficacious despite the fact that it has a short plasma half-life (30 min), which suggests that SU5416 has long-lasting inhibitory activity in vivo. The goal of the present study was to determine the basis for the prolonged activity of SU5416. The results indicate that a short (3 h) exposure to 5 microM SU5416 (to mimic plasma levels of the compound as measured in patients who were receiving SU5416 therapy) produced long-lasting (at least 72 h) inhibition of the VEGF-dependent proliferation of HUVECs in culture, which indicate that SU5416 has long-lasting inhibitory activity in vitro as well as in vivo. SU5416 treatment of HUVECs did not affect surface expression of Flk-1/KDR or the affinity of the receptor for VEGF. Instead, the durability of the in vitro activity of SU5416 was shown to be attributable to its long-lasting ability to specifically inhibit VEGF-dependent phosphorylation of Flk-1/KDR and subsequent downstream signaling, although SU5416 is not an irreversible inhibitor of Flk-1/KDR tyrosine kinase activity. The long-lasting inhibition of cellular responses to VEGF was attributable to the accumulation of SU5416 in cells, as shown using radiolabeled compound, such that inhibitory cellular concentrations of SU5416 are maintained long after the removal of the compound from the medium. The long-lasting inhibitory activity of SU5416 in vitro is consistent with the finding that SU5416 has demonstrated evidence of biological activity in clinical studies when administered twice a week despite a short plasma half-life.

3T3 Cells↗
PubMed1999

The effect of extracellular pH on angiogenesis in vitro.

The microenvironment of the majority of solid tumours in which new vessels must grow and survive is acidic. Whilst recent reports suggest a role of the low tumour pH in the invasive and metastatic potential of tumour cells, little is known as to its impact on angiogenesis. The three-dimensional in vitro rat aortic ring model was used to study the effects of low extracellular pH (pH(e)) on microvascular growth. The spontaneous angiogenic response in collagen gels was seen to be highly dependent on the pH of the culture medium, with optimal outgrowth at pH 7.4, and a marked delay in microvascular growth at pH 6.9. This inhibition of vascular development was reversible. The absence of similar effects of medium pH on monolayer outgrowths of endothelial cells from rat aortic rings suggested an effect of pH(e) on aspects specific to three-dimensional growth. Vascular endothelial growth factor and basic fibroblast growth factor, whilst having limited effects at pH 7.4, were seen to reduce the time to onset of vessel outgrowth at pH 7.1, and lead to an initial growth rate similar to that observed at pH 7.4 in the absence of growth factors. Thus, the low environmental pH encountered by endothelial cells in solid tumours would not necessarily be detrimental to neovascularisation: a prominent in vitro angiogenic response is still observed at low pH(e) when stimulated by exogenous growth factors, high concentrations of which would be present in vivo.

Journal Article↗
PubMed1998

Ultrasound and MR imaging of fibromatosis colli (sternomastoid tumor of infancy).

The sonographic and CT findings of fibromatosis colli (sternomastoid tumor of infancy) have been described, but the MRI appearance has been reported in only one case in which the mass resolved over time. This case describes the detailed MRI findings in a biopsy-proven case of fibromatosis colli; the signal intensity of the mass on T2-weighted images was slightly less than on gradient-recalled T1-weighted images, consistent with the presence of some fibrous tissue within the muscle mass. The involved portion of the muscle was better defined on MRI than sonography. MRI was helpful in demonstrating the signal characteristics of the mass; localizing the mass to within the sternocleidomastoid muscle; and demonstrating clear surrounding fascial planes with lack of associated lymphadenopathy, airway compression, vascular encasement, bone involvement or intracranial/intraspinal extension associated with other neck masses.

Biopsy, Needle↗
PubMed1997

Fibrotic healing of adult and late gestation fetal wounds correlates with increased hyaluronidase activity and removal of hyaluronan.

The lack of scarring and fibrosis in healing fetal skin wounds may relate to a prolonged presence of hyaluronan (HA). It has been suggested that fetal wounds may lack hyaluronidase, but the hyaluronidase levels in fetal wounds remain unknown. The size of HA influences its biological action, especially in relation to angiogenesis, which is also reduced in fetal wound healing. The present study determined the levels and size of HA, as well as hyaluronidase levels, in fetal and adult lamb wounds. Wire mesh cylinders, or polyvinyl acetate sponges, were placed subcutaneously in fetal lambs at 75, 100 or 120 days gestation. Wound fluid and wound tissue were harvested 3, 7 or 14 days later. Samples were digested with papain and both HA and hyaluronidase activity were determined in a competitive ELISA assay. Size distribution of HA was estimated using a Sephacryl S1000 column and fractions were collected for HA determination. Adult wound fluid HA remained low (4-5 micrograms/ml) over the 14 days. Fetal fluids were similar on day 3, but increased to 15-25 micrograms/ml by day 7. In 75/100-day wounds, HA remained elevated at 14 days, but in 120-day fluids decreased to levels similar to adult fluid. The HA in all fluids was polydisperse with a main peak at 200 kDa. Hyaluronidase levels were detected in all samples, reaching a peak 7 days post-wounding. In adult wound fluids hyaluronidase was much higher than the fetal wound fluids. These data suggest that lower hyaluronidase levels in fetal wounds may underlie the different pattern of HA deposition seen in fetal wounds.

Amniotic Fluid↗