Biomedical subjects

S C Watkins

Publications and source records attributed to S C Watkins.

At least 55 records · Page 3Linked to original sources

PubMed2001

Differential effects of overexpression of two forms of ephrin-A5 on neonatal rat cardiomyocytes.

Eph receptors constitute the largest family of receptor tyrosine kinases. Multiple transcripts of ephrin-A5, the cognate ligand of the EphA3 receptor, were found in neonatal rat cardiomyocytes. Two cDNA clones encoding the full-length ephrin-A5 (ephrin-A5 alpha) and a 27-amino acid deletion form (ephrin-A5 beta) were isolated. To examine the role of ephrin-A5 in cardiomyocytes, the cDNAs were inserted into adenoviral vectors, termed Ad.ephrin-A5 alpha and Ad.ephrin-A5 beta, respectively, and overexpressed in cardiomyocytes. The effect of ephrin-A5 on cardiomyocyte gene expression was investigated using a cDNA expression array and Western blot analysis. The results showed that both ephrin-A5 alpha and ephrin-A5 beta downregulated cyclin D2, cyclin-dependent kinase-4 proteins, and their cognate receptor EphA3, which were associated with reduced bromodeoxyuridine incorporation in cardiomyocytes. Whereas ephrin-A5 alpha and ephrin-A5 beta also induced differential gene expression, only ephrin-A5 beta significantly upregulated the transcription of brain natriuretic peptide and downregulated ras-related protein RAB2, protein kinase C inhibitor protein-1, clusterin, and insulin-like growth factor-binding protein. The results suggest that the two forms of ephrin-A5 share similar function while differ in regulating different sets of genes in cardiomyocytes.

Adenoviridae↗

PubMed2001

The density of social networks and fertility decisions: evidence from South Nyanza district, Kenya.

Demographers have argued increasingly that social interaction is an important mechanism for understanding fertility behavior. Yet it is still quite uncertain whether social learning or social influence is the dominant mechanism through which social networks affect individuals' contraceptive decisions. In this paper we argue that these mechanisms can be distinguished by analyzing the density of the social network and its interaction with the proportion of contraceptive users among network partners. Our analyses indicate that social learning is most relevant with high market activity; in regions with only modest market activity, however, social influence is the dominant means by which social networks affect women's contraceptive use.

Birth Rate↗

PubMed2001

p27Kip1 localizes to detergent-insoluble microdomains within lymphocyte membranes.

BACKGROUND: Low levels of the cyclin-dependent kinase inhibitor p27Kip1 are associated with poor prognosis in cancer. It is unclear whether this is related strictly to p27Kip1-mediated cell cycle inhibition or to other, possibly extranuclear, roles of this protein. In this study, we examined p27Kip1 expression in quiescent and activated lymphocytes. T-cell membranes have been shown to possess sphingolipid and cholesterol-rich microdomains that are insoluble in non-ionic detergents. These "rafts" provide a scaffold for signaling proteins. Signal transduction coincides with coalescence of these microdomains into larger complexes. METHODS: Localization of p27Kip1 was studied by electron and confocal microscopy. Association of p27Kip1 with membrane microdomains in unstimulated and stimulated lymphocytes was determined using Western blots analysis of isolated membranes variably treated with detergents. RESULTS: We demonstrated that p27Kip1 was present in clusters associated with the plasma membrane in normal lymphocytes. The solubility profile of p27Kip1 in isolated membranes indicated that it was localized to raft structures. When lymphocytes were stimulated, however, p27Kip1 was excluded from aggregated raft complexes. CONCLUSIONS: This study identifies, for the first time, the localization of p27 within a membrane microdomain associated with signaling. Because some cell surface signaling complexes lose p27Kip1 upon cellular activation, p27Kip1 may play a functional role in modulating membrane signaling.

Antigens, CD↗

PubMed2000

Tumor-induced apoptosis of T cells: amplification by a mitochondrial cascade.

We have recently reported that apoptosis of T cells induced by squamous cell carcinoma of the head and neck (SCCHN) is partly Fas dependent. This tumor-induced T-cell death is mediated by the activities of caspase-8 and caspase-3 and is partially inhibited by antibodies to either Fas or Fas ligand. We report here that in contrast to apoptosis induced by agonistic anti-Fas antibody (Ab), the tumor-induced apoptotic cascade in Jurkat cells is significantly amplified by a mitochondrial loop. The involvement of mitochondria in tumor-induced apoptosis of T cells was demonstrated by changes in mitochondrial permeability transition as assessed by 3,3'-dihexiloxadicarbocyanine staining, by cleavage of cytosolic BID and its translocation to the mitochondria, by release of cytochrome c to the cytosol, and by the presence of active subunits of caspase-9 in Jurkat T cells cocultured with tumor cells. To further elucidate the significance of mitochondria in tumor-induced T-cell death, we investigated the effects of various inhibitors of the mitochondrial pathway. Specific antioxidants, as well as two inhibitors of mitochondria permeability transition, bongkrekic acid and cyclosporin A, significantly blocked the DNA degradation induced in Jurkat T cells by SCCHN cells. However, these inhibitors had no effect on cells triggered by anti-Fas Ab. Furthermore, a cell-permeable inhibitor of caspase-9, Ac-LEHD.CHO, which did not inhibit T-cell apoptosis induced by anti-Fas Ab, markedly inhibited apoptosis induced by etoposide or by coculture of Jurkat with SCCHN cells. These findings demonstrate that apoptotic cascades induced in Jurkat T lymphocytes by anti-Fas Ab or tumor cells are differentially susceptible to a panel of inhibitors of mitochondrial apoptotic events. It appears that besides the Fas-mediated pathway, additional mitochondria-dependent cascades are involved in apoptosis of tumor-associated lymphocytes. Inhibition of mitochondria-dependent cascades of caspase activation should be considered to enhance the success of immunotherapy or vaccination protocols in cancer.

Anti-Bacterial Agents↗

PubMed2000

Myocardial extracellular matrix remodeling in transgenic mice overexpressing tumor necrosis factor alpha can be modulated by anti-tumor necrosis factor alpha therapy.

Myocardial fibrosis caused by maladaptive extracellular matrix (ECM) remodeling is implicated in the dysfunction of the failing heart. Matrix metalloproteinases (MMPs) regulate ECM remodeling, and are regulated by cytokines. Transgenic mice with cardiac-specific overexpression of tumor necrosis factor alpha (TNF-alpha) (TNF1.6) develop heart failure. We hypothesized that modulation of TNF-alpha and/or MMP activity might alter the myocardial ECM remodeling process and the development of heart failure. To test this hypothesis, we took advantage of the TNF1.6 mice and studied soluble and total collagens and collagen type profiling by using hydroxyproline quantification, Sircol collagen assay, Northern blot analysis, and immunohistochemistry and studied myocardial function by using echocardiography. Progressive ventricular hypertrophy and dilation in the TNF1.6 mice were accompanied by a significant increase in MMP-2 and MMP-9 activity, an increase in collagen synthesis, deposition, and denaturation, and a decrease in undenatured collagens. In young TNF1.6 mice, these changes in the ECM were associated with marked diastolic dysfunction as demonstrated by significantly reduced transmitral Doppler echocardiographic E/A wave ratio. Anti-TNF-alpha treatment with adenoviral vector expressing soluble TNF-alpha receptor type I attenuated both MMP-2 and MMP-9 activity, prevented further collagen synthesis, deposition and denaturation, and preserved myocardial diastolic function in young, but not old, TNF1.6 mice. The results suggest a critical role of TNF-alpha and MMPs in myocardial matrix remodeling and functional regulation and support the hypothesis that both TNF-alpha and MMPs may serve as potential therapeutic targets in the treatment of heart failure.

Animals↗

PubMed2000

Vaginal mucosa serves as an inductive site for tolerance.

These data demonstrate that tolerance can be induced by vaginal Ag exposure. In these experiments, mice were given vaginal agarose gel suppositories containing either 5 mg OVA or saline for 6 h. Mice were given suppositories either during the estrous (estrogen dominant) or diestrous (progesterone dominant) stage of the estrous cycle. Mice were restrained during the inoculation period to prevent orovaginal transmission of the Ag. After 1 wk, mice were immunized s. c. with OVA in CFA. After 3 wk, mice were tested for delayed-type hypersensitivity responses by measuring footpad swelling and measuring in vitro proliferation of lymphocytes to Ag. Using ELISA, the magnitude of the serum Ab response was also measured. In some mice, FITC conjugated to OVA was used to track the dissemination of the protein into the systemic tissues. The magnitude of footpad swelling was significantly reduced in mice receiving OVA-containing suppositories during estrus compared with mice receiving saline suppositories. Concomitant decreases in the Ag-specific proliferative response were also observed in lymph node lymphocytes and splenocytes. Conversely, mice inoculated during diestrus did not show a decreased response to Ag by either footpad response or in vitro proliferation. Serum Ab titers in the estrus-inoculated mice did not decrease significantly. These data demonstrate that the reproductive tract can be an inductive site for mucosally induced tolerance. However, unlike other mucosal sites such as the lung and gastrointestinal tract, reproductive tract tolerance induction is hormonally regulated.

Administration, Intravaginal↗

PubMed2000

The mechanism of unresponsiveness to circulating tumor antigen MUC1 is a block in intracellular sorting and processing by dendritic cells.

Immunity to tumor Ags in patients is typically weak and not therapeutic. We have identified a new mechanism by which potentially immunogenic glycoprotein tumor Ags, such as MUC1, fail to stimulate strong immune responses. MUC1 is a heavily glycosylated membrane protein that is also present in soluble form in sera and ascites of cancer patients. We show that this soluble protein is readily taken up by dendritic cells (DC), but is not transported to late endosomes or MHC class II compartments for processing and binding to class II MHC. MUC1 uptake is mediated by the mannose receptor, and the protein is then retained long term in early endosomes without degradation. Long-term retention of MUC1 does not interfere with the ability of DC to process and present other Ags. We also demonstrate inhibited processing of another important glycoprotein tumor Ag, HER-2/neu. This may, therefore, be a frequent obstacle to presentation of tumor Ags and an important consideration in the design of cancer vaccines. It should be possible to overcome this obstacle by providing DC with a form of tumor Ag that can be better processed. For MUC1 we show that a 140-aa-long synthetic peptide is very efficiently processed by DC.

Amino Acid Sequence↗

PubMed2000

Empirical assessments of social networks, fertility and family planning programs: nonlinearities and their implications.

Empirical studies of the diffusion of modern methods of family planning have increasingly incorporated social interaction within nonlinear models such as logits. But they have not considered the full implications of these nonlinear specifications. This paper considers the implications of using nonlinear models in empirical analyses of the impact of family programs, modulated by social interaction, on reproductive behavior. Three implications of nonlinear models, in comparison with linear models, are developed. 1) With nonlinear models, there may be both low and high contraceptive-use equilibria (i.e., the ultimate level of use of modern family planning that a population can be expected to reach after the effects of a sustained change in a family planning program have worked through the population) rather than just one equilibrium as in linear models. If there are multiple equilibria, then one striking and important result is that a transitory large program effort may move a community from sustained low- to high-level contraceptive use. 2) With nonlinear models, the extent to which a social interaction multiplies program efforts depends on whether the community is at a low or high level of contraceptive use rather than being independent of the level of contraceptive use as in linear models. 3) With nonlinear models, intensified social interaction can retard or enhance the diffusion of family planning, in contrast to only enhancing diffusion as within linear models. To clarify these implications, for comparison a simple and more transparent linear model is also discussed. Illustrative estimates are presented of simple linear and nonlinear models for rural Kenya that demonstrate that some of these effects may be considerable.

Contraception↗

PubMed2000

The recruitment of Raf-1 to membranes is mediated by direct interaction with phosphatidic acid and is independent of association with Ras.

The serine/threonine kinase Raf-1 is an essential component of the MAPK cascade. Activation of Raf-1 by extracellular signals is initiated by association with intracellular membranes. Recruitment of Raf-1 to membranes has been reported to be mediated by direct association with Ras and by the phospholipase D product phosphatidic acid (PA). Here we report that insulin stimulation of HIRcB fibroblasts leads to accumulation of Ras, Raf-1, phosphorylated MEK, phosphorylated MAPK, and PA on endosomal membranes. Mutations that disrupt Raf-PA interactions prevented recruitment of Raf-1 to membranes, whereas disruption of Ras-Raf interactions did not affect agonist-dependent translocation. Expression of a dominant-negative Ras mutant did not prevent insulin-dependent Raf-1 translocation, but inhibited phosphorylation of MAPK. Finally, the PA-binding region of Raf-1 was sufficient to target green fluorescent protein to membranes, and its overexpression blocked recruitment of Raf-1 to membranes and disrupted insulin-dependent MAPK phosphorylation. These results indicate that agonist-dependent Raf-1 translocation is primarily mediated by a direct interaction with PA and is independent of association with Ras.

Animals↗

PubMed2000

Alterations in bcl-2 and caspase gene family protein expression in human temporal lobe epilepsy.

OBJECTIVE: To address the role of cell death regulatory genes of the bcl-2 and caspase families in the neuropathology of human epilepsy using tissue extracted from patients undergoing temporal lobectomy for intractable seizures. METHODS: Using Western blotting and immunohistochemistry, the authors investigated the expression of bcl-2, bcl-xL, bax, caspase-1,and caspase-3 in temporal cortex samples from patients who had undergone temporal lobectomy surgery for intractable epilepsy (n = 19). Nonepileptic postmortem tissue from a brain bank served as control (n = 6). RESULTS: Western blot analysis demonstrated significant increases in levels of bcl-2 and bcl-xL protein in seizure brain compared to control. Cleavage of caspase-1 was evidenced by a reduction in levels of the 45 kDa proenzyme form and an increase in levels of the p10 fragment. Levels of the 32 kDa proenzyme form of caspase-3 were elevated in seizure patients, as were levels of the 12 kDa cleaved fragment. Bcl-2, bax, and caspase-3 immunoreactivity was increased predominantly in cells with the morphologic appearance of neurons, whereas bcl-xL immunoreactivity was increased in cells with the appearance of glia. DNA fragmentation was detected in some but not all sections from epileptic brain samples. CONCLUSIONS: Cell death regulatory genes of the bcl-2 and caspase families may play a role in ongoing neuropathologic processes in human epilepsy, and offer novel targets as an adjunct to anticonvulsant therapy.

Adolescent↗

PubMed2000

CC chemokine receptor-7 on dendritic cells is induced after interaction with apoptotic tumor cells: critical role in migration from the tumor site to draining lymph nodes.

Dendritic cells (DCs) are very potent antigen-presenting cells and play critical roles in regulating immune responses in cancer. The migrating of DCs from the tumor site to the lymphoid organs is believed to be one of the critical events. To examine this important DC function in tumor situations, bone marrow-derived DCs, cultured for 6 days with granulocyte macrophage colony-stimulating factor and interleukin 4, were inoculated at the tumor site. We have shown (Y. Nishioka et al., Cancer Res., 59: 40354041, 1999) that DCs can migrate from tumor site to the draining lymph nodes within 24 h (approximately 0.1% of administrated DCs). The DCs then form clusters with adjacent lymphoid cells, which produce IFN-gamma (1500-3200pg/10(6) cells/48 h) in response to tumor stimulation. The number of the DCs migrating into lymph nodes were greater when they were inoculated into the tumor rather than the skin. Coculture of DCs and apoptotic tumor cells resulted in decreased expression of CC chemokine receptor (CCR) 1 and increased CCR7 expression at mRNA level without alteration in other phenotypical markers on DCs. Chemotaxis assay showed that CCR7 ligands, macrophage inflammatory protein 3beta and secondary lymphoid-tissue chemokine significantly (P < 0.05) induced the migration of DCs when cocultured with apoptotic tumor cells. To directly examine the involvement of CCR7 expression in DC migration, we investigated the functions of DCs genetically modified to express high levels of CCR7. CCR7 transduction promotes DC migration in response to relevant ligands in vitro and in vivo. These results suggest that the CCR7 expression of DCs is enhanced with direct contact with apoptotic tumor cells and may have a critical role for DC migrating to regional lymph nodes. The means to promote DC delivery to tumor and to nodal sites represent novel targets for the biological therapy of cancer.

Animals↗

PubMed2000

Constitutive activation of Stat3 signaling abrogates apoptosis in squamous cell carcinogenesis in vivo.

Field cancerization predisposes the upper aerodigestive tract mucosa to the formation of multiple primary tumors, when exposed to environmental carcinogens. Up-regulation of epidermal growth factor receptor occurs early in squamous cell carcinogenesis and is critical for the loss of growth control in a variety of human cancers, including head and neck squamous cell carcinomas. In these tumor cells in culture, epidermal growth factor receptor stimulation initiates signaling via persistent activation of selective STAT proteins. To determine the timing of Stat3 activation in head and neck carcinogenesis, we studied the expression and constitutive activation of Stat3 in tumors and normal mucosa from patients with head and neck cancer compared with mucosa from controls without cancer. Stat3 was up-regulated and constitutively activated in both primary human head and neck tumors as well as in normal mucosa from these cancer patients compared with control normal mucosa from patients without cancer. In vivo liposome-mediated gene therapy with a Stat3 antisense plasmid efficiently inhibited Stat3 activation, increased tumor cell apoptosis, and decreased Bcl-x(L) expression in a head and neck xenograft model. These findings provide evidence that constitutively activated Stat3 is an early event in head and neck carcinogenesis that contributes to the loss of growth control by an anti-apoptotic mechanism.

Animals↗

PubMed2000

Endothelial nitric oxide synthase protects aortic allografts from the development of transplant arteriosclerosis.

BACKGROUND: Inducible nitric oxide synthase (iNOS) is up-regulated in rejecting allografts and is protective against allograft arteriosclerosis; it suppresses neointimal smooth muscle cell accumulation and inhibits adhesion of platelets and leukocytes to the endothelium. However, the functional importance of endothelial NOS (eNOS) in the rejecting allografts remains unclear. METHODS: We examined the effects of selective eNOS deficiency in aortic allografts in a murine chronic rejection model using grafts from eNOS knockout (KO) mice (C57BL/6 background; H2b) and normal C3H (H2K) as recipients. Grafts from wild-type C57BL/6 mice served as controls. Grafts from iNOS KO mice served as a second group of controls where the contribution from iNOS was eliminated but eNOS was preserved. Aortic grafts were harvested and analyzed at days 10-14, 18-22, and 26-30 after transplantation. RESULTS: Endothelial NOS-deficient grafts showed significantly increased intima/media ratios at days 26-30 compared to controls. Immunostaining demonstrated that in eNOS KO grafts, eNOS was not detectable whereas iNOS was expressed prominently in infiltrating recipient mononuclear cells. In control grafts, eNOS expression was preserved in the endothelium even by day 30, and associated with a decrease in intimal thickening. We further demonstrated that early overexpression of iNOS by ex vivo gene transfer completely prevented the development of arteriosclerosis associated with eNOS deficiency. CONCLUSIONS: We found that eNOS plays a protective role in allografts, and that in eNOS-deficient allografts, early overexpression of iNOS is capable of preventing the development of allograft arteriosclerosis. In allografts with dysfunctional vascular endothelium and impaired eNOS activity as a result of ischemia or native arteriosclerotic disease, iNOS gene therapy may serve to improve their long-term survival and function.

Animals↗

PubMed2000

Maturation and trafficking of monocyte-derived dendritic cells in monkeys: implications for dendritic cell-based vaccines.

Human dendritic cells (DC) have polarized responses to chemokines as a function of maturation state, but the effect of maturation on DC trafficking in vivo is not known. We have addressed this question in a highly relevant rhesus macaque model. We demonstrate that immature and CD40 ligand-matured monocyte-derived DC have characteristic phenotypic and functional differences in vitro. In particular, immature DC express CC chemokine receptor 5 (CCR5) and migrate in response to macrophage inflammatory protein-1alpha (MIP-1alpha), whereas mature DC switch expression to CCR7 and respond exclusively to MIP-3beta and 6Ckine. Mature DC transduced to express a marker gene localized to lymph nodes after intradermal injection, constituting 1.5% of lymph node DC. In contrast, cutaneous DC transfected in situ via gene gun were detected in the draining lymph node at a 20-fold lower frequency. Unexpectedly, the state of maturation at the time of injection had no influence on the proportion of DC that localized to draining lymph nodes, as labeled immature and mature DC were detected in equal numbers. Immature DC that trafficked to lymph nodes underwent a significant up-regulation of CD86 expression indicative of spontaneous maturation. Moreover, immature DC exited completely from the dermis within 36 h of injection, whereas mature DC persisted in large numbers associated with a marked inflammatory infiltrate. We conclude that in vitro maturation is not a requirement for effective migration of DC in vivo and suggest that administration of Ag-loaded immature DC that undergo natural maturation following injection may be preferred for DC-based immunotherapy.

Adoptive Transfer↗

PubMed2000

Lyophilization of cationic lipid-protamine-DNA (LPD) complexes.

Cationic lipid-based gene delivery systems have shown promise in transfecting cells both in vitro and in vivo. However, these systems tend to form aggregates in liquid formulation during storage, which has limited their clinical applications. As a result, lyophilization of these systems has recently become a subject of increasing interest. In this paper, lyophilization of LPD, a novel cationic lipid-based gene delivery system, was studied. Both particle size and transfection efficiency could be preserved in the presence of sufficient amount of appropriate lyoprotectant. A series of monosaccharides and disaccharides, including dextrose, galactose, mannose, lactose, maltose, sucrose and trehalose, were evaluated for their lyoprotective effect and disaccharides showed more superior protection to monosaccharides. The effect of different freezing protocols for lyophilization was also evaluated and no significant difference was found. However, for freeze-thawing, fast freezing caused less aggregation. Finally, nonlyophilized LPD and LPD lyophilized with 10% sucrose were stored at different temperatures and their stability was followed for eight weeks. Lyophilized LPD could be stored at room temperature without significant change in particle size or loss of transfection efficiency.

Animals↗

PubMed2000

Quantitative biochemical and ultrastructural comparison of mitochondrial permeability transition in isolated brain and liver mitochondria: evidence for reduced sensitivity of brain mitochondria.

Opening of the mitochondrial permeability transition pore has increasingly been implicated in excitotoxic, ischemic, and apoptotic cell death, as well as in several neurodegenerative disease processes. However, much of the work directly characterizing properties of the transition pore has been performed in isolated liver mitochondria. Because of suggestions of tissue-specific differences in pore properties, we directly compared isolated brain mitochondria with liver mitochondria and used three quantitative biochemical and ultrastructural measurements of permeability transition. We provide evidence that brain mitochondria do not readily undergo permeability transition upon exposure to conditions that rapidly induce the opening of the transition pore in liver mitochondria. Exposure of liver mitochondria to transition-inducing agents led to a large, cyclosporin A-inhibitable decrease in spectrophotometric absorbance, a loss of mitochondrial glutathione, and morphologic evidence of matrix swelling and disruption, as expected. However, we found that similarly treated brain mitochondria showed very little absorbance change and no loss of glutathione. The absence of response in brain was not simply due to structural limitations, since large-amplitude swelling and release of glutathione occurred when membrane pores unrelated to the transition pore were formed. Additionally, electron microscopy revealed that the majority of brain mitochondria appeared morphologically unchanged following treatment to induce permeability transition. These findings show that isolated brain mitochondria are more resistant to induction of permeability transition than mitochondria from liver, which may have important implications for the study of the mechanisms involved in neuronal cell death.

Animals↗

PubMed2000

Expression of inducible nitric oxide synthase and interleukin-12 in experimental necrotizing enterocolitis.

BACKGROUND: Previous investigators have relied on administration of pro-inflammatory cytokines or invasive surgical procedures to reproduce the morphologic changes of necrotizing enterocolitis (NEC) in rats. However, these artificial insults do not mimic the human disease. We developed a reproducible model of NEC in rats that more closely resembles human NEC and determined the pattern of inflammatory cytokine expression in this model. MATERIALS AND METHODS: Newborn rats were randomized into four groups. Groups 1 and 2 were breast-fed, while Groups 3 and 4 were gavaged with formula thrice daily. In addition, Groups 2 and 4 were subjected to 3 min of hypoxia thrice daily, prior to each feeding. The rats were killed on day 4 and the distal 2 cm of terminal ileum was harvested for morphological studies and analysis of inflammatory cytokine mRNA expression. RESULTS: Nearly 70% of formula-fed neonatal rats displayed moderate or severe morphological abnormalities resembling human NEC. Breast-fed pups had normal histology. The terminal ileum from rats with abnormal histology demonstrated increased inducible nitric oxide synthase (iNOS) expression, decreased interleukin-12 (IL-12) mRNA expression, and enterocyte apoptosis. There was a trend toward upregulation of IFN-gamma mRNA, but no difference in expression of TNF-alpha mRNA. Hypoxia did not significantly alter intestinal morphology or mRNA expression. CONCLUSIONS: Formula-fed neonatal rats, with or without hypoxia, exhibit morphological changes in the intestinal epithelium similar to those seen in patients with acute NEC. The mechanism likely involves upregulation of iNOS mRNA, enterocyte apoptosis, and decreased IL-12 production in the intestinal epithelium. This model may offer a simple reproducible method for inducing experimental NEC.

Animals↗

PubMed2000

Nitric oxide induces angiogenesis and upregulates alpha(v)beta(3) integrin expression on endothelial cells.

Nitric oxide (NO) has been implicated as a mediator of angiogenesis. However, its precise role in angiogenesis and its mechanism of action have not been established. We performed in vivo and in vitro angiogenesis assays using NO donor S-nitroso-N-acetylpenicillamine (SNAP) and NO synthase inhibitor N-iminoethyl-l-ornithine (L-NIO). SNAP significantly increased and L-NIO significantly suppressed capillary ingrowth into subcutaneously implanted Matrigel plugs in mice. For the in vitro angiogenesis assay, human umbilical vein endothelial cells (HUVECs) (4 x 10(4) cells/well) were treated with placebo, SNAP (100 microM), or L-NIO (100 microM) and cultured on Matrigel for 18 h. The typical capillary networks formed on Matrigel by HUVECs as a result of cell migration and differentiation were quantified by computer-assisted image analysis as a measure of angiogenesis. Treatment of HUVECs with SNAP significantly increased the capillary network area compared with control, 8701 +/- 693 vs 6258 +/- 622 area units (P < 0.05), whereas L-NIO significantly decreased the capillary area (4540 +/- 342, P < 0.05). Furthermore, we have shown with a blocking monoclonal antibody that formation of capillary networks on Matrigel is mediated by the functional expression of the alpha(v)beta(3) integrin, which plays a role in facilitating endothelial cell adhesion to basement membrane matrix and endothelial cell migration. After an 18-h culture, flow cytometry revealed that SNAP significantly upregulated and L-NIO significantly downregulated in a concentration-dependent manner alpha(v)beta(3) integrin expression on endothelial cells. In conclusion, NO induces angiogenesis in vivo and in vitro by promoting endothelial cell migration and differentiation into capillaries. One possible mechanism might involve the upregulation of alpha(v)beta(3) integrin on endothelial cells, a critical mediator of cell-matrix adhesion and migration.

Animals↗