Biomedical subjects

Julia M Polak

Publications and source records attributed to Julia M Polak.

15 recordsLinked to original sources

PubMed2003

Effect of nitric oxide donor nitroglycerin on bone mineral density in a rat model of estrogen deficiency-induced osteopenia.

Nitric oxide (NO) may modulate estrogen's anabolic effects on bone homeostasis by restraining osteoclast-mediated bone resorption and stimulation of osteoblast activity. Accordingly, NO donated by organic nitrates, including nitroglycerin, is thought to protect against bone loss associated with estrogen deficiency. In this study, we have explored this phenomenon. Thirty-two 12-week-old female Wistar rats were divided into four groups prior to bilateral ovariectomy or a sham operation. The ovariectomised rats received (1). vehicle control (OVX control), (2). 17-beta-estradiol (OVX+E2), or (3). transdermal nitroglycerin (OVX+NG) for 4 weeks. Femoral and tibial bone mineral density (BMD), serum alkaline phosphatase and urine deoxypyridinoline and NO metabolites were analysed at the end of the study period together with failure torque and torsional rigidity of the tibiae and cellular localisation of the NO-synthase (NOS) isoforms. In OVX+E2 group, proximal and distal femoral and proximal tibial BMD exceeded that of the Sham controls. Nitroglycerin prevented BMD loss at these three sites at levels comparable to that of the Sham controls. Deoxypyridinoline excretion did not change except in the OVX-E2 group that showed an expected reduction when compared to the Sham and OVX controls. There were no treatment-related differences in total alkaline phosphatase or urinary NO metabolites. Tibial failure torque was comparable between the groups but both OVX+E2 and OVX+NG groups showed decreased torsional rigidity compared with the OVX controls. Endothelial and inducible NOS were found in osteoblast-like cells associated with calcifying cartilage spicules in the distal femoral metaphysis. These data confirm previous findings and show that nitroglycerin counteracts the estrogen deficiency-induced osteopenia in the ovariectomised rat model. Organic nitrates may thus be beneficial in conditions where bone turnover is compromised such as in osteoporosis.

Animals↗

PubMed2003

Induction of inducible nitric oxide synthase, argininosuccinate synthase, and GTP cyclohydrolase I in arthritic joints of human tumor necrosis factor-alpha transgenic mice.

OBJECTIVE: Transgenic mice that express human tumor necrosis factor-alpha (Tg197 h-TNF-alpha) develop polyarthritis at 3 to 4 weeks of age leading to severe joint destruction at 8 to 10 weeks of age. Studies have suggested that inducible nitric oxide synthase (iNOS) activity can modulate the progression of arthritis. We investigated the induction of iNOS together with argininosuccinate synthase (AS) and GTP cyclohydrolase I (GTPCH), 2 of the rate-limiting enzymes for high output NO generation, in the Tg197 h-TNF-alpha transgenic model of arthritis. METHODS: We used 4 and 8-week-old Tg197 h-TNF-alpha transgenic mice and wild-type CBA C57B1/6 control mice to investigate the expression of iNOS with respect to that of AS, GTPCH, and 3-nitrotyrosine by quantitative RT-PCR and immunocytochemistry. Urinary NO metabolites were analyzed using a chemiluminescence assay. RESULTS: Inducible NOS, AS, and GTPCH mRNA was found in all study groups; however, only iNOS mRNA showed a clear increase in 4-week-old Tg197 h-TNF-alpha transgenics in comparison to age matched wild-type controls. Abundant iNOS protein expression was found in macrophages and vascular smooth muscle cells in hyperplastic synovium and pannus. AS expression was found in vascular endothelium and fibroblasts of the inflammatory synovium and pannus. GTPCH immunoreactivity was mostly restricted to macrophages in inflammatory synovium. Localization of 3-nitrotyrosine overlapped with that of iNOS, indicating formation of reactive nitrogen species. Consistent with the high output NO generation, there was a 5-fold increase in urinary NO metabolites in 8-week-old Tg197 h-TNF-alpha transgenic mice. CONCLUSION: We characterized the Tg197 h-TNF-alpha transgenic model of inflammatory arthritis in terms of high output NO-generating pathway, and showed that both AS and GTPCH are intimately associated with inflammatory arthritis. The concomitant induction of AS and GTPCH with that of iNOS suggests that they may be important modulators of arthritis, and that they may represent novel targets for modulation of disease activity.

Animals↗

PubMed2002

Nitric oxide in the development of obliterative bronchiolitis in a heterotopic pig model.

BACKGROUND: Inflammation, epithelial cell injury, and development of fibrosis and airway obliteration are the major histological features of posttransplant obliterative bronchiolitis (OB). The expression of inducible nitric oxide synthase (iNOS) in the damaged epithelium, accompanied by peroxynitrite, suggests that endogenous nitric oxide (NO) mediates the epithelial destruction preceding obliteration. To elucidate the role of NO in this cascade, heterotopic bronchial allografts were studied in pigs. METHODS: Allografts or autografts were harvested serially 3-90 days after transplantation and processed for histology and immunocytochemistry for iNOS, nitrotyrosine, a marker of peroxynitrite formation, and superoxide dismutase (SOD). RESULTS: During initial ischemic damage to the epithelium, iNOS, nitrotyrosine, and SOD were found to be strongly expressed in the epithelium of all implants as well as later, after partial recovery, parallel to onset of epithelial destruction and subsequent airway obliteration in allografts. The levels of expression of iNOS in fibroblasts during the early phase of obliteration paralleled the onset of fibrosis. Constant expression of iNOS and SOD, but not nitrotyrosine, occurred in autografts and allografts with blocked alloimmune response. CONCLUSIONS: These findings suggest that an excessive amount of NO promotes posttransplant obliterative bronchiolitis by destroying airway epithelium and stimulating fibroblast activity. SOD may provide protection by binding reactive molecules and preventing peroxynitrite formation.

Animals↗

PubMed2002

Cloning and tissue distribution of three murine alpha/beta hydrolase fold protein cDNAs.

We have cloned 3 novel murine cDNAs encoding proteins containing an alpha/beta hydrolase fold; a catalytic domain found in a very wide range of enzymes. These proteins belong to the prosite UPF0017 uncharacterized protein family and we have named them lung alpha/beta hydrolase 1, 2, and 3 (LABH) since they were cloned from lung cDNA. All have 9 coding exons, encoding 412, 425, and 411 residue proteins respectively (46-48 kDa); LABH1 being closely related to LABH3 having 45% identity. All 3 proteins have a single predicted amino-terminus transmembrane domain. An alignment of family members from different phyla enabled the identification of the LABH1 catalytic triad as Ser211, Asp337, and His366. mRNA expression levels of LABH1 and 3 were highest in liver and LABH2 highest in testis. These findings suggest that the LABH proteins consist of a novel family of membrane bound enzymes whose function has yet to be determined.

Amino Acid Sequence↗

PubMed2002

Third-generation biomedical materials.

Whereas second-generation biomaterials were designed to be either resorbable or bioactive, the next generation of biomaterials is combining these two properties, with the aim of developing materials that, once implanted, will help the body heal itself.

Absorbable Implants↗

PubMed2002

ET(A) and ET(B) receptors modulate the proliferation of human pulmonary artery smooth muscle cells.

We determined the distribution of ET(A) and ET(B) receptors in pulmonary arteries from pulmonary hypertensive patients and control subjects, using in vitro autoradiography, and investigated their role in mediating the proliferative effects of endothelin-1 (ET-1) on distal human pulmonary artery smooth muscle cells (PASMCs). Distal arteries possessed more medial [(125)I]-ET-1 binding sites (105 +/- 10 versus 45 +/- 6 amol/mm(2); p < 0.001) and a greater proportion of ET(B) receptors than proximal arteries (36 +/- 3% versus 3 +/- 1%; p < 0.001). Receptor density in distal arteries and lung parenchyma was twofold greater (p < 0.05) in pulmonary hypertensive patients than in control subjects. ET-1 (10(-9)-10(-7) mol/L) stimulated DNA synthesis (147 +/- 10% of control subjects; p < 0.05) and attenuated the antiproliferative action of cicaprost and forskolin on PASMCs, these effects being mediated via ET(A) and ET(B) receptors. Serum-stimulated proliferation was attenuated by inhibiting either endogenous ET-1 release with phosphoramidon (10(-5) mol/L) or its action with PD145065 (10(-5) mol/L). Cicaprost (10(-10)-10(-7) mol/L) inhibited ET-1 release from PASMCs (49 +/- 16% of control after 24 h; p < 0.001) and increased intracellular cAMP levels, whereas ET(B) receptor stimulation selectively reduced cAMP levels. In conclusion, ET(A) and ET(B) receptors are differentially distributed in human pulmonary arteries. Both receptors promote the proliferation of PASMCs in vitro and may contribute to vascular remodeling in pulmonary hypertension.

Adult↗

PubMed2002

iNOS gene upregulation is associated with the early proliferative response of human lung fibroblasts to cytokine stimulation.

Increased release of oxidants has been implicated in the pathogenesis of pulmonary fibrosis. Previous work in the rat showed that formation of the early fibrotic lesion is associated with increased expression of inducible nitric oxide synthase (iNOS) in pulmonary fibroblasts. The aim of this study was to test the hypothesis that NO is involved in the activation of pulmonary fibroblasts. The effects of endogenous and exogenous NO on proliferation of human pulmonary fibroblasts were investigated by administration of cytomix or SNAP, respectively. At low concentrations, both treatments increased cell numbers, an effect attenuated by iNOS inhibitor or NO scavenger. Induction of iNOS was confirmed by measurement of nitrate/nitrite production and by immunodetection. Quantitative RT-PCR showed an increase in iNOS mRNA as early as 3 h after stimulation. These results support the hypothesis and show that upregulation of the iNOS gene is an early event in the proliferative response of human lung fibroblasts to inflammatory stimuli.

Blotting, Western↗

PubMed2002

Embryonic stem cells.

The capacity of embryonic stem cells for virtually unlimited self-renewal and differentiation capacity has opened up the prospect of widespread applications in biomedical research and regenerative medicine. For the latter, the cells provide hope that it will be possible to overcome the problems of donor tissue shortage and also, by making the cells immunocompatible with the recipient, implant rejection. Four years after the first derivation of human pluripotent cell lines from pre-implantation embryos, a great deal has been learnt about their biology and how differentiation can be encouraged towards particular cell lineages. However, considerable research is needed, not least into means to enrich and purify derivative cell lineages, before clinical trials can be considered.

Cell Differentiation↗

PubMed2002

Immunocytochemical evidence for inducible nitric oxide synthase and cyclooxygenase-2 expression with nitrotyrosine formation in human hibernating myocardium.

BACKGROUND: Myocardial hibernation may result from repetitive episodes of transient ischaemia leading to prolonged dysfunction. Inducible nitric oxide synthase (iNOS) expression has been demonstrated in animals following brief, non-lethal ischaemia-reperfusion injury. We therefore, hypothesised that in human hibernating myocardium: 1). iNOS would be present; 2). the reaction of nitric oxide and superoxide would form the strong oxidant peroxynitrite; 3) that this process would be accompanied by the expression of cyclooxygenase-2 (Cox-2) which interacts with NOS and whose products could further affect myocardial function. METHOD AND RESULTS: In sixteen patients with coronary artery disease (CAD), left ventricular biopsies were obtained from chronically dysfunctional segments subtended by a stenotic artery (> 75 %) and shown to be viable by (18)F-fluorodeoxyglucose positron emission tomography. Comparison was made with myocardial biopsies (n = 8) from normally contracting myocardium in patients undergoing coronary surgery, from unused transplant donors and at post-mortem. Regional wall motion score improved in all patients 6 months post-revascularisation (from 2.7 +/- 0.7 to 1.5 +/- 0.5; p < 0.001), confirming hibernation. Immunocytochemistry localized reactivity to iNOS, Cox-2 and nitrotyrosine (a marker of peroxynitrite formation) to cardiomyocytes from hibernating segments. No difference in reactivity to endothelial NOS was seen between hibernating and control cardiomyocytes. CONCLUSION: Cox-2 and iNOS are co-expressed in hibernating myocardium with nitrotyrosine suggesting nitric oxide production and peroxynitrite formation. We propose that this is secondary to ischaemia-reperfusion and that the products of these enzymes may have consequences for myocardial contractile function and survival.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed2002

Reciprocal age-related changes in GAP-43/B-50, substance P and calcitonin gene-related peptide (CGRP) expression in rat primary sensory neurones and their terminals in the dorsal horn of the spinal cord and subintima of the knee synovium.

Age-related changes in the expression of the growth associated protein GAP-43/B-50, and the neuropeptides substance P and calcitonin gene-related peptide (CGRP) were investigated in the sensory neurones of rat dorsal root ganglia, dorsal horns of the spinal cord and subintimal knee synovium. The two time-points studied were 2 months (young adults) and 14-month (aged)-old Sprague Dawley rats. Dorsal root ganglia: In young adults, 40 and 35% of the L4-L5 dorsal root ganglion neurones were positive for GAP-43/B-50 with a 1.5 fold increase in frequency in aged rats at the L5 ganglion. GAP-43/B-50 was strongly expressed by the non-neuronal satellite cells of some medium and many large sized neurones in aged rats. There were marked reciprocal shifts between small and medium sized sensory neurones in respect to their substance P and CGRP expression profiles. Dorsal horn of the spinal cord: there was a 1.3 fold decrease of substance P at L5 level and a 1.3 and 1.5 fold decrease of CGRP at L4-L5 levels in aged rats, respectively. Synovial membrane: There was a 2.3 fold increase in GAP-43/B-50 and a 2.5 fold decrease of CGRP with no changes in substance P expression. These results indicate that (i) primary sensory neurones undergo age-related changes already in early stages of aging, (ii) aging may result in a reduction of substance P and CGRP axonal transport, and (iii) reduced numbers of CGRP containing synovial perivascular fibres may imply a deficient regulation of the synovial microvasculature and therefore metabolic homeostasis of the joint in aged subjects.

Aging↗

PubMed2002

Derivation of type II alveolar epithelial cells from murine embryonic stem cells.

Embryonic stem (ES) cell pluripotency is being investigated increasingly to obtain specific cell lineages for tissue engineering. However, the possibility that ES cells can give rise to lung tissue has not been tested. We hypothesized that lung epithelial cells (type II pneumocytes) can be derived in vitro from murine ES cells. After withdrawal of leukemia inhibitory factor (LIF) and formation of embryoid bodies in maintenance medium for 10, 20, and 30 days, differentiating ES cells were kept in the same medium or transferred to serum-free small airway growth medium (SAGM) for a further 3 or 14 days of culture. The presence of type II pneumocytes in the resulting mixed cultures was demonstrated by reverse transcriptase-polymerase chain reaction (RT-PCR) of surfactant protein C (SPC) mRNA, immunostaining of SPC, and electron microscopy of osmiophilic lamellar bodies only at 30 days sampling time. SAGM appeared to be more favorable for type II cell formation than ES medium. No SPC transcripts were found in differentiating cells grown under the same conditions without formation of embryoid bodies. These findings could form the basis for the enrichment of ES cell-derived cultures with type II pneumocytes, and provide an in vitro system for investigating mechanisms of lung repair and regeneration.

Animals↗

PubMed2002

Differentiating embryonic stem cells: GAPDH, but neither HPRT nor beta-tubulin is suitable as an internal standard for measuring RNA levels.

Embryonic stem (ES) cells are pluripotent cell lines that possess virtually unlimited self-renewal and differentiation capacity. Such characteristics make them potentially an invaluable cell source for diverse tissue-engineering applications. In vitro ES cell differentiation occurs spontaneously in three-dimensional structures termed "embryoid bodies" that mimic postimplantation embryonic tissue. HPRT, beta-tubulin, and GAPDH are commonly used as internal RNA standards in ES cell-derived gene transcription studies so that corrected sample mRNA levels can be obtained for (semi) quantitative gene expression data. However, if reliable data is to be obtained, it is essential that such housekeeping gene expression remains constant, and this has not been demonstrated for differentiating ES cell cultures, which represent a mixed and changing population of cells with time in culture. Therefore, in the present study, we tested the suitability of these housekeeping genes to act as true internal standards for differentiating murine ES cells cultured as embryoid bodies. PCR-amplified gene-specific products were quantified from digital images of ethidium bromide-stained gels using a computer software package. Both HPRT and beta-tubulin mRNA levels varied markedly in spontaneously differentiating and growth factor-supplemented (TGF-beta) ES cell cultures (p < 0.001, ANOVA), while GAPDH expression remained relatively constant (p > 0.2). Our results demonstrate the importance of fully validating housekeeping gene expression in in vitro ES cell gene transcription studies and suggest that GAPDH may be a suitable candidate to act as an internal RNA standard, while both HPRT and beta-tubulin appear to be inappropriate. Finally, we demonstrate enhanced mesodermal differentiation of ES cell-derived cultures by treatment with TGF-beta through significant upregulation of Brachyury T expression, with a concomitant decrease in expression of the undifferentiated ES cell marker Oct-4.

Animals↗

PubMed2002

Tissue engineering and ENT surgery.

Tissue engineering is the development of biological substitutes for the repair and regeneration of damaged tissues. We explain the principles of this emerging field of biotechology. The present and potential applications of tissue engineering technologies in ENT surgery are then reviewed.

Animals↗

PubMedDate not supplied

Pelvic nerve plexus trauma at radical and simple hysterectomy: a quantitative study of nerve types in the uterine supporting ligaments.

OBJECTIVE: Using neuropeptide and enzyme markers to autonomic nerves, we sought to demonstrate and quantify the nerve types contained within the uterosacral ligaments (USLs) and cardinal ligaments (CLs) that are divided during radical hysterectomy (RH). METHODS: Cross-sectional biopsies were collected from the lateral third of the USL and the CL in 24 women who had an RH for cervical cancer, and from the uterine insertion of these ligaments in 11 women who had a simple hysterectomy for benign disease. We applied indirect immunofluorescence with FITC-conjugated secondary antibodies, using polyclonal primary antibodies to neuropeptide markers that predominate within somatic and autonomic nerves, to show different populations of the following nerve types within the biopsies: neuropeptide Y (NPY) and tyrosine hydroxylase (TH) for sympathetic nerves; vasoactive intestinal polypeptide (VIP) for parasympathetic nerves; substance P (SP) for nociceptive and sensory-motor nerves; and calcitonin gene-related peptide (CGRP) for sensory and sensory-motor nerves. The percentage area of immunoreactivity (PAI), determined by a computer-assisted image analyzer attached to a fluorescent microscope, was used as an objective quantitative measure of nerve density. Confocal microscopy was used to determine the composition and spatial arrangement of nerve fibers in the ligaments. RESULTS: The PAI was greater for all markers tested in both the USL and CL (P <.001) in RH compared with simple hysterectomy biopsies. For RH specimens, the PAI was greater for the sympathetic, sensory, and sensory-motor nerve markers in the USL compared with the CL (P <.01), but the PAI for VIP was similar (P >.05). Conversely, excluding the large trunks and associated ganglia, the free nerve fiber PAI in the CL was greater than that of the USL for all nerve markers (P <.001). The staining of peripheral autonomic ganglia and associated fibers, for NPY and TH, indicates that some sympathetic nerves are preganglionic with their cell bodies within the pelvic plexus. CONCLUSIONS: Significantly more autonomic nerves are transected in the more lateral division of the uterine supporting ligaments during a radical hysterectomy than during a simple hysterectomy. Sympathetic, parasympathetic, sensory, and sensory-motor nerve types are present within the CL and USL. The proportions of each nerve type differ between the two ligaments, and sympathetic nerves in the USL are the single largest nerve type. The uterine supporting ligaments are a major pathway for autonomic nerves to the pelvic organs.

Biopsy↗

PubMedDate not supplied

Cigarette smoke decreases inducible nitric oxide synthase in lung epithelial cells.

Cigarette smoking has been associated with decreased exhaled nitric oxide (NO). To investigate the mechanism of this decrease, the effects of a cigarette smoke extract were evaluated a murine lung epithelial cell line (LA-4), a human lung epithelial cell line (A549), and primary cultures of human lung epithelial cells induced to produce NO by cytokines. NO production was evaluated by measuring nitrite, a stable end product of NO, in cell culture supernatant fluids. Cigarette smoke extract caused a reduction in the cytokine-induced nitrite concentrations in the culture supernatant fluids from all 3 cell types (P < .01, all comparisons). To further investigate these observations, immunohistochemistry demonstrated a decrease in cytokine-induced inducible NO synthase (iNOS) protein expression and iNOS mRNA after cigarette smoke extract exposure in LA-4 cells. However, iNOS mRNA half-life was not altered by the smoke extract, suggesting that the smoke extract decreased NO by decreasing iNOS mRNA transcription. These findings demonstrate that cigarette smoke extract decreases iNOS expression and NO production from lung epithelial cells.

Animals↗