Modulation of the baboon (Papio anubis) uterine endometrium by chorionic gonadotrophin during the period of uterine receptivity

This study was undertaken to determine the modulation of uterine function by chorionic gonadotrophin (CG) in a nonhuman primate. Infusion of recombinant human CG (hCG) between days 6 and 10 post ovulation initiated the endoreplication of the uterine surface epithelium to form distinct epithelial plaques. These plaque cells stained intensely for cytokeratin and the proliferating cell nuclear antigen. The stromal fibroblasts below the epithelial plaques stained positively for α-smooth muscle actin (αSMA). Expression of αSMA is associated with the initiation of decidualization in the baboon endometrium. Synthesis of the glandular secretory protein glycodelin, as assessed by Western blot analysis, was markedly up-regulated by hCG, and this increase was confirmed by immunocytochemistry, Northern blot analysis, and reverse transcriptase-PCR. To determine whether hCG directly modulated these uterine responses, we treated ovariectomized baboons sequentially with estradiol and progesterone to mimic the hormonal profile of the normal menstrual cycle. Infusion of hCG into the oviduct of steroid-hormone-treated ovariectomized baboons induced the expression of αSMA in the stromal cells and glycodelin in the glandular epithelium. The epithelial plaque reaction, however, was not readily evident. These studies demonstrate a physiological effect of CG on the uterine endometrium in vivo and suggest that the primate blastocyst signal, like the blastocyst signals of other species, modulates the uterine environment prior to implantation.

Cloning, expression, and distribution of a Ca(2+)-activated K+ channel beta-subunit from human brain.

We have cloned and expressed a Ca(2+)-activated K+ channel beta-subunit from human brain. The open reading frame encodes a 191-amino acid protein possessing significant homology to a previously described subunit cloned from bovine muscle. The gene for this subunit is located on chromosome 5 at band q34 (hslo-beta). There is no evidence for alternative RNA splicing of this gene product. hslo-beta mRNA is abundantly expressed in smooth muscle, but expression levels are low in most other tissues, including brain. Brain subregions in which beta-subunit mRNA expression is relatively high are the hippocampus and corpus callosum. The coexpression of hslo-beta mRNA together with hslo-alpha subunits in either Xenopus oocytes or stably transfected HEK 293 cells give rise to Ca(2+)-activated potassium currents with a much increased calcium and/or voltage sensitivity. These data indicate that the beta-subunit shows a tissue distribution different to that of the alpha-subunit, and in many tissues there may be no association of alpha-subunits with beta-subunits. These beta-subunits can play a functional role in the regulation of neuronal excitability by tuning the Ca2+ and/or the voltage dependence of alpha-subunits.

Induction of cytotoxic T lymphocytes by intramuscular immunization with plasmid DNA is facilitated by bone marrow-derived cells.

Striated muscle is the predominant site of gene expression after i.m. immunization of plasmid DNA, but it is not clear if myocytes or professional antigen-presenting cells (APCs) of hematopoietic origin present the encoded antigens to class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL). To address this issue, CTL responses were assessed in mice engrafted with immune systems that were partially MHC matched with antigen-producing muscle cells. Spleen cells (sc) from immunocompetent F1 H-2bxd mice were infused into H-2b or H-2d mice carrying the severe combined immunodeficiency (scid) mutation, creating F1sc-->H-2b and F1sc-->H-2d chimeras, respectively. Immunization with DNA plasmids encoding the herpes simplex virus gB or the human immunodeficiency virus gp120 glycoproteins elicited antiviral CTL activity. F1sc-->H-2d chimeras responded to an H-2d-restricted gp120 epitope but not an H-2b restricted gB epitope, whereas F1sc-->H-2b chimeras responded to the H-2b but not the H-2d restricted epitope. This pattern of epitope recognition by the sc chimeras indicated that APCs of recipient (scid) origin were involved in initiation of CTL responses. Significantly, CTL responses against epitopes presented by the mismatched donor class I molecules were elicited if F1 bone marrow cells and sc were transferred into scid recipients before or several days to weeks after DNA immunization. Thus, bone marrow-derived APCs are sufficient for class I MHC presentation of viral antigens after i.m. immunization with plasmid DNA. Expression of plasmid DNA by these APCs is probably not a requirement for CTL priming. Instead, they appear to present proteins synthesized by other host cells.

Role of the p21 cyclin-dependent kinase inhibitor in limiting intimal cell proliferation in response to arterial injury.

Arterial injury induces a series of proliferative, vasoactive, and inflammatory responses that lead to vascular proliferative diseases, including atherosclerosis and restenosis. Although several factors have been defined which stimulate this process in vivo, the role of specific cellular gene products in limiting this response is not well understood. The p21 cyclin-dependent kinase inhibitor affects cell cycle progression, senescence, and differentiation in transformed cells, but its expression in injured blood vessels has not been investigated. In this study, we report that p21 protein is induced in porcine arteries following balloon catheter injury and suggest that p21 is likely to play a role in limiting arterial cell proliferation in vivo. Vascular endothelial and smooth muscle cell growth was arrested through the ability of p21 to inhibit progression through the G1 phase of the cell cycle. Following injury to porcine arteries, p21 gene product was detected in the neointima and correlated inversely with the location and kinetics of intimal cell proliferation. Direct gene transfer of p21 using an adenoviral vector into balloon injured porcine arteries inhibited the development of intimal hyperplasia. Taken together, these findings suggest that p21, and possibly related cyclin-dependent kinase inhibitors, may normally regulate cellular proliferation following arterial injury, and strategies to increase its expression may prove therapeutically beneficial in vascular diseases.

Immunologic basis of transplant-associated arteriosclerosis.

Although immunosuppressive therapy minimizes the risk of graft failure due to acute rejection, transplant-associated arteriosclerosis of the coronary arteries remains a significant obstacle to the long-term survival of heart transplant recipients. The participation of specific inflammatory cell types in the genesis of this lesion was examined in a mouse model in which carotid arteries were transplanted across multiple histocompatibility barriers into seven mutant strains with immunologic defects. An acquired immune response--with the participation of CD4+ (helper) T cells, humoral antibody, and macrophages--was essential to the development of the concentric neointimal proliferation and luminal narrowing characteristic of transplant arteriosclerosis. CD8+ (cytotoxic) T cells and natural killer cells were not involved in the process. Arteries allografted into mice deficient in both T-cell receptors and humoral antibody showed almost no neointimal proliferation, whereas those grafted into mice deficient only in helper T cells, humoral antibody, or macrophages developed small neointimas. These small neointimas and the large neointimas of arteries grafted into control animals contained a similar number of inflammatory cells; however, smooth muscle cell number and collagen deposition were diminished in the small neointimas. Also, the degree of inflammatory reaction in the adventitia did not correlate with the size of the neointima. Thus, the reduction in neointimal size in arteries allografted into mice deficient in helper T cells, humoral antibody, or macrophages may be accounted for by a decrease in smooth muscle cell migration or proliferation.

Myofibroblasts differentiate from fibroblasts when plated at low density.

Myofibroblasts, defined by their expression of smooth muscle alpha-actin, appear at corneal and dermal incisions and promote wound contraction. We report here that cultured fibroblasts differentiate into myofibroblasts by a cell density-dependent mechanism. Fibroblasts seeded at low density (5 cells per mm2) produced a cell culture population consisting of 70-80% myofibroblasts, 5-7 days after seeding. In contrast, fibroblasts seeded at high density (500 cells per mm2) produced cultures with only 5-10% myofibroblasts. When the myofibroblast-enriched cultures were subsequently passaged at high density, the smooth muscle alpha-actin phenotype was lost within 3 days. Furthermore, initially 60% of the low density-cultured cells incorporated BrdUrd compared to 30% of cells passaged at high density. Media from myofibroblast-enriched cultures had more latent and active transforming growth factor beta (TGF-beta) than did media from fibroblast-enriched cultures. Although there was a trend towards increased numbers of myofibroblasts after addition of exogenous TGF-beta, the results did not reach statistical significance. We conclude that myofibroblast differentiation can be induced in fibroblasts by plating at low density. We propose a cell density-dependent model of myofibroblast differentiation during wounding and healing in which at least two factors interact: loss of cell contact and the presence of TGF-beta.

Cocaine: mechanism of inhibition of a muscle acetylcholine receptor studied by a laser-pulse photolysis technique.

Effects of cocaine on the muscle nicotinic acetylcholine receptor were investigated by using a chemical kinetic technique with a microsecond time resolution. This membrane-bound receptor regulates signal transmission between nerve and muscle cells, initiates muscle contraction, and is inhibited by cocaine, an abused drug. The inhibition mechanism is not well understood because of the lack of chemical kinetic techniques with the appropriate (microsecond) time resolution. Such a technique, utilizing laser-pulse photolysis, was recently developed; by using it the following results were obtained. (i) The apparent cocaine dissociation constant of the closed-channel receptor form is approximately 50 microM. High carbamoylcholine concentration and, therefore, increased concentrations of the open-channel receptor form, decrease receptor affinity for cocaine approximately 6-fold. (ii) The rate of the receptor reaction with cocaine is at least approximately 30-fold slower than the channel-opening rate, resulting in a cocaine-induced decrease in the concentration of open receptor channels without a concomitant decrease in the channel-opening or -closing rates. (iii) The channel-closing rate increases approximately 1.5-fold as the cocaine concentration is increased from 20 to 60 microM but then remains constant as the concentration is increased further. The results are consistent with a mechanism in which cocaine first binds rapidly to a regulatory site of the receptor, which can still form transmembrane channels. Subsequently, a slow step (t1/2 approximately 70 ms) leads to a receptor form that cannot form transmembrane channels, and acetylcholine receptor-mediated signal transmission is, therefore, blocked. Implications for the search for therapeutic agents that alleviate cocaine poisoning are mentioned.

Parkinson's disease and gastrointestinal dysfunctions: morphological, neurochemical and functional modifications of the enteric nervous system associated with central dopaminergic impairment

Parkinson’s disease (PD) is frequently associated with gastrointestinal (GI) symptoms, mostly represented by abdominal distension, constipation and defecatory dysfunctions. Despite GI dysfunctions have a major impact on the clinical picture of PD, there is currently a lack of information on the neurochemical, pathological and functional correlates of GI dysmotility associated with PD. Moreover, there is a need of effective and safe pharmacological therapies for managing GI disturbances in PD patients. The present research project has been undertaken to investigate the relationships between PD and related GI dysfunctions by means of investigations in an animal model of PD induced by intranigral injection of 6-hydroxydopamine (6-OHDA). The use of the 6-OHDA experimental model of PD in the present program has allowed to pursue the following goals: 1) to examine the impact of central dopaminergic denervation on colonic excitatory cholinergic and tachykininergic neuromotility by means of molecular, histomorphologic and functional approaches; 2) to elucidate the role of gut inflammation in the onset and progression of colonic dysmotility associated with PD, characterizing the degree of inflammation and oxidative damage in colonic tissues, as well as identifying the immune cells involved in the production of pro-inflammatory cytokines in the gut; 3) to evaluate the impact of chronic treatment with L-DOPA plus benserazide on colonic neuromuscular activity both in control and PD animals. The results suggest that central nigrostriatal dopaminergic denervation is associated with an impaired excitatory cholinergic neurotransmission and an enhanced tachykininergic control, resulting in a dysregulated smooth muscle motor activity, which likely contributes to the concomitant decrease in colonic transit rate. These motor alterations might result from the occurrence of a condition of gut inflammation associated with central intranigral denervation. The treatment with L-DOPA/BE following central dopaminergic neurodegeneration can restore colonic motility, likely through a normalization of the cholinergic enteric neurotransmission, and it can also improve the colonic inflammation associated with central dopaminergic denervation.

Inflamação na aterosclerose : impacto na doença coronária

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Implications physiopathologiques de la Nestine lors du remodelage pulmonaire et cardiaque à la suite de l’infarctus du myocarde, du diabète et de l’hypertension pulmonaire

Il est reconnu que la protéine filamenteuse intermédiaire Nestine est exprimée lors du processus de cicatrisation et du remodelage fibrotique. De plus, nous avons identifié l’expression de la Nestine au sein de deux populations distinctes qui sont directement impliquées dans les réponses de fibroses réparative et réactive. Ainsi, une population de cellules souches neurales progénitrices résidentes du coeur de rat adulte exprime la Nestine et a été identifiée à titre de substrat de l’angiogenèse et de la neurogenèse cardiaque. Également, la Nestine est exprimée par les myofibroblastes cicatriciels cardiaques et il a été établi que la protéine filamenteuse intermédiaire joue un rôle dans la prolifération de ces cellules. Ainsi, l’objectif général de cette thèse était de mieux comprendre les évènements cellulaires impliqués dans la réponse neurogénique des cellules souches neurales progénitrices résidentes cardiaques Nestine(+) (CSNPRCN(+)) lors de la fibrose réparative cardiaque et d’explorer si l’apparition de fibroblastes Nestine(+) est associée avec la réponse de fibrose réactive secondaire du remodelage pulmonaire. Une première publication nous a permis d’établir qu’il existe une régulation à la hausse de l’expression de la GAP43 (growth associated protein 43) et que cet événement transitoire précède l’acquisition d’un phénotype neuronal par les CSNPRCN(+) lors du processus de cicatrisation cardiaque chez le rat ayant subi un infarctus du myocarde. De plus, la surimposition de la condition diabétique de type 1, via l’injection unique de Streptozotocine chez le rat, abolit la réponse neurogénique des CSNPRCN(+), qui est normalement induite à la suite de l’ischémie cardiaque ou de l’administration de 6-hydroxydopamine. Le second article a démontré que le développement aigu de la fibrose pulmonaire secondaire de l’infarctus du myocarde chez le rat est associé avec une augmentation de l’expression protéique de la Nestine et de l’apparition de myofibroblastes pulmonaires Nestine(+). Également, le traitement de fibroblastes pulmonaires avec des facteurs de croissances peptidiques pro-fibrotiques a augmenté l’expression de la Nestine par ces cellules. Enfin, le développement initial de la condition diabétique de type 1 chez le rat est associé avec une absence de fibrose réactive pulmonaire et à une réduction significative des niveaux protéiques et d’ARN messager de la Nestine pulmonaire. Finalement, la troisième étude représentait quant à elle un prolongement de la deuxième étude et a alors examiné le remodelage pulmonaire chronique chez un modèle établi d’hypertension pulmonaire. Ainsi, les poumons de rats adultes mâles soumis à l’hypoxie hypobarique durant 3 semaines présentent un remodelage vasculaire, une fibrose réactive et une augmentation des niveaux d’ARN messager et de la protéine Nestine. De plus, nos résultats ont démontré que la Nestine, plutôt que l’alpha-actine du muscle lisse, est un marqueur plus approprié des diverses populations de fibroblastes pulmonaires activés. Également, nos données suggèrent que les fibroblastes pulmonaires activés proviendraient en partie de fibroblastes résidents, ainsi que des processus de transition épithélio-mésenchymateuse et de transition endothélio-mésenchymateuse. Collectivement, ces études ont démontré que des populations distinctes de cellules Nestine(+) jouent un rôle majeur dans la fibrose réparative cardiaque et la fibrose réactive pulmonaire.

Runx2 and Vascular Calcification

Thesis (Ph.D.)--University of Washington, 2016-06

Advances in vascular tissue engineering

Coronary and peripheral artery bypass grafting is commonly used to relieve the symptoms of vascular deficiencies, but the Supply Of autologous artery or vein may not be sufficient or suitable for multiple bypass or repeat procedures, necessitating the use of other materials. Synthetic materials are suitable for large bore arteries but often thrombose when used in smaller arteries. Suitable replacement grafts must have appropriate characteristics, including resistance to infection, low immunogenicity and good biocompatability and thromboresistance, with appropriate mechanical and physiological properties and cheap and fast manufacture. Current avenues of graft development include coating synthetic grafts with either biological chemicals or cells with anticoagulatory properties. Matrix templates or acellular tubes of extracellular matrix (such as collagen) may be coated or infiltrated with cultured cells. Once placed into the artery, these grafts may become colonised by host cells and gain many of the properties of normal artery. Tissue-engineered blood vessels may also be formed from layers of human vascular cells grown in culture. These engineered vessels have many of the characteristics of arteries formed in vivo. Artificial arteries may be also be derived from peritoneal granulation tissue in body bioreactors by adapting the body's natural wound healing response to produce a hollow tube. (C) 2003 Elsevier Inc. All rights reserved.

Leukaemia inhibitory factor retards the progression of atherosclerosis

Objective: Our previous studies showed that the pleiotropic cytokine leukaemia inhibitory factor (LIF) inhibits the de novo formation of experimental atherosclerotic lesions. The present study examined whether LIF also inhibits progression of pre-existing atheroma. Methods: Balloon angioplasty was performed on the right carotid arteries of 18 rabbits immediately before placing animals on a cholesterol-enriched diet. After 4 weeks, at which time the intima:media ratio (IN) was 0.99+/-0.12 (n=6), osmotic minipumps containing LIF (n=6) or saline control n=6) were inserted into the peritoneal cavity of each of the remaining rabbits for a further 4 weeks. Arteries were then harvested for analysis. Results: Continuous administration of LIF for the final 4 weeks of an 8-week cholesterol-enriched diet completely inhibited lesion progression in injured carotid arteries (I:M 1.05+/-0.16) compared with the saline-treated group at 8 weeks (1.62+/-0.13; P

Discovery of an MIT-like atracotoxin family: Spider venom peptides that share sequence homology but not pharmacological properties with AVIT family proteins

This project identified a novel family of six 66-68 residue peptides from the venom of two Australian funnel-web spiders, Hadronyche sp. 20 and H. infensa: Orchid Beach (Hexathelidae: Atracinae), that appear to undergo N- and/or C-terminal post-translational modifications and conform to an ancestral protein fold. These peptides all show significant amino acid sequence homology to atracotoxin-Hvf17 (ACTX-Hvf17), a non-toxic peptide isolated from the venom of H. versuta, and a variety of AVIT family proteins including mamba intestinal toxin 1 (MIT1) and its mammalian and piscine orthologs prokineticin 1 (PK1) and prokineticin 2 PK2). These AVIT family proteins target prokineticin receptors involved in the sensitization of nociceptors and gastrointestinal smooth muscle activation. Given their sequence homology to MITI, we have named these spider venom peptides the MIT-like atracotoxin (ACTX) family. Using isolated rat stomach fundus or guinea-pia ileum organ bath preparations we have shown that the prototypical ACTX-Hvf17, at concentrations up to 1 mu M, did not stimulate smooth muscle contractility, nor did it inhibit contractions induced by human PK1 (hPK1). The peptide also lacked activity on other isolated smooth muscle preparations including rat aorta. Furthermore, a FLIPR Ca2+ flux assay using HEK293 cells expressing prokineticin receptors showed that ACTX-Hvf17 fails to activate or block hPK1 or hPK2 receptors. Therefore, while the MIT-like ACTX family appears to adopt the ancestral disulfide-directed beta-hairpin protein fold of MIT1, a motif believed to be shared by other AVIT family peptides, variations in the amino acid sequence and surface charge result in a loss of activity on prokineticin receptors. (c) 2005 Elsevier Inc. All rights reserved.

Agonists and antagonists of protease activated receptors (PARs)

Protease activated receptors (PARs) are a category of G-protein coupled receptors (GPCRs) implicated in the progression of a wide range of diseases, including thrombosis, inflammatory disorders, and proliferative diseases. Signal transduction via PARs proceeds via an unusual activation mechanism. Instead of being activated through direct interaction with an extracellular signal like most GPCRs. they are self-activated following cleavage of their extracellular N-terminus by serine proteases to generate a new receptor N-terminus that acts as an intramolecular ligand by folding back onto itself and triggering receptor activation. Short synthetic peptides corresponding to this newly exposed N-terminal tethered ligand can activate three of the four known PARs in the absence of proteases. and such PAR activating peptides (PAR-APs) have served as templates for agonist/antagonist development. In fact much of the evidence for involvement of PARs in diseases has relied upon use of PAR-APs. often of low potency and uncertain selectivity. This review summarizes current structures of PAR agonists and antagonists, the need for more selective and more potent PAR ligands that activate or antagonize this intriguing class of receptors, and outlines the background relevant to PAR activation, assay methods, and physiological properties anticipated for PAR ligands.