Biomedical subjects

E Ponte

Publications and source records attributed to E Ponte.

At least 19 recordsLinked to original sources

PubMed2003

Effects of protein kinase C inhibition and activation on proliferation and apoptosis of bovine retinal pericytes.

AIMS/HYPOTHESIS: Drop-out of capillary pericytes occurs early and selectively in diabetic retinopathy. High glucose concentrations decrease replication and increase apoptosis of cultured pericytes. Since glucose activates protein kinase C, we investigated the effects of modulating this intracellular mediator on replication, cell cycle and apoptosis of cultured bovine retinal pericytes. METHODS: Pericytes cultured in 5.6 or 28 mmol/l glucose were exposed to a protein kinase C activator (phorbol 12-myristate 13-acetate) and/or a selective inhibitor of its beta2 isoform (LY379196). Cells were counted after 7 days. Proliferation by the tetrazolium to formazan assay and DNA synthesis by 5-bromo-2'-deoxyuridine incorporation were measured at day 4. Cell cycle by flow cytometry and apoptosis by ELISA were assessed at day 2. RESULTS: High glucose reduced pericyte replication and increased apoptosis. Protein kinase C activation increased proliferation, while inhibition of its beta2 isoform decreased it. Cell cycle was accelerated by protein kinase C activation and delayed by inhibition. Apoptosis was enhanced by protein kinase C inhibition and reduced by activation. CONCLUSIONS/INTERPRETATION: Protein kinase C inhibition amplifies the anti-proliferative and pro-apoptotic effects of high glucose on cultured pericytes, whereas stimulation reduces apoptosis and promotes proliferation both in physiological glucose and high glucose. Protein kinase C inhibition, proposed for the treatment of diabetic macular edema and proliferative retinopathy, might accelerate pericyte dropout in earlier stages when these cells are still present in retinal capillaries.

Animals↗

PubMed2003

A study of capillary pericyte viability on extracellular matrix produced by endothelial cells in high glucose.

AIMS/HYPOTHESIS: Thickening of the basement membrane and selective loss of pericytes occur early in diabetic retinopathy. As we showed previously that pericyte adhesion is impaired on extracellular matrix produced by endothelial cells in high hexose concentrations, we aimed to verify if altered adhesion could influence pericyte viability and replication. METHODS: Conditioned extracellular matrices were obtained by growing human umbilical vein endothelial cells in media containing 28 mmol/l D-glucose, with or without the inhibitors of protein glycation thiamine or aminoguanidine, and D-galactose or L-glucose up to 28 mmol/l. Having removed the endothelium, bovine retinal pericytes were grown on these matrices and, in separate experiments, on laminin, fibronectin or type IV collagen. Pericyte viability and replication were measured by cell counts and DNA synthesis after 7 days, cell cycle traversal after 2 days and apoptosis after 18 h, 2 days and 7 days. RESULTS: Pericyte counts and DNA synthesis were reduced on matrices produced in high D-glucose and D-galactose, whilst matrix obtained in L-glucose reduced DNA synthesis but not counts. Both thiamine and aminoguanidine corrected reduced pericyte viability when added to high D-glucose. Cell cycle and apoptosis were not affected by growing pericytes on different conditioned matrices. Laminin, fibronectin and type IV collagen did not modify pericyte replication. CONCLUSIONS/INTERPRETATIONS: Reduced pericyte counts could depend on impaired initial adhesion to the extracellular matrix produced by endothelium in high hexose concentrations, rather than impaired replication or viability. Altered cell-matrix interactions might facilitate pericyte dropout in diabetic retinopathy, independently of the effects of high glucose on pericyte replication.

Apoptosis↗

PubMed2002

Pericyte adhesion is impaired on extracellular matrix produced by endothelial cells in high hexose concentrations.

AIMS/HYPOTHESIS: Thickening of the basement membrane and selective loss of pericytes are early events in diabetic retinopathy. We aimed at checking whether pericyte interaction with extracellular matrix produced by endothelial cells is influenced by the hexose concentrations in which endothelial cells are cultured. METHODS: Conditioned extracellular matrixes were obtained by growing human umbilical vein endothelial cells in media containing 28 mmol/l hexoses (D-glucose, D-galactose, L-glucose), which undergo different intracellular processing, before and after adding the inhibitors of protein glycation thiamine or aminoguanidine. Having removed the endothelium, bovine retinal pericytes were grown on such matrixes and, in separate experiments, on laminin, fibronectin or type IV collagen. Pericyte adhesion was determined by cell counts 18 h after seeding. RESULTS: Reduced adhesion was observed on matrixes produced in high D-glucose, high D-galactose and high L-glucose. Both thiamine and aminoguanidine restored impaired pericyte adhesion when added to high D-glucose and high D-galactose, but not L-glucose. Laminin, fibronectin and type IV collagen did not consistently modify pericyte adhesion. CONCLUSIONS/INTERPRETATIONS: Pericyte adhesion is impaired on extracellular matrix produced by endothelium in high hexose concentrations. This could result from excess protein glycation, corrected by aminoguanidine and thiamine, rather than altered glycoprotein composition.

Cell Adhesion↗

PubMed2002

Failure of angiotensin II and insulin to stimulate transforming growth factor-beta1. Release from cultured bovine retinal pericytes.

BACKGROUND: Activation of the renin-angiotensin system (RAS) may induce cardiovascular and renal fibrosis in hypertension and diabetes. This fibrogenic effect is mainly mediated by Transforming Growth Factor-B1 (TGF-B1), a multifunctional citokyne released by endothelial, vascular smooth muscle and renal mesangial cells, that is able to increase extracellular matrix deposition. Retinal capillary pericytes have functions similar to those of mesangial cells, including ability to synthesize and release TGF-B1 and produce extracellular matrix. An intraocular RAS was described in the human eye and may produce effects similar to those observed in the heart and kidney, which could be mediated by TGF-B1. In particular, TGF-B1 might be involved in thickening of the capillary basement membrane in diabetic microangiopathy. We therefore aimed at evaluating the possible effects of Angiotensin-II on TGF-B1 secretion by cultured retinal pericytes (BRP). METHODS: BRP cultures were incubated with Angiotensin-II or insulin (known to play a permissive effect on TGF-B1 release from mesangial cells) or Angiotensin-II + insulin at final concentrations of 10-10, 10-8, 10-6, 10-4 mol/L. RESULTS: Baseline TGF-B1 concentrations in the supernatants of pericyte cultures were 6 139 +/- 1 919 pg/mL/106 cells; no changes of TGF-B1 concentrations resulted from adding increasing amounts of Ang II, insulin or both. CONCLUSIONS: Though confirming that cultured bovine retinal pericytes spontaneously release TGF-B1, Angiotensin-II did not produce any stimulatory effects of in our experimental system

Analysis of Variance↗

PubMed2001

Diabetes mellitus and oral disease.

The oral complications of diabetes mellitus are generally considered to be no more than inevitable annoyances and, as such, have been the subject of relatively few scientific and clinical studies. This review highlights both the known correlations between oral disease and diabetes mellitus, such as those involving supporting tissues and oral mucosa, and those that are still a matter of debate, such as dental caries. In all cases, we carefully analyzed the pathogenesis of the disorder with a view to establishing a possible therapeutic approach, an aspect that currently appears to be overlooked. In view of the fact that the treatment of certain oral conditions, in particular periodontal disease, has been reported to improve metabolic control in diabetic patients, we conclude that diabetes mellitus-related oral diseases deserve adequate recognition and further investigation.

Dental Caries↗

PubMed2001

Addition and correction: the NF-kappa B-like DNA binding activity observed in Dictyostelium nuclear extracts is due to the GBF transcription factor.

We have previously reported that a NF-kappa B transduction pathway was likely to be present in the cellular slime mold Dictyostelium discoideum. This conclusion was based on several observations, including the detection of developmentally regulated DNA binding proteins in Dictyostelium nuclear extracts that bound to bona fide kappa B sequences. We have now performed additional experiments which demonstrate that the protein responsible for this NF-kappa B-like DNA binding activity is the Dictyostelium GBF (G box regulatory element binding factor) transcription factor. This result, along with the fact that no sequence with significant similarity to components of the mammalian NF-kappa B pathway can be found in Dictyostelium genome, now almost entirely sequenced, led us to reconsider our previous conclusion on the occurrence of a NF-kappa B signal transduction pathway in Dictyostelium.

Published Erratum↗

PubMed2001

Increased c-met expression during ductal beta cell neogenesis in experimental autoimmune diabetes.

C-met immunoreactivity and its co-expression with duct-associated insulin were evaluated in pancreata of non-obese diabetic (NOD) and low-dose streptozotocin (Id-STZ) mice. Diabetic NOD and non-diabetic NOD at the age of 4-8, 15-22 and 30-41 weeks and Balb/c mice at the same age intervals were studied. Ld-STZ mice were studied at day 12 and 24 after STZ administration. A stronger ductal c-met immunoreactivity and a significantly higher number of c-met positive ducts were found in diabetic NOD vs both non-diabetic NOD and Balb/c mice of comparable age. In non-diabetic NOD, the ductal c-met immunoreactivity progressively increased with age and was significantly higher than controls. In 1d-STZ mice a significantly increased ductal c-met immunoreactivity was detected both at day 12 and 24 vs untreated mice. C-met positive ductal cells were also positive for insulin although insulin positive c-met negative ducts were present. This study showed an increased c-met expression and the co-expression of c-met and duct-associated insulin, in both NOD and 1d-STZ mice.

Animals↗

PubMed2001

Caval thrombosis in a young athlete.

We here describe a clinical case of caval thrombosis of sudden and unexpected onset, in an athlete without signs of venous insufficiency. Pre-existing caval hypoplasia was observed through imaging examinations. Following a review of the literature, the authors interpret the pathophysiological mechanism of thrombosis as such: a combination of intense physical activity and the malformation led to an overwhelming venous flow and a consequent congestion. Caval thrombosis distal to the malformation is an absolute indication to lifelong oral anticoagulant therapy.

Adult↗

PubMed2001

Venous thromboembolism and melanoma.

The association between venous thromboembolism and cancer has been known for many years. The clotting system may be activated because of enhanced platelet aggregation and adhesion or by stimulation from the endothelial cells or the tumour cells themselves. Furthermore, thrombosis may develop as a result of prolonged immobilization, surgery, chemotherapy or hormone therapy. We present a case of a patient who underwent surgery for excision of a Clark level II melanoma on the back who developed pulmonary metastasis four years after the operation. The metastasis manifested with very severe venous thromboembolism which, despite anticoagulant therapy and the placement of a vena caval filter, led to the patient's death from pulmonary embolism. The case is uncommon, in terms of both the rarity with which venous thromboembolism is associated to melanoma, and the severity with which it manifested.

Humans↗

PubMed2000

Severe developmental defects in Dictyostelium null mutants for actin-binding proteins.

The actin cytoskeleton is implicated in many cellular processes, such as cell adhesion, locomotion, contraction and cytokinesis, which are central to any development. The extent of polymerization, cross-linking, and bundling of actin is regulated by several actin-binding proteins. Knock-out mutations in these proteins have revealed in many cases only subtle, if any, defects in development, suggesting that the actin system is redundant, with multiple proteins sharing overlapping functions. The apparent redundancy may, however, reflect limitations of available laboratory assays in assessing the developmental role of a given protein. By using a novel assay, which reproduces conditions closer to the natural ones, we have re-examined the effects of disruption of many actin-binding proteins, and show here that deletion of alpha-actinin, interaptin, synexin, 34-kDa actin-bundling protein, and gelation factor affect to varying degrees the efficiency of Dictyostelium cells to complete development and form viable spores. No phenotypic defects were found in hisactophilin or comitin null mutants.

Agar↗

PubMed2000

Cell-cell signaling and adhesion in phagocytosis and early development of Dictyostelium.

Cell-cell signaling and adhesion regulate transition from the unicellular to the multicellular stage of development in the cellular slime mold Dictyostelium. Essential gene networks involved in these processes have been identified and their interplay dissected. Heterotrimeric G protein-linked signal transduction plays a key role in regulating expression of genes mediating chemotaxis or cell adhesion, as well as coordinating actin-based cell motility during phagocytosis and chemotaxis. Two classes of cell adhesion molecules, one cadherin-like and the second belonging to the IgG superfamily, contribute to the strength of adhesion in Dictyostelium aggregates. The developmental role of genes involved in motility and adhesion, and their degree of redundancy, have been re-assessed by using novel developmental assay conditions which are closer to development in nature.

Animals↗

PubMed2000

Digital necrosis in a woman over eighty.

Chronic vascular disorders of the upper limbs are not frequently encountered in women; in addition, advanced age of onset tends to suggest the presence of common diseases such as arteriosclerosis or microembolization. Nonetheless, diagnosis is not always straightforward and in some cases both-laboratory and instrumental findings can do no more than arouse a diagnostic suspicion. We present a case of acro-localized disorder, which proved difficult to diagnose and which we believe may be attributed to a slowly progressing form of scleroderma of the extremities which, in our case, determined positivity of anticentromere autoantibodies.

Aged↗

PubMed1999

Atherosclerosis and inflammation. Patterns of cytokine regulation in patients with peripheral arterial disease.

Inflammatory phenomena at sites of atherosclerotic plaques are increasingly thought to be major determinants of the progression and clinical outcome of atherosclerotic disease. Therefore, attention is being paid to systemic markers/mediators which may reflect the inflammatory activity in the plaques. This study evaluates the pattern of the main proinflammatory cytokines tumor necrosis factor-alpha (TNFalpha), interleukin-1beta (IL-1beta), and interleukin-6 (IL-6), their soluble receptors/antagonist, and a variety of inflammatory markers, in patients with peripheral arterial disease (PAD). Eight patients with PAD suffering from claudicatio intermittens (CI), eight with critical limb ischemia (CLI) and eight controls (C) were studied. Blood samples were collected at baseline in all groups and. for C and CI, immediately after and 4 h after a 30-min treadmill test. Baseline: no differences in cytokine plasma levels were detected among the three groups. In contrast, soluble receptors of TNF (type I and II) and of IL-6, and IL-1beta receptor antagonist (IL-1ra) were increased in CI and CLI patients, as compared to C. Of note, IL-Ira correlated with the occurrence and stage of the disease in a highly significant proportion of the patients, reaching a predictive value for the disease of P < 0.0001. The opposite trend was observed for the soluble receptor of IL-1beta. Notably, in the patients no alterations could be found in white blood cell counts, expression of CD11c adherence molecule by circulating monocytes or, in vitro. O2- release from zymosan-activated neutrophils. Moreover, plasma levels of platelet activating factor (PAF), of neutrophil elastase and of the acute phase reactants C-reactive protein (CRP) and alpha1-acid glycoprotein were not found to be significantly altered. In contrast, the acute-phase proteins alpha1-antitrypsin (alpha1AT) and haptoglobin (HG) were found to be increased. Effect of treadmill: IL-1beta and TNFalpha remained at baseline levels following exercise, and IL-6 dropped to undetectable levels. Among cytokine antagonists, again the most relevant changes concerned the IL-1ra, which was significantly increased immediately after the treadmill test, both in CI and C, and returned to baseline levels after 4 h. In contrast, soluble TNFalpha, IL-1beta and IL-6 receptors, PAF, and the other markers of leukocyte activation were not found to be altered. Soluble TNFalpha and IL-6 receptors were shown to inhibit the biological effects of their ligands. Similarly, IL-1ra and the acute phase proteins alpha1AT and HG have been reported to exert anti-inflammatory functions. The increased plasma levels of these agents, together with low levels of inflammatory cytokines and other pro-inflammatory mediators such as PAF and alpha1-acid glycoprotein, appear to draw an undescribed picture, so far, of upregulation of a composite systemic anti-inflammatory mechanism in atherosclerotic patients. IL-1ra appears to be a reliable marker of the state of activation of this mechanism. These results may provide a basis for developing new insights into the pathogenesis of the atherosclerotic disease.

Acute-Phase Proteins↗

PubMed1999

Evidence for the presence of an NF-kappaB signal transduction system in Dictyostelium discoideum.

The Rel/NF-kappaB family of transcription factors and regulators has so far only been described in vertebrates and arthropods, where they mediate responses to many extracellular signals. No counterparts of genes coding for such proteins have been identified in the Caenorhabditis elegans genome and no NF-kappaB activity was found in Saccharomyces cerevisiae. We describe here the presence of an NF-kappaB transduction pathway in the lower eukaryote Dictyostelium discoideum. Using antibodies raised against components of the mammalian NF-kappaB pathway, we demonstrate in Dictyostelium cells extracts the presence of proteins homologous to Rel/NF-kappaB, IkappaB and IKK components. Using gel-shift experiments in nuclear extracts of developing Dictyostelium cells, we demonstrate the presence of proteins binding to kappaB consensus oligonucleotides and to a GC-rich kappaB-like sequence, lying in the promoter of cbpA, a developmentally regulated Dictyostelium gene encoding the Ca(2+)-binding protein CBP1. Using immunofluorescence, we show specific nuclear translocation of the p65 and p50 homologues of the NF-kappaB transcription factors as vegetatively growing cells develop to the slug stage. Taken together, our results strongly indicate the presence of a complete NF-kappaB signal transduction system in Dictyostelium discoideum that could be involved in the developmental process.

Animals↗

PubMed1999

Expression of Reg and cytokeratin 20 during ductal cell differentiation and proliferation in a mouse model of autoimmune diabetes.

OBJECTIVE: To evaluate the existence of beta-cell differentiation and proliferation in the low-dose streptozotocin (ld-STZ) mouse model of autoimmune diabetes. DESIGN: We studied the expression of Reg protein and cytokeratin 20 (CK20), the presence of proliferative phenomena (judged by the incorporation of bromodeoxyuridine (BrdU)), and the co-expression of Reg, CK20 or BrdU with insulin. MATERIALS AND METHODS: Diabetes was induced in male C57Bl6/J mice by administration of ld-STZ. The animals were killed at days 10 and 23 from the beginning of the induction of disease. Five animals were used at each time point and each group was evaluated for blood glucose concentrations, insulitis, expression of Reg and CK20 pancreatic proteins and BrdU incorporation, together with staining for insulin by immunohistochemistry and laser confocal microscopy. RESULTS: All mice treated with ld-STZ were hyperglycemic and histological investigation showed a mild or severe insulitis both at day 10 and at day 23. At day 10, immunochemistry revealed an intense expression of Reg and CK20 in pancreatic ducts in ld-STZ mice, but not in control mice. Reg and CK20 immunoreactive cells were also positive for insulin. In contrast, at day 23, pancreatic sections reacted weakly with anti-Reg and anti-CK20 antibody; co-localization with insulin was observed for both Reg and CK20. The incorporation of BrdU was observed only in insulin-positive cells in pancreatic sections from mice killed at day 10. CONCLUSIONS: These observations show an islet regeneration mechanism in response to an autoimmune attack, and that the ld-STZ mouse is a suitable model in which to evaluate intervention strategies.

Animals↗

PubMed1999

Insulin prophylaxis down-regulates islet antigen expression and islet autoimmunity in the low-dose Stz mouse model of diabetes.

The aims of this study were to evaluate in an autoimmune diabetes animal model [low-dose streptozotocin (LD-STZ) mouse] (a) the efficacy of a prophylactic insulin treatment as a diabetes prevention tool, and (b) its possible mechanisms through both the insulitis evaluation and islets antigen expression. Diabetes was induced in male C57Bl6/J mice with STZ (50 mg/kg b/w for five consecutive days); insulin (1 U/day) was injected subcutaneously for ten consecutive days before the induction of diabetes and for a further ten days. Seventy-one male C57Bl6/J mice were grouped as follows: Group 1 (n = 25) made diabetic with i.p. STZ, Group 2 (n = 21) made diabetic with i.p. STZ and injected subcutaneously with insulin, Group 3 (n = 15) injected with insulin, while Group 4 (n = 10) comprised normal animals as controls. The animals of each group were killed at two intervals: half of them at day 12 and the remainder at day 24 from the beginning of the STZ treatment. A significant reduction of glycemia levels and insulitis severity was observed between mice of Group 1 vs. Group 2 at day 12 and day 24. Down-regulation of islet antigen expression (insulin, A2B5, GM2-1, ICA Ag) was achieved even without a complete metabolic suppression of beta-cell activity. In conclusion, prophylactic insulin treatment is effective to reduce glycemia levels and insulitis severity and down-regulates islet antigen expression in the LD-STZ model.

Animals↗

PubMed1999

[Advanced stage vasculopathies of the lower extremities. Medical and management aspects in amputees].

AIM: The paper reports the experience of an operating unit for the rehabilitation of amputees. The unit includes doctors and physiatrists working at the 6th Division of Internal Medicine and the Centre for Motor Rehabilitation at the "Ospedali Riuniti" in Trieste. METHODS: Subjects were selected on the basis of their psychophysical characteristics and coexistent pathologies. All subjects took part in a hospitalised rehabilitation programme lasting for 60-90 days. This period was followed by outpatient controls that continued for one year after discharge. Clinical conditions, self-sufficiency and quality of life were monitored and studied using special questionnaires. RESULTS: Of a total of 45 amputees, 27 were not regarded as suitable and 9 died while in hospital. Eighteen were selected for prosthesis; 14 completed the programme and 11 were followed-up for one year as planned. One patient did not attend the follow-up, 1 patient had a stroke and 1 patient died from myocardial infarction. CONCLUSIONS: Eleven of the 45 amputees included in the study were successfully treated with prothesis. The medical, nursing and economic commitment appears to have been considerable. The expectations of all subjects were high and remained high during rehabilitation in hospital, but deteriorated rapidly at home with the onset of depressive type symptoms. This may have been due to disappointment following their high expectations and the fact that post-discharge assistance was not always appropriate. The high cost-benefit ratio underlines the importance of the careful selection of candidates for rehabilitation.

Amputation, Surgical↗

PubMed1998

Detection of subtle phenotypes: the case of the cell adhesion molecule csA in Dictyostelium.

Dictyostelium amoebae aggregate into a multicellular organism by cAMP-driven chemotaxis and cell-cell adhesion. Cell adhesion is mediated by an EDTA-sensitive and an EDTA-resistant adhesion system. The latter is developmentally regulated and triggered by homophilic interactions of the membrane glycoprotein csA; on disruption of the encoding gene, EDTA-resistant contacts fail to form. Nevertheless, csA-null cells under usual laboratory conditions aggregate normally and complete development. By using experimental conditions that reproduce more closely the habitat of Dictyostelium amoebae, evidence is provided that csA is required for development and that its expression confers a selective advantage to populations of wild-type cells over csA-null mutants. The latter display reduced cell-cell adhesion, increased adhesiveness to the substratum, and slower motility, which lead to their sorting out from aggregating wild-type cells. It is proposed that the experimental conditions commonly used in the laboratory are not stringent enough to assess the developmental role of csA and other proteins. The assay described can be used to detect subtle phenotypes, to reexamine the developmental role of apparently nonessential genes, and to test the validity of recent models on emergence and maintenance of apparent genetic redundancy.

Animals↗